CN107828819B - Method for constructing recombinant adenovirus by using ANP or IgANP gene, recombinant adenovirus and application - Google Patents

Abstract

The invention discloses a method for constructing recombinant adenovirus by using ANP or IgANP gene, and the recombinant adenovirus and application. The present invention uses ANP as the target gene to construct a recombinant adenovirus vector, which expresses ANP polypeptide in tongue cancer cell SCC9, and adds an immunoglobulin signal peptide sequence before the ANP sequence to construct a secreted ANP polypeptide. Recombinant adenovirus infected tongue cancer cells, and RT-PCR confirmed that ANP or IgANP could be expressed in tongue cancer cells SCC9. The relative activity detection of MTT cells and cell scratch experiments confirmed that ANP has the ability to inhibit tongue cancer in vitro. Cell SCC9 activity and migration, and the effect of IgANP is more obvious, thereby inhibiting the growth of tongue cancer cells. The method of the invention has high efficiency and large yield, and provides an unreported gene therapy system for anti-tumor cell migration and growth.

Description

A kind of method of utilizing ANP or IgANP gene to construct recombinant adenovirus and recombinant adenovirus Poison and Application

technical field

The present invention relates to a method for constructing recombinant adenovirus, in particular to a method for constructing recombinant adenovirus by using ANP or IgANP gene, and the application of the recombinant adenovirus and the preparation of a drug for inhibiting the activity of tongue cancer cells.

Background technique

Tongue cancer is a common malignant tumor in the oral and maxillofacial region, more than 98% of which are squamous cell carcinoma, and its biological characteristics are mainly local infiltration growth and cervical lymph node metastasis. Patients with tongue squamous cell carcinoma have a higher rate of cervical lymph node metastasis, and are prone to early cervical lymph node metastasis. The prognosis of patients with cervical lymph node metastasis is significantly worse than that of patients without cervical lymph node metastasis. one. Therefore, how to inhibit cancer cell metastasis and improve the prognosis of treatment is particularly important.

Significant advances have been made in the research and treatment of human cancer over the past few decades. However, the effect of traditional cancer treatment is not ideal. Most chemotherapy drugs can kill cancer cells but also kill healthy rapidly dividing cells, and also suppress the immune system. At the same time, there are also cancer drug resistance and tumor areas. Insufficient drug concentration, etc. This greatly reduces their therapeutic efficacy, and the reason for this is related to the lack of specificity of the drug to cancer cells. Anticancer peptides have become one of the targets of new research due to the continuous search for new therapeutic drugs, especially those that can evade drug resistance, inhibit metastasis and have no other important side effects. Anticancer peptides are a new class of small molecule polypeptide anticancer drugs, which can kill cancer cells by destroying the structure of tumor cell membranes or inhibiting the proliferation and migration of cancer cells and the formation of tumor blood vessels, while the toxicity to normal cells is low. A hot spot in the research of new anti-tumor drugs.

Atrial natriuretic peptide (ANP) is a multifunctional active peptide consisting of 28 amino acid residues, also known as atrial natriuretic factor, atrial diuretic peptide, atrial natriuretic peptide or atrial peptide. Atrial diuretic peptide, an active polypeptide substance with natriuretic and diuretic properties, was first isolated from the atrial tissue of rats and humans. Inhibition of cell proliferation, etc., has also been widely recognized for its important role in maintaining blood pressure, water and sodium balance and in the pathophysiological process of cardiovascular disease. In recent studies, it has been found that it can directly kill tumor cells or strongly inhibit the synthesis of cancer cell DNA, thereby inhibiting the growth of cancer cells without producing toxic reactions to normal human cells.

Existing routes of administration include oral administration, respiratory administration, and transdermal administration. The low absorption of protein and polypeptide drugs in the gastrointestinal tract and the degradation of the drugs by enzymes are the two major obstacles for oral polypeptide drugs. Therefore, finding a suitable absorption site to avoid enzymatic degradation in the gastrointestinal tract and first-pass effect in the liver is the key to solving the problem. When peptide drugs are administered in the respiratory tract, they also face the threat of peptide degradation by aminopeptidase in the nasal mucosa, and the clearance of nasal cilia will also affect the absorption of peptide drugs. Transdermal administration can avoid the direct degradation of protease and the scavenging effect of tissues and organs, but this method must overcome the barrier of the skin stratum corneum. In order to promote the entry of polypeptide drugs into the skin, iontophoresis technology is currently used, but most of this system is still In the laboratory stage, there are still some problems, such as: the current through the skin, which is limited by the intensity and duration due to the physiological conditions of the skin; the determination of the mechanism of promoting penetration; the selection of transdermal drugs; the introduction device is more practical and convenient , economics, etc. need further research.

According to the strategy of inhibiting tumor cell metastasis and improving the prognosis of cancer treatment, the present invention provides a new targeted drug for inhibiting tumor cell metastasis with clinical application prospect.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to construct a recombinant adenovirus vector with ANP or secreted ANP (IgANP) as the target gene, use the recombinant adenovirus vector as a tongue cancer gene therapy system, and utilize the gene therapy expressing ANP or secreted ANP for gene therapy. The method can play a role in inhibiting the activity and migration of tongue cancer cells.

For realizing the purpose of the present invention, the present invention provides the following technical solutions:

In a first aspect, the present invention provides a construction method utilizing ANP or secreted ANP gene recombinant adenovirus, comprising the following steps:

1) Obtaining the proANP gene fragment:

a. Extraction of total cell RNA;

b. Obtaining cDNA;

c, proANP gene amplification: the primers used in the proANP gene amplification system are as follows:

Sene: CGGGTACCCCATGAGCTCCTTCTCCACCACC;

Antisene: GCTCTAGAGCTCAGTACCGGAAGCTGTTACAGC;

Overlapping PCR primer 1: CTTCCAAATGGTCCAGCAGCAAATTCTTGAAATCCATCAGGTCTGCG;

Overlapping PCR primer 2: CGCAGACCTGATGGATTTCAAGAATTTGCTGGACCATTTGGAAG;

2) Acquisition of ANP and IgANP gene fragments:

a. Use PCR reaction to obtain ANP gene from proANP fragment, and the primers used are as follows:

ANP sens sequence: CGGAATTCCGGCCACCATGAGCCTGCGGAGATCCAGC;

ANP anti sequence: GCTCTAGAGCTCAGTACCGGAAGCTGTTACAGC;

b, overlapping PCR reaction to obtain IgANP gene:

First, a PCR reaction was used to synthesize the Igκ signal peptide gene that could be used for overlapping PCR. The primers used were as follows:

Igκsens sequence:

CGGAATTCCGGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGG;

Igκanti sequence:

GCAGCTGGATCTCCGCAGGCTGTCACCAGTGGAACCTGGAACCCAGAGCAGCAGTACCC.

And, using PCR reaction to obtain SecANP gene that can be used for overlapping PCR from the proANP fragment, the primers used are as follows:

SecANP sens sequence: AGCCTGCGGAGATCCAGC;

ANP anti sequence: GCTCTAGAGCTCAGTACCGGAAGCTGTTACAGC;

Finally, using the above-mentioned Igκ signal peptide gene and ANP gene as templates, obtain IgANP gene fragment, and the primers used are SecANP sens and Igκanti;

3) Construction of pAdTrack-IgANP or pAdTrack-ANP shuttle vector: IgANP or ANP is connected with pAdTrack-CMV vector;

4) Construction of pAd-IgANP or pAd-ANP adenovirus backbone vector: pAdTrack-IgANP or pAdTrack-ANP plasmid Pme I single-enzyme digestion, get the recovered product and transform it into BJ5183 competent state together with pAd-easy plasmid, extract the plasmid namely pAd- IgANP or pAd-ANP;

5) Preparation of recombinant adenovirus Ad-IgANP or Ad-ANP: After the extracted pAd-IgANP or pAd-ANP plasmid was linearized with Pac I single enzyme digestion, 293-A cells were transfected with Lipofectamine2000, and the green color was observed by fluorescence microscope Fluorescent protein reporter gene expression, after 7 days, a large number of cells can be seen floating, and the fluorescence begins to weaken. At this time, a part of the supernatant is aspirated and transferred to a sterile 15mL centrifuge tube, and the remaining medium is repeatedly pipetted. The cells were transferred to a centrifuge tube together with the culture medium; the supernatant was collected by centrifugation at 1000 rpm for 5 min, which was the recombinant adenovirus Ad-IgANP or Ad-ANP obtained after packaging.

The second aspect of the present invention provides a recombinant adenovirus prepared by the above method, which expresses ANP or secretory ANP, the sequence of the ANP is shown in SEQ ID NO: 1, and the sequence of the secretory ANP is shown in SEQ ID NO: 2 .

SLRRSSCFGGRMDRIGAQSGLGCNSFRY (SEQ ID NO: 1);

METDTLLLWVLLLWVPGSTGDSLRRSSCFGGRMDRIGAQSGLGCNSFRY (SEQ ID NO: 2).

Due to the degeneracy of codons, the sequence of the ANP gene of the present invention can also include any nucleotide sequence that can encode the amino acid sequence shown in SEQ ID NO: 1, and the sequence of the secreted ANP gene can also include any nucleotide sequence that can encode the amino acid sequence shown in SEQ ID NO: 1. Nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2.

Preferably, the nucleotide sequence of the ANP gene it carries is:

AGCCTGCGGAGATCCAGCTGCTTCGGGGGCAGGATGGACAGGATTGGAGCCCAGAGCGGACTGGGCTGTAACAGCTTCCGGTACTGA (SEQ ID NO: 3);

Preferably, the nucleotide sequence of the secretory ANP gene carried by it is:

ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACAGCCTGCGGAGATCCAGCTGCTTCGGGGGCAGGATGGACAGGATTGGAGCCCAGAGCGGACTGGGCTGTAACAGCTTCCGGTACTGA (SEQ ID NO: 4).

Preferably, the shuttle plasmid used for constructing the recombinant adenovirus is pAdTrack-CMV vector;

Preferably, the viral backbone plasmid used for constructing the recombinant adenovirus is pAdeasy-1;

Preferably, the packaging cells used for constructing the recombinant adenovirus are mammalian cells, preferably human embryonic kidney 293-A cells.

In a third aspect, the present invention provides a recombinant adenovirus plasmid obtained by homologous recombination of a recombinant shuttle plasmid carrying an ANP or secreted ANP gene and a viral backbone plasmid;

Preferably, the recombinant shuttle plasmid is composed of a Track vector carrying ANP or secreted ANP gene, which is expressed as pAdTrack-IgANP or pAdTrack-ANP in the present invention;

Preferably, the viral backbone plasmid is pAdeasy-1;

Preferably, the homologous recombination occurs in E. coli, preferably E. coli BJ5183.

In a fourth aspect, the application of the recombinant adenovirus prepared by the above-mentioned method for constructing a recombinant adenovirus by using ANP or secreted ANP gene in the preparation of a drug for inhibiting the activity of tongue cancer cells is provided.

The present invention uses recombinant adenovirus vector to express ANP polypeptide in tongue cancer cell SCC9, and at the same time adds an immunoglobulin signal peptide sequence before the ANP sequence to construct an experimental group of secreted ANP polypeptide (the following labels are all IgANP). Through RT-PCR, it was confirmed that ANP could be expressed in tongue cancer cells SCC9, and by MTT cell relative activity detection and cell scratch experiments, it was confirmed that ANP had the effect of inhibiting tongue cancer cell SCC9 activity and migration in vitro, and IgANP effect is more obvious. Thereby inhibiting the growth of tongue cancer cells.

Description of drawings

Fig. 1 The electrophoresis of proANP gene fragments obtained by PCR;

Figure 2 IgANP (ANP) double-enzyme digestion electrophoresis;

Figure 3 PCR detection of integration electrophoresis in Ad-ANP genome;

Figure 4 PCR detection of integration electrophoresis in Ad-IgANP genome;

Figure 5 RT-PCT detects the expression of ANP and IgANP in SCC9;

Fig. 6 Scratch experiment of ANP and IgANP;

Fig. 7 Scratch experiment migration analysis diagram of ANP and IgANP;

Figure 8 The effect of different treatments on the relative activity of human tongue squamous cell carcinoma cells SCC9 detected by MTT;

Figure 9 Flow cytometer detects cell cycle diagram;

Detailed ways

The features and advantages of the present invention can be further understood from the following detailed description in conjunction with the accompanying drawings. The examples provided are merely illustrative of the methods of the present invention, and are not intended to limit the remainder of the present disclosure in any way.

[Example 1] Obtaining the target gene ANP and IgANP

1. Obtaining the proANP gene fragment

1.1 Primer Design

The gene sequence was obtained according to the GeneBank No. NM_006172.3 of the human ANP gene, in which the CDS region has 456 bp, and the sequence is as follows:

ATGAGCTCCTTCTCCACCACCACCGTGAGCTTCCTCCTTTTACTGGCATTCCAGCTCCTAGGTCAGACCAGAGCTAATCCCATGTACAATGCCGTGTCCAACGCAGACCTGATGGATTTCAAGAATTTGCTGGACCATTTGGAAGAAAAGATGCCTTTAGAAGATGAGGTCGTGCCCCCACAAGTGCTCAGTGAGCCGAATGAAGAAGCGGGGGCTGCTCTCAGCCCCCTCCCTGAGGTGCCTCCCTGGACCGGGGAAGTCAGCCCAGCCCAGAGAGATGGAGGTGCCCTCGGGCGGGGCCCCTGGGACTCCTCTGATCGATCTGCCCTCCTAAAAAGCAAGCTGAGGGCGCTGCTCACTGCCCCTCGGAGCCTGCGGAGATCCAGCTGCTTCGGGGGCAGGATGGACAGGATTGGAGCCCAGAGCGGACTGGGCTGTAACAGCTTCCGGTACTGA。

Primer Premier 5 was used to design the following primers according to the above gene sequences (Table 1). In the ANP amplification primers, the upstream primer adds a Kpn I restriction site, and the downstream primer adds an Xba I restriction site. ANP overlapping PCR primers, fragment 1 upstream primer and fragment 2 downstream primer are identical to ANP amplification primers.

Amplification primer Sene: CGGGTACCCCATGAGCTCCTTCTCCACCACC; restriction site: Kpn I.

Antisene: GCTCTAGAGCTCAGTACCGGAAGCTGTTACAGC; restriction site: Xba I

Detection primer Sene: ACGCAGACCTGATGGATTTC.

Antisene: GCTTCTTCATTCGGCTCACT.

Overlap PCR primer 1: CTTCCAAATGGTCCAGCAGCAAATTCTTGAAATCCATCAGGTCTGCG.

Overlap PCR primer 2: CGCAGACCTGATGGATTTCAAGAATTTGCTGGACCATTTGGAAG.

1.2 RT-PCR

Extraction of total cellular RNA

①Collect cells, add 1mL Trizol reagent and mix;

②Add 200μL of chloroform, vortex to make it well mixed, until the solution is emulsified into white, put on ice for 10min;

③ Centrifuge at 12000rpm/min at 4°C for 10min, take out the centrifuge tube, the solution is in three layers, and the supernatant contains RNA. Aspirate the supernatant into a nuclease-free 2mL Ep tube. Be careful not to aspirate the middle white layer. Add an equal volume of isopropanol to the Ep tube. Slowly invert the Ep tube up and down several times to mix well, and place it at -20°C for 10 minutes;

④ Centrifuge at 12000rpm/min at 4℃ for 10min. A small amount of white precipitate will appear at the bottom of the Ep tube. Carefully remove the supernatant. Add 1 mL of absolute ethanol, and use a pipette to blow up the precipitate at the bottom of the Ep tube;

⑤ Centrifuge at 7500rpm/min at 4°C for 5min, discard the supernatant, place it on a clean bench, and let it air dry. The final white precipitate was translucent. Add 20μL of RNase-free water to dissolve;

1.3 Obtaining cDNA

293-T and SCC-9 cells RNA reverse transcription system: AMV (reverse transcriptase) 1 μL, dNTP 3 μL, RNase inhibitor 0.5 μL, 5×Buffer 4 μL, Oligo(dT) 1 μL, RNA Template 10.5 μL.

Reverse transcription program: 30°C for 10 minutes, 42°C for 50 minutes, 95°C for 50 minutes, and 10°C for 5 minutes. The reverse transcription reaction was performed on a PCR machine.

1.4 Obtaining the proANP gene fragment

293-T and SCC-9 cell cDNA PCR system: ddH ₂ O 12.3 μL, PCR Buffer 10×2 μL, dNTP Mixture 2.5 μL, rTaq enzyme 0.2 μL, Primer F 1 μL, Primer R 1 μL, Template 1 μL.

The PCR program was set as follows: 94°C for 5 min; 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 5 min, 16°C for 15 min.

PCR reaction products were electrophoresed on a 1% agarose gel (Figure 1).

2. Primer design for obtaining ANP and IgANP gene fragments

According to the amino acid sequence SLRRSSCFGGRMDRIGAQSGLGCNSFRY of human ANP, the corresponding base sequence is as follows:

AGCCTGCGGAGATCCAGCTGCTTCGGGGGCAGGATGGACAGGATTGGAGCCCAGAGCGGACTGGGCTGTAACAGCTTCCGGTACTGA

According to the amino acid sequence METDTLLLWVLLLWVPGSTGD of the Igκ signal peptide, the corresponding base sequence is obtained as follows:

ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGAC

The following primers were designed using Primer Premier 5 according to the above gene sequence. In the ANP amplification primers, the upstream primer has an EcoR I restriction site, a KOZAK sequence fragment and ATG, and the downstream primer has an Xba I restriction site. Among the SecANP amplification primers used for linking with the Igκ signal peptide, the upstream primer selects the first 18 nucleotides of the ANP gene sequence, and the downstream primer is the same as the downstream primer in the ANP amplification primer. In the amplification primer of the Igκ signal peptide used for ligation with SecANP, the EcoR I restriction site and KOZAK sequence were added to the upstream primer, and the partial gene sequence of the Igκ signal peptide and the partial gene sequence of ANP were selected for the downstream primer.

ANP sens sequence: CGGAATTCCGGCCACCATGAGCCTGCGGAGATCCAGC; enzyme cleavage site: EcoR I.

ANP anti sequence: GCTCTAGAGCTCAGTACCGGAAGCTGTTACAGC; enzyme cleavage site: Xba I.

SecANP sens sequence: AGCCTGCGGAGATCCAGC.

Igκsens sequence: CGGAATTCCGGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGG; restriction site: EcoR I.

Igκanti sequence: GCAGCTGGATCTCCGCAGGCTGTCACCAGTGGAACCTGGAACCCAGAGCAGCAGTACCC.

ANP was obtained from the proANP fragment by PCR reaction, reaction system: 10×buffer 2.5 μL, ANP sens 1 μL, ANP anti 1 μL, Taq enzyme 0.2 μL, dNTP 2 μL, ddH ₂ O 18.1 μL, template 0.2 μL. The system for synthesizing Igκ signal peptide by overlapping PCR reaction is as follows: 10×buffer 2.5μL, Igκsens 2μL, Igκanti 2μL, Taq enzyme 0.2μL, dNTP 2μL, ddH ₂ O 16.3μL. The ANP system that can be used for overlapping PCR is obtained from the proANP fragment by PCR reaction as follows: 10×buffer 2.5 μL, SecANP sens 1 μL, ANPanti 1 μL, Taq enzyme 0.2 μL, dNTP 2 μL, ddH2O 18.1 μL, template 0.2 μL, synthesized by overlapping PCR reaction The ANP system containing the signal peptide is as follows: 5×buffer (pfu) 5 μL, Igκanti 5 μL, SecANP 5 μL, Taq enzyme 0.3 μL, Pfu enzyme 0.2 μL, dNTP 2 μL, ddH ₂ O 7.5 μL. All PCR reaction procedures were the same, all settings were as follows: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 5 min, 16°C for 5 min.

[Example 2] Preparation of recombinant adenovirus carrying ANP or secreted ANP gene

1 Construction of recombinant adenovirus gene vector

1.1 Construction and identification of pMD-20T-IgANP or pMD-20T-ANP vector

IgANP or ANP and pMD-20T carrier connection system: Solution I 5μL, pMD-20T carrier 0.5μL, overlapping IgANP or ANP recovery product 2.6μL, ddH ₂ O 1.9μL.

The ligation system was placed in a thermostat at 16°C overnight. Kpn I, Xba I double enzyme digestion identification, the reaction product is electrophoresed by 1% agarose gel (as shown in Figure 2). Sequencing and identification were carried out by the Wuhan Sequencing Department of Sangon Bioengineering (Shanghai) Co., Ltd.

1.2 Construction and identification of pAdTrack-IgANP or pAdTrack-ANP shuttle vector

IgANP or ANP and pAdTrack-CMV vector ligation system: T4 ligase Buffer 10×2 μL, T4 ligase 2 μL, IgANP or ANP recovery product 2 μL, pAdTrack-CMV 4 μL, ddH ₂ O 10 μL.

The ligation system was placed in a thermostat at 16°C overnight. Transform DH5α Escherichia coli, extract the plasmid, Kpn I, Xba I double digestion identification. The reaction product was electrophoresed on a 1% agarose gel.

1.3 Construction and identification of pAd-IgANP or pAd-ANP adenovirus backbone vector

pAdTrack-IgANP or pAdTrack-ANP plasmid Pme I single digestion system: ddH ₂ O 29.5 μL, B Buffer 10×5 μL, pAdTrack-IgANP or pAdTrack-ANP 15 μL, Pme I 0.5 μL. Placed in a constant temperature water bath at 37°C for single-enzyme digestion reaction identification, and the reaction product was electrophoresed on a 1% agarose gel. Gel recovery was performed with reference to the DNA gel recovery kit of TIAN GEN (see Figure 2, lanes 1, 2, and 3 are pAdTrack-IgANP, and lanes 4 and 5 are pAdTrack-ANP). The recovered product was transformed into BJ5183 competent cells together with pAd-easy-1 plasmid, and the plasmid was extracted as pAd-IgANP or pAd-ANP.

Put the extracted pAd-IgANP or pAd-ANP plasmid into a 37°C constant temperature water bath for single digestion with Pac I. Pac I single digestion system: ddH ₂ O 9.8 μL, Pac I Buffer 10×2 μL, pAd-IgANP Or pAd-ANP 8 μL, Pac I 0.2 μL. The reaction product was electrophoresed on a 1% agarose gel. Placed in a constant temperature water bath at 37°C for single-enzyme digestion reaction identification, and the reaction product was electrophoresed on a 1% agarose gel.

1.4 Extraction of medium amount of plasmid

Referring to the high-purity medium-quantity plasmid extraction instructions of TIAN GEN company, the product was identified by 1% agarose gel electrophoresis.

1.5 Single-enzyme digestion ethanol precipitation recovery

The extracted pAd-IgANP plasmid was digested with Pac I in a constant temperature water bath at 37°C for 3 hours. Pac I single digestion system: ddH ₂ O 9.8 μL, Pac I Buffer 10×2 μL, Plasmid 8 μL, Pac I 0.2 μL. The steps of ethanol precipitation and recovery of enzyme digestion products are as follows:

① After taking out the digested system, transfer it to a 1.5 mL Ep tube, and add ddH ₂ O to the system to make up to 200 μL;

②Add 1/10 mentioned sodium acetate (3mol/L, pH=5.2) and mix well with the system;

③ Add 2 times the volume of pre-cooled absolute ethanol, and mix by inversion. Place at -20℃, 30min;

④ Centrifuge at 12,000 rpm for 10 min at 4°C to collect DNA pellets;

⑤ Open the mouth of the tube, discard the supernatant, add 1 mL of 70% ethanol to the Ep tube, and bounce the precipitate to wash thoroughly. Centrifuge at 12,000 rpm, 4°C for 5 min, discard the supernatant, and air dry the ethanol at room temperature;

⑥ Add 10 μL ddH ₂ O to dissolve the DNA.

2. Preparation of recombinant adenovirus Ad-IgANP or Ad-ANP

2.1 Transfection of 293-A cells with recombinant adenovirus vector pAd-IgANP

293-A cells were cultured in DMEM containing 10% calf serum in a 37°C, 5% CO ₂ incubator. After the recombinant pAd-IgANP or pAd-ANP plasmid was linearized by Pac I single enzyme digestion, 293-A cells were transfected with Lipofectamine2000, and the expression of the green fluorescent protein reporter gene was observed by fluorescence microscope. After 7 days, a large number of cells floating and fluorescence were observed. start to weaken. At this time, aspirate a part of the supernatant and transfer it to a sterile 15mL centrifuge tube. The remaining medium is repeatedly pipetted and beaten the unfloated cells at the bottom of the dish, and then transferred to the centrifuge tube together with the medium; 1000 rpm, 5min, centrifuge The supernatant was collected, aliquoted into 1.5 mL EP tubes, each tube was 1 mL, and stored at -20°C, which was recombinant adenovirus Ad-IgANP or Ad-ANP.

2.2 Amplification of IgANP-containing adenovirus

①Cultivation and passage of 293A cells, inoculate 293A cells in a 60mm dish with about 90% confluence in a 10cm dish, the medium used is high-glucose DMEM (10%FBS), and the culture conditions are 37℃, 5%CO ₂ .

②When the confluence rate of 293A cells is about 80%-90%, change to fresh medium, inoculate virus, and inoculate 300 μL of virus supernatant in a 10cm dish.

③The cell morphology has changed, from the 293A cells that were initially infected with the virus, which showed the characteristics of polygonal epithelial cells, began to become round, showing the shape of grape bunches, and on the sixth day, a large number of cells could be seen floating (about 50% of the cells were floating) , the poisoning can be carried out at this time.

In the first step, aspirate a part of the supernatant and transfer it to a sterile 15mL centrifuge tube. The remaining medium is repeatedly pipetted and beat the unfloated cells at the bottom of the dish, and then transfer it to the centrifuge tube together with the medium;

The second step, 1000 rpm, 5min, centrifuged to collect the supernatant, aliquoted into 1.5mL EP tubes, one 1mL tube, and stored at -20°C;

The third step, add 1mL PBS to resuspend the cells;

The fourth step, seal the tube cover with parafilm, tie the centrifuge tube with a string, and put the centrifuge tube into the liquid nitrogen tank vertically. After more than ten seconds, you can take it out without hearing the boiling sound of liquid nitrogen;

The fifth step is to stir the frozen cell liquid in warm water at 37°C repeatedly to make it thaw rapidly;

Step 6, Steps 4 and 5 are repeated three times;

The seventh step, 13,000 rpm, 10min centrifugation to collect the supernatant, aliquot into 1.5mL EP tubes, 0.5mL per tube, and store at -20°C.

In the eighth step, 50 μL of the supernatant collected in the second step and the cell lysate in the step 7 were taken for PCR detection of the integration of the target gene in the genome.

2.3 PCR identification of target gene in recombinant adenovirus

Adenovirus PCR identification system containing ANP gene (25μL in total)

Virus detection PCR identification system in supernatant and cell lysate: rTaq enzyme 0.2 μL, supernatant (cell lysate) 2 μL, ANPsence 0.25 μL, ANPanti 0.25 μL, dNTP (2.5 mM) 2 μL, 10×Buffer 2.5 μL, H ₂ O 17.8 μL.

PCR identification procedure of adenovirus containing ANP gene: 94℃ for 5min; 94℃ for 30s, 55℃ for 30s, 72℃ for 30s, 35 cycles; 72℃ for 5min, 16℃ for 5min. After PCR, perform agarose gel electrophoresis (as shown in Figure 3, 1: water; 2: Ad-ANP supernatant; 3: Ad-ANP cell lysate; 4: Ad-egfp supernatant; 5: Ad -egfp cell lysate; 6: pAd-ANP plasmid as a positive control. The size of 125bp is the ANP target band)

Adenovirus PCR identification system containing IgANP gene (25μL in total)

Virus detection in supernatant and cell lysate: rTaq enzyme 0.2 μL, supernatant (cell lysate) 2 μL, Igsence 0.25 μL, ANPanti 0.25 μL, dNTP (2.5 mM) 2 μL, 10×Buffer 2.5 μL, H ₂ O 17.8 μL.

PCR identification procedure of adenovirus containing IgANP gene: 94℃ for 5min; 94℃ for 30s, 55℃ for 30s, 72℃ for 30s, 35 cycles; 72℃ for 5min, 16℃ for 5min. After PCR, perform agarose gel electrophoresis (as shown in Figure 4, 1: pAd-IgANP plasmid as positive control; 2: Ad-egfp supernatant; 3: Ad-egfp cell lysate; 4: Ad-IgANP supernatant Clear liquid; 5: Cell lysate containing a large amount of virus obtained from Ad-IgANP plasmid packaging; 6: Water. The size of 196bp is the IgANP target band)

2.4 Rapid Adenovirus Titer Assay

①Inoculate 293A cells in a 96-well plate, about 7000 cells per well, test one virus gradient in a group of twelve wells, and one virus is repeated three times, and placed in a 37°C, 5% _CO2 incubator Cultivate for 24h;

②When the cell confluence is 70%-80%, replace with fresh medium, add 90 μL of virus solution to each well. Add 10 μL to the first well. After mixing, pipette 10 μL and transfer it to the next well. After mixing, transfer 10 μL backwards according to the previous steps until the 11th well. The aspirated 10 μL is discarded directly. The first four dilution gradients can be carried out in 1.5mL EP tubes and placed in a 37°C, 5% _CO2 incubator for 24h;

③ Observe under an inverted fluorescence microscope, and observe the number of green fluorescent cells in order from high concentration to low concentration. When it is found that there is no green fluorescence in the well after a certain well, this well is used as a metering well (counted as 1U). There is no green fluorescence in front of this well, and this well is also used as a metering well, and then the virus titer is calculated.

The titer of the virus solution=(1U×dilution factor of the metering well relative to the first well)/the volume of the virus added to the first well.

The virus titer of 10:1 dilution is 1U×10 ^n-1 /0.01mL=10 ⁿ⁺¹ U/mL, that is, when the 8th well is the measuring well, the virus titer=10 ⁹ U/mL. The calculated titers of Ad-ANP and Ad-IgANP were both 10 ⁹ U/mL.

2.5 Detection of recombinant adenovirus expression in SCC9 cells

Total RNA extraction:

①Inoculate the cells in a 35mm dish and culture at 37°C, 5% _CO2 for 24 hours;

②When the cell confluence is about 80%, change to fresh medium, add 40μL of virus solution (cell lysate), shake and mix well, and incubate at 37°C, 5% CO ₂ for 48 hours;

③Extraction of total RNA

In the first step, aspirate the medium, wash it once with 1×PBS, add 1 mL of RNAiso Plus (TakaRa) to the dish, and let it stand for 5 minutes at room temperature;

The second step, 12000g, 4℃, 5min, collect the supernatant, transfer it to a 1.5mL EP tube, and add about 200μL of chloroform;

The third step, the vortex shaker shakes and mixes;

The fourth step, 12000g, 4℃, 15min, collect the supernatant and transfer it to a new EP tube, about 600μL;

The fifth step, add 600 μL isopropanol;

The sixth step, after standing at room temperature for 10 minutes, 12000g, 4 ℃, 5 minutes, centrifuge to collect the precipitate, discard the supernatant;

The seventh step, add 1 mL of absolute ethanol, invert the EP tube to wash the precipitate;

The eighth step, 12000g, 4°C, 5min centrifugation to collect the precipitate and discard the supernatant;

The ninth step, the tube is buckled on the sterile filter paper, and the ethanol is drained;

The tenth step, dissolve the RNA with 20 μL of free-RNAase ddH ₂ O.

④ Reverse transcription to obtain cDNA

The first step, configure the reverse transcription system (1): OligodT Primer (50 μM) 0.5 μL, dNTP Mixture (10 mM) 0.5 μL, template RNA 1 μg, RNase free dH ₂ O supplemented to 5 μL.

In the second step, the system (1) was kept at 65 °C to make the secondary structure of the RNA open to the primary structure, and the incubation time was 5 min, and it was rapidly cooled on ice;

The third step is to prepare reverse transcription system (2): DNase I (70U/μL) 0.2 μL, Primescript II RTase (200U/μL) 0.5 μL,

RNase Inhibitor (40U/μL) 0.25 μL, 5×Primescript II Buffer 0.5 μL, RNasefree dH ₂ O 3.55 μL.

The fourth step, the system is placed at 42 ° C, 30-60min (actual operation is 40min)

The fifth step is to inactivate DNase I at 95°C for 5min, and cool on ice.

cDNA detection: PCR identification

The first step, adenovirus PCR identification system containing ANP gene (total 25μL)

Virus detection PCR system in supernatant and cell lysate: rTaq enzyme 0.2 μL, supernatant (cell lysate) 2 μL, ANPsence 0.25 μL, ANPanti 0.25 μL, dNTP (2.5 mM) 2 μL, 10×Buffer 2.5 μL, H ₂ O 17.8 μL.

PCR identification procedure of adenovirus containing ANP gene: 94℃ for 5min; 94℃ for 30s, 55℃ for 30s, 72℃ for 30s, 35 cycles; 72℃ for 5min, 16℃ for 5min.

Adenovirus PCR identification system containing IgANP gene (25μL in total)

Virus detection in supernatant and cell lysate: rTaq enzyme 0.2 μL, supernatant (cell lysate) 2 μL, Igsence 0.25 μL, ANPanti 0.25 μL, dNTP (2.5 mM) 2 μL, 10×Buffer 2.5 μL, H ₂ O 17.8 μL.

PCR identification procedure of adenovirus containing IgANP gene: 94℃ for 5min; 94℃ for 30s, 55℃ for 30s, 72℃ for 30s, 35 cycles; 72℃ for 5min, 16℃ for 5min.

Detection of intracellular reference actin: rTaq enzyme 0.2 μL, cell lysate 2 μL, qactinsence 0.25 μL, qactinanti 0.25 μL, dNTP (2.5 mM) 2 μL, 10×Buffer 2.5 μL, H ₂ O 17.8 μL. The PCR identification procedure of cell internal reference actin: 94°C for 5 min; 94°C for 30s, 60°C for 30s, 72°C for 30s, 35 cycles; 72°C for 5min, 16°C for 5min. The PCR products were electrophoresed on a 1% agarose gel, as shown in Figure 5, and both ANP and Ig-ANP recombinant adenoviruses were expressed in SCC9 cells.

[Example 3] Detection of tumor suppressor effect

1. Cell Scratch Experiment

①Cell preparation, add SCC9 cells in a 60mm dish with a confluence of about 70%, add virus at a multiplicity of infection of 100 (add 30 μL of virus with a pfu of about 1×10 ⁹ ), and place it at 37°C, 5% CO ₂ . Cultivated in an incubator.

② Draw two parallel lines at the bottom of each well of the 12-well plate with a marker in advance, and suspend the cells in the 60mm dish yesterday with trypsin. A 60mm dish with a confluence of about 70%-80% can be covered with 4 wells of 12-well plate (cells infected with virus grow slowly, adding virus at 70% confluence will not cause too many cells during plating), place the plated 12-well plate at 37°C, Cultured in a 5% CO ₂ incubator;

③ For cell scratch treatment, take an iron ruler that has been burnt with an alcohol lamp, and after it cools, place it on the hole, fix it by hand, and use a pipette with a 200 μL pipette tip along the iron ruler. Draw lines through the holes, one line per hole. After painting, discard the supernatant medium, add 1 mL of 1×PBS along the wall, wash the floating cells generated during scratches, wash about 1-3 times, add 1 mL of medium with 1% serum content, and take pictures under an inverted microscope for 0 h The scratch state is fixed, and the intersection of the scratch and the pre-marked stroke is fixed, and the cells are placed in a 37°C, 5% CO ₂ incubator for culture. Photographs were taken every 12-24 hours for three days (as shown in Figure 6, the wound healing in the recombinant adenovirus-expressing ANP and Ig-ANP groups was slow);

④ Collect the pictures, analyze the pictures with IPP (image pro plus), and obtain the cell movement rate of each group. After taking the average value, divide the experimental group and the blank control to obtain the ratio of the experimental group/control group, and observe the cell movement rate. In the condition of being inhibited in different time periods (as shown in Figure 7), both ANP and IgANP have better ability to inhibit the migration of tongue cancer cell SCC9, and IgANP has a better ability to inhibit the migration of tongue cancer cell SCC9.

2. Detection of relative activity of MTT cells

① Prepare cells: the cell type is SCC9

Use a hemocytometer to determine the cell concentration to ensure that the cells in the 96-well plate can be 40% confluent on the second day, 5000 SCC9 cells per well, and 100 μL of medium per well. Incubate at 37°C, 5% CO ₂ for 24h.

② Replace the medium with fresh medium, add virus solution (cell lysate), use adriamycin as a positive control, put it at 37° C., 5% CO ₂ , and cultivate for 48 hours.

③ Add 15 μL MTT (5 mg/mL, final concentration 0.75 mg/mL) to each well and place in the incubator to incubate for 4 h. Discard the supernatant and need to be careful not to aspirate the formazan. Add 150 μL of DMSO and shake on a shaker for 10 min. Use a microplate reader to detect with a detection wavelength of 570. After obtaining the value, calculate the inhibition rate.

Inhibition rate = (OD ₅₇₀ value of the control group - OD ₅₇₀ value of the experimental group)/OD ₅₇₀ value of the control group (as shown in Figure 8), compared with the EGFP control group, Ad-ANP and Ad-IgANP had better effects on SCC9. high inhibition rate.

3. Cell cycle detection

①Cell plating: Count the cells the afternoon before the experiment, and adjust the number of cells to about 1×10 ⁵ /mL. Spread the diluted cell suspension evenly into a 24-well plate, 500 μL per well, about 50,000 cells. Carefully place the seeded cell culture plate in the incubator overnight.

②Add virus: Add 20 μL of Ad-ANP or Ad-IgANP to each well respectively, and the negative control is Ad-EGFP, and culture in an incubator for 48 hours.

③ Cell fixation: cells were collected by centrifugation, the supernatant was discarded, and the cells were washed twice with pre-cooled PBS. First add 300μL of PBS to resuspend the cells, then add 700μL of pre-cooled absolute ethanol (preferably at -20°C) to make the ethanol concentration 70%, fix at 4°C overnight, or at -20°C for long-term fixation.

④ Cell staining: collect cells by centrifugation, wash cells once with 1 mL of PBS, add 80 μL of PBS containing 100 μg/mL propidium iodide (PI), 0.25% Triton-100, and incubate at 4°C for 30 min in the dark.

⑤ Detection by flow cytometry: PI was detected by FL2 channel. The detection steps were performed according to standard procedures using a flow cytometer (Figure 9). As shown in the figure, M1 (the percentage of cells in the G0/G1 phase) was significantly increased compared with the control group, while M2 (the percentage of cells in the G2 phase) was decreased compared with the control group, indicating that ANP can inhibit the cells in the G0/G1 phase. stagnation.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

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Claims (2)

1. A recombinant adenovirus expressing ANP or secreted ANP, characterized in that it carries an ANP or secreted ANP gene, and the nucleotide sequence of the ANP gene is shown in SEQ ID NO: 3, and the secreted ANP The nucleotide sequence of the ANP gene is shown in SEQ ID NO: 4; The shuttle plasmid used for constructing the recombinant adenovirus is pAdTrack-CMV; the viral backbone plasmid used for constructing the recombinant adenovirus is pAdeasy-1; the packaging cell used for constructing the recombinant adenovirus is human embryonic kidney 293A cells; It expresses ANP or secreted ANP, the amino acid sequence of the ANP is shown in SEQ ID NO: 1, and the amino acid sequence of the secreted ANP is shown in SEQ ID NO: 2; The construction method of the recombinant adenovirus expressing ANP or secreted ANP gene comprises the following steps: 1) Obtaining the proANP gene fragment: a. Extraction of total cell RNA; b. Obtaining cDNA; c, proANP gene amplification: the primers used in the proANP gene amplification system are as follows: Sene: CGGGTACCCCATGAGCTCCTTCTCCACCACC; restriction site: Kpn I; Antisene: GCTCTAGAGCTCAGTACCGGAAGCTGTTACAGC; restriction site: Xba I; Overlapping PCR primer 1: CTTCCAAATGGTCCAGCAGCAAATTCTTGAAATCCATCAG GTCTGCG; Overlapping PCR primer 2: CGCAGACCTGATGGATTTCAAGAATTTGCTGGACCAT TTGGAAG; 2) Obtaining ANP and IgANP gene fragments: a. Use PCR reaction to obtain ANP gene from proANP fragment, and the primers used are as follows: ANP sens sequence: CGGAATTCCG GCCACCATGAGCCTGCGGAGATCCAGC; restriction site: EcoR I; ANP anti sequence: GCTCTAGAGC TCAGTACCGGAAGCTGTTACAGC; restriction site: Xba I; b, overlapping PCR reaction to obtain IgANP gene: First, a PCR reaction was used to synthesize the Igκ signal peptide gene that could be used for overlapping PCR. The primers used were as follows: Igκ sens sequence: CGGAATTCCGGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGG; restriction site: EcoR I; Igκ anti-sequence: GCAGCTGGATCTCCGCAGGCTGTCACCAGTGGAACCTGGAACCCAGAGCAGCAGTACCC; And, using PCR reaction to obtain SecANP gene that can be used for overlapping PCR from the proANP fragment, the primers used are as follows: SecANP sens sequence: AGCCTGCGGAGATCCAGC; ANP anti sequence: GCTCTAGAGC TCAGTACCGGAAGCTGTTACAGC; Finally, using the above-mentioned Igκ signal peptide gene and ANP gene as templates, the IgANP gene fragment was obtained, and the primers used were SecANP sens and Igκ anti; 3) Construction of pMD-20T-IgANP or pMD-20T-ANP vector IgANP or ANP and pMD-20T vector ligation system: pMD-20T vector, overlapping IgANP or ANP were digested by Kpn I and Xba I respectively to recover the product, and the ligation system was placed in a 16°C thermostat overnight; 4) Construction of pAdTrack-IgANP or pAdTrack-ANP shuttle vector: IgANP or ANP Kpn I , Xba I double digestion recovery product is connected with Kpn I , Xba I double digestion Track vector; 5) Construction of pAd-IgANP or pAd-ANP adenovirus backbone vector: pAdTrack-IgANP or pAdTrack-ANP plasmid Pme I was digested with single enzyme, and the recovered product was transformed with pAd-easy-1 plasmid into competent BJ5183, and the plasmid was extracted as pAd-IgANP or pAd-ANP; 6) Preparation of recombinant adenovirus Ad-IgANP or Ad-ANP: After the extracted pAd-IgANP or pAd-ANP plasmid was linearized with Pac I single enzyme digestion, 293-A cells were transfected with Lipofectamine2000, and the green color was observed by fluorescence microscope Fluorescent protein reporter gene expression, after 7 days, a large number of cells can be seen floating, and the fluorescence begins to weaken. At this time, a part of the supernatant is aspirated and transferred to a sterile 15mL centrifuge tube, and the remaining medium is repeatedly pipetted. The cells were transferred to a centrifuge tube together with the culture medium; the supernatant was collected by centrifugation at 1000 rpm for 5 min, which was the recombinant adenovirus Ad-IgANP or Ad-ANP obtained after packaging. 2. Application of the recombinant adenovirus according to claim 1 in the preparation of a drug for inhibiting the activity of tongue cancer cells.

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