TW201247219A

TW201247219A - Method for inhibiting Src kinase activity

Info

Publication number: TW201247219A
Application number: TW100118337A
Authority: TW
Inventors: Jian-Wei Chen; Ching-Hsien Chen; Sung-Liang Yu; Gee-Chen Chang
Original assignee: Univ Nat Chunghsing
Priority date: 2011-05-25
Filing date: 2011-05-25
Publication date: 2012-12-01
Also published as: TWI418360B

Abstract

A method for inhibiting Src kinase activity is disclosed. The method includes the following steps. A Src kinase which contains SH2, SH3 and kinase domain is provided. A peptide that contains amino acid Y172 and P301/P304 shown as SEQ ID NO: 1 is utilized to react with the Src kinase. The peptide specifically binds to kinase and SH3 domain of Src kinase and suppresses Src kinase activity.

Description

201247219 六、發明說明：【發明所屬之技術領域】本發明是有關於一種抑制酪胺酸激酶的方法，特別是有關於一種抑制Src酪胺酸激酶的方法。【先前技術】癌症轉移（metastasis)是導致癌症病患死亡的主要原因，其是為癌細胞經由侵襲(invasion)周遭正常組織，亦或經由體内循環及淋巴系統而轉移到身體其他部位。癌細胞侵襲是轉移的起始步驟，非侵襲性的腫瘤細胞經由降低細胞黏附因子，失去細胞極性並使細胞骨架重組而造成上皮· 間葉轉變作用（epithelial-mesenchymal transition，EMT)，成為具轉移能力的癌細胞。在生物體中’許多訊息傳遞路徑包括Src (由sarcoma 一詞而來）赂胺酸激酶(Src kinase)等的活化，會導致特定的轉錄因子大量表現而引發上皮·間葉轉變作用，造成癌細胞的轉移而使癌症加重。若能了解癌症轉移的機制，將能提供癌症治療重要之發展。目前已發現Src酪胺酸激酶是促使癌症轉移的重要因子之一，並使其成為癌症治療上的標靶分子。在許多種癌細胞中’特別是高度轉移癌症’ Src酪胺酸激酶的表現量和活性有增高的情形。Src酪胺酸激酶的活性會受到§rc結合蛋白質（Src-binding proteins)所調控，而大部分的調控是增進其活性，卻很少有蛋白質是抑制Src酪胺酸激酶的活性。因此，至今並未發展出專一性阻斷其活性的抗癌藥物，進而使許多藥物抗癌效果不佳並且導致副作用產生。 201247219 【發明内容】因此’本發明提供專一性私j , 具有抑癌活性的胺基酸序列。卩制Sn^氨酸激酶活性與本發明之-態樣是在提供— 序列辨識編號1所示之序列的㈣ ^ , 的第172個胺基酸、第301個胺基酸與第304個胺基酸所構成 ^ lL L A 苒成之二級結構，且此多肽係專一性結合至Src酪氨酸激醢夕、紅a广紙微啤之教酶區與Src同源3區（Src homology 3 ; SH3)。依據本七明之-實施方式，其中上述之加赂氨酸激酶包括細胞型Src酪氨酸激酶或病毒型如酪氨酸激酶。，本發明之另-態樣是在提供—種抑制Μ祕酸激酶活性的方法。此方法包含提供Src赂胺酸激酶，纟包含如同源2區（Src homology 2; SH2)、Src同源3區以及激酶區，利用如序列辨識編號丨所示序列之多肽與Src酪胺酸激酶於〇_4〇〇C反應5分鐘至16小時，使此多肽的第172個胺基酸、第301個胺基酸與第3〇4個胺基酸所構成之三級結構專性結合至激酶區以及Src同源3區，藉以抑制此Src 酷·胺酸激酶之活性。依據本發明之一實施方式，其中上述之Src酪氨酸激酶〇括細胞型Src赂氨酸激酶或病毒型src酷氨酸激酶。依據本實施方式之一實施例，其中上述之Src酪胺酸激酶活性為一受質之磷酸化反應。依據本實施方式之另一實施例，其中上述之多肽與Src 赂胺酸激酶於反應12小時。、 201247219 本發明之又一態樣是在提供一種抑制癌細胞之上皮_ 間質轉化作用的方法。包含構築一重組質體，其包含如序列辨識編號2所示的去氧核糖核酸片段，將此重組質體導入癌細胞，以獲得一轉型株。此去氧核糖核酸片段在轉型株中產生如序列辨識編號1所示之多肽，該多肽包含如序列辨識編號1所示之序列的第172個胺基酸、第301個胺基酸與第304個胺基酸所構成之三級結構，此多肽抑制轉型株中Src酪氨酸激酶之磷酸化活性，進而抑制上皮_間質轉化作用。依據本發明之一實施方式，其中癌細胞為肺癌細胞。本發明之再一態樣是在提供一種藥學組成物，包含上述所述之多肽以及一藥學上可接受之載體。本發明之又一態樣是在提供一種癌細胞轉移性之檢驗方法。包含檢測一待測細胞之類DnaJ熱休克蛋白（DnaJ-like heat shock protein ; HLJ1)之第172個胺基酸之磷酸化程度，以及檢測類DnaJ熱休克蛋白之第301個胺基酸與第 304個胺基酸之胺基酸種類’以獲得待測細胞之一構酸化程度以及二胺基酸種類。接著，將待測細胞之磷酸化程度與一健康細胞之一參考磷酸化程度比較，以及將二胺基酸種類之任一者與健康細胞之一參考胺基酸種類比較，以獲得一比較結果。然後，判斷比較結果，其中當磷酸化程度係高於之參考磷酸化程度’且二胺基酸種類之任一者係不同於前述之參考胺基酸種類，則判斷待測細胞具有癌細胞轉移性。根據上述可知，本發明提出可專一性結合至Src酪胺 201247219 酸激酶之三級結構結合區域，此結構具有進行藥物分子模擬及合成的抗癌藥開發的潛力。【實施方式】本發明鑑定出類DnaJ熱休克蛋白（HLJ1)及Src酪氨酸激酶在癌細胞中的交互作用與其胺基酸結合區域。其顯示類DnaJ熱休克蛋白之第172個胺基酸(係為酪胺酸，本文以Y172表示）與第301個和304個胺基酸(係皆為脯胺酸，本文以P301/P304表示）可專一性結合Src酪氨酸激酶並抑制其活化。首先利用微陣列（microarray)與電腦預測分析尋得類 DnaJ熱休克蛋白與Src酪氨酸激酶可能有交互作用之關係，再利用免疫共沉澱(Immunoprecipitation)、麵胺基硫轉移酶(glutathione S transferase; GST)沉殿試驗等技術鑑定出類DnaJ熱休克蛋白及Src酪氨酸激酶在癌細胞中的交互作用與其胺基酸結合區域。類DnaJ熱休克蛋白為熱休克蛋白40 (heat shock protein 40 ; Hsp40)的一員，胺基酸序列如序列辨識編號i 所示，其為腫瘤抑制蛋白並與癌細胞侵襲的能力有關，可以抑制腫瘤的生長與轉移。類DnaJ熱休克蛋白之Y172與 P301/P304胺基酸分子所形成的立體結構（空間）能專一性地分別結合至Src酷·乱酸激酶的激酶區（kinase)和§rc同源 3區（SH3)，此交互作用可以抑制src酪氨酸激酶的活性而干擾其填酸化下游分子的能力，包括局部黏附激酶(focal adhesion kinase ; FAK)與 β_連環蛋白（(3_catenin)，進而阻斷 201247219201247219 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to a method for inhibiting tyrosine kinase, and more particularly to a method for inhibiting Src tyrosine kinase. [Prior Art] Metastases are the main cause of death in cancer patients, and they are transferred to normal tissues of the cancer cells by invasion, or to other parts of the body via circulation and lymphatic system. Invasion of cancer cells is the initial step of metastasis. Non-invasive tumor cells become metastatic by reducing cell adhesion factors, losing cell polarity and recombining the cytoskeleton to cause epithelial-mesenchymal transition (EMT). The ability of cancer cells. In many organisms, many message transmission pathways, including Src (from the word sarcoma), activate Src kinase, which leads to a large number of specific transcription factors that trigger epithelial-mesenchymal transition and cause cancer. The transfer of cells makes the cancer worse. Understanding the mechanisms of cancer metastasis will provide important developments in cancer treatment. Src tyrosine kinase has been found to be one of the important factors contributing to cancer metastasis and has become a target for cancer therapy. In many cancer cells, the expression and activity of Src tyrosine kinase, particularly high-metastatic cancer, are increased. The activity of Src tyrosine kinase is regulated by §rc-binding proteins, and most of the regulation is to increase its activity, while few proteins inhibit the activity of Src tyrosine kinase. Therefore, anticancer drugs which specifically block their activities have not been developed so far, and many drugs have poor anticancer effects and cause side effects. 201247219 SUMMARY OF THE INVENTION Therefore, the present invention provides an amino acid sequence having a specific cancer suppressing activity. The succinylkinic acid, the 301th amino acid and the 304th amine are provided in the sequence of (4)^, which provides the sequence shown in SEQ ID NO: 1. The base acid constitutes the secondary structure of the lL LA ,, and the polypeptide is specifically bound to the Src tyrosine stimulating enzyme, the red a wide paper micro beer and the Src homology 3 region (Src homology 3 ; SH3). According to the embodiment of the present invention, wherein the above-described kallikrein kinase comprises a cell type Src tyrosine kinase or a viral type such as a tyrosine kinase. In another aspect of the invention, there is provided a method of inhibiting the activity of a scorpion acid kinase. The method comprises providing a Src citrate kinase comprising a region such as a homologous 2 region (Src homology 2; SH2), a Src homolog 3 region, and a kinase region, and a polypeptide having a sequence such as the sequence number 丨 and a Src tyrosine kinase The reaction is carried out at 〇4〇〇C for 5 minutes to 16 hours, and the tertiary structure of the 172th amino acid, the 301th amino acid and the 3rd 4th amino acid of the polypeptide is specifically bound to The kinase domain and the Src homology 3 region are thereby inhibited the activity of this Src-cool acid kinase. According to an embodiment of the present invention, the above Src tyrosine kinase comprises a cell type Src kinase or a viral src tyrosine kinase. According to an embodiment of the present invention, the above Src tyrosine kinase activity is a substrate phosphorylation reaction. According to another embodiment of the present invention, the polypeptide is reacted with Src citrate kinase for 12 hours. 201247219 A further aspect of the invention provides a method of inhibiting epithelial-mesenchymal transition of cancer cells. A recombinant plasmid comprising a deoxyribonucleic acid fragment as shown in SEQ ID NO: 2 is introduced, and the recombinant plastid is introduced into a cancer cell to obtain a transformed strain. This DNA fragment produces a polypeptide as shown in SEQ ID NO: 1 in a transformed strain comprising the 172th amino acid, 301th amino acid and 304th of the sequence shown in SEQ ID NO: 1. The tertiary structure consisting of amino acids inhibits the phosphorylation activity of Src tyrosine kinase in the transformed strain, thereby inhibiting epithelial-mesenchymal transition. According to an embodiment of the invention, the cancer cell is a lung cancer cell. A further aspect of the invention provides a pharmaceutical composition comprising the polypeptide described above and a pharmaceutically acceptable carrier. Yet another aspect of the present invention is to provide a test method for cancer cell metastasis. The method comprises the steps of detecting the phosphorylation degree of the 172th amino acid of DnaJ-like heat shock protein (HLJ1), and detecting the 301th amino acid of the DnaJ heat shock protein. The amino acid type ' of 304 amino acids' is such that the degree of acidification of one of the cells to be tested and the type of diamine acid are obtained. Next, the degree of phosphorylation of the test cell is compared with the degree of reference phosphorylation of one of the healthy cells, and any one of the diamine acid species is compared with the reference amino acid species of one of the healthy cells to obtain a comparison result. . Then, judging the comparison result, wherein when the degree of phosphorylation is higher than the reference phosphorylation degree' and any one of the diamine acid species is different from the aforementioned reference amino acid species, it is judged that the test cell has cancer cell metastasis Sex. Based on the above, the present invention proposes a specific binding to the tertiary structural binding region of Src tyramine 201247219 acid kinase, which has the potential to develop anticancer drugs for drug molecular modeling and synthesis. [Embodiment] The present invention identifies an interaction between a DnaJ-like heat shock protein (HLJ1) and a Src tyrosine kinase in cancer cells and an amino acid binding region thereof. It shows the 172th amino acid of DnaJ-like heat shock protein (which is tyrosine, which is represented by Y172) and the 301th and 304 amino acids (all of which are valerine, which is represented by P301/P304 It can specifically bind to Src tyrosine kinase and inhibit its activation. Firstly, microarray and computer predictive analysis were used to find out the possible interaction between DnaJ heat shock protein and Src tyrosine kinase, and then use immunoprecipitation and glutathione S transferase. GST) Shen Dian test and other techniques identified the interaction of DnaJ-like heat shock proteins and Src tyrosine kinases in cancer cells with their amino acid binding regions. The DnaJ-like heat shock protein is a member of heat shock protein 40 (Hsp40), and the amino acid sequence is shown in sequence identification number i, which is a tumor suppressor protein and is involved in the ability of cancer cells to invade and inhibit tumors. Growth and transfer. The steric structure (space) formed by the Y172 and P301/P304 amino acid molecules of the DnaJ heat shock protein can specifically bind to the kinase and Src homology 3 regions of Src cool acid kinase, respectively. SH3), this interaction can inhibit the activity of src tyrosine kinase and interfere with its ability to acidate downstream molecules, including focal adhesion kinase (FAK) and β_catenin ((3_catenin), which blocks 201247219

Src酪氨酸激酶的訊息傳遞作用。類DnaJ熱休克蛋白的 Y172可與Src酪氨酸激酶之激酶區域結合而被Src酪氨酸激酶誘導磷酸化，此種結合可以降低Src酪氨酸激酶所調控的癌細胞侵襲作用，對類DnaJ熱休克的抑癌活性相當重要；同時，類DnaJ熱休克蛋白的p3〇1/p3〇4具有Src同源 3區結合序列（binding m〇tif)，不僅能夠抑制Src酪氨酸激酶的活化，也能干擾Src酪氨酸激酶與其他受質的交互作用，進而阻斷Src酪氨酸激酶在癌細胞内的訊息傳遞。類 DnaJ熱休克蛋白藉此結合序列，在立體空間上可專一性地結合至Src路氨酸激酶，降低癌細胞上皮_間質轉化作用。本發明提供專一性抑制Src酪氨酸激酶活性與具有抑癌活性的胺基酸序列，可將此序列應用於藥物合成，進行分子模擬’發展出類似於序列辨識編號1所示之γΐ72與 Ρ301/Ρ304分子結構的化合物或多肽，具有專一性的Src酪氨酸激酶結合能力，既能有效抑制Src酪氨酸激酶的活性與訊息傳遞，又能減少抗癌藥物的副作用，進而開發新的抗癌藥物及其衍生物。除非特別指明’本發明所指抑制Src酪胺酸激酶活性，其包含細胞型Src酪胺酸激酶(cellular Src ; c-Src)和病毒型 Src酪胺酸激酶(Virai Src ; v_Src)。細胞型Src酪胺酸激酶廣泛存在於真核生物組織中，包含有三個功能區塊，分別是Src同源3區（SH3)、Src同源2區（SH2)和激酶(kinase) 區。而病毒型Src酪胺酸激酶為突變型Src酪胺酸激酶，其結構與細胞型Src酷·胺酸激酶相似，然而病毒型§rc赂胺酸激酶缺乏一可抑制其活性的胺基酸，使得病毒型Src 酪胺酸激酶呈現持續活化的狀態，因此若在正常細胞大量 201247219 表現此致癌蛋白（oncogenic protein) ’可使細胞轉變成為癌化細胞。於此亦提供一抑制癌細胞轉移的分子路徑，而可應用於檢驗癌細胞之轉移性’包括檢測一待測細胞之類DnaJ 熱休克蛋白之第172個胺基酸之磷酸化程度’以及檢測該類DnaJ熱休克蛋白之第301個胺基酸與第304個胺基酸之胺基酸種類，將待測細胞之磷酸化程度及胺基酸種類與健康細胞之參考磷酸化程度和參考胺基酸比較。若其中當磷酸化程度係高於之參考磷酸化程度，且胺基酸種類之任一者不同於參考胺基酸種類，則判斷待測細胞具有癌細胞轉移性。以下利用數個實施方式以說明類DnaJ熱休克蛋白及 Src酪氨酸激酶交互作用及結合區域之試驗測試，以建立此交互作用區域可提供未來抗癌藥物開發或癌轉移判別之應用基礎。實施例一：製備重組質體及轉型株及蛋白質結合之分析 1、質體之構築 V5標誌之類DnaJ熱休克蛋白的產生係利用引子對序列辨識編號3 (具5^所//1切位）及序列辨識編號4 (具切位）進行增幅’得到可編碼產生類DnaJ熱休克蛋白序列之去氧核醣核酸片段(序列辨識編號2)。聚合酶連鎖反應之反應條件為94°C反應1分鐘、55°C反應1分鐘、72°C反應2分鐘 201247219 進行35個循環。將增幅出之片段構築入載體pGEM-T Easy (Promega, Madison，WI，USA; pGEM-HLJl)，可編碼產生類 D n a J熱休克蛋白序列之片段再次選殖入持續型哺乳動物表現載體 pEF6/V5-HisTOPO (Invitrogen Corp.，Carlsbad, CA)。組胺酸標誌之類DnaJ熱休克蛋白的產生係自質體 pGEM-HLJl截切出可編碼產生類DnaJ熱休克蛋白序列之片段（1.2kb) ’再次選殖入質體PQE30，以構築一誘導型表現載體表現組胺酸標誌之類DnaJ熱休克蛋白，其利用 QIAexpressionist system (Qiagen，Hilden，Germany)表現蛋白於細菌中。類DnaJ熱休克蛋白之突變蛋白質係利用點突變 (Stratagene，La Jolla，CA)之方式，藉由突變的引子對增幅所需之片段。Y64F突變蛋白質係利用引子對序列辨識編號 5及序列辨識編號6 ; Y142F突變蛋白質係利用引子對序列辨識編號7及序列辨識編號8 ; Y172F突變蛋白質係利用引子對序列辨識編號9及序列辨識編號1 〇 ; Y296F突變蛋白質係利用引子對序列辨識編號11及序列辨識編號12 ; P301/304A突變蛋白質係利用引子對序列辨識編號13及序列辨識編號14。而質體pEVXcSrc為包含可編碼產生細胞型src酷胺酸激酶序列片段之質體；質體pMv-Src為包含可編碼產生病毒型Src酪胺酸激酶序列片段之質體；質體pGEX2T_SrcSH2為包含可編碼產生Src同源2區序列片段之質體；質體 pGEX2T-SrcSH3為包含可編碼產生Src同源3區序列片段之質體；質體pGEX2T-Srckinase為包含可編碼產生Src激酶區 201247219 序列片段之質體；質體pGEX2T-Src為包含可編碼產生全長 Src酪胺酸激酶序列片段之質體。 2、質體轉染低侵襲性腺癌細胞株CL1-0和高侵襲性腺癌細胞株 CL1-5分別培養於含10%胎牛血清（fetal bovine serum, FBS ; Life Technologies，Inc” Carlsbad, California)和 1 %盤尼西林-鏈黴素(penicillin-streptomycin ; Life Technologies， Inc” Carlsbad, California)之Dulbecco’s modified Eagle’s medium (DMEM)培養液中，於37°C、5%二氧化碳培養。非惡性人類表皮HEK293細胞株培養於含10%胎牛血清之 DMEM培養液’於37°C、5°/。二氧化碳培養。使細胞表現V5標誌之類DnaJ熱休克蛋白係將質體 pEF6/V5-HisTOPO-HLJl 或 pEF6/V5-HisTOPO 以 lipofectamine (Invitrogen，Carlsbad, CA)轉染入CL1-5細胞株，質體轉染依照廠商提供之流程進行。轉染後的細胞培養於含 10 pg/mL blasticidine (Sigma，St Louis, MO)之培養液，2至3週後，篩選轉染成功的細胞，將帶有轉染質體的細胞培養於含5 pg/mLblasticidin之培養液。建立穩定型類DnaJ熱休克蛋白沉默之細胞係分別將質體 pSilencer4.1-CMVpuro-HLJl-shRNA 和 pSilencer4.1-CMVpuro-control-shRNA轉染入 CL1-0細胞以分別產生類D n a J熱休克蛋白沉默細胞和對照組細胞。而以小干擾RNA(small interfering RNA; siRNA)暫時性轉染入細胞而使HLJ1沉默，係自Ambion Inc (Austin, TX) 201247219 購買類DnaJ熱休克蛋白特異性小干擾RNA和陰性對照小干擾 RNA (scrambled siRNA)，利用 RNAiFect Transfection Reagent (Qiagen，Crawley, UK.)將小干擾RNA分別轉染入細胞’小干擾RNA轉染依照廠商提供之流程進行。 3、蛋白質結合試驗收取細胞後，以裂解緩衝液(50mMTris/HCl(pH7.4)， 1% (v/v) Triton X 100, 10% glycerol, 150 mM NaCl, 1 mM EDTA, 20 ug/ml leupeptin, 1 mM PMSF (phenylmethylsulfonyl fluoride), 20 pg/ml aprotinin，20 pg/ml pepstatin)處理細胞，再以離心力15,000 x g，於40C 離心15分鐘。進行免疫沉澱分析，先收集離心後的上清液並與蛋白質A-瓊脂醣珠(protein A-Sepharose beads)進行前培養以除去非特異性結合的蛋白質。再將處理過的細胞萃取液與蛋白質A-瓊脂醣珠於4°C培養隔夜，此蛋白質A-瓊脂醣珠上帶有單株抗體可被後續實驗的一級抗體所辨識。培養後進行離心，以含0.5%1^〇11丨(161?-40之生理緩衝液(？117.4)清洗沉澱物(pellet)6至8次，再重新懸浮於十二烧基硫酸鈉（Sodium dodecyl sulfate，SDS)樣品緩衝液中。接著，以西方墨點法分析，首先將蛋白質溶液以SDS-PAGE (SDS-polyacrylamide)膠體電泳分離，再轉潰至PVDF (Polyvinylidene fluoride)膜，依照不同的免疫沉殿目標蛋白，選用適當的一級抗體（anti-V5、anti-Src、anti-pTyr、 anti-HLJl 或 anti-GFP)，再以帶有 HRP (horseradish peroxidase)的二級抗體辨識，利用Enhanced 201247219The signaling role of Src tyrosine kinase. Y172, a DnaJ-like heat shock protein, binds to the kinase domain of the Src tyrosine kinase and is phosphorylated by Src tyrosine kinase, which reduces the invasion of cancer cells regulated by Src tyrosine kinase, against DnaJ The anti-cancer activity of heat shock is quite important. At the same time, p3〇1/p3〇4 of DnaJ-like heat shock protein has a Src homology 3 region binding sequence (binding m〇tif), which can not only inhibit the activation of Src tyrosine kinase, It also interferes with the interaction of Src tyrosine kinase with other receptors, thereby blocking the transmission of Src tyrosine kinases in cancer cells. The DnaJ-like heat shock protein binds to the sequence and specifically binds to Src-pathase kinase in a stereoscopic space, thereby reducing the epithelial-mesenchymal transition of cancer cells. The present invention provides an amino acid sequence which specifically inhibits Src tyrosine kinase activity and has antitumor activity, and can be applied to drug synthesis for molecular simulation to develop γΐ72 and Ρ301 similar to sequence identification number 1. /Ρ304 molecular structure of compounds or peptides, with specific Src tyrosine kinase binding ability, can effectively inhibit the activity and signaling of Src tyrosine kinase, but also reduce the side effects of anticancer drugs, and then develop new resistance Cancer drugs and their derivatives. Unless specifically indicated by the invention to inhibit Src tyrosine kinase activity, it comprises a cell type Src tyrosine kinase (cellular Src; c-Src) and a viral Src tyrosine kinase (Virai Src; v_Src). The cell type Src tyrosine kinase is widely present in eukaryotic tissues and contains three functional blocks: the Src homology 3 region (SH3), the Src homology 2 region (SH2), and the kinase region. The viral Src tyrosine kinase is a mutant Src tyrosine kinase, and its structure is similar to that of the cell type Src-cool acid kinase. However, the viral type §rc's kinase lacks an amino acid which inhibits its activity. The viral Src tyrosine kinase is in a state of sustained activation, so if the normal cell count 201247219 shows that the oncogenic protein 'can transform the cells into cancer cells. This also provides a molecular pathway for inhibiting cancer cell metastasis, and can be applied to test the metastasis of cancer cells, including detecting the phosphorylation degree of the 172th amino acid of DnaJ heat shock protein such as a test cell, and detecting The 301 amino acid of the DnaJ heat shock protein and the amino acid type of the 304th amino acid, the degree of phosphorylation of the cells to be tested and the degree of reference phosphorylation of the amino acid species and healthy cells and the reference amine Base acid comparison. If the degree of phosphorylation is higher than the degree of reference phosphorylation, and any of the amino acid species is different from the reference amino acid species, it is judged that the cells to be tested have cancer cell transferability. Several embodiments are used below to demonstrate experimental testing of DnaJ-like heat shock proteins and Src tyrosine kinase interactions and binding regions to establish an application basis for future anticancer drug development or cancer metastasis discrimination. Example 1: Preparation of recombinant plastids and transformation strains and protein binding analysis 1. Construction of plastids DnaJ heat shock protein production line such as V5 marker using primer pair sequence identification number 3 (with 5^//1 cleavage And sequence identification number 4 (with cleavage) for amplification to obtain a deoxyribonucleic acid fragment (SEQ ID NO: 2) encoding a DnaJ-like heat shock protein sequence. The reaction conditions of the polymerase chain reaction were 1 minute at 94 ° C, 1 minute at 55 ° C, and 2 minutes at 72 ° C. 201247219 For 35 cycles. The amplified fragment was constructed into the vector pGEM-T Easy (Promega, Madison, WI, USA; pGEM-HLJ1), and the fragment encoding the D na J heat shock protein sequence was re-selected into the sustained mammalian expression vector pEF6. /V5-HisTOPO (Invitrogen Corp., Carlsbad, CA). The production of DnaJ heat shock protein such as histidine marker is truncated from the plastid pGEM-HLJ1 to encode a fragment encoding the DnaJ heat shock protein sequence (1.2 kb) 'Re-selection into the plastid PQE30 to construct an induction The type expression vector exhibits a DnaJ heat shock protein such as a histidine marker which expresses the protein in bacteria using the QIAexpressionist system (Qiagen, Hilden, Germany). The mutant protein of the DnaJ-like heat shock protein utilizes a point mutation (Stratagene, La Jolla, CA) to amplify the desired fragment by a mutated primer pair. The Y64F mutant protein line uses sequence pair identification number 5 and sequence identification number 6; the Y142F mutant protein line uses primer pair sequence identification number 7 and sequence identification number 8; Y172F mutant protein line uses primer pair sequence identification number 9 and sequence identification number 1 Y; Y296F mutant protein line using sequence pair identification number 11 and sequence identification number 12; P301/304A mutant protein system using primer pair sequence identification number 13 and sequence identification number 14. The plastid pEVXcSrc is a plastid containing a fragment encoding a cell type src valine kinase; the plastid pMv-Src is a plastid containing a fragment encoding a viral Src tyrosine kinase; the plastid pGEX2T_SrcSH2 is included The plastid encoding the Src homology 2 region sequence fragment can be encoded; the plastid pGEX2T-SrcSH3 is a plastid containing a fragment encoding a Src homologous 3 region; the plastid pGEX2T-Srckinase is a sequence encoding the Src kinase region 201247219 The plastid of the fragment; the plastid pGEX2T-Src is a plastid comprising a fragment encoding a full length Src tyrosine kinase sequence. 2. The plastid transfected low-invasive adenocarcinoma cell line CL1-0 and the highly invasive adenocarcinoma cell line CL1-5 were cultured in 10% fetal bovine serum (FBS; Life Technologies, Inc.) Carlsbad, California, respectively. The medium was cultured in Dulbecco's modified Eagle's medium (DMEM) in 1% penicillin-streptomycin (Life Technologies, Inc., Carlsbad, California) at 37 ° C, 5% carbon dioxide. The non-malignant human epidermal HEK293 cell line was cultured in DMEM medium containing 10% fetal bovine serum at 37 ° C, 5 ° /. Carbon dioxide culture. DnaJ heat shock protein line such that the cell expresses the V5 marker. The plastid pEF6/V5-HisTOPO-HLJl or pEF6/V5-HisTOPO was transfected into the CL1-5 cell line with lipofectamine (Invitrogen, Carlsbad, CA), and the plasmid was transfected. According to the process provided by the manufacturer. The transfected cells were cultured in a culture medium containing 10 pg/mL blasticidine (Sigma, St Louis, MO). After 2 to 3 weeks, the successfully transfected cells were selected, and the cells bearing the transfected plastids were cultured in the containing. 5 pg/mL blasticidin culture. A stable cell line of DnaJ heat shock protein silencing was constructed to transfect plastid pSilencer4.1-CMVpuro-HLJl-shRNA and pSilencer4.1-CMVpuro-control-shRNA into CL1-0 cells to generate D na J-like heat, respectively. Shock protein silenced cells and control cells. HLJ1 was silenced by transient transfection of small interfering RNA (siRNA) into cells, purchased from Ambion Inc (Austin, TX) 201247219. DnaJ heat shock protein-specific small interfering RNA and negative control small interfering RNA were purchased. (scrambled siRNA), small interfering RNA was transfected into cells using RNAiFect Transfection Reagent (Qiagen, Crawley, UK.) 'Small interfering RNA transfection was performed according to the manufacturer's protocol. 3. Protein binding assay After the cells were harvested, lysis buffer (50 mM Tris/HCl (pH 7.4), 1% (v/v) Triton X 100, 10% glycerol, 150 mM NaCl, 1 mM EDTA, 20 ug/ml The cells were treated with leupeptin, 1 mM PMSF (phenylmethylsulfonyl fluoride), 20 pg/ml aprotinin, 20 pg/ml pepstatin), and centrifuged at 40 C for 15 minutes at a centrifugal force of 15,000 xg. For immunoprecipitation analysis, the supernatant after centrifugation was collected and precultured with protein A-Sepharose beads to remove non-specifically bound proteins. The treated cell extract was further cultured overnight with protein A-Sepharose beads at 4 ° C, and the monoclonal antibody with the monoclonal antibody on the A-Sepharose beads was recognized by the primary antibody of the subsequent experiment. After the culture, centrifugation was carried out, and the pellet was washed 6 to 8 times with 0.5% 1 〇 11 丨 (161?-40 physiological buffer (?117.4), and resuspended in sodium dodecyl sulfate (Sodium). Dodecyl sulfate, SDS) in the sample buffer. Then, by Western blot analysis, the protein solution was first separated by SDS-PAGE (SDS-polyacrylamide) colloidal electrophoresis, and then collapsed to PVDF (Polyvinylidene fluoride) membrane, according to different Immune-suppressed target protein, using appropriate primary antibody (anti-V5, anti-Src, anti-pTyr, anti-HLJl or anti-GFP), and then identified by HRP (horseradish peroxidase) secondary antibody, using Enhanced 201247219

Chemiluminescence System (Amersham)摘測結果。進行體外結合分析（in vitro binding assay)，表現純化帶有麩胺基硫轉移酶的全長細胞型Src或胺酸蛋白、Src同源2 區、Src同源3區或激酶區之融合蛋白，分別將此些融合蛋白與表現有V5標誌類DnaJ熱休克蛋白之HEK293細胞蛋白萃取液，於1% Nonidet P-40的裂解緩衝液，4°C反應12小時，之後以l%NonidetP-40的裂解緩衝液清洗結合複合物6 次。結合反應之溫度與時間視情況而定，可於0-40。(：反應5 分鐘至16小時，反應溫度愈低，則反應時間愈長；反之，反應溫度愈高，則反應時間愈短。結合反應完成之後，以西方墨點法分析，過程如上所述，在此係利用anti-V5抗體和anti-GST抗體辨識結合結果。進行麵胺基硫轉移酶沉殿分析(pull down assay)，首先以 0.2 mg/mL之IPTG (isopropyl-D- thiogalactopyranoside)誘導表現組胺酸標誌之類DnaJ熱休克蛋白，利用親和層析法以次 II 基乙酸鎳樹脂（nickel-nitrilotriacetic acid (Ni-NTA) resin ; Qiagen)純化蛋白質，純化依照廠商提供之流程進行。純化後的組胺酸標誌之類DnaJ熱休克蛋白分別與各麩胺基硫轉移酶標誌之Src酪胺酸激酶蛋白質於4。(：反應12小時。反應之溫度與時間視情況而定，可於0-40°C反應5分鐘至16小時，反應溫度愈低，則反應時間愈長；反之，反應溫度愈高，則反應時間愈短。反應過後，再以西方墨點法分析，過程如上所述，在此係利用anti-His抗體和anti-GST 抗體辨識結合結果。 12 201247219 4、冷光表現分析分別將1.1 kb含上皮約黏蛋白及2.1 kb含鋅指轉錄因子啟動子之片段構築入帶冷光基因的質體中。將2 X 1〇4個細胞移入24孔盤並培養16小時，利用lipofectamine (Invitrogen, Carlsbad, CA)將上述冷光質體與pEF6/V5-HisTOPO-HLJl 或對照組質體轉染入細胞，在此同時並將帶有β-半乳糖苷酶（β-galactosidase)之質體（pSV_p_Gal，Promega，Madison， WI)共同轉染入細胞。48小時後，收取細胞，以冷光分析套組（Tropix，Bedford，MA)測量冷光酶（luciferase)活性；以 Galacto-Light化學發光分析套組(Tropix, Bedford, MA)分析 β-半乳糖音活性。利用多孔掃描分光光度計（Victor3; PerkinElmer，Boston，MA)偵測光訊號，質體轉染效率以β· 半乳糖苷酶光訊號數值作基準，計算各組別之相對冷光表現。 5、轉移和侵襲分析利用 Transwell 細胞培養盤(Transwell chambers;孔徑 8 μιη; Costar，Cambridge，MA)進行轉移和侵襲分析，培養盤上有一聚碳酸酯濾膜(polycarbonate filters)。在轉移分析中，5 X 103細胞種於濾膜上並培養12小時，以棉花棒擦拭濾膜’再以曱醇固定’以Giemza溶液（Sigma，StLouis，MO) 染色，利用光學顯微鏡計算轉移細胞數。在侵襲分析中，將濾膜覆蓋上一層基質膠（Becton Dickinson，Franklin Lakes，NJ)，2 X 1〇4細胞種於基質膠上並培養20小時，利用光學顯微鏡(放大倍率為1〇〇)計算貼附於濾膜較低表面的細胞數。 13 201247219 本發明之重組質體構築、細胞轉染作用及轉形株之篩選，為此技術領域中具有通常知識者，依照現有發展的知識與技術即可輕易完成，故此處不另多贅述。實施例二：類DnaJ熱休克蛋白與Src酪氨酸激酶之交互作用： 1、類DnaJ熱休克蛋白可與Src酪氨酸激酶結合：請參照第1(A)圖與第1(B)圖，其為類DnaJ熱休克蛋白與Src酪胺酸激酶之交互作用圖。第1(A)圖是將V5標誌 (V5-tagged)之類DnaJ熱休克蛋白質體(PEF6/V5-HLJ1)和綠螢光蛋白標諸（green fluorescent protein-tagged ; GFP_tagged) 之Src酪胺酸激酶質體（pEGFP-Srcwt)共同轉染入CL 1 -0肺癌細胞中，使細胞同時表現V5標誌之類DnaJ熱休克蛋白和綠螢光蛋白標誌之Src酪胺酸激酶，經免疫共沉澱後，再以西方墨點法觀察其連結情形，結果顯示類DnaJ熱休克蛋白與Src酪胺酸激酶有交互作用之情形。第1(B)圖為進一步測試内源性之類DnaJ熱休克蛋白和Src酪胺酸激酶的交互作用’肺癌細胞中的内源性類DnaJ熱休克蛋白和Src酪胺酸激酶同時也具有交互作用。 2、全長類DnaJ熱休克蛋白結合至Src酪氨酸激酶之Src 同源3區和激酶區：凊參照第2(A)圖與第2(B)圖，其為類DnaJ熱休克蛋白與Src酪胺酸激酶之功能區塊交互作用圖。第2(A)圖表示Src酪胺酸激酶的三個功能區塊，分別是src同源3區、 201247219Chemiluminescence System (Amersham) results. In vitro binding assay for the purification of full-length cell type Src or amino acid protein, Src homology 2 region, Src homology 3 region or kinase region fusion protein with glutamine-based thiotransferase, respectively These fusion proteins were reacted with HEK293 cell protein extract showing V5 marker DnaJ heat shock protein in 1% Nonidet P-40 lysis buffer at 4 ° C for 12 hours, followed by lysis with 1% Nonidet P-40. The buffer was washed and bound to the complex 6 times. The temperature and time of the binding reaction may be from 0 to 40, as the case may be. (: 5 minutes to 16 hours of reaction, the lower the reaction temperature, the longer the reaction time; conversely, the higher the reaction temperature, the shorter the reaction time. After the completion of the binding reaction, the Western blot method is used, as described above. Here, the anti-V5 antibody and the anti-GST antibody were used to identify the binding results. The face-down analysis of the face amine-based thiotransferase was first performed with IPTG (isopropyl-D-thiogalactopyranoside) at 0.2 mg/mL. DnaJ heat shock protein such as histidine marker, purified protein by affinity chromatography with nickel-nitrilotriacetic acid (Ni-NTA resin; Qiagen), purified according to the manufacturer's protocol. The DnaJ heat shock protein such as the histidine marker is separately labeled with the Src tyrosine kinase protein labeled with each glutamine-based thiotransferase. (: 12 hours of reaction. The temperature and time of the reaction are determined according to the conditions, and may be 0. -40 ° C reaction for 5 minutes to 16 hours, the lower the reaction temperature, the longer the reaction time; conversely, the higher the reaction temperature, the shorter the reaction time. After the reaction, the Western blot analysis The procedure is as described above, in which the anti-His antibody and the anti-GST antibody are used to identify the binding result. 12 201247219 4. Cold light performance analysis A fragment of a 1.1 kb epithelial-containing mucin and a 2.1 kb zinc-containing transcription factor promoter, respectively. Constructed into a plastid with a luminescent gene. 2×1〇4 cells were transferred to a 24-well plate and cultured for 16 hours, and the above-mentioned luminescence plastids were combined with pEF6/V5-HisTOPO-HLJl or lipofectamine (Invitrogen, Carlsbad, CA). The plastids of the control group were transfected into the cells, and the plastids with β-galactosidase (pSV_p_Gal, Promega, Madison, WI) were co-transfected into the cells. After 48 hours, the cells were collected. Cells were assayed for luciferase activity in a luminescence assay kit (Tropix, Bedford, MA); beta-galactose activity was analyzed in a Galacto-Light chemiluminescence assay kit (Tropix, Bedford, MA). The photometer (Victor3; PerkinElmer, Boston, MA) detects the optical signal, and the plastid transfection efficiency is based on the β-galactosidase optical signal value to calculate the relative cold light performance of each group. 5. Metastasis and invasion analysis Transfer and invasion assays were performed using Transwell cell culture plates (Transwell chambers; pore size 8 μιη; Costar, Cambridge, MA) with a polycarbonate filter on the plate. In the transfer assay, 5 X 103 cells were seeded on the filter and incubated for 12 hours, and the filter was wiped with cotton swabs and fixed with sterols. The cells were stained with Giemza solution (Sigma, StLouis, MO) and the transferred cells were counted using an optical microscope. number. In the invasion assay, the filter was covered with a layer of Matrigel (Becton Dickinson, Franklin Lakes, NJ), 2 X 1 4 cells were seeded on Matrigel and cultured for 20 hours using an optical microscope (magnification 1 〇〇) Calculate the number of cells attached to the lower surface of the filter. 13 201247219 The recombinant plasmid construction, cell transfection, and screening of the transformed strain of the present invention can be easily accomplished by the knowledge and technology of the prior art, and therefore will not be further described herein. Example 2: Interaction of DnaJ-like heat shock proteins with Src tyrosine kinase: 1. DnaJ-like heat shock proteins can bind to Src tyrosine kinase: Please refer to Figure 1(A) and Figure 1(B) It is an interaction diagram between DnaJ-like heat shock proteins and Src tyrosine kinase. Figure 1(A) shows DcJ heat shock protein bodies (PEF6/V5-HLJ1) such as V5-tagged (G5-tagged) and SRC tyrosine labeled with green fluorescent protein-tagged (GFP_tagged). The kinase plastid (pEGFP-Srcwt) was co-transfected into CL 1 -0 lung cancer cells, and the cells simultaneously expressed DnaJ heat shock protein such as V5 marker and Src tyrosine kinase marked by green fluorescent protein. After co-immunoprecipitation Then, the Western blotting method was used to observe the linkage, and the results showed that the DnaJ-like heat shock protein interacted with Src tyrosine kinase. Figure 1(B) shows further interactions between endogenous DnaJ heat shock proteins and Src tyrosine kinases. Endogenous DnaJ heat shock proteins and Src tyrosine kinases in lung cancer cells also interact. effect. 2. The full-length DnaJ heat shock protein binds to the Src homology 3 region and kinase region of Src tyrosine kinase: 凊 Refer to Figure 2(A) and Figure 2(B), which are DnaJ-like heat shock proteins and Src Functional block interaction map of tyrosine kinase. Figure 2(A) shows three functional blocks of Src tyrosine kinase, src homology 3, 201247219

Src同源2區和激酶區。於此生產麩胺基硫轉移酶標誌 (GST-tagged)之Src同源3區、Src同源2區和激酶區蛋白’ 以及純化出麩胺基硫轉移酶標誌之全長Src酪胺酸激酶和組胺酸標誌'(histidine-tagged ; His-tagged)之類 DnaJ 熱休克蛋白，以確認類DnaJ熱休克蛋白與Src酪胺酸激酶具直接交互作用之功能區塊。以組胺酸標誌之類DnaJ熱休克與各個不同之麩胺基硫轉移酶標誌的Src功能區塊進行麩胺基硫轉移酶沉澱分析(pull down assay)。第2(B)圖是經麵胺基硫轉移酶沉澱分析後，以西方墨點法檢視作用結果，其顯示類DnaJ熱休克蛋白會與Src酷·胺酸激酶的Src同源3區和激酶區連結。 3、類DnaJ熱休克蛋白之Y172和P301/304為Src酪氨酸激酶的重要結合位：利用蛋白質三級結構預測軟體KinasePhos 1.1 version 和礙酸胜肽(phospho-peptide)資料庫 PhosphoSite，預測 Src 酪胺酸激酶於類DnaJ熱休克蛋白進行磷酸化的酪胺酸 (tyrosine)位置，預測結果顯示類DnaJ熱休克蛋白之Y64、 Y142、Y172和Y296被磷酸化的可能性高。接著，各別將上述各點的酷胺酸置換成笨丙胺酸(phenylalanine)以建構各類DnaJ熱休克之V5標誌突變蛋白質質體，分別表示為 Y64F、Y142F 和 Y172F、Y296F。請參照第3(A)圖至第3(C)圖，其為類DnaJ熱休克蛋白Y172F突變蛋白質與Src酪胺酸激酶之交互作用圖。第 3(A)圖指出Y64F、Y142F和Y172F之突變點於HLJ1序列之位置。分別將各突變蛋白質質體與pMv-Src質體轉染入 15 201247219 HEK293細胞株，使細胞分別共同表現各突變蛋白質和病毒型Src酪胺酸激酶，轉染48小時後，再以免疫共沉殿和西方墨點法觀察結果，其中WT代表轉染野生型（wildtype) 類DnaJ熱休克蛋白質體之細胞。結果顯示類DnaJ熱休克蛋白Y64F和Y142F之突變蛋白質與Src酪胺酸激酶的連結能力和野生型類DnaJ熱休克蛋白蛋白質與src酪胺酸激酶的連結能力相似，然而類DnaJ熱休克蛋白Y172F突變蛋白質與Src酷·胺酸激酶的連結能力明顯下降，表示類 DnaJ熱休克蛋白之ΥΠ2對類DnaJ熱休克蛋白-Src酪胺酸激酶間的交互作用扮演重要角色。接著’第3(B)圖為進行src酪胺酸激酶於各類DnaJ熱休克蛋白突變蛋白質之磷酸化作用試驗，分別將各突變蛋白質質體與pMv-Src質體共同轉染入HEK293細胞株，使細胞分別共同表現各突變蛋白質和病毒型Src酪胺酸激酶’再以免疫共沉澱和西方墨點法檢視各突變蛋白質的磷酸化程度。第3(C)圖為各突變蛋白質磷酸化程度之量化結果，其中WT代表表現野生型類DnaJ熱休克蛋白之細胞， v-Src/WT為共同表現病毒型Src酪胺酸激酶和野生型類 DnaJ熱休克蛋白突變蛋白質之細胞。v_Src/wT、Y64F和 Y296F的細胞組別均顯著有酪胺酸酶磷酸化的情形，然而表現Y142F和Y172F突變蛋白質的細胞，其磷酸化程度與 v-Src/WT相比分別下降至65%和28%。根據前述結果，類DnaJ熱休克蛋白會直接與Src酪胺酸激酶之Src同源區3連結’利用另一蛋白質三級結構預測工具「 SH3 hunter 」（網址： http://cbm.bio.uniroma2.it/SH3-Hunter/)分析類 DnaJ 熱休 201247219 克蛋白序列。Src同源3區連結位置的共同基序（consensus motif)為「PXXP」’而分析結果顯示可能的連結位置是位於類DnaJ熱休克蛋白之C端區域。依據預測結果，類Dnaj熱休克蛋白可能之Src同源3區連結位置為第301個和第304個胺基酸，將此兩個脯胺酸(proline)置換成丙胺酸(aianine)以建構突變蛋白質P301/304A。請參照第4圖’其為類DnaJ熱休克蛋白P301/304A突變蛋白質與Src酪胺酸激酶之交互作用圖，其中顯示 P301/304A之突變點於類DnaJ熱休克蛋白序列之位置。突變蛋白質P301/304A與Src酪胺酸激酶進行麩胺基硫轉移酶沉澱分析後，再以西方墨點法觀察結果。圖中的wt表示組胺酸標誌之野生型類DnaJ熱休克蛋白，mut表示組胺酸標誌之P301/P304A突變蛋白質。野生型類DnaJ熱休克蛋白和P301/304A突變蛋白質分別與麵胺基硫轉移酶、麩胺基硫轉移標这、之Src同源3區或全長Src赂胺酸激酶進行連結反應’結果P301/304A突變蛋白質幾乎無法與Src 酪胺酸激酶之Src同源3區連結，而P301/304A突變蛋白質與全長Src酪胺酸激酶的連結能力比起野生型類DnaJ熱休克蛋白約下降50%，表示類DnaJ熱休克蛋白上之P301 和P304為Src酿胺酸激酶之Src同源3區的連結位置。綜合上述結果，請參照第5圖，其為類DnaJ熱休克蛋白與Src赂胺酸激酶結合示意圖。類DnaJ熱休克蛋白的 Y172可與Src酪氨酸激酶之激酶區結合並被Src酪氨酸激酶誘導磷酸化，而P301/P304具有Src酪胺酸激酶之Src 同源3區結合序列，能與其結合。 17 201247219 實施例三：類DnaJ熱休克蛋白表現對Src絡氨酸激轉碟酸化之影響：Src homology 2 region and kinase region. This produces a GST-tagged Src homology 3 region, a Src homology 2 region and a kinase region protein', and a full-length Src tyrosine kinase that purifies the glutamine-based thiotransferase marker and A DnaJ heat shock protein such as histidine-tagged (Hisidine-tagged; His-tagged) to confirm a functional block in which DnaJ heat shock protein interacts directly with Src tyrosine kinase. The glutamate thiotransferase precipitation down assay was performed with DnaJ heat shock such as the histidine marker and the Src functional block of each of the different glutamine thiotransferase markers. Figure 2(B) shows the results of Western blot analysis after precipitation analysis by face amine thiotransferase, which shows that DnaJ-like heat shock protein and Src homologous acid kinase Src homology 3 region and kinase District link. 3. Y172 and P301/304, which are DnaJ-like heat shock proteins, are important binding sites for Src tyrosine kinase: predicting Src by predicting software KinasePhos 1.1 version and PhosphoSite database with protein tertiary structure The tyrosine position of tyrosine kinase phosphorylated by DnaJ-like heat shock proteins predicted that Y64, Y142, Y172 and Y296, which are DnaJ-like heat shock proteins, are highly phosphorylated. Next, the valine acid at each of the above points was replaced with phenylalanine to construct various V5 marker mutant protein bodies of DnaJ heat shock, which were represented as Y64F, Y142F, Y172F and Y296F, respectively. Please refer to Figures 3(A) to 3(C) for the interaction between DnaJ-like heat shock protein Y172F mutant protein and Src tyrosine kinase. Figure 3(A) indicates the point of mutation of Y64F, Y142F and Y172F at the position of the HLJ1 sequence. The mutant protein plastids and pMv-Src plastids were transfected into 15 201247219 HEK293 cell lines, respectively, and the cells were expressed together with each mutant protein and viral Src tyrosine kinase. After transfection for 48 hours, immunosuppression was performed. The results of the observations by the Temple and Western blotting methods, in which WT represents cells transfected with wild type DnaJ heat shock protein bodies. The results showed that the mutated proteins of DnaJ-like heat shock proteins Y64F and Y142F were linked to Src tyrosine kinase and the binding ability of wild-type DnaJ heat shock protein and src tyrosine kinase was similar, whereas DnaJ-like heat shock protein Y172F mutation The ability of the protein to bind to Src-cool-amino acid kinase was significantly reduced, indicating that the DnaJ-like heat shock protein ΥΠ2 plays an important role in the interaction between the DnaJ-like heat shock protein-Src tyrosine kinase. Next, 'Fig. 3(B) shows the phosphorylation of src tyrosine kinase in various DnaJ heat shock protein mutant proteins, and the respective mutant protein plastids and pMv-Src plastids were co-transfected into HEK293 cell line. The cells were separately expressed for each mutant protein and the viral Src tyrosine kinase', and the degree of phosphorylation of each mutant protein was examined by immunoprecipitation and Western blotting. Figure 3(C) shows the quantification of the degree of phosphorylation of each mutant protein, where WT represents cells expressing wild-type DnaJ heat shock proteins, and v-Src/WT is a common expression of viral Src tyrosine kinase and wild type DnaJ heat shock protein mutant protein cells. The cell groups of v_Src/wT, Y64F and Y296F were significantly phosphorylated by tyrosinase, whereas the cells expressing Y142F and Y172F mutant proteins decreased to 65% compared with v-Src/WT, respectively. And 28%. Based on the above results, the DnaJ-like heat shock protein is directly linked to the Src homologous region 3 of Src tyrosine kinase 'utilizing another protein tertiary structure prediction tool "SH3 hunter" (website: http://cbm.bio.uniroma2 .it/SH3-Hunter/) Analytical class DnaJ heat break 201247219 gram protein sequence. The consensus motif of the Src homology 3 region linkage position is "PXXP" and the analysis revealed that the possible linkage position is located in the C-terminal region of the DnaJ-like heat shock protein. Based on the predicted results, the Dna heat shock protein-like Src homologous 3 region is linked to the 301th and 304th amino acids, and the two prolines are replaced by aianine to construct the mutation. Protein P301/304A. Please refer to Fig. 4, which is an interaction diagram of the DnaJ-like heat shock protein P301/304A mutant protein and Src tyrosine kinase, which shows that the mutation point of P301/304A is located at the position of the DnaJ-like heat shock protein sequence. The mutant protein P301/304A and Src tyrosine kinase were subjected to glutamate thiotransferase precipitation analysis, and the results were observed by Western blotting. The wt in the figure indicates the histidine-labeled wild-type DnaJ heat shock protein, and the mut indicates the histidine-labeled P301/P304A mutant protein. The wild-type DnaJ heat shock protein and the P301/304A mutant protein were linked to the surface amine-based thiotransferase, glutamine-based sulfur transfer target, Src homologous region 3 or full-length Src citrate kinase, respectively. The 304A mutant protein is almost incapable of linking to the Src homology region 3 of Src tyrosine kinase, and the binding ability of the P301/304A mutant protein to the full-length Src tyrosine kinase is reduced by about 50% compared to the wild-type DnaJ heat shock protein. P301 and P304 on the DnaJ-like heat shock protein are the linkage sites of the Src homology 3 region of the Src-TAC kinase. To summarize the above results, please refer to Fig. 5, which is a schematic diagram showing the binding of DnaJ-like heat shock protein to Src's kinase. Y172 of the DnaJ-like heat shock protein binds to the kinase domain of Src tyrosine kinase and is phosphorylated by Src tyrosine kinase, whereas P301/P304 has the Src homology 3 region binding sequence of Src tyrosine kinase Combine. 17 201247219 Example 3: Effect of DnaJ-like heat shock protein expression on Src tyrosine-induced dish acidification:

Src 酷胺酸激酶之 Y418 的自體碟酸化 (autophosphorylation)為Src赂胺酸酶活化所需之條件，而局部黏附激酶(FAK)與β-連環蛋白（β-catenin)是Src調節訊息途徑中的因子，且與癌細胞侵襲和上皮-間質轉化作用相關，以免疫共沉澱分析局部黏附激酶或β-連環蛋白與src 在類DnaJ熱休克蛋白沉默(HLJ1-silenced)細胞中的交互作用。請參照第6(A)圖至第6(C)圖，其為類DnaJ熱休克蛋白對Src酪胺酸激酶磷酸化之影響。第6(A)圖指出在類 DnaJ熱休克蛋白沉默的細胞中，局部黏附激酶之酪胺酸磷酸化明顯的上升，第6(B)圖中的β_連環蛋白之酪胺酸磷酸化情形在類DnaJ熱休克蛋白沉默的細胞中也有提升。第6(C)圖為在類DnaJ熱休克蛋白沉默的細胞中再度表現野生型類DnaJ熱休克蛋白或p3〇i/3〇4A突變蛋白，觀察PY418表現情形。ρΥ4ΐ8的表現於野生型類DnaJ熱休克蛋白再度表現時會降低，也就是細胞型Src酪胺酸激酶活性會受到抑制’然而若是再度表現類DnaJ熱休克蛋白 P301/304A突變蛋白，則又可觀察到ργ418的表現。綜合以上結果’顯示在類DnaJ熱休克蛋白的表現下， ’酷胺酸被磷酸化情形和Six路胺酸激酶與局部黏附激酶或 β-連環蛋白連結的能力會下降，而且類DnaJ熱休克蛋白與Autophosphorylation of S418 tyrosine kinase Y418 is required for Src's activation, while local adhesion kinase (FAK) and β-catenin (β-catenin) are Src regulatory signaling pathways. Factor, and associated with cancer cell invasion and epithelial-mesenchymal transition, immunoprecipitation was used to analyze the interaction of local adhesion kinase or β-catenin with src in DnaJ-like heat shock protein silencing (HLJ1-silenced) cells. Please refer to Figures 6(A) to 6(C) for the effect of DnaJ-like heat shock proteins on phosphorylation of Src tyrosine kinase. Figure 6(A) shows that tyrosine phosphorylation of local adhesion kinase is markedly elevated in cells with DnaJ-like heat shock protein silencing, and tyrosine phosphorylation of β-catenin in panel 6(B) There is also an increase in cells that are silenced by DnaJ-like heat shock proteins. Figure 6(C) shows the re-expression of wild-type DnaJ heat shock protein or p3〇i/3〇4A mutant protein in cells with DnaJ-like heat shock protein silencing, and observed the PY418 expression. The expression of ρΥ4ΐ8 is reduced when the wild-type DnaJ heat shock protein is re-expressed, that is, the cell type Src tyrosine kinase activity is inhibited. However, if the DnaJ heat shock protein P301/304A mutant protein is re-expressed, it can be observed. To the performance of ργ418. Taken together, the above results show that under the expression of DnaJ-like heat shock proteins, 'the ability of phosphorylation to phosphorylate and the ability of Six L-acid kinase to link to local adhesion kinase or β-catenin is decreased, and DnaJ-like heat shock protein versus

Src路胺酸激酶之Src同源3區的交互作用會對酪胺酸激酶之活性造成影響。 201247219 實施例四：類DnaJ熱休克蛋白抑制癌細胞之上皮_間質轉化作用：習知辞指轉錄因子（Slug)和上皮鈣黏蛋白（E-cadherin) 為調節上皮·間質轉化作用（epitheHai_mesenchymai transition; EMT)之重要因子，而類DnaJ熱休克蛋白可調控鋅指轉錄因子和上皮鈣黏蛋白的mRNA表現。請參照第7(A)圖至第7(C)圖，其為類DnaJ熱休克蛋白對癌細胞之上皮-間質轉化作用的表現分析圖。第7(A) 圖與第（B)圖為將全長鋅指轉錄因子或上皮辦黏蛋白啟動子分別建構入含冷光基因（luciferase)的質體中，以冷光表現觀察類DnaJ熱休克蛋白對鋅指轉錄因子和上皮鈣黏蛋白轉錄活性的影響。結果顯示在不同癌細胞株表現類DnaJ 熱休克蛋白可顯著提升上皮|弓黏蛋白之轉錄活性，但會降低鋅指轉錄因子的轉錄活性。第7(C)圖為利用西方墨點法檢測細胞中鋅指轉錄因子蛋白質表現，在類Dnaj熱休克蛋白沉默的CL1-0細胞中’可觀察到鋅指轉錄因子蛋白質之表現；而在表現類DnaJ熱休克蛋白的CL1_5細胞中，鋅指轉錄因子蛋白質之表現顯著降低。請參照第8圖，其為類DnaJ熱休克蛋白P301/304A突變蛋白質對癌細胞之上皮-間質轉化作用的表現分析圖。在類DnaJ熱休克蛋白沉默的細胞中再度表現野生型類Dnaj 熱休克蛋白或P301/304A突變蛋白質，觀察鋅指轉錄因子表現和上皮-間葉轉變作用標記蛋白表現情形。波形蛋白 (Vimentin)為上皮-間葉轉變作用標記蛋白之一，其表現於野生型類DnaJ熱休克蛋白再度表現時會降低，然而若是再度表現類DnaJ熱休克蛋白P301/304A突變蛋白質，則會觀 201247219 察到鋅指轉·子和波形蛋自的表現，而上朗黏蛋現會下降。因此’類D n a J熱休克蛋白抑制癌細胞轉移是藉由降癌細胞的上皮-間質轉化作用，而類DnaJ熱休克蛋白與酷胺酸激酶之Src同源3區的交互作用能抑制上皮·間葉轉變作用而防止癌細胞的侵襲及轉移。實施例五·類DnaJ熱休克蛋白抑制癌細胞的侵繫和轉移：利用基質膠(Matrigel)侵襲分析觀察類DnaJ熱休克蛋白突變對癌細胞侵襲能力的影響。先分別將不同的類DnaJ 熱休克蛋白突變蛋白質與病毒型Src酪胺酸激酶共轉染入低侵襲性CL1-0細胞株’之後將細胞種於基質膠上，24小時後觀察結果。請參照第9(A)圖與第9(B)圖，其為癌細胞侵襲與轉移分析圖。第9(A)圖顯示野生型類DnaJ熱休克蛋白和Y296F 突變蛋白質均可抑制病毒型Src酪胺酸激酶引發的癌細胞侵襲’然而Y172F突變蛋白質會造成細胞的侵襲能力增強。第9(B)圖為P301/304A突變蛋白質亦會增高癌細胞的侵襲和轉移能力，與野生型類DnaJ熱休克蛋白相比，類DnaJ 熱休克蛋白之P301/304A突變蛋白細胞的侵襲和轉移情形增高2.3倍。利用將類DnaJ熱休克蛋白沉默癌細胞株注射入小鼠體内，觀察癌細胞之轉移。分別將兩株類DnaJ熱休克蛋白沉默癌細胞株(siHLJl-Ι和siHLJl-2)注射入六週大非肥胖型糖尿病免疫缺失（n〇n obeses diabetic-severe combined 20 201247219 immunodeficiency ; NOD/SCID)小鼠的左肺，注射量為 2 x 104個細胞於含10 ng基質膠的生理緩衝液（phosphate buffered saline ; PBS)中。小鼠於植入癌細胞50天後犧牲，將肺和肝取出，固定於10%福馬林(formalin)中並切片，以免疫組織染色觀察結果。請參照附件1 ’其為小鼠肺和肝之免疫組織染色照片。由附件1之免疫組織染色照片結果顯示，兩株類Dnaj熱休克蛋白沉默細胞株（siHUl-Ι與siHLJl-2)均形成腫瘤（如白色箭頭所示），並造成癌細胞轉移至右肺甚至肝臟，表示類 DnaJ熱休克蛋白可抑制癌細胞之轉移能力。 ^根據本實施方式’類DnaJ熱休克蛋白及Src酪胺酸激酶的胺基酸結合區域提供一個調控癌細胞侵入之分子機制並發現抑癌活性的分子結構，可以應用於新標靶性治療的開發與癌症分子病理學的研究，而有助於發展新的抗癌藥物與癌轉移診斷。 —雖然本發明已以實施方式揭露如上，然其並非用以限疋本發明，在本發明所屬技術領域中任何具有通常知識者、’在不脫離本發明之精神和範圍内，當可作各種之更動與潤飾，因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。【圖式簡單說明】 Λ為讓本發明之上述和其他目的、特徵、優點盥實施例旎更明顯易懂，所附圖式之說明如下： ’、第1(A)圖與第1(Β)圖為類DnaJ熱休克蛋白與Src酪胺酸激酶之交互作用圖。 21 201247219 第2(A)圖與第2(B)圖為類DnaJ熱休克蛋白與Src酪胺酸激酶之功能區塊交互作用圖。第3(A)圖至第3(C)圖為類DnaJ熱休克蛋白Y172F突變蛋白質與Src酪胺酸激酶之交互作用圖。第4圖為類DnaJ熱休克蛋白P301/304A突變蛋白質與 Src赂胺酸激酶之交互作用圖。九第5圖為類DnaJ熱休克蛋白與Src酪胺酸激酶結合示思圖。酿μ $ 6(A)圖至第6(C)圖為類DnaJ熱休克蛋白對Src酪胺 •磷醆化之影響。第 7 (A)圖至第7(C)圖為類DnaJ熱休克蛋白對癌細胞第1質轉化作用的表現分析圖。殤細跑圖為類DnaJ熱休克蛋白P301/304A突變蛋白質對第之上皮-間質轉化作用的表現分析圖。與第9(B)圖為癌細胞侵襲與轉移分析圖。 1為小鼠肺和肝之免疫組織染色照片。【主要- 無元件义號說明】 22 201247219 序列表 <110>國立中興大學 <120> —種抑制Src酪胺酸激酶之方法 <160>14 <210>1 <211>337The interaction of the Src homology 3 region of Src glutamate kinase affects the activity of tyrosine kinase. 201247219 Example 4: DnaJ-like heat shock protein inhibits cancer cell epithelium _ mesenchymal transition: The conventional transcription factor (Slug) and epithelial cadherin (E-cadherin) regulate epithelial-mesenchymal transition (epitheHai_mesenchymai) Transition; EMT) is an important factor, while DnaJ-like heat shock proteins regulate the mRNA expression of zinc finger transcription factors and epithelial cadherin. Please refer to Fig. 7(A) to Fig. 7(C), which are graphs showing the expression of DnaJ heat shock protein on epithelial-mesenchymal transition of cancer cells. Figure 7(A) and (B) show that the full-length zinc finger transcription factor or epithelial mucin promoter is constructed into a luciferase-containing plastid, and the DnaJ heat shock protein pair is observed in cold light. Zinc refers to the transcription factor and the effect of epithelial cadherin transcriptional activity. The results showed that DnaJ heat shock proteins in different cancer cell lines can significantly increase the transcriptional activity of epithelial-epilin, but reduce the transcriptional activity of zinc finger transcription factors. Figure 7(C) shows the expression of zinc finger transcription factor protein in cells by Western blotting. The expression of zinc finger transcription factor protein was observed in CL1-0 cells silencing Dnaj heat shock protein; In the CL1_5 cells of the DnaJ-like heat shock protein, the performance of the zinc finger transcription factor protein was significantly reduced. Please refer to Fig. 8, which is a graph showing the expression of DnaJ heat shock protein P301/304A mutant protein on cancer cell epithelial-mesenchymal transition. Wild-type Dnaj heat shock protein or P301/304A mutant protein was re-expressed in DnaJ-like heat shock protein-silenced cells, and zinc finger transcription factor expression and epithelial-mesenchymal transition marker protein expression were observed. Vimentin is one of the epithelial-mesenchymal transition marker proteins, which is expressed when the wild-type DnaJ heat shock protein is re-expressed. However, if the DnaJ heat shock protein P301/304A mutant protein is re-expressed, View 201247219 The performance of the zinc finger turn and the wave egg is observed, and the Shang Lang sticky egg will now drop. Therefore, 'Dna J heat shock protein inhibits cancer cell metastasis by reducing epithelial-mesenchymal transition of cancer cells, while interaction of DnaJ-like heat shock protein with Src homologous region 3 of valine kinase inhibits epithelium · Mesenchymal transition to prevent invasion and metastasis of cancer cells. Example 5: DnaJ heat shock protein inhibits invasion and metastasis of cancer cells: Matrigel invasion assay was used to observe the effect of DnaJ-like heat shock protein mutation on cancer cell invasion ability. Different DnaJ heat shock protein mutant proteins and viral Src tyrosine kinase were separately transfected into the low-invasive CL1-0 cell line, and the cells were seeded on Matrigel. The results were observed 24 hours later. Please refer to Figure 9(A) and Figure 9(B) for the analysis of cancer cell invasion and metastasis. Figure 9(A) shows that both wild-type DnaJ heat shock proteins and Y296F mutant proteins can inhibit the invasion of cancer cells induced by viral Src tyrosine kinase. However, the Y172F mutant protein will increase the invasive ability of cells. Figure 9(B) shows that the P301/304A mutant protein also increases the invasion and metastasis ability of cancer cells. Compared with the wild-type DnaJ heat shock protein, the invasion and metastasis of P301/304A mutant protein cells of DnaJ-like heat shock protein The situation increased by 2.3 times. The metastasis of cancer cells was observed by injecting a DnaJ-like heat shock protein silencing cancer cell line into a mouse. Two DnaJ heat shock protein silencing cancer cell lines (siHLJl-Ι and siHLJl-2) were injected into six-week non-obese diabetic immunodeficiency (n〇n obeses diabetic-severe combined 20 201247219 immunodeficiency; NOD/SCID) The left lung of the mice was injected in an amount of 2 x 104 cells in phosphate buffered saline (PBS) containing 10 ng of Matrigel. The mice were sacrificed 50 days after the implantation of the cancer cells, and the lungs and liver were taken out, fixed in 10% formalin and sectioned, and the results were observed by immunohistochemical staining. Please refer to Annex 1 ' for photomicrographs of immune tissues of mouse lung and liver. The results of immunohistochemical staining of Annex 1 showed that two Dnaj heat shock protein silencing cell lines (siHUl-Ι and siHLJl-2) formed tumors (as indicated by the white arrow) and caused cancer cells to metastasize to the right lung and even The liver, which expresses a DnaJ-like heat shock protein, inhibits the metastatic ability of cancer cells. According to the present embodiment, the amino acid-binding region of DnaJ-like heat shock protein and Src tyrosine kinase provides a molecular mechanism for regulating cancer cell invasion and discovers a molecular structure of tumor suppressor activity, which can be applied to new target treatments. Development of research with molecular pathology of cancer, and contribute to the development of new anticancer drugs and cancer metastasis diagnosis. </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> </ RTI> <RTIgt; The scope of protection of the present invention is defined by the scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features, advantages and embodiments of the present invention will be more apparent and understood. The description of the drawings is as follows: ', 1(A) and 1(Β) The picture shows the interaction between DnaJ-like heat shock proteins and Src tyrosine kinase. 21 201247219 Figures 2(A) and 2(B) show the functional block interactions between DnaJ-like heat shock proteins and Src tyrosine kinase. Figures 3(A) to 3(C) are diagrams showing the interaction of the DnaJ-like heat shock protein Y172F mutant protein with Src tyrosine kinase. Figure 4 is a diagram showing the interaction between the DnaJ heat shock protein P301/304A mutant protein and Src's kinase. Figure 5 is a diagram showing the binding of DnaJ-like heat shock proteins to Src tyrosine kinase. Brewing μ $ 6(A) to 6(C) shows the effect of DnaJ-like heat shock proteins on Src tyramine • Phosphate. Figures 7(A) to 7(C) are graphs showing the performance of DnaJ-like heat shock proteins on the first quality conversion of cancer cells. The fine run chart is a graph showing the performance of the epithelial-mesenchymal transition of the DnaJ heat shock protein P301/304A mutant protein. Figure 9(B) shows the analysis of cancer cell invasion and metastasis. 1 is a photomicrograph of immunohistochemistry of mouse lung and liver. [Main - No element description] 22 201247219 Sequence table <110> National Chung Hsing University <120> - Method for inhibiting Src tyrosine kinase <160>14 <210>1 <211>337

<212>PRT <213> <220> <223>類DnaJ熱休克蛋白之胺基酸序列 <400〉1<212>PRT <213><220><223> DnaJ heat shock protein amino acid sequence <400>1

Met Gly Lys Asp Tyr Tyr Cys lie Leu Gly lie Glu Lys Gly Ala 1 5 10 15Met Gly Lys Asp Tyr Tyr Cys lie Leu Gly lie Glu Lys Gly Ala 1 5 10 15

Ser Asp Glu Asp lie Lys Lys Ala Tyr Arg Lys Gin Ala Leu Lys 20 25 30Ser Asp Glu Asp lie Lys Lys Ala Tyr Arg Lys Gin Ala Leu Lys 20 25 30

Phe His Pro Asp Lys Asn Lys Ser Pro Gin Ala Glu Glu Lys Phe 35 40 45Phe His Pro Asp Lys Asn Lys Ser Pro Gin Ala Glu Glu Lys Phe 35 40 45

Lys Glu Val Ala Glu Ala Tyr Glu Val Leu Ser Asp Pro Lys Lys 50 55 60Lys Glu Val Ala Glu Ala Tyr Glu Val Leu Ser Asp Pro Lys Lys 50 55 60

Arg Glu lie Tyr Asp Gin Phe Gly Glu Glu Gly Leu Lys Gly Gly 65 70 75Arg Glu lie Tyr Asp Gin Phe Gly Glu Glu Gly Leu Lys Gly Gly 65 70 75

Ala Gly Gly Thr Asp Gly Gin Gly Gly Thr Phe Arg Tyr Thr Phe 80 85 90Ala Gly Gly Thr Asp Gly Gin Gly Gly Thr Phe Arg Tyr Thr Phe 80 85 90

His Gly Asp Pro His Ala Thr Phe Ala Ala Phe Phe Gly Gly Ser 95 100 105His Gly Asp Pro His Ala Thr Phe Ala Ala Phe Phe Gly Gly Ser 95 100 105

Asn Pro Phe Glu lie Phe Phe Gly Arg Arg Met Gly Gly Gly Arg 110 115 120Asn Pro Phe Glu lie Phe Phe Gly Arg Arg Met Gly Gly Gly Arg 110 115 120

Asp Ser Glu Glu Met Glu lie Asp Gly Asp Pro Phe Ser Ala Phe 125 130 135Asp Ser Glu Glu Met Glu lie Asp Gly Asp Pro Phe Ser Ala Phe 125 130 135

Gly Phe Ser Met Asn Gly Tyr Pro Arg Asp Arg Asn Ser Val Gly 140 145 150Gly Phe Ser Met Asn Gly Tyr Pro Arg Asp Arg Asn Ser Val Gly 140 145 150

Pro Ser Arg Leu Lys Gin Asp Pro Pro Val lie His Glu Leu Arg 155 160 165Pro Ser Arg Leu Lys Gin Asp Pro Pro Val lie His Glu Leu Arg 155 160 165

Val Ser Leu Glu Glu lie Tyr Ser Gly Cys Thr Lys Arg Met Lys 170 175 180 lie Ser Arg Lys Arg Leu Asn Ala Asp Gly Arg Ser Tyr Arg Ser 185 190 195Val Ser Leu Glu Glu lie Tyr Ser Gly Cys Thr Lys Arg Met Lys 170 175 180 lie Ser Arg Lys Arg Leu Asn Ala Asp Gly Arg Ser Tyr Arg Ser 185 190 195

Glu Asp Lys lie Leu Thr lie Glu lie Lys Lys Gly Trp Lys Glu 200 205 210Glu Asp Lys lie Leu Thr lie Glu lie Lys Lys Gly Trp Lys Glu 200 205 210

Gly Thr Lys lie Thr Phe Pro Arg Glu Gly Asp Glu Thr Pro Asn 201247219 215 220 225 Ser lie Pro Ala Asp lie Val Phe lie lie Lys Asp Lys Asp His 230 235 240 Pro Lys Phe Lys Arg Asp Gly Ser Asn lie lie Tyr Thr Ala Lys 245 250 255 lie Ser Leu Arg Glu Ala Leu Cys Gly Cys Ser lie Asn Val Pro 260 265 270 Thr Leu Asp Gly Arg Asn lie Pro Met Ser Val Asn Asp lie Val 275 280 285 Lys Pro Gly Met Arg Arg Arg lie lie Gly Tyr Gly Leu Pro Phe 290 295 300 Pro Lys Asn Pro Asp Gin Arg Gly Asp Leu Leu lie Glu Phe Glu 305 310 315 Val Ser Phe Pro Asp Thr lie Ser Ser Ser Ser Lys Glu Val Leu 320 325 330 Arg Lys His Leu Pro Ala Ser 335 <210>SEQ ID NO: 2 <211>1014 <212>DNA <213> < 220 > CDS <223>類DnaJ熱休克蛋白之基因序列 <400>2 atggggaaag aaaaaggctt gcagaggaaa agagaaatat ggacaaggag tttttcggag gattctgaag ggatatccaa attcatgaac atttctcgaa accattgaga gatgaaacac ccaaaattta gcattgtgtg gtaaatgata actattattg accgaaaaca aatttaaaga atgatcagtt gtaccttccg ggtccaaccc aaatggaaat gagacaggaa ttagagtatc aaaggctaaa ttaaaaaagg caaatagtat aaagggatgg gctgctcaat ttgtgaaacc cattttggga attgagaaag agccctcaaa tttcatccgg ggtcgcagaa gcttatgaag tggggaggaa gggttgaaag gtacaccttt catggcgatc ctttgaaatt ttctttggaa agatggtgat ccttttagtg ttctgtgggg ccatcccgcc acttgaagag atatatagtg cgctgatgga aggagttaca gtggaaagaa ggcaccaaaa tccagcagac attgttttta atcaaatata atttatactg taatgtacca acactggatg cggaatgagg agaagaatta gagcttcaga tgaagatatt 60 acaagaacaa atctcctcag 120 tattgagtga tcctaaaaag 180 gaggagcagg aggtactgat 240 ctcatgctac atttgctgca 300 gacgaatggg tggtggtaga 360 cctttggttt cagcatgaat 420 tcaaacaaga tcctccagtt 480 gttgtaccaa acggatgaag 540 gatctgagga caaaattctt 600 ttacttttcc aagagaagga 660 tcattaaaga caaagatcat 720 ctaaaattag tttacgagag 780 gaagaaacat acctatgtca 840 ttggatatgg gctgccattt 900 201247219 ccaaaaaatc ctgaccaacg tggtgacctt ctaatagaat ttgaggtgtc cttcccagat 960 actatatctt cttcatccaa agaagtactt aggaaacatc ttcctgcctc atag 1014 <210>SEQ ID NO:3 <211>30 <212>DNA <213>人工序列 < 220 > primer <223〉進行PCR增幅類DnaJ熱休克蛋白基因片段的引子（forward) <400>3 cgcggatcca tggggaaaga ctattattgc 30 <210>SEQ ID NO: 4 <211>29 <212>DNA <213>人工序列 < 220 > primer <223〉進行PCR增幅類DnaJ熱休克蛋白基因片段的引子（reversed) <400>4 gctctagaat tctatgaggc aggaagatg 29 <210>SEQ ID NO: 5 <211>44 <212>DNA <213>人工序列 < 220 > primer <223〉進行PCR增幅類DnaJ熱休克蛋白Y64F突變蛋白質基因片段的引子（forward) <400>5 gagtgatcct aaaaagagag aaatatttga tcagtttggg gagg 44 <210>SEQ ID NO: 6 <211>44 <212>DNA <213>人工序列 201247219 < 220 > primer <223〉進行PCR增幅類DnaJ熱休克蛋白Y64F突變蛋白質基因片段的引子（reversed) <400>6 cctccccaaa ctgatcaaat atttctctc tttttaggatc actc 44 <210>SEQ ID NO: 7 <211>37 <212>DNA <213>人工序列 < 220 > primer <223〉進行PCR增幅類DnaJ熱休克蛋白Y142F突變蛋白質基因片段的引子（forward) <400>7 ctttggtttc agcatgaatg gatttccaag agacagg 37 <210>SEQIDNO:8 <211>38 <212>DNA <213>人工序列 < 220 > primer <223〉進行PCR增幅類DnaJ熱休克蛋白YM2F突變蛋白質基因片段的引子（reversed) <400>8 cctgtctctt ggaaatccat tcatgctgaa accaaag 38 <210>SEQ ID NO: 9 <211>48 <212>DNA <213>人工序列 < 220 > primer <223>進行PCR增幅類DnaJ熱休克蛋白Y172F突變蛋白質基因片段的引子（forward) <400>9 atgaacttag agtatcactt gaagagatat ttagtggttg taccaaac 48 <210>SEQ ID NO: 10 201247219 <211 >48 <212>DNA <213>人工序列 < 220 > primer <223〉進行PCR增幅類DnaJ熱休克蛋白Y172F突變蛋白質基因片段的引子（reversed) <400〉10 tttggtacaa ccactaaata tctcttcaag tgatactcta agttcatg 48 <210>SEQIDNO: 11 <211>48 <212>DNA <213>人工序列 < 220 > primer <223〉進行PCR增幅類DnaJ熱休克蛋白Y296F突變蛋白質基因片段的引子（forward) <400〉11 tttggtacaa ccactaaata tctcttcaag tgatactcta agttcatg 48 <210>SEQ ID NO: 12 <211>40 <212>DNA <213>人工序列 < 220 > primer <223〉進行PCR增幅類DnaJ熱休克蛋白Y296F突變蛋白質基因片段的引子（reversed) <400>12 ggaatgagga gaagaattat tggatttggg ctgccatttc 4〇 <210>SEQIDNO: 13 <211>43 <212>DNA <213>人工序列 < 220 > primer <223>進行PCR增幅類DnaJ熱休克蛋白P301/304A突變蛋白質基因片段的引子（forward) <400〉13 201247219 attggatatg ggctgccatt tgcaaaaaat gctgaccaac gtg 43 <210>SEQ ID NO: 14 <211>29 <212>DNA <213 >人工序列 < 220 > primer <223>進行PCR增幅類DnaJ熱休克蛋白P301/304A突變蛋白質基因片段的引子（reversed) <400>14 cacgttggtc agcatttttt gcaaatggca gcccatatcc 40Gly Thr Lys lie Thr Phe Pro Arg Glu Gly Asp Glu Thr Pro Asn 201247219 215 220 225 Ser lie Pro Ala Asp lie Val Phe lie lie Lys Asp Lys Asp His 230 235 240 Pro Lys Phe Lys Arg Asp Gly Ser Asn lie lie Tyr Thr Ala Lys 245 250 255 lie Ser Leu Arg Glu Ala Leu Cys Gly Cys Ser lie Asn Val Pro 260 265 270 Thr Leu Asp Gly Arg Asn lie Pro Met Ser Val Asn Asp lie Val 275 280 285 Lys Pro Gly Met Arg Arg Arg lie lie Gly Tyr Gly Leu Pro Phe 290 295 300 Pro Lys Asn Pro Asp Gin Arg Gly Asp Leu Leu lie Glu Phe Glu 305 310 315 Val Ser Phe Pro Asp Thr lie Ser Ser Ser Ser Lys Glu Val Leu 320 325 330 Arg Lys His Leu Pro Ala Ser 335 <210> SEQ ID NO: 2 <211>1014 <212>DNA<213><220> CDS <223> Gene sequence of DnaJ heat shock protein <400>2 atggggaaag Aaaaaggctt gcagaggaaa agagaaatat ggacaaggag tttttcggag gattctgaag ggatatccaa attcatgaac atttctcgaa accat tgaga gatgaaacac ccaaaattta gcattgtgtg gtaaatgata actattattg accgaaaaca aatttaaaga atgatcagtt gtaccttccg ggtccaaccc aaatggaaat gagacaggaa ttagagtatc aaaggctaaa ttaaaaaagg caaatagtat aaagggatgg gctgctcaat ttgtgaaacc cattttggga attgagaaag agccctcaaa tttcatccgg ggtcgcagaa gcttatgaag tggggaggaa gggttgaaag gtacaccttt catggcgatc ctttgaaatt ttctttggaa agatggtgat ccttttagtg ttctgtgggg ccatcccgcc acttgaagag atatatagtg cgctgatgga aggagttaca gtggaaagaa ggcaccaaaa tccagcagac attgttttta atcaaatata atttatactg taatgtacca acactggatg cggaatgagg agaagaatta gagcttcaga tgaagatatt 60 acaagaacaa atctcctcag 120 tattgagtga tcctaaaaag 180 gaggagcagg aggtactgat 240 ctcatgctac atttgctgca 300 gacgaatggg tggtggtaga 360 cctttggttt cagcatgaat 420 tcaaacaaga tcctccagtt 480 gttgtaccaa acggatgaag 540 gatctgagga caaaattctt 600 ttacttttcc aagagaagga 660 tcattaaaga caaagatcat 720 ctaaaattag tttacgagag 780 gaagaaacat acctatgtca 840 ttggatatgg gctgccattt 900 201247219 ccaaaaaatc ctgaccaacg tggtgacctt ctaatagaat Ttgaggtgtc c Ttcccagat 960 actatatctt cttcatccaa agaagtactt aggaaacatc ttcctgcctc atag 1014 <210> SEQ ID NO: 3 <211>30 <212>DNA<213>Artificial sequence<220>primer <223> Forward of the shock protein gene fragment <400>3 cgcggatcca tggggaaaga ctattattgc 30 <210> SEQ ID NO: 4 <211>29 <212>DNA <213>Artificial sequence<220 > primer < 223>Reversed of PCR amplification type DnaJ heat shock protein gene fragment <400>4 gctctagaat tctatgaggc aggaagatg 29 <210> SEQ ID NO: 5 <211>44 <212>DNA <213> Artificial sequence < 220 > primer <223>>223 for PCR amplification of DnaJ heat shock protein Y64F mutant protein gene fragment <400>5 gagtgatcct aaaaagagag aaatatttga tcagtttggg gagg 44 <210> SEQ ID NO: 6 <211>44 <212>DNA<213>Artificial sequence 201247219 <220 >primer <223> The primer for performing PCR amplification of DnaJ heat shock protein Y64F mutant protein gene fragment (reversed) <400>6 cctccccaaa ctgatcaaat atttctctc tttttaggatc actc 44 <210> SEQ ID NO: 7 <211>37 <212>DNA<213>Artificial sequence<220>primer<223> The forward of the DnaJ heat shock protein Y142F mutant protein gene fragment <400>7 ctttggtttc agcatgaatg gatttccaag agacagg 37 <210> SEQ ID NO: 8 <211>38 <212>DNA <213> artificial sequence< 220 >primer <223> The primer for performing PCR amplification of DnaJ heat shock protein YM2F mutant protein gene fragment <400>8 cctgtctctt ggaaatccat tcatgctgaa accaaag 38 <210> SEQ ID NO: 9 <211>48 <212>DNA <213> Artificial sequence <220 >primer <223> Introduction of PCR amplification of DnaJ heat shock protein Y172F mutant protein gene fragment <400>9 atgaacttag agtatcactt gaagagatat ttagtggttg taccaaac 48 <210> SEQ ID NO: 10 201247219 <211 > 48 <212>DNA <213> Artificial Sequence < 220 > Primer < 223 > PCR amplification of DnaJ heat shock protein Y Reverted 172F mutant protein gene fragment <400>10 tttggtacaa ccactaaata tctcttcaag tgatactcta agttcatg 48 <210> SEQ ID NO: 11 <211>48 <212>DNA <213>Artificial sequence<220 > primer <223> A forward of a PCR-amplified DnaJ heat shock protein Y296F mutant protein gene fragment <400>11 tttggtacaa ccactaaata tctcttcaag tgatactcta agttcatg 48 <210> SEQ ID NO: 12 <211>40 <212>;DNA<213>Artificialsequence<220>primer<223> Reinforced PCR amplification of DnaJ heat shock protein Y296F mutant protein gene fragment <400>12 ggaatgagga gaagaattat tggatttggg ctgccatttc 4〇<210&gt ; SEQ ID NO: 13 < 211 > 43 < 212 > DNA < 213 > Artificial Sequence < 220 > Primer < 223 > Performing PCR Amplification of DnaJ Heat Shock Protein P301/304A Mutant Protein Gene Fragment (forward) <400>13 201247219 attggatatg ggctgccatt tgcaaaaaat gctgaccaac gtg 43 <210> SEQ ID NO: 14 <211>29 <212>DNA <213 > Column < 220 > primer < 223 > PCR was performed DnaJ heat shock protein-based growth P301 / 304A mutant protein gene fragment primer (reversed) < 400 > 14 cacgttggtc agcatttttt gcaaatggca gcccatatcc 40

Claims

^47219 Patent Application Range: The 17th isolated polypeptide of the sequence, which comprises an amino acid such as the acid number shown in SEQ ID NO: 1, an 301th amino acid and a 304th amine tyrosine. a ~-order structure, and the tertiary structure is specifically bound to the kinase domain of Src Ridge and the Src homology 3 region. The invention relates to the isolated polypeptide according to the first aspect of the patent, wherein the tyrosine kinase comprises a cell type SrC tyrosine kinase or a viral type Src = a method for inhibiting Src tyrosine kinase activity. Including: src gamma-gammonic acid kinase, wherein the tyrosine kinase comprises a homologous region 2, a Src homology 3 region, and a kinase region; and an amino acid kinase such as the sequence of the sequence identification number i (ΜΟΥ反应5 minutes to ', V 172 amino acids, 3〇1徊睑A 16 hours, the third amino acid of the polypeptide and the 304th amine acid The third-order structure is specific to the human body? The 兮仏妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆妆A method for the activity of an enzyme, wherein the Src lysine y acid: a sulphuric acid kinase or a viral Sreluine 1 has a cell type Src brit 201247219 5. The Src tyrosine kinase is inhibited according to the third aspect of the patent application. An active method wherein the Src tyrosine kinase activity is a substrate phosphorylation reaction. According to the method of claim 3, the method for inhibiting Src tyrosine-enzyme activity, wherein the polypeptide is reacted with the Src tyrosine kinase at 4 ° C for 12 hours. The method for epithelial-mesenchymal transition comprises: constructing a recombinant plastid comprising a deoxyribonucleic acid fragment as shown in SEQ ID NO: 2, introducing the recombinant plastid into a cancer cell to obtain a transformed strain; An oligoribonucleic acid fragment produces a polypeptide as shown in SEQ ID NO: 1 in the transformed strain, the polypeptide comprising the 172th amino acid, the 301th amino acid, and the 304th, as shown in SEQ ID NO: 1. a tertiary structure composed of an amino acid; and the polypeptide inhibits the phosphorylation activity of the Src tyrosine kinase in the transformed strain, thereby inhibiting the epithelial-mesenchymal transition. 8. According to the scope of claim 6 A method for inhibiting epithelial-mesenchymal transition of a cancer cell, wherein the cancer cell is a lung cancer cell. 9. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 2, and a polypeptide Pharmacy Accepted vector 2 201247219 ίο. A test method for cancer cell metastasis, comprising: detecting the phosphorylation degree of the 172th amino acid of DnaJ heat shock protein such as a test cell, and detecting the DnaJ heat shock a 301th amino acid of the protein and an amino acid species of the 304th amino acid to obtain a degree of phosphorylation of one of the cells to be tested and a diamine acid species; the degree of phosphorylation of the cell to be tested is One of the healthy cells is compared with the degree of phosphorylation, and any one of the diamine acid species is compared with one of the reference cells of the healthy cell to obtain a comparison result; and the comparison result is judged, wherein The degree of phosphorylation is higher than the degree of reference phosphorylation, and any one of the diamino acid species is different from the reference amino acid species, and it is judged that the test cell has cancer cell metastasis.