DNA molecule for coding porcine alpha interferon and expression and purification method of recombinant protein of porcine alpha interferon

An interferon and coding technology, applied in the field of protein purification in the large-scale production of recombinant interferon, can solve the problems of complex steps and low antiviral activity of recombinant porcine alpha-interferon, and achieve a short purification period and disulfide bond. Correct pairing, cost-saving effect

Inactive Publication Date: 2016-07-13

北京大北农科技集团股份有限公司动物医学研究中心 +3

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The method steps are complicated, and the recombinant porcine interfe...

Abstract

The invention relates to the field of biological pharmacy, and mainly relates to a DNA molecule for coding porcine alpha interferon and an expression and purification method of a recombinant protein of the porcine alpha interferon. Recombinant escherichia coli BL21/pET-21a-rPoIFN[alpha] is taken as a production bacterial strain, an expression protein of which has a coding sequence optimized through a codon bias in escherichia coli and is suitable for expression in escherichia coli. According to the expression and purification method, plenty of target protein in an inclusion body form can be obtained in precipitation of broken bacteria, and a large amount of recombinant porcine alpha interferon with high purity can be obtained through inclusion body denaturation, renaturation, affinity chromatography, and dialysis displacement of a buffer. The recombinant porcine alpha interferon is high in biological activity, and is convenient to apply with reduced production cost.

Application Domain

BacteriaPeptide preparation methods +4

Technology Topic

Expression proteinAlpha interferon +14

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Experimental program(2)
Effect test(1)

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[0046] Example 1 Construction, expression and purification of recombinant porcine alpha interferon

[0047] (1) Optimization of porcine alpha interferon gene and construction of seed bacteria: The porcine alpha interferon gene was optimized and modified according to the codon preference of Escherichia coli. The modified gene was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and constructed into pET21a On the vector, the recombinant expression plasmid pET-21a-rPoIFNα was obtained. Its nucleotide sequence is shown in SEQ ID No. 1, including 519 bases, 1-516 bases encode 172 amino acids, and its theoretical molecular weight is 19.8kDa. The expression plasmid pET-21a-rPoIFNα was transformed into E. coli BL21 competent cells to obtain the expressing seed bacteria BL21/pET-21a-rPoIFNα.

[0048] (2) Seed bacteria recovery: Inoculate the seed bacteria frozen at -20°C on the ampicillin-resistant LB solid medium by streaking with an inoculating loop, and cultivate for 8 hours in a constant temperature incubator at a temperature of 37°C. ;

[0049] (3) Seed bacteria culture: When the seed bacteria on the LB solid medium grows a single colony, pick a single colony and inoculate it into 5 ml of ampicillin-resistant liquid LB medium, and culture it in a constant temperature shaker for 6 hours at a speed of 220 r/min, and the incubation temperature was 37 °C. Determination of OD of bacterial liquid by UV spectrophotometer 600 When it is 0.6, the bacterial solution was transferred into 2L ampicillin-resistant liquid LB medium at a ratio of 1:150, and cultured in a constant temperature shaker for 5 hours, the rotation speed was 220 r/min, and the culture temperature was 37 °C;

[0050] (4) Induction: determine the OD of the cultured bacterial solution by UV spectrophotometer 600 When it is 0.8, add sterile isopropyl thiogalactoside (IPTG) to a final concentration of 1 mM, and continue to induce expression in a constant temperature shaker at 37 °C for 5 h;

[0051] (5) Bacteria harvest by centrifugation: at a speed of 6000r/min, centrifuge at 4°C for 10min and collect the bacterial pellet;

[0052] (6) Ultrasonic breakage of bacteria: use 60ml of 1×PBS buffer (NaCl8g/L, KCl0.2g/L, Na 2 HPO 4 2.9g/L, KH 2 PO 4 0.2g/L, pH8.0) suspended bacterial precipitation, lysed bacteria with ultrasonic breaker, ultrasonic conditions were ultrasonic 5S, interval 10S, repeated 200 times, ultrasonic power was 300W;

[0053] (7) Collect the precipitate by centrifugation: Centrifuge at 12000 r/min for 15 min at 4°C to collect the precipitate, remove the bacterial debris on the surface of the precipitate with a glass rod, and wash the remaining precipitate with washing buffer (Triton-1000.5 %, Tris 50 mM, NaCl 300 mM, pH 8.0) suspension, the total amount of inclusion body protein in the precipitate after bacterial fragmentation accounts for more than 50% of the total bacterial protein content (such as figure 1 shown);

[0054] (8) Centrifuge again to collect the precipitate: Centrifuge at 12000r/min for 15 minutes at 4°C, collect the precipitate, which is the porcine interferon inclusion body protein, and weigh the inclusion body;

[0055] (9) Solubilization of inclusion body protein: Add inclusion body protein to lysis buffer (Gua-HCl6M, glycerol 10%, Tris50mM, NaCl100mM, pH8.0) at 1:30 (W/V) by weight of the precipitate, at 4°C Stir and dissolve under conditions for 8h;

[0056] (10) Collection of inclusion bodies by centrifugation: Centrifuge at 12,000 r/min for 15 minutes at 4°C, and collect the supernatant, which is the dissolved inclusion body protein;

[0057] (11) Refolding inclusion body protein by dilution method: slowly drop the dissolved inclusion body liquid into the renaturation buffer (Tris 100mM, L-ArgHCl 400Mm, reduced glutathione 5mM, oxidized glutathione) at 4°C. 6ml of inclusion body dissolving solution can be added to 1L renaturation buffer system, stirring for renaturation for 12h;

[0058] (12) Purification of inclusion body renaturation protein by affinity chromatography: Wash the nickel affinity chromatography column with 5 column volumes of double distilled water; then use 5 column volumes of binding buffer (Tris 50mM, NaCl 100mM, pH 8.0) Equilibrate the chromatography column; pass the sample filtered with a 0.22 μm pore size filter through the chromatography column; use 4 column volumes of PBS to flow through the chromatography column to wash off part of the protein that is not bound to the chromatography column; Debuffer (50mM and 200mM MImidazole, 1×PBS, pH8.0) to elute and collect the target protein (specific peaks are shown in figure 2 shown), this is the renatured purified protein of rPoIFNα, and the purity of rPoIFNα was checked by SDS-PAGE (as shown in image 3 shown); rinse the column with double-distilled water, and store the column with 20% ethanol.

Example Embodiment

[0059] Example 2 Determination of antiviral biological activity of recombinant porcine alpha interferon protein

[0060] Biological activity was measured using a cytopathic (CPE) inhibition-based microinhibition assay. Using the Marc-145 cell-porcine reproductive and respiratory syndrome virus (PRRSV) system, the highest dilution of interferon that inhibited 50% of cytopathic effects was defined as 1 unit of interferon activity.

[0061] (1) Inoculate Marc-145 cells into 96-well plates (100 μl/well), and culture for 6-8 hours until all adherent cells are adhered to 10 4 pcs/hole or so;

[0062] (2) Discard the culture medium, and dilute the prepared recombinant porcine alpha interferon protein sample in 8 gradients. The dilution is DMEM medium containing 10% FBS. Each gradient is set to 8 replicates, and 100 μl of virus is added to each well. Diluent.

[0063] (3) After culturing at 37°C for 18 hours, add PRRSV virus solution (300TCID50/well) diluted with serum-free DMEM medium, and set a positive control group with virus without interferon and a negative without interferon and virus. control group.

[0064] (4) When more than 90% of the cells in the positive control group have cytopathic changes, observe them under a microscope (such as Figure 4 shown). After calculation, the activity of this batch of recombinant porcine alpha interferon protein is 3 × 10 6.5 U/mg.

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DNA molecule for coding porcine alpha interferon and expression and purification method of recombinant protein of porcine alpha interferon

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