Preparation method of recombinant tumor specificity antiapoptotic factors with activity and application of products thereof

[0036] detailed description

[0037] (1) Directly obtain Apoptin gene fragments by artificial synthesis; construct the prokaryotic expression vector pGEX-A containing GST-Apoptin gene;

[0038] (2) Fermentation, high-efficiency expression, dissolution, renaturation and purification of GST-Apoptin fusion protein, specifically:

[0039] (2.1) Activation, fermentation of engineering bacteria and recovery of inclusion bodies. Inoculate engineering bacteria stored at -70°C on a nutrient agar plate medium containing AP, and incubate at 37°C for more than 12 hours; pick a single colony to inoculate AP-containing In LB liquid medium, culture in a shaker at 37°C for more than 12 hours, inoculate the bacterial solution into the fermentor at a ratio of 1:10, and cultivate at 37°C; after the bacterial solution concentration reaches 2.0 OD, add IPTG inducer; induce Cultivate for 4 hours, while continuing to add a small amount of filler solution, the ammonia automatically adjusts the pH value of 7.0. The cells were collected by centrifugation, and the cells were suspended in phosphate buffer (PB, pH 7.2). Under ice bath conditions, the cells were crushed by high-pressure homogenization and repeated 4 times. Centrifuge the broken bacteria and collect the precipitate, which is the inclusion body ( image 3 );

[0040] (2.2) Dissolution and renaturation of inclusion bodies Weigh an appropriate amount of inclusion bodies, add the inclusion body dissolving solution (2%), and dissolve it with magnetic stirring for 4 hours at room temperature; centrifuge at 4°C, 9000 rpm, 15 min, and collect the supernatant; press 1 :Slowly add the refolding solution at a ratio of 8 and dissolve it under magnetic stirring at room temperature for 4h; refolding at 4℃ for more than 12h; ultrafiltration and concentration for more than 20 times, immediately purify or store at -20℃ for later use.

[0041] (2.3) Equilibrate the Separose 12 chromatography column with a balance solution for the purification of tumor-specific apoptosis factors; load the concentrated refolding solution at a rate of 3% of the bed volume at a flow rate of 2ml/min; collect the second peak step by step; After sodium dodecyl sulfate (SDS) electrophoresis identification, take the protein fraction with a molecular weight of 39kD and high purity; put the above sample on the FF chromatography column that has been equilibrated with the balance solution (or stored at -20°C for later use), and press the bed volume Load the sample at a ratio of 75%. After the sample is left in the column for 1 hour, it is eluted with the equilibrium solution; the elution peak is collected and stored at -20°C for identification.

[0042] (2.4) Purified tumor-specific apoptotic factors are identified by SDS electrophoresis or high performance liquid method to determine the purity of the protein, and the purity is above 95% ( Figure 4 ), using ultraviolet spectrophotometer to determine the protein content, about 0.5mg/ml; using limulus reagent method to determine the endotoxin content in the protein sample, 1:200 qualified.

[0043] The range and preparation of the above liquid formulas are as follows:

[0044] The nutrient agar medium plate is to weigh the nutrient agar at a concentration of 1.5%, add an appropriate amount of water for injection, autoclave, and when the medium is cooled to 45°C, add ampicillin (AP), the final concentration is 100μg/ml) , And then pour the agar into a petri dish. After the agar is solidified, store it at 4°C for later use (1 to 2 weeks available).

[0045] LB liquid medium is prepared in the following proportions in 1000ml units: autoclave after dissolution.

[0046] Peptone 10g

[0047] Yeast extract 5g

[0048] NaCl 1g

[0049] The fermentation broth is prepared in the following proportions in units of 20L, and autoclaved in the fermentation tank.

[0050] Peptone 200g

[0051] Yeast extract 100g

[0052] NaCl 20g

[0053] CaCl 2g

[0054] NH 4 Cl 30g

[0055] Glucose 100g (high pressure alone, 8 pounds)

[0056] Sodium dihydrogen phosphate. 2H 2 O 30g

[0057] Disodium hydrogen phosphate. 12H 2 O 120g

[0058] The replenishing fluid is prepared in the following proportions in units of 1000ml.

[0059] Peptone 100g

[0060] Yeast extract 100g

[0061] Glucose 200g (700ml of water dissolved in a single high pressure, 8 pounds)

[0062] 1mol IPTG (1000 times) is, filter sterilized, store at -20℃.

[0063] IPTG 0.48g

[0064] Water 20ml

[0065] Ampicillin (AP, 5000 times), the application concentration is 100μg/ml

[0066] AP 500mg

[0067] Water 5ml

[0068] 20mmol PB buffer (pH7.2) is prepared in the following proportions in 1000ml units.

[0069] Sodium dihydrogen phosphate. 2H 2 O 3.12g

[0070] Disodium hydrogen phosphate. 12H 2 O 7.16g

[0071] EDTA 0.37g

[0072] 20mmol PB buffer (pH8.0) is prepared in the following proportions in 1000ml units.

[0073] Sodium dihydrogen phosphate. 2H 2 O 3.12g

[0074] Disodium hydrogen phosphate. 12H 2 O 7.16g

[0075] EDTA 0.37g

[0076] The inclusion body dissolving solution is prepared in the following proportions in 100ml units.

[0077] 20mmol PB buffer (pH8.0) 100ml

[0078] Sodium creatine (1.0%-3.0%) 1.0~3.0g, take 1.5g

[0079] Triethanolamine (10-30mM) 0.13~0.66ml, take 0.33ml

[0080] DTT(10-30mM) 0.15~0.45g, take 0.31g

[0081] The refolding solution is prepared in the following proportions in units of 1000ml.

[0082] 20mmol PB buffer (pH8.0) 1000ml

[0083] DTT(10-30mM) 1.5~4.5g

[0084] The balance solution is prepared in the following proportions in units of 1000ml.

[0085] 20mmol PB buffer (pH7.2) 1000ml

[0086] Sodium creatine (0.1%-1.0%) 0.1~1.0g, take 1.0g

[0087] Triethanolamine (1mM) 0.132ml

[0088] The inclusion body washing solution is prepared in the following ratio in units of 1000ml.

[0089] 20mmol PB buffer (pH7.2) 1000ml

[0090] TritonX-100(0.1%) 0.1ml

[0091] Urea (2mol) 120g

[0092] (3) The chemical modification of GST-Apoptin fusion protein, that is, the activation of GST-Apoptin fusion protein.

[0093] (3.1) Experimental materials: medical folic acid, the above-mentioned purified recombinant tumor-specific apoptosis factor, glutaraldehyde,

[0094] (3.2) Experimental method: Use the glutaraldehyde method to connect the recombinant tumor-specific apoptosis factor and folic acid, and use the coupling buffer to prepare the connection system of the recombinant tumor-specific apoptosis factor and folic acid in the following ratio.

[0095] 0.2mg/ml recombinant tumor-specific apoptosis factor 1ml

[0096] 1.0mg/ml folic acid 1ml

[0097] Glutaraldehyde (125 times the original solution) 16μl (0.2%)

[0098] Place the prepared connection system at room temperature (above 25°C) and avoid light for 3h; dialyze the connection solution against 1000ml phosphate buffer solution at 4°C for more than 12h;

[0099] (3.3) Sterilize by filtration and store at 4°C for later use. Perform the following activity identification.

[0100] (4) The new recombinant tumor-specific apoptosis factor induces tumor cell apoptosis in vitro

[0101] (4.1) Experimental materials:

[0102] (4.1.1) Recombinant tumor-specific apoptosis factor PB solution [20mmol PB buffer (pH7.2), 0.1% sodium creatine, 1mM triethanolamine];

[0103] (4.1.2) Human melanoma cell line A375 (quoted from the Academy of Military Medical Sciences);

[0104] (4.2) Test method: The 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) method was used to determine the apoptin-induced apoptosis of tumor cells.

[0105] (4.2.1) Routinely subculture A375 cells;

[0106] (4.2.2) Take well-growing cells and prepare 4×10 3 Cells/ml of cell suspension;

[0107] (4.2.3) Add cell suspension to 96-well cell culture plate, 0.1ml/well, 37℃, 5% CO 2 Cultivate overnight

[0108] (4.2.4) Add the above activated apoptin, 0.1ml/well, starting from 50μg/ml, 2 times dilution, 4 wells for each titer;

[0109] (4.2.5) 37°C, 5% CO 2 Cultivate for 72h;

[0110] (4.2.6) Aspirate and discard the culture supernatant, and wash the cells twice with phosphate buffer;

[0111] (4.2.7) Add MTT (1mg/ml) prepared with phenol red-free RPMI1640, 50μl/well, 37℃, 5% CO 2 Cultivate for 3h;

[0112] (4.2.8) Aspirate and discard the culture supernatant, wash the cells twice with phosphate buffer;

[0113] (4.2.9) Add dimethyl sulfoxide (DMSO), 50μl/well, 37°C, 10min;

[0114] (4.2.10) Detect the A value of each hole with EL340560nm wavelength. The test set up a normal cell control and a non-activated apoptin control.

[0115] (4.3) Results 10μg/mL activated apoptin can cause more than 50% of tumor cells to undergo apoptosis ( Figure 5 ). There was no difference between the cells in the inactivated apoptin group and the normal control group.

[0116] (5) Anti-tumor effects of new recombinant tumor-specific apoptosis factors in vivo

[0117] (5.1) Experimental materials:

[0118] Male BALB/c mice (body weight 18-20g) were purchased from the Animal Department of China Medical University. Mouse S-180 sarcoma cells were introduced from the Medical Laboratory Department of the General Hospital of Shenyang Military Region, and the mice were passaged through the abdominal cavity to ensure their tumorigenicity. Recombinant tumor-specific apoptosis factor PB solution [20mmol PB buffer (pH7.2), 0.1% sodium creatine, 1mM triethanolamine];

[0119] (5.2) Experimental method

[0120] Prepare S-180 ascites sarcoma cells with saline to make 2×10 9 /L cell suspension, inoculated subcutaneously in the right axilla of the mouse, 0.2ml/mouse. Regular feeding of all animals [4]. After the mouse tumors grew to 5 mm, they were randomly divided into a recombinant tumor-specific apoptosis factor treatment group, a GST-Apoptin fusion protein control group, and a normal saline group, with 8 mice in each group. Treatment group: mice were injected subcutaneously into the right side of the armpit, 50μg (0.1mL)/mouse; the control group and saline group were injected with the same amount and volume of GST-Apoptin fusion protein and saline. Each group of mice was injected once a day; the mice were routinely raised, and the general living conditions and tumor size of the mice were observed daily. The body weight was measured every 6 days. The cervical vertebrae were sacrificed 3 weeks after inoculation, and the tumor tissues were separated and weighed.

[0121] (5.3) Results

[0122] (5.3.1) The general condition of the experimental mice. After the mice were inoculated with S-180 sarcoma cells, the mice in each group were fed normally, and the general physiological conditions did not change significantly within 1 week of inoculation. After 1wk, compared with the treatment group, the mice in the GST-Apoptin fusion protein control group and the saline group had reduced food intake, weight loss, weight loss, tarnished fur, and significantly increased tumor masses; by the second week, the saline group had a small The mice began to die; the mice in the treatment group were generally in good condition.

[0123] (5.3.2) Kill the mice alive after 3 weeks, take the tumor tissue and weigh the tumor, the tumor weight of the treatment group is (0.56±0.41) g, and the tumor inhibition rate is 60.06%; the tumor weight of the GST-Apoptin fusion protein control group is (1.36±0.55)g, the tumor inhibition rate was 4.22%. Taking the tumor weight of the saline group as the base, the calculated tumor inhibition rate between the treatment group and the GST-Apoptin fusion protein control group was significantly different (p<0.01) ( Image 6 ).

[0124] The beneficial effects of the present invention are: high-efficiency expression in Escherichia coli, good stability, high purity and high activity, and significant therapeutic effects. In particular, the implementation of the present invention has no special requirements for production equipment, and is suitable for large-scale and industrialization. It is of great significance to overcome tumors and extend human life.

[0125] Sequence 1 GST-Apoptin fusion protein amino acid sequence:

[0126] Chicken anemia virus

[0127] Met Asn Ala Leu Gln Glu Asp Thr Pro Pro Gly Pro Ser Thr Val Phe Arg Pro Pro Thr Ser Ser Arg

[0128] 1 5 10 15 20

[0129] Pro Leu Glu Thr Pro His Cys Arg Glu Ile Arg Ile Gly Ile Ala Gly Ile Thr Ile Thr Leu Ser Leu Cys

[0130] 25 30 35 40 45

[0131] Gly Cys Ala Asn Ala Arg Ala Pro Thr Leu Arg Ser Ala Thr Ala Asp Asn Ser Glu Ser Thr Gly Phe

[0132] 50 55 60 65 70

[0133] Lys Asn Val Gln Asp Leu Arg Thr Asp Gln Pro Lys Pro Pro Ser Lys Lys Arg Ser Cys Asp Pro Ser

[0134] 75 80 85 90

[0135] Glu Tyr Arg Val Ser Glu Leu Lys Glu Ser Leu Ile Thr Thr Thr Thr Pro Ser Arg Pro Arg Thr Ala Arg

[0136] 95 100 105 110 115

[0137] Arg Arg Ile Arg Leu

[0138] 120

[0139] Sequence 2 GST-Apoptin fusion protein nucleotide sequence

[0140] 5’-GAA TTC ATG AAC GCT CTC CAA GAA GAT ACT CCA CCC GGA CCA TCA ACG 48

[0141] GTG TTC AGG CCA CCA ACA AGT TCA CGG CCG TTG GAA ACC CCT CAC TGC 96

[0142] AGA GAG ATC CGG ATT GGT ATC GCT GGA ATT ACA ATC ACT CTA TCG CTG 144

[0143] TGT GGC TGC GCG AAT GCT CGC GCT CCC ACG CTA AGA TCT GCA ACT GCG 192

[0144] GAC AAT TCA GAA AGC ACT GGT TTC AAG AAT GTG CAG GAC TTG AGG ACC 240

[0145] GAT CAA CCC AAG CCT CCC TCG AAG AAG CGA TCC TGC GAC CCC TCC GAG 288

[0146] TAC AGG GTA AGC GAG CTA AAA GAA AGC TTG ATT ACC ACT ACT CCC AGC 336

[0147] CGA CCC CGA ACC GCA AGA AGG CGT ATA AGA CTG TAA GTC GAC-3’ 378