CN1256347C - Fusion protein of kininogen D5 and tumor necrosis factor related apoptosis-inducing ligand(D5-TRAIL), its preparation and use thereof - Google Patents

Abstract

The present invention provides fusion protein (Kininogen D5-TRAIL fusion protein) for a human kininogen fifth structural domain and a tumor necrosis factor related apoptosis-inducing ligand, a DNA sequence for encoding the fusion protein, a carrier containing the DNA sequence, host cells containing the carrier, a method for preparing the fusion protein by using genetic engineering and a medical composition containing the fusion protein. The Kininogen D5-TRAIL fusion protein of the present invention has the function of inhibiting tumor new vessel endothelial cell growth of Kininogen D5 and has the function of TRAIL induced tumor apoptosis. The Kininogen D5-TRAIL fusion protein can be used for preparing medicine for treating diseases, such as tumors, etc. Sequence table number explanation is as following: SEQ ID NO. 1 represents a Kininogen D5 encoding nucleotide sequence; SEQ ID NO. 2 represents a Kininogen D5 protein amino acid sequence; SEQ ID NO. 3 represents a TRAIL encoding nucleotide sequence; SEQ ID NO. 4 represents a TRAIL protein amino acid sequence; SEQ ID NO. 5 represents a fusion protein nucleotide sequence; SEQ ID NO. 6 represents a fusion protein amino acid sequence; SEQ ID NO. 7 represents the characteristic nucleotide sequence of pBV-TRAIL; SEQ ID NO. 8-37 represents the nucleotide sequence of various primers and joints.

Description

Fusion protein of the fifth domain of kininogen-tumor necrosis factor-related apoptosis-inducing ligand, its preparation method and application

technical field

The invention relates to the fields of DNA recombination technology and medicine. More specifically, the present invention relates to a fusion protein of human kininogen fifth domain (Kininogen D5) and human tumor necrosis factor-related apoptosis-inducing ligand (hereinafter referred to as TRAIL), a DNA sequence encoding the fusion protein, comprising the DNA The carrier of the sequence, the host cell containing the carrier, the method for preparing the fusion protein by genetic engineering, and the application of the fusion protein in the treatment of diseases such as tumors.

Background technique

Kininogen (Kininogen) has been discovered for nearly two decades, and its main function is anticoagulation, so it plays an important role in fibrinolysis, thrombosis, inflammation, etc. Human kininogen consists of 644 amino acids. The amino acid sequence is shown in GenBank No.: ABB59550. The product encoded by its gene is excised from one of its structural regions (ie D4) under the action of kallikrein, and the remaining D1, 2, 3 and D5, 6 are connected by a disulfide bond to form kininogen (Kininogen), and the nucleotide sequence is shown in GenBank No.: AH005302. In recent years, it has been found that it has anti-tumor effects (mainly concentrated in the Kininogen D5 part), and it has been proved that the N-terminal 402-626 amino acids of kininogen (ie, the 24-248 amino acids of Kiininogen D5) or part of the sequence are composed of Protein [Blood, 2000; 95(2): 543-550], or polypeptide [Arterioscler Thromb Vasc Biol, 2001; 21(9): 1427-1433] can inhibit the proliferation, migration and induce apoptosis of endothelial cells It produces the effect of inhibiting tumor angiogenesis in vivo, thus having anti-tumor function.

In 1995, it was reported abroad that a gene encoding an anti-tumor protein was screened from a human expressed sequence tag (EST), and the protein encoded by the gene was called tumor necrosis factor-related apoptosis-inducing ligand (tumor necrosis factor). -related apoptosis-inducing ligand (TRAIL), also known as apoptotin-2 ligand (Apo-2L for short), is a member of the tumor necrosis factor (tumor necrosis factor, TNF) family, which activates the intrinsic apoptosis of cells It can effectively induce apoptosis of tumor and transformed cells without affecting normal cells. The coding sequence of the human TRAIL gene has 843 nucleotides (SEQ ID NO.3). The nucleotide sequence is shown in GenBank No.: NM_003810, and the encoded protein TRAIL consists of 281 amino acids (SEQ ID NO.4). It has been proved that TRAIL is a typical type II transmembrane protein, and the extracellular region starting from the 95th or 114th position of its N-terminal can form a free soluble tumor apoptin protein, which also has the effect of tumor apoptosis. The gene is currently available from Biotech (http://www.invivogen.com).

Since Kininogen D5 and TRAIL inhibit tumor growth in different stages, the two are fused and expressed with a suitable amino acid linker [Protein Eng, 2003; 15(11): 871-879], and the resulting recombinant fusion protein It can not only inhibit tumor by inducing tumor cell apoptosis through TRAIL, but also resist tumor by inhibiting the growth of neovascular endothelial cells through Kiininogen D5. Therefore, the fusion protein obtained by the present invention can enhance the anti-tumor effect under the synergy of the two, and can also reduce the trouble and pain caused by the separate administration of the two to patients, and provide new treatments for diseases such as anti-tumor. drug or preparation. Therefore, there is an urgent need in this field to develop new drugs with both biological activities of Kininogen D5 and TRAIL. In addition, there is an urgent need in this field to develop a production process with low cost and/or simple steps.

Contents of the invention

An object of the present invention is to provide a new drug having both biological activities of Kiininogen D5 and TRAIL, which is a fusion protein of Kiininogen D5 and TRAIL.

Another object of the present invention is to provide DNA encoding the fusion protein, a vector containing the DNA sequence, and a host cell containing the vector.

Another object of the present invention is to provide a method for producing the KininogenD5-TRAIL fusion protein with low cost and/or simple steps.

In a first aspect of the present invention, a fusion protein is provided, comprising:

(a) a kininogen fifth domain element having the amino acid sequence of a human kininogen fifth domain or an active fragment thereof;

(b) a tumor necrosis factor-related apoptosis-inducing ligand element, which has an amino acid sequence of a human tumor necrosis factor-related apoptosis-inducing ligand or an active fragment thereof; and

(c) A linker sequence of 1-20 amino acids located between the kininogen fifth domain element and the tumor necrosis factor-related apoptosis-inducing ligand element.

More preferably, the fifth domain element of kininogen has the amino acid sequence of positions 1-266, 24-153, or 60-148 in SEQ ID NO.2;

The tumor necrosis factor-related apoptosis-inducing ligand element has the amino acid sequences of positions 1-281, 95-281, and 114-281 in SEQ ID NO.4;

Moreover, the linking sequence is a linker peptide containing 4-10 amino acids.

More preferably, the fusion protein includes the amino acid sequence shown in SEQ ID NO.6.

In the second aspect of the present invention, there is provided an isolated DNA molecule encoding the above-mentioned fusion protein of the present invention. Preferably, the DNA molecule encodes a fusion protein comprising the amino acid sequence shown in SEQ ID NO.6. More preferably, said DNA molecule comprises the nucleotide sequence shown in SEQ ID NO.5.

In the third aspect of the present invention, there are provided vectors containing the above-mentioned DNA molecules and host cells containing the above-mentioned vectors.

In a fourth aspect of the present invention, a method for producing a fusion protein of the present invention is provided, comprising the steps of:

cultivating the above-mentioned host cells under conditions suitable for expressing the fusion protein, thereby expressing the fusion protein; and isolating the fusion protein.

In the fifth aspect of the present invention, a pharmaceutical composition is provided, which includes a pharmaceutically acceptable carrier or excipient or diluent, and an effective amount of the fusion protein of the present invention.

In the sixth aspect of the present invention, the use of the fusion protein of the present invention is provided, which is used to prepare a drug for treating tumors.

Description of drawings

表示：TRAIL氨基酸序列；

表示：Kininogen D5氨基酸序列；……表示：氨基酸连接臂序列；

表示：缺失部分氨基酸。Figure 1 shows the different splicing patterns of different lengths of Kininogen D5, amino acid tethers and TRAIL. in:

Indicates: TRAIL amino acid sequence;

Indicates: amino acid sequence of Kininogen D5; ... indicates: amino acid tether sequence;

Indicates: missing some amino acids.

Figure 2 shows the electrophoresis analysis (SDS-PAGE) of a characteristic KininogenD5-TRAIL fusion protein recombinant expression and purification process. Among them, A: protein molecular weight marker; B: prokaryotic recombinant heat-induced expression of Kininogen D5-TRAIL engineering bacteria broken supernatant; C: metal-affinity purified recombinant KininogenD5-TRAIL fusion protein activity peak; D: metal-affinity eluate: E: further purified Kininogen D5-TRAIL; F: active components of expression supernatant after ammonium sulfate salting out; G: broken supernatant of bacteria before induced expression of Kininogen D5-TRAIL.

Detailed ways

After extensive and in-depth research, the inventors fused the Kininogen D5 gene and the TRAIL gene to produce a fusion protein of Kininogen D5-TRAIL connected by suitable amino acid linking arms. The fusion protein has the biological functions of both, it can inhibit the growth of tumor neovascular endothelial cells through the N-terminus of Kininogen D5 to resist tumors, and can also induce tumor cell apoptosis through TRAIL to inhibit tumors. Therefore, the Kininogen D5-TRAIL fusion protein obtained in the present invention can enhance the anti-tumor effect under the synergy of the two, and can also reduce the trouble and pain caused by the separate administration of the two to patients, and provide anti-tumor and other treatments. new compounds. The present invention has been accomplished on this basis.

definition

As used herein, the terms "fusion protein of kininogen fifth domain and tumor necrosis factor-related apoptosis-inducing ligand", "Kininogen D5-TRAIL fusion protein", etc. A protein fused with the amino acid sequence of the domain element and the amino acid sequence of the tumor necrosis factor-related apoptosis-inducing ligand element, wherein there may or may not be a linking peptide sequence between the two. Furthermore, the fusion protein may or may not have an initial methionine or signal peptide.

As used herein, the term "kininogen fifth domain element" in a fusion protein refers to a portion of the amino acid sequence in said fusion protein that is identical to the native or variant full-length kininogen fifth domain or its activity. Fragments have substantially the same amino acid sequence and have substantially the same biological activity as the fifth domain of native kininogen. The preferred kininogen fifth domain element is the fifth domain of human kininogen, more preferably the full-length fifth domain of human kininogen or an active fragment thereof, such as the 1-th in SEQ ID NO.2 The amino acid sequence at position 266, position 24-153, or position 60-148.

As used herein, the term "tumor necrosis factor-related apoptosis-inducing ligand element" or "TRAIL element" in the fusion protein can be used interchangeably and refers to a part of the amino acid sequence in the fusion protein that is different from the native or variant The full-length tumor necrosis factor-related apoptosis-inducing ligand or its active fragment has substantially the same amino acid sequence, and has substantially the same biological activity as the natural tumor necrosis factor-related apoptosis-inducing ligand. A preferred TRAIL element is a human tumor necrosis factor-related apoptosis-inducing ligand, more preferably a full-length tumor necrosis factor-related apoptosis-inducing ligand or an active fragment thereof, such as positions 1-281, The amino acid sequences of positions 95-281 and 114-281.

The sequences of the fifth domain of kininogen and the tumor necrosis factor-related apoptosis-inducing ligand can be derived from humans or non-human animals. However, the human native sequence is preferred.

As used herein, the terms "linker peptide" or "amino acid linker" are used interchangeably and refer to a short peptide or linker between the amino acid sequence of the kininogen fifth domain element and the amino acid sequence of the TRAIL element. amino acid. The length of the connecting peptide is usually 1-20 amino acids, preferably 3-10 amino acids, most preferably 4-6 amino acids. The skilled person can follow conventional methods in this field [for example, see PNAS 1998; 95:5929-5934; Protein Eng, 2000; 13(5):309-312; Protein Eng, 2003; 15(11):871-879 and other documents] Design linker peptides. Usually, the connecting peptide does not affect or seriously affects the formation of correct folding and spatial conformation of the amino acid sequence of the fifth domain element of kininogen and the amino acid sequence of the TRAIL element.

Preferred examples of connecting peptides include (but are not limited to): In order to facilitate protein folding into mutually independent domains, it is appropriate to use sequences such as SGGGGSGGGG as connecting arms (positions 170-181 in SEQ ID NO.6); It is beneficial for proteases to cut Kininogen D5-TRAIL into two independent protein molecules, and the enzyme cleavage site of active factor X (IEGR) can be used as a connecting arm, similarly, chymotrypsin 1, papain, plasmamin, thrombin, trypsin, etc. The site can also be designed as an amino acid linker; in order to facilitate purification, 6His can be used as a linker to purify the Clareticulin-TRAIL fusion protein by metal affinity chromatography; the combination of the above three schemes can also be designed as a new amino acid link Arms such as the NVVVHQAHHHHHHEFTYK tether are fused with a protease cleavage site (NIa protease) and a metal affinity chromatography site 6His.

The DNA sequence encoding the fusion protein of the present invention can be completely artificially synthesized. The coding DNA sequence of the fifth domain of kininogen and/or tumor necrosis factor-related apoptosis-inducing ligand can also be obtained by PCR amplification or synthesis, and then spliced together to form the DNA sequence coding the fusion protein of the present invention.

After obtaining the DNA sequence encoding the new fusion protein of the present invention, it is connected into a suitable expression vector and then transformed into a suitable host cell. Finally, the transformed host cells are cultured, and the novel fusion protein of the present invention is obtained by separation and purification.

As used herein, the term "vector" includes plasmids, cosmids, expression vectors, cloning vectors, viral vectors, and the like. Representative states include (but are not limited to): vectors that can be expressed in eukaryotic cells such as CHO and COS series, vectors that can be expressed in Saccharomyces cerevisiae or Pichia pastoris, and vectors that can be expressed in insect cells such as silkworm Vectors expressed in and prokaryotic expression vectors.

In the present invention, various vectors known in the art such as commercially available vectors can be used. For example, a commercially available vector is selected, and then the nucleotide sequence encoding the new fusion protein of the present invention is operably linked to the expression control sequence to form a protein expression vector.

As used herein, "operably linked" refers to the condition that some portion of a linear DNA sequence is capable of affecting the activity of other portions of the same linear DNA sequence. For example, a signal peptide (secretion leader) DNA is operably linked to a polypeptide DNA if the signal peptide DNA is expressed as a precursor and is involved in the secretion of the polypeptide; if a promoter controls the transcription of the sequence, it is operably linked to A coding sequence; a ribosome binding site is operably linked to a coding sequence if it is placed in a position to enable its translation. Generally, "operably linked to" means adjacent, and with respect to a secretory leader it means adjacent in reading frame.

In the present invention, the term "host cell" includes prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis, and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cell is a eukaryotic cell, more preferably a silkworm cell.

After the transformed host cell is obtained, the cell can be cultured under conditions suitable for expressing the fusion protein of the present invention, thereby expressing the fusion protein. Recombinant proteins can be isolated and purified by various separation methods taking advantage of their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include (but are not limited to): conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), Adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The Kininogen D5-TRAIL fusion protein of the present invention not only has the function of Kininogen D5 to inhibit the growth of tumor neovascularization endothelial cells, but also has the function of TRAIL to induce tumor cell apoptosis.

In another aspect of the present invention, a pharmaceutical composition is also provided. The pharmaceutical composition of the present invention includes an effective amount of the novel Kininogen D5-TRAIL fusion protein of the present invention, and at least one pharmaceutically acceptable carrier, diluent or excipient. In preparing these compositions, the active ingredient is usually mixed with an excipient, or diluted with an excipient, or enclosed within a carrier in the form of a capsule or sachet. When the excipient acts as a diluent, it can be a solid, semi-solid or liquid material which acts as a vehicle, carrier or medium for the active ingredient. Thus, the composition can be in the form of tablets, pills, powders, solutions, syrups, sterile injectable solutions, and the like. Examples of suitable excipients include: lactose, dextrose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, and the like. The formulation may also include: wetting agents, emulsifiers, preservatives (such as methyl and propylparaben), sweeteners, and the like.

The compositions can be presented in unit or multiple dosage form. Each dosage form contains a predetermined amount of active substance calculated to produce the desired therapeutic effect, together with suitable pharmaceutical excipients.

The formulated pharmaceutical composition can be administered by conventional routes, including but not limited to: intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral or topical administration.

When using the pharmaceutical composition, a safe and effective amount of the fusion protein of the present invention or its antibody is administered to humans, wherein the safe and effective amount is usually at least about 1 μg/kg body weight, and in most cases no more than about 8 mg/kg Body weight, preferably the dose is about 1 microgram/kg body weight to about 1 mg/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

In addition, the fusion protein of the present invention can also be used in combination with other therapeutic drugs, including (but not limited to): various cytokines, such as IFN, TNF, IL-2, etc.; various tumor chemotherapy drugs, such as 5-Fu , methotrexate and other drugs that affect nucleic acid biosynthesis, nitrogen mustard, cyclophosphamide and other alkylating agents, doxorubicin, actinomycin D and other drugs that interfere with the transcription process and prevent RNA synthesis, vincristine, camptotheca Various drugs such as alkalis that affect protein synthesis and certain hormones.

In summary, the main advantages of the present invention are as follows:

(1) Kininogen D5-TRAIL fusion protein, which not only has the function of Kininogen D5 to inhibit the growth of tumor neovascular endothelial cells, but also has the function of TRAIL to induce tumor cell apoptosis, is a new drug for treating tumors.

(2) Convenient administration. Since the effective doses of the two are close in most cases, the simultaneous administration of the fusion protein facilitates the ratio of single drugs and reduces the pain of the subjects.

(3) Targeted. While Kininogen D5 inhibits tumor angiogenesis, it can deliver TRAIL to the local tumor to exert its effect; similarly, when TRAIL acts on tumor cells, Kininogen D5 can deliver Kininogen D5 to tumor tissue to exert its inhibitory effect on angiogenesis.

(4) Enhance protein stability. After the two are fused and expressed, the half-life in vitro is significantly longer, and the stability in aqueous solution changes from about one week each to one to two months after fusion (both at 4°C); it is believed that the half-life in vivo will also be appropriate. extend.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions.

Example 1

Establish a cDNA library and screen to obtain TRAIL and Kininogen D5 genes

1. Preparation of total RNA from human peripheral blood mononuclear cells

Separate human peripheral blood lymphocytes with lymphocyte separation medium according to routine, use RPMI-1640 medium (GIBCO company), add 10% newborn calf serum (Hangzhou Sijiqing biological company), penicillin and streptomycin and culture until adherent, The mononuclear cells were obtained, and then stimulated with 10 μg/L endotoxin of Escherichia coli R595 for 2 h to activate the mononuclear cells, collected ¹⁰ cells, and extracted total RNA with a total RNA extraction kit (Qiagen Company) to obtain human peripheral Total RNA of blood mononuclear cells.

2. Establish cDNA library and screen TRAIL gene

The total RNA obtained above was purified with an Oligo-dT column (Qiagen Company) to obtain total mRNA, and the cDNA synthesis kit (Clontch Company) was used to synthesize the first and second strands of cDNA according to the instructions, and then ligated after adding the EcoR I linker into a λgt10 vector, and packaged into a λ phage library using a λ phage packaging kit (Clontech Company), to construct a λgt10 cDNA library with a titer of 6×10 ⁶ .

The λgt10 cDNA library was plated on a 1×10 ⁵ colony/LB plate, and repeated impressions were made on a 20×20 cm nitrocellulose filter. Random primers were designed using TRAIL theoretical sequences, gene probes were prepared, and libraries were hybridized and screened. Duplicate membranes containing colonies were plated in plate selection buffer (50mmol/L Tris-HCl ^pH7.5 , 1mol/L NaCl, 0.1% sodium pyrophosphate, 0.2% polyvinylpyrrolidone and 0.2% Ficoll) were hybridized overnight at 65°C. Plate screening at 65°C, washed twice with 2×SSC (0.3mol/L NaCl, 30mmol/L sodium citrate, pH7.0), and 0.1% SDS buffer. Then it was in close contact with the film at -40°C and screened for 40 hours.

Double positive samples were selected from the main plate to pick out the corresponding bacterium colony, and transferred to the LB plate of the buffer solution (100mmol/L NaCl, 10mmol/L MgSO ₄ , 50mmol/L Tris-HCl, pH7.5) added with gelatin, 12 positive phage plaques were obtained. 12 purified lambda DNAs were digested with Not I, electrophoresed on 1% agarose gel, and hybridized with the probe by Southern blotting. The clone with the highest radioactive intensity (about 1.7kp) was selected for DNA purification when hybridizing with the above probes. The inserts of these clones were subcloned into the Not I site of pSPORT1 (GIBCO). The DNA sequence of the insert in the plasmid is determined. From the results of sequence analysis, the insert sequence in one of the plasmids was 1769bp, including 87bp 5'untranslated region, 843bp open reading frame and 3'untranslated region (containing polyadenylation signal or polyA tail) , this fragment is the TRAIL gene, the coding sequence of which is shown in SEQ ID NO.3. According to the translation initiation site requirements defined by Kozak, the 88th-90th nucleotide (ATG, encoding methionine) is the translation initiation site, and the 281 amino acid sequences encoded by the largest open reading frame thus obtained are shown as SEQ ID NO.4.

3. Screening of the Kininogen D5 gene

Using a method similar to steps 1 and 2, screen the human cardiomyocyte cDNA library to obtain the coding sequence of the Kininogen D5 gene, such as SEQ ID NO.1, and the deduced encoded 266 amino acid sequence, such as SEQ ID NO.2.

Example 2

The coding sequence of Kiininogen D5 was obtained by reverse transcription-polymerase chain reaction (i.e. RT-PCR)

1. RT obtained the first strand of Kiininogen D5 cDNA

Using human cardiomyocyte total RNA purchased from Clontech (USA) as a template and P1 as a primer, reverse transcriptase catalyzes the synthesis of the first strand of Kiininogen D5 cDNA.

2. Polymerase chain reaction (PCR) amplification to obtain the coding sequence of Kininogen D5

Using the above-mentioned first strand of cDNA as a template, P1 and P2 as primers, Tag DNA polymerase catalyzes the synthesis and amplification of the coding sequence of Kininogen D5. After sequence analysis, the obtained DNA sequence was consistent with the coding sequence of Kiininogen D5 shown in the sequence registered in GenBank (GenBank No.: AH005302), that is, the coding nucleotide sequence of Kiininogen D5 was obtained. Primers:

P1: CTG TTTA GAT CTC ACT GAT G (SEQ ID NO.30)

P2: AGA AAT ACC CAT AGC CAC (SEQ ID NO.31)

Example 3

The coding sequence of TRAIL was obtained by RT-PCR

1. Preparation of total RNA from human peripheral blood mononuclear cells

(1) Separating lymphocytes from human peripheral blood according to conventional methods, and then obtaining mononuclear cells by the adherence method;

(2) The mononuclear cells were activated with bacterial endotoxin to stimulate the cells to express more genes and increase the abundance of RNA, and the total RNA was extracted with an RNA extraction kit (Qiagen Company).

2. Reverse transcription (RT) to obtain the first strand of TRAIL cDNA

Using the above total RNA as a template and P3 as a primer, reverse transcriptase catalyzes the synthesis of the first strand of TRAIL cDNA.

3. Polymerase chain reaction (PCR) amplification to obtain TRAIL coding sequence

Using the above-mentioned first strand of cDNA as a template, P3 and P4 as primers, Tag DNA polymerase catalyzes the synthesis and amplification of the TRAIL coding sequence. After sequence analysis, the obtained DNA sequence was consistent with the TRAIL coding sequence shown in the sequence registered in GenBank (NM_003810), that is, the TRAIL coding nucleotide sequence was obtained.

P3: GAC TTA CAG CAG TCA GAC TCT GAC (SEQ ID NO.32)

P4: TAT TGC TTT TTC TTT CCA GGT CAG (SEQ ID NO.33)

Example 4

Construct expression vector of Kininogen D5-TRAIL fusion protein

Using the rebuilt pBV220 as a vector (the original plasmid was donated by the Institute of Virology, Chinese Academy of Preventive Medicine), a recombinant plasmid expressing amino acids 114-281 of TRAIL was constructed, and the constructed prokaryotic expression vector expressing amino acids 114-281 of TRAIL was named is pBV-TRAIL. The recombinant expression of the corresponding fusion protein of the present invention is carried out in the same way. The specific implementation steps are as follows:

1. Design and synthesize primers and oligonucleotide double strands

Design and amplify the primers P5 and P6 encoding the 114th-281st amino acid of TRAIL, and its sequence is:

P5: 5'AC G AAT TC A CA A TG G TGA GAG AAA GAG GTC-3' (SEQ ID NO.34);

P6: 5'-AC G GAT CC T TAG CCA ACT AAA AAG-3' (SEQ ID NO.35)

P5 optimizes the distance between the SD sequence and the foreign gene to 8 nucleotides (italics), and introduces the protein synthesis start codon ATG and the EcoR I restriction site GAATTC; P6 introduces the BamH I restriction site GGATCC .

Synthetic oligonucleotide double strands, the sequence is as follows (the sequence of the complementary strand is not given):

C TGA GCA CAT CAGCAG GAC GCA CTG ACC ACC AIG AAG GTG ACG CTC TTA AAA ATT AA G CCCTGA AGA AGG GCA GCA TTC AAA GCA GAA GGC TTT GGG GTG TGT GAT ACG AAACGA AGC ATT GGT TAA AAA TTA AGG AG G AAT TCA CA-3′(SEQ ID NO.36)P7: 5′-AA A GAT CT C TCA CCT ACC AAA CAA TGC CCC CCT GCA AAA AATAAA TTC ATA TAA AAA ACA TAC AGA TAA CCA TCT GCG GTG ATA AAT TATCTC TGG CGG TGT TGA CAT AAA

C TGA GCA CAT CAGCAG GAC GCA CTG ACC ACC AIG AAG GTG ACG CTC TTA AAA ATT AA G CCCTGA AGA AGG GCA GCA TTC AAA GCA GAA GGC TTT GGG GTG TGT GAT ACG AAACGA AGC ATT GGT TAA AAA TTA AGG AG G AAT TC A CA -3' (SEQ ID NO.36)

Among them, the italic part of the P7 sequence is the λPLPR tandem promoter sequence, the shaded part is the cI repressor protein binding site, the bold part is the optimized SD sequence, the 5' end introduces the Bgl II restriction site AGATCT, and the 3' end introduces EcoR I Restriction site GAATTC;

P8: 5'-AA G GAT CC G TCG ACC TGC AGC CAA GCT TGG CTG TTT TGG CGGATG AGA GAA GAT TTT C AG-3' (SEQ ID NO. 37)

Among them, the 5' end of the P8 sequence introduced the BamH I restriction site GGATCC, and the 3' introduced the Xmn I restriction site GAANNNNTTC.

2. Construct the expression vector pBV-TRAIL, as follows:

Using P5 and P6 as primers, and using the TRAIL gene of Example 3 as a template, the TRAIL gene coding sequence was amplified by PCR, and the product was digested with EcoR I and BamH I (tool enzymes were all purchased from NEB, USA). Digest P7 with Bgl II and EcoR I, and digest P8 with BamH I and Xmn I. The pBV220 plasmid was digested with Bgl II and Xmn I.

The above fragments were digested with agarose gel to recover fragments of corresponding size, and the fragments were ligated with T4 DNA ligase, and the resulting recombinant plasmid was the expression vector of tumor apoptin, named pBV-TRAIL.

Compared with _pBV220 , pBV-TRAIL has the following characteristics: the TRAIL coding gene is controlled by the upstream lambda phage _PLPR tandem promoter, the optimized ribosome binding sequence (SD sequence), the insertion of the EcoR I restriction site and the translation initiation codon At the same time, the distance between the ATG and the SD sequence was optimized to be 8 nucleotides; a BamH I restriction site was inserted downstream of the gene encoding tumor apoptin, etc. This plasmid is the same as pBV220. It contains cIts857 binding sequence in the promoter sequence; it has ampicillin resistance gene; transcription termination sequence rrnB; the plasmid contains cIts857 gene encoding temperature-sensitive protein factor, and the encoded product is inactivated at 42°C. Thus, the repression of the promoter is lost, and the downstream foreign gene controlled by the promoter is expressed. The characteristic nucleotide sequences such as upstream control sequence, optimized SD sequence, TRAIL coding sequence and translation termination sequence are shown in SEQ ID NO.7.

Using the same method as above, use the sequences listed in Table 1 as primers (the listed primers only partially enumerate the amplification of the 18 fusion proteins listed in Figure 1), and use the corresponding genes obtained above as templates to amplify the corresponding coding DNA fragments , Restriction endonuclease digestion, T4 DNA ligase ligated into the carrier that has been digested in the same way, transformed competent cells, screened clones containing recombinant plasmids, and confirmed by sequencing.

Table 1 Nucleotide sequence list of primers

The first group: the Kiininogen D5 in the encoded fusion protein is at the N-terminus, as shown in Figure 1. A-I (p represents the primer, K represents the Kiininogen D5, T represents TRAIL, and the middle number represents the beginning or the beginning of the encoded amino acid Termination position, f indicates forward primer, r indicates reverse primer)

1 pK1f 5′-gcg aat tca cat atg aaa agg cct cca gg-3′ SEQ ID NO.8 2 pK24f 5′-gcg aat tca tgg taa gtc cac ccc aca c-3′ SEQ ID NO.9 3 pK60f 5′-gcg aat tca tga aac ata atc ttg gcc atg-3′ SEQ ID NO.10 4 pK148r 5′-aag gat ccg ctt gcc aaa tgc tct g-3′ SEQ ID NO.11 5 pK153r 5′-aag gat cca ctg tct tca gaa gag ctt g-3′ SEQ ID NO.12 6 PK266r 5′-aag gat cca gaa agg cca tca gtg ag-3′ SEQ ID NO.13 7 pT1f 5′-aac tgc aga tgg cta tga tgg agg tc-3′ SEQ ID NO.14 8 pT95f 5′-aac tgc aga cct ctg agg aaa cca tt-3′ SEQ ID NO.15 9 pT114f 5′-aac tgc agg tga gag aaa gag gtc ctc-3′ SEQ ID NO.16 10 pT281r 5′-tca agc tta gcc aac taa aaa ggc-3′ SEQ ID NO.17

The second group: amplify TRAIL in the encoded fusion protein at the N-terminus, a-i as shown in Figure 1 (in order to distinguish the first group of primers, add 1 after the primer name shown)

11 pT1f1 5′-gcg aat tca cat atg gct atg atg gag gtc-3′ SEQ ID NO.18 12 pT95f1 5′-gcg aat tca cat atg acc tct gag gaa acc att-3′ SEQ ID NO.19 13 pT114f1 5′-gcg aat tca cat atg gtg aga gaa aga gg-3′ SEQ ID NO.20 14 pT281r1 5′-aag gat cca act aaa aag gcc ccg-3′ SEQ ID NO.21 15 pK1f1 5′-aac tgc aga tga aaa ggc ctc cag g-3′ SEQ ID NO.22 16 pK24f1 5′-aac tgc agg taa gtc cac ccc aca c-3′ SEQ ID NO.23 17 pK60f1 5′-aac tgc aga aac ata atc ttg gcc atg-3′ SEQ ID NO.24

18 pK148r1 5′-tca agc tta gct tgc caa atg ctc tg-3′ SEQ ID NO.25 19 pK153r1 5′-tca agc tta act gtc ttc aga aga gct tg-3′ SEQ ID NO.26 20 pK266r1 5′-tca agc tta aga aag gcc atc agt g-3′ SEQ ID NO.27

The third group: the coding sequence of the amino acid linker, which can form double-stranded DNA after simple denaturation and renaturation

pL1 5′-ga tcc ggc gga ggc ggg agc ggc ggg ggc gga agc ctg ca-3′ SEQ ID NO.28 pL2 5′-ggc ttc cgc ccc cgc cgc tcc cgc ctc cgc cg-3′ SEQ ID NO.29

A series of recombinant plasmids expressing different lengths of Kininogen D5-TRAIL fusion proteins were thus obtained. In particular, the 18 kinds of KininogenD5-TRAIL recombinant fusion proteins prepared with the listed primers as shown in Figure 1 can directly show the biological effects of killing tumor cells in vitro and inhibiting the growth of transplanted tumors in vivo.

A preferred nucleotide sequence for expressing the Kininogen D5-TRAIL fusion protein is shown in SEQ ID NO.5, and the deduced amino acid sequence is shown in SEQ ID NO.6.

Example 5

Transform Escherichia coli to establish engineering bacteria

A series of recombinant expression plasmids such as pBV-TRAIL obtained in Example 4 were transformed into Escherichia coli BL21 [genotype: hsdS gal (λcIts857ind1 Sam7 nin5 lacUV5-T7)] (available from Shanghai Sangong Bioengineering Co., Ltd.) as usual, from Plasmid DNA was isolated from ampicillin-resistant colonies, identified by enzyme digestion, and confirmed by sequencing. The positive clones obtained were engineering bacteria expressing corresponding proteins.

Example 6

Preparation of Kininogen D5-TRAIL fusion protein

Various media are as follows: LB medium is used for test tube seed culture, 2×YT medium is used for secondary seed culture, semi-synthetic medium is used for fermentation, and fed batches are added to culture.

LB medium formula (g/L): peptone: yeast powder: NaCl=10:5:5;

2×YT medium formula (g/L): peptone: yeast powder: NaCl=16:10:5;

Formula in semi-synthetic fermentation medium (g/L): peptone: yeast powder: KH ₂ PO ₄ : K ₂ HPO ₄ : Na ₂ HPO ₄ .12H ₂ O: (NH ₄ ) ₂ SO ₄ : NH ₄ Cl=5 :5:2:4:7:1.2:0.2;

Various trace element solutions, concentration (g/L): MnSO ₄ .5H _{2 O:} CaCl ₂ .6H ₂ O: _{Na 2} MoO ₄ .2H 2 O _: ZnCl ₂ : CuSO ₄ .5H ₂ O: H ₃ BO ₄ :FeSO ₄ .7H ₂ O:CaCl ₂ .2H ₂ O:MgSO ₄ .7H ₂ O=0.001:0.004:0.002:0.002:0.001:0.0005:0.02:0.02:0.3;

Fermentation feed formula (g/L): glucose:yeast powder:peptone:MgSO ₄ .7H ₂ O=200:70:70:5.7.

The pH of the medium was adjusted to 7.0 throughout. After high-temperature sterilization of various media, ampicillin was added to a final concentration of 100 μg/ml.

After the primary seeds were cultured overnight, they were transferred to the secondary seeds at a ratio of 1:50, and the fermenter was inoculated into the secondary seeds cultivated overnight by 5% of the working volume. The cultivation is carried out in two stages, 5-7 hours at 32°C; 4-5 hours at 42°C. The constant flow pump adds feed, and the dissolved oxygen is controlled between 30-50%. Obtain the wet thallus of about 20-30 g/liter fermented liquid.

Sonicate the fermented cells, centrifuge and take the supernatant to pass through a 0.22 μm filter membrane; use NTA Supperflow (Qiagen Company) or TALON Metal Affinity Rsins (Clontech Company) for metal affinity chromatography, elution with 10 mmol/L imidazole, pH7.0 Miscellaneous protein, then eluted recombinant protein with 100mmol/L imidazole, pH7.0; the eluate was passed through CM cellulose to collect active components; further purified by Sephacryl S-200, high-purity recombinant fusion protein could be obtained .

Results: Taking prokaryotic recombinant expression of a fusion protein (TRAIL _114-281 -Kininogen D5 _60-148 ) shown in Figure 1-i as an example, the engineered bacteria were induced to express by heat, and after ultrasonic disruption, centrifuged The separated supernatant was subjected to SDS-PAGE. It can be seen that the expressed fusion protein accounted for more than 10% of the total protein in the supernatant, and it was electrophoresis pure after further purification. The results are shown in FIG. 2 . The calculated molecular weight is 30.6kD, consistent with the theoretical value. The deduced theoretical isoelectric point is 10.2. Similar results can be obtained after induction and expression of other single TRAIL, Kininogen D5 or fusion of the two, and recombinant proteins with corresponding molecular weights can be obtained.

Example 7

Determination of the biological activity of the fusion protein

In this example, the TRAIL activity and Kiininogen D5 activity of the fusion protein were determined.

Activity of TRAIL: In vitro, human pancreatic ductal carcinoma 1990 cells, human large cell lung cancer NCI-460, mouse melanoma B16, etc. can be used as target cells to determine the biological activity of TRAIL, and in vivo to inhibit human colon cancer in nude mice HCT-8 xenografts and other mouse xenografts that are sensitive to TRAIL in vitro and can form tumors in vivo are used as models to verify the biological function of TRAIL.

Activity of Kininogen D5: The inhibitory effect of Kininogen D5 on newborn endothelial cells (human umbilical vein, bovine aorta, rat lung-derived capillaries, etc.) cultured in the presence of basic epithelial growth factor (bFGF) was observed in vitro. The in vivo observation of Kiininogen D5 on the growth of new blood vessels in the chicken embryo capsule, the growth of new blood vessels in the cornea of rabbits, and the transplantation of human colon cancer HCT-8 in nude mice were used as models to verify the biological function of Kiininogen D5.

The specific test operation is as follows:

1. TRAIL activity analysis: killing various tumor cells

Various target cells were obtained from the Institute of Cells, Chinese Academy of Sciences or Shanghai Changhai Hospital. Mainly human pancreatic ductal carcinoma 1990, 8898 strains, human large cell lung cancer NCI-460, human colon cancer HCT-8, human gastric cancer M85 strain, human neuroblastoma SK-N-SH strain, human laryngeal carcinoma Hep-2 strain , human nasopharyngeal carcinoma CNE-2 strain, human endothelial cell CEV304 strain, human fibroblast cell line, human colon cancer AT-29, human ovarian cancer 3AO, mouse fibroblast L929 strain, human glioma U251, human breast cancer, Human liver cancer HepG-2, SMMU7721, human hematological tumors (U937, Jukart, HL60, etc.), melanoma B16-MB, Ehrlich ascites tumor, Lewis sarcoma, etc.

Taking 1990 cells as an example, seed the cultured cells into 96-well cell culture plates at 2× ¹⁰⁸ cells/L, incubate in a 5% CO ₂ incubator at 37°C for 4-6 hours, discard the supernatant; Add to the cell plate after serial dilution; observe the effect of adding 1.5 μg/ml actinomycin D on the killing cells of various factors at the same time; the definition of activity unit: make 50% of the cells in the well of the culture plate die as a unit, and the titer That is, the reciprocal of the dilution of the sample at which 50% killing is achieved. Quantitative analysis by crystal violet method: discard the supernatant of adherent cells, stain with crystal violet fixative (5g/L crystal violet, 80mL/L formaldehyde, 1g/L NaCl, 200mL/L ethanol) for 15 minutes, wash with distilled water to remove crystals Violet, air-dried, the stained cells are living cells. Add 200 μL of 330 mL/L acetic acid to each well, rotate the shaker to dissolve the crystal violet, set the enzyme-linked analyzer to read the A value at a wavelength of 595 nm, and take the blank well without adding cells as the A background. Suspension cells were determined by the MTT method to determine the number of viable cells. The activity titer is linearly regressed with the logarithm of sample dilution and the mean of A595nm to obtain constants A and B, and (A control-A background)/2 is the 50% killing end point (Y). Calculate the activity unit of the sample according to Y=A+BlgX, that is, the X value. At the same time, the cells showed typical apoptotic morphology under the action of TRAIL, and the typical changes of the cells were observed within 2 hours under the action of 10 mg/L tumor apoptin.

2. Culture of neonatal bovine aortic endothelial cells

Cut the neonatal bovine aorta section about 10cm long under sterile conditions, and cut the aorta neatly in a sterile laminar flow chamber, and ligate the arterial branches with string; Rinse the aortic lumen to completely remove the remaining blood cells; ligate one end of the aorta with a string and pour about 10ml of digestive solution (0.2% collagenase type II, using DMEM medium as the solvent), and let stand for digestion at room temperature for 20 minutes; take out the digestive solution , wash the inner wall of the aortic tube with 0.1mol/L PBS, collect the washing solution and mix it with the digestive solution, put it into a sterile 50ml centrifuge tube; centrifuge at 500×g for 10 minutes, discard the supernatant; ), centrifuge at 500×g for 10 minutes, and repeat once; suspend in DMEM culture solution containing 20% FBS and 200 μg/ml ECGF, add plastic cell culture flasks, and place in a 37°C, 5% _CO2 cell incubator for cultivation; microscopic examination After a large number of cells adhered to the wall and survived (about 3 days), the medium was changed, and the medium was changed every 2 days thereafter. After the cells grew into a confluent monolayer, they were routinely subcultured.

3. Culture of rat lung-derived capillary endothelial cells

Preoperative treatment: Take a SD rat weighing about 150g, inject 3ml of 0.5% heparin sodium solution into the intraperitoneal cavity, fumigate the animal with ether in a sealed cylinder, immerse in 75% alcohol for 3 minutes for disinfection; take the lung for surgery: open the chest, cut the left Inject 10ml of PBS solution into the ventricle and right ventricle, flush the pulmonary blood vessels until the left ventricular outflow is colorless, cut off the edge of the lung lobe, wash it in PBS solution, cut the cut lung tissue into small pieces of about 2×2mm, and put it in the containing Soak in DMEM culture solution with 20% calf serum for 5 minutes; place small pieces of lung tissue in a sterile, dry plastic cell culture bottle, and incubate for 1.5 hours at 37°C in a 5% CO ₂ incubator to make the lung tissue The block adheres to the wall; upright culture: carefully add 10ml of DMEM containing 20% FBS and 200μg/ml ECGF, wash the tissue block away from the bottle wall, and culture in a 37°C, 5% CO ₂ incubator; carefully remove the tissue block and replace The medium was changed once every 2 days, and the cells were subcultured after the cells grew to the confluence of the monolayer.

4. Fusion protein inhibits various cell proliferation tests

Various cells (mouse-derived pulmonary capillary endothelial cells, neonatal bovine aortic endothelial cells, B16 melanoma cells) grow confluent (in a culture flask), digest with 0.125% trypsin, suspend and adjust in DMEM containing 20% FBS The cell concentration was 5×10 ⁴ /ml, inoculated into 96-well cell culture plate at 100 μl/well, and cultured in a 37°C, 5% CO ₂ incubator. After observing the cell attachment with an inverted phase-contrast microscope (about 4 to 5 hours), give the sample: the initial protein concentration is 2 μg/well, and a 2-fold serial dilution is made. Place in a 37°C, 5% CO ₂ incubator. After 48 hours, add 15 μl/well of MTT solution and incubate for 4 hours at 37°C in 5% CO ₂ ; add 100 μl/well of MTT method reaction termination solution and place at 37°C for 1 hour. Mix each well well and remove air bubbles, then install an enzyme-linked analyzer to detect the absorbance at 595/630nm.

5. Fusion protein inhibits endothelial cell proliferation test

Prepare neonatal bovine aortic endothelial cells and mouse lung-derived capillary endothelial cell suspensions by the aforementioned method, adjust the concentration to 6×10 ⁴ /ml, inoculate 50 μl/well into 96-well cell culture plates, and place in 37°C 5% _CO2 incubator culture. After the cells adhere to the wall (about 4 to 5 hours), give samples: set six concentration gradients of 0, 2.5, 5.0, 10, 20, and 40 μl/ml per well, and set 10 parallel wells for each concentration gradient. After 48 hours, add 15 μl/well of MTT solution, and incubate for 4 hours at 37°C in a 5% CO ₂ incubator. Add 100 μl/well stop solution, and incubate at 37°C for 1 hour. Mix each well well and remove air bubbles, then install an enzyme-linked analyzer to detect the 595nm/630nm dual-wave absorption value. Calculate the 50% inhibitory concentration of the fusion protein.

6. Inhibition test of fusion protein on neovascularization induced by rabbit cornea

The sample was coated in a sustained-release substance (EVA, namely ELvax40, a copolymer of ethylene and ethylene acetate), and implanted into the cornea of rabbits to observe its inhibition of the growth of induced blood vessels. The sample is released at a rate of 0.1-1 μg/d, and the release can last for 100 days.

Soak 50mg of EVA in 75% alcohol for 10 minutes, put it in 2ml of dichloromethane solution after disinfection, stir to dissolve it, add 8mg of bacterial endotoxin and experimental samples, stir well to make it evenly mixed, and let it stand in the EVA solution for 1 hour , form an EVA film, cut into small pieces, each weighing 1 mg, irradiate with ultraviolet rays for 30 minutes, and sterilize for later use. After the rabbit was anesthetized intravenously (3% sodium pentobarbital, 40 μg/kg body weight), a radial incision (2-3 mm long and 0.3-0.5 mm deep) was made at the outer 1/3 of the cornea, and a lamellar separator was used to The corneal laminae were separated to form a 3×3mm cavity in the cornea, and a piece of treated EVA was filled. The corneal growth of the rabbits was observed every day, and the results were recorded and analyzed on the 10th day after operation.

7. Inhibition test of fusion protein on neovascularization of chicken embryo allantoic membrane

Samples were embedded in EVA as before, but no endotoxin was added. Take 9-day-old chicken embryos, wash them with sterile dH ₂ O, wipe them with 1/4000 formaldehyde, dry them in the air, and incubate them in a 37°C incubator. On the next day, put the chicken embryos under the egg inspection lamp to find the embryo head, and draw a 1×1.5 cm rectangular area with a crayon at the projected part of the eggshell between the two anterior yolk veins 1 cm in front of the embryo head. Drill a small hole of about 1.0-2.0 mm at the end of the air chamber of the chicken embryo and penetrate the shell membrane of the air chamber. At the window opening position of the eggshell, after disinfecting with 75% alcohol, use a small file to cut through the eggshell along the edge of the rectangular area (do not damage the eggshell membrane below), and use ophthalmic forceps to clamp the edge of the rectangular eggshell, parallel Lift up to expose the egg shell membrane below, and pour off the egg shell dust. Use the injection needle to cut a small hole with a diameter of 1mm along the direction of the shell membrane fiber, without injuring the underlying allantoic membrane (CAM). Add a little sterile saline on the shell membrane window, then use ophthalmic tweezers to gently lift up the egg shell membrane on the edge of the small hole, let the liquid immerse between the egg shell membrane and CAM, and cut off the long square after the CAM sinks Inner shell membrane. Place the EVA embedding block directly on the avascular area on the CAM. Seal the egg window with sterilized transparent tape, seal the small hole of the air cell with paraffin, and incubate at 37°C. Three days later, use ophthalmic tweezers to remove the egg shell and egg shell membrane above the CAM plane, and be careful not to damage the CAM blood vessels. Add an appropriate amount of methanol and acetone equal amounts of fixative solution dropwise on the CAM plane, and fix at room temperature for 15 minutes. After the intravascular blood on the CAM is coagulated, cut the CAM in the test area and place it in a dish filled with dH ₂ O. After unfolding it with an elbow straw, pour out the dH ₂ O in the dish and spread it on the bottom of the dish. Flatten the CAM, turn it over and paste it on the filter paper, take pictures to record the results, and analyze the results.

8. Fusion protein inhibits transplanted tumors in mice

Expansion of various tumor cells in vivo and in vitro: The tumor cells cryopreserved in liquid nitrogen were revived and cultured or inoculated into the peritoneal cavity of Kunming mice (10 ⁷ cells/mouse). After 10 days, the mice were sacrificed, and the ascites fluid of the mice was aspirated under aseptic conditions, and the cell concentration was adjusted to 10 ⁸ /ml with PBS. 0.2 ml of the cell suspension was inoculated subcutaneously in experimental mice. Randomly grouped, 6 mice in each group, subcutaneously inoculated mice were given 0 (PBS, control group), 10, 100, 1000 μg (sample)/kg (mouse body weight) respectively, administered every other day for 2 weeks. One week after drug withdrawal, the mice were sacrificed, the tumors were peeled off and weighed, the average tumor weight and tumor inhibition rate of each group were calculated, and the results were analyzed. The tumor inhibition rate was calculated by the following formula:

Results: Table 2 shows the results of transplanted tumors of human colon cancer cell HCT-8.

Table 2 HCT-8 test result table protein structure 10μg/kg tumor inhibition rate (%) 100μg/kg tumor inhibition rate (%) 1000μg/kg tumor inhibition rate (%) Figure 1-A* - - - Figure 1-B 30.8 39.2 44.2 Figure 1-C 25.6 34.9 40.6 Figure 1-D* - - - Figure 1-E 20.8 31.3 40.5 Figure 1-F 25.6 33.3 41.9 Figure 1-G* - - - Figure 1-H 33.7 42.6 53.9 Figure 1-I 40.7 49.8 62.6 Figure 1-a* - - - Figure 1-b* - - - Figure 1-c* - - - Figure 1-d 23.5 28.6 36.1 Figure 1-e 22.4 33.6 41.5 Figure 1-f 21.7 34.7 44.8 Figure 1-g 23.8 31.5 45.6 Figure 1-h 29.6 41.7 55.4

Figure 1-i 34.6 47.9 63.4 Kininogen D51-180aa 20.6 31.6 37.8 TRAIL114-281aa 23.6 36.7 40.9

*: These six groups were not measured because: the expression level of the fusion protein was low during recombinant expression, and a large amount of purified protein could not be obtained

It can be seen from Table 2 that the recombinantly expressed fusion protein has a better inhibitory effect on colon cancer cells. For other transplanted tumors, such as human large cell lung cancer NCI-H460, human pancreatic cancer SW1990, 8898 strain, human gastric cancer M85, human laryngeal cancer Hep-2, human nasopharyngeal cancer CNE-2, human colon cancer AT-29, human glia Similar results can be obtained in plasmoma U251, human liver cancer HepG-2, melanoma B16-MB, Ehrlich ascites tumor, Lewis sarcoma and other models.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

<110> The Second Military Medical University of the Chinese People's Liberation Army

<120>Kininogen Fifth Domain-Tumor Necrosis Factor-related Apoptosis-Inducing Ligand Fusion Protein and Its Preparation and Application

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<170>Patent In version 3.1

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<223> fusion protein

<400>6

Met Lys His Asn Leu Gly His Gly His Lys His Glu Arg Asp Gln Gly

1 5 10 15

His Gly His Gln Arg Gly His Gly Leu Gly His Gly His Glu Gln Gln

20 25 30

His Gly Leu Gly His Gly His Lys Phe Lys Leu Asp Asp Asp Leu Glu

35 40 45

His Gln Gly Gly His Val Leu Asp His Gly His Lys His Lys His Gly

50 55 60

His Gly His Gly Lys His Lys Asn Lys Gly Lys Lys Asn Gly Lys His

65 70 75 80

Asn Gly Trp Lys Thr Glu His Leu Ala Ser Gly Ser Gly Gly Gly Gly

85 90 95

Ser Gly Gly Gly Gly Ser Leu Gln Val Arg Glu Arg Gly Pro Gln Arg

100 105 110

Val Ala Ala His Ile Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu Ser

115 120 125

Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys Ile Asn Ser

130 135 140

Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu His Leu

145 150 155 160

Arg Asn Gly Glu Leu Val Ile His Glu Lys Gly Phe Tyr Tyr Ile Tyr

165 170 175

Ser Gln Thr Tyr Phe Arg Phe Gln Glu Glu Ile Lys Glu Asn Thr Lys

180 185 190

Asn Asp Lys Gln Met Val Gln Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro

195 200 205

Asp Pro Ile Leu Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser Lys

210 215 220

Asp Ala Glu Tyr Gly Leu Tyr Ser Ile Tyr Gln Gly Gly Ile Phe Glu

225 230 235 240

Leu Lys Glu Asn Asp Arg Ile Phe Val Ser Val Thr Asn Glu His Leu

245 250 255

Ile Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu Val Gly

260 265 270

<210>7

<211>1288

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<222>(1)..(1288)

<223>Characteristic nucleotide sequence of pBV-TRAIL

<400>7

aaattcttca acgctaactt tgagaatttt tgtaagcaat gcggcgttat aagcatttaa 60

tgcattgatg ccattaaata aagcaccaac gcctgactgc cccatcccca tcttgtctgc 120

acgtgcgtcc tcaagctgct cttgtgttaa tggtttcttt tttgtgctca tacgttaaat 180

ctatcaccgc aagggataaa tatctaacac cgtgcgtgtt gactatttta cctctggcgg 240

tgataatggt tgcatgtact aaggaggttg tatggaacaa cgcataaccc tgaaagatta 300

tgcaatgcgc tttgggcaaa ccaagacagc taaagatctc tcacctacca aacaatgccc 360

ccctgcaaaa aataaattca tataaaaaac atacagataa ccatctgcgg tgataaatta 420

tctctggcgg tgttgacata aataccactg gcggtgatac tgagcacatc agcaggacgc 480

actgaccacc atgaaggtga cgctcttaaa aattaagccc tgaagaaggg cagcattcaa 540

agcagaaggc tttggggtgt gtgatacgaa acgaagcatt ggttaaaaat taaggaggaa 600

ttcacaatgg tgagagaaag aggtcctcag agagtagcag ctcacataac tgggaccaga 660

ggaagaagca aacacattgtc ttctccaaac tccaagaatg aaaaggctct gggccgcaaa 720

ataaactcct gggaatcatc aaggagtggg cattcattcc tgagcaactt gcacttgagg 780

aatggtgaac tggtcatcca tgaaaaaggg ttttactaca tctattccca aacatacttt 840

cgatttcagg aggaaataaa agaaaacaca aagaacgaca aacaaatggt ccaatatatt 900

tacaaataca caagttatcc tgaccctata ttgttgatga aaagtgctag aaatagttgt 960

tggtctaaag atgcagaata tggactctat tccatctatc aagggggaat atttgagctt 1020

aaggaaaatg acagaatttt tgtttctgta acaaatgagc acttgataga catggaccat 1080

gaagccagtt tttttggggc ctttttagtt ggctaaggat ccgtcgacct gcagccaagc 1140

ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa tcagaacgca 1200

gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcgggtggt cccacctgac 1260

cccatgccga actcagaagt gaaacccc 1288

<210>8

<211>29

<212>DNA

<213> Artificial sequence

<400>8

gcgaattcac atatgaaaag gcctccagg 29

<210>9

<211>28

<212>DNA

<213> Artificial sequence

<400>9

gcgaattcat ggtaagtcca ccccaacac 28

<210>10

<211>33

<212>DNA

<213> Artificial sequence

<400>10

gcgaattc atgaaacat aatcttggcc atg 33

<210>11

<211>26

<212>DNA

<213> Artificial sequence

<400>11

aaggatccgc ttgccaaat gctctg 26

<210>12

<211>28

<212>DNA

<213> Artificial sequence

<400>12

aaggatccac tgtcttcaga agagcttg 28

<210>13

<211>26

<212>DNA

<213> Artificial sequence

<400>13

aaggatccag aaaggccatc agtgag 26

<210>14

<211>26

<212>DNA

<213> Artificial sequence

<400>14

aactgcagat ggctatgatg gaggtc 26

<210>15

<211>26

<212>DNA

<213> Artificial sequence

<400>15

aactgcagac ctctgaggaa accatt 26

<210>16

<211>27

<212>DNA

<213> Artificial sequence

<400>16

aactgcaggt gagagaaaga ggtcctc 27

<210>17

<211>24

<212>DNA

<213> Artificial sequence

<400>17

tcaagcttag ccaactaaaa aggc 24

<210>18

<211>30

<212>DNA

<213> Artificial sequence

<400>18

gcgaattcac atatggctat gatggaggtc 30

<210>19

<211>33

<212>DNA

<213> Artificial sequence

<400>19

gcgaattcaca tatgacctc tgaggaaacc att 33

<210>20

<211>29

<212>DNA

<213> Artificial sequence

<400>20

gcgaattcaca tatggtgag agaaagagg 29

<210>21

<211>24

<212>DNA

<213> Artificial sequence

<400>21

aaggatccaa ctaaaaaggc cccg 24

<210>22

<211>25

<212>DNA

<213> Artificial sequence

<400>22

aactgcagat gaaaaggcct ccagg 25

<210>23

<211>25

<212>DNA

<213> Artificial sequence

<400>23

aactgcaggta agtccaccc cacac 25

<210>24

<211>27

<212>DNA

<213> Artificial sequence

<400>24

tcaagcttag cttgccaaa tgctctg 27

<210>25

<211>32

<212>DNA

<213> Artificial sequence

<400>25

aactgcagaa acataatctt ggccatg 27

<210>26

<211>29

<212>DNA

<213> Artificial sequence

<400>26

Tcaagcttaa ctgtcttcag aagagcttg 29

<210>27

<211>25

<212>DNA

<213> Artificial sequence

<400>27

tcaagcttaa gaaaggccat cagtg 25

<210>28

<211>40

<212>DNA

<213> Artificial sequence

<400>28

gatccggcgg aggcgggagc ggcgggggcg gaagcctgca 40

<210>29

<211>32

<212>DNA

<213> Artificial sequence

<400>29

ggcttccgcc cccgccgctc ccgcctccgc cg 32

<210>30

<211>21

<212>DNA

<213> Artificial sequence

<400>30

ttttaaaggg cccgcgcgtt g 21

<210>31

<211>21

<212>DNA

<213> Artificial sequence

<400>31

attggcgcg gcaagctcag c 21

<210>32

<211>24

<212>DNA

<213> Artificial sequence

<400>32

gacttacagc agtcagactc tgac 24

<210>33

<211>24

<212>DNA

<213> Artificial sequence

<400>33

tattgctttt tctttccagg tcag 24

<210>34

<211>30

<212>DNA

<213> Artificial sequence

<400>34

acgaattcac aatggtgaga gaaagaggtc 30

<210>35

<211>24

<212>DNA

<213> Artificial sequence

<400>35

acggatcctt agccaactaa aaag 24

<210>36

<211>274

<212>DNA

<213> Artificial sequence

<400>36

aaagatctct cacctaccaa acaatgcccc cctgcaaaaa ataaattcat ataaaaaaca 60

tacagataac catctgcggt gataaattat ctctggcggt gttgacataa ataccactgg 120

cggtgatact gagcacatca gcaggacgca ctgaccacca tgaaggtgac gctcttaaaa 180

attaagccct gaagaagggc agcattcaaa gcagaaggct ttggggtgtg tgatacgaaa 240

cgaagcattg gttaaaaatt aaggaggaat tcaca 274

<210>37

<211>60

<212>DNA

<213> Artificial sequence

<400>37

aaggatccgt cgacctgcag ccaagcttgg ctgttttggc ggatgagaga agattttcag 60

Claims (10)

1. fused protein is characterized in that it is made up of following elements:

(a) prokinin the 5th structural domain element, this element has the aminoacid sequence of people's prokinin the 5th structural domain or its active fragments, promptly has the aminoacid sequence of 1-266 position, 24-153 position or 60-148 position among the SEQ ID NO.2;

(b) tumor necrosin relative death inducing ligand element, this element has the aminoacid sequence of apoptosis induction ligand related to human tumor necrosis factor or its active fragments, promptly has the aminoacid sequence of 95-281 position among the SEQ ID NO.4 or 114-281 position; And

(c) 1-20 between prokinin the 5th structural domain element and tumor necrosin relative death inducing ligand element amino acid whose catenation sequence.

2. fusion rotein as claimed in claim 1 is characterized in that, described catenation sequence contains 4-10 amino acid.

3. fusion rotein as claimed in claim 1 is characterized in that described fusion rotein has the aminoacid sequence shown in the SEQ IDNO.6.

4. an isolated DNA molecule is characterized in that, the described fused protein of its coding claim 1.

5. dna molecular as claimed in claim 4 is characterized in that, it has the nucleotide sequence shown in the SEQ ID NO.5.

6. a carrier is characterized in that, it contains the described dna molecular of claim 4.

7. a host cell is characterized in that, it contains the described carrier of claim 6.

8. method that produces the described fusion rotein of claim 1, it is characterized in that step is as follows: (1) sets up the cDNA library, and screening obtains TRAIL and Kininogen D5 gene; (2) RT-PCR obtains the encoding sequence of KininogenD5 and the encoding sequence of TRAIL; (3) make up Kininogen D5-TRAIL expression vector; (4) transformed into escherichia coli is set up engineering bacteria; (5) preparation Kininogen D5-TRAIL fused protein; (6) biologic activity of fused protein is determined.

9. a pharmaceutical composition is characterized in that, by pharmaceutically acceptable carrier or vehicle or thinner, and the described fusion rotein of the claim 1 of significant quantity is formed.

10. the purposes of the described fusion rotein of claim 1 is characterized in that, is used to prepare the medicine for the treatment of tumour.

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