CN105349562A - Recombinant vector and recombinant strain for expressing PPV (porcine parvovirus) VP2 protein and applications of recombinant vector and recombinant strain - Google Patents

Abstract

The invention provides a recombinant vector expressing porcine parvovirus VP2 protein, a recombinant bacterium and an application thereof, belonging to the field of biotechnology. The recombinant vector expressing porcine parvovirus VP2 protein is to insert the gene encoding porcine parvovirus VP2 protein into the prokaryotic expression vector pEASY-blunt? Obtained after E1. The invention also provides a recombinant bacterium expressing porcine parvovirus VP2 protein, which is obtained after introducing the recombinant vector into Escherichia coli. The recombinant vector expressing porcine parvovirus VP2 protein of the present invention can solublely express VP2 protein after being introduced into Escherichia coli, the VP2 protein has hemagglutination and excellent antigenicity, and is expected to be applied to prepare porcine parvovirus vaccine. The recombinant bacterium of the invention can efficiently prepare porcine parvovirus VP2 protein, has low production cost, simple operation, and better biological safety without manipulation of porcine parvovirus.

Description

Recombinant vector expressing porcine parvovirus VP2 protein, recombinant bacterium and application thereof

technical field

The invention belongs to the field of biotechnology, and in particular relates to a recombinant vector expressing porcine parvovirus VP2 protein, a recombinant bacterium and an application thereof.

Background technique

Porcine parvovirus (porcine parvovirus, PPV) is one of the main pathogens that cause reproductive disorders in sows, especially in primiparous sows without immunity. Delayed estrus and weak offspring are the main features. After Cartwright isolated PPV for the first time in 1967, the virus spread and became popular all over the world. After my country's reform and opening up in the 1980s, with the gradual increase of large-scale pig farms, PPV infection in my country became more and more serious, with a positive detection rate of more than 90%, which brought huge economic losses to my country's pig industry. PPV belongs to the Parvoviridae family and the genus Parvovirus. The PPV genome encodes three structural proteins: VP1, VP2, and VP3, and three nonstructural proteins: NS1, NS2, and NS3. Among them, VP2 protein is the main structural protein that constitutes the capsid of PPV virus, and is the most important immunoprotective antigen.

Many studies and clinical practice have shown that vaccination is a very effective method to control the prevalence of PPV. After immunization, pigs mainly obtain protection against PPV infection by generating a humoral immune response. The vaccines used to prevent PPV in my country are mainly inactivated vaccines, that is, PPV is cultured on pig-derived cell lines such as ST cells, and the infectious virus is obtained by separating and removing cell impurities, and then inactivated with chemical reagents and mixed with adjuvants to prepare into a vaccine. PPV inactivated vaccine has the advantages of good safety and no need for low temperature storage. However, inactivated vaccines also have the disadvantages of high production cost, long production time, large labor force, and unstable immune effect; in addition, inactivated chemical reagents and residual virus DNA may be dangerous to immunized pigs. New PPV subunit vaccines have been extensively studied, and VP2 not only has good immunogenicity after expression in vitro, but also can induce strong immune protection. At present, the expression system used to study porcine parvovirus subunit vaccine antigen is mainly the expression system of insect cells infected by baculovirus. The expressed protein has high solubility and hemagglutination, can produce virus-like particles, and has a good immune protection effect. Its production cost is several times higher than that of PPV inactivated vaccine, and its production operation is more complicated and requires higher quality of personnel. Usually, compared with the eukaryotic system, the prokaryotic expression system has higher protein expression, lower production cost, simpler production operation, and lower requirements for personnel quality. However, in the prior art, the VP2 prepared by the prokaryotic expression system The protein is an inclusion body that does not have solubility and hemagglutination, and its immunogenicity is poor, and the immune index after immunization is not easy to evaluate with the general HI test.

Contents of the invention

The object of the present invention is to provide a recombinant vector expressing porcine parvovirus VP2 protein. After the recombinant vector is introduced into Escherichia coli, the VP2 protein can be solublely expressed. The VP2 protein has hemagglutination and excellent antigenicity.

Another object of the present invention is to provide a recombinant bacterium for preparing porcine parvovirus VP2 protein, capable of soluble expression of VP2 protein, the VP2 protein has hemagglutination and excellent antigenicity, and can be used for preparing porcine parvovirus vaccine.

Another object of the present invention is to provide a method for preparing porcine parvovirus VP2 protein, which can efficiently prepare VP2 protein, has low production cost, is easy to operate, and has better biological safety without manipulation of porcine parvovirus. From practical considerations, it is expected to replace the antigen used in the production of inactivated vaccines.

The object of the present invention adopts following technical scheme to realize.

The recombinant vector expressing the porcine parvovirus VP2 protein is obtained by inserting the porcine parvovirus VP2 protein coding gene into the prokaryotic expression vector pEASY-bluntE1.

In the present invention, the porcine parvovirus VP2 protein coding gene is derived from porcine parvovirus PPV-JS strain.

The invention also provides a recombinant bacterium expressing porcine parvovirus VP2 protein, which is obtained after introducing the recombinant vector into Escherichia coli.

The Escherichia coli is BL-21.

The invention also provides the application of the recombinant bacteria in preparing porcine parvovirus VP2 protein.

In the present invention, the application includes the step of inducing the recombinant bacteria to express porcine parvovirus VP2 protein.

The preferred technical scheme is that when the OD600 of the recombinant bacterial culture reaches 0.4-0.6, IPTG is used to induce the expression of the porcine parvovirus VP2 protein.

In a preferred technical solution, the final concentration of IPTG added to the recombinant bacterial culture is 0.05-0.2mM.

The recombinant vector expressing porcine parvovirus VP2 protein of the present invention can solublely express VP2 protein after being introduced into Escherichia coli, the VP2 protein has hemagglutination and excellent antigenicity, and is expected to be applied to prepare porcine parvovirus vaccine. The recombinant bacterium of the invention can efficiently prepare porcine parvovirus VP2 protein, has low production cost, simple operation, and better biological safety without manipulation of porcine parvovirus.

Description of drawings

Figure 1 shows the amplification results of the VP2 protein-coding gene, in which lane M is DL2000Marker, and the remaining lanes are PCR amplification products.

Figure 2 shows the results of double enzyme digestion identification of recombinant plasmid p-PPV-VP2, wherein lane M is DL2000Marker, and lane 1 is positive recombinant plasmid p-PPV-VP2.

Figure 3 shows the expression of VP2 protein in BL-21-VP2 and control bacteria BL-21-p. Marker: ThermoScientific PageRuler pre-stained Ladder; 1: Supernatant of lysate of control bacteria BL-21-p; 2: Supernatant of lysate of BL-21-VP2; 3: Precipitation of lysate of control bacteria BL-21-p; 4: BL- 21-VP2 Lysate Precipitation.

Figure 4 shows the hemagglutination test results of each sample. The number of the wells is shown on the left of each row, and the concentration of samples in each row decreases sequentially from left to right.

Figure 5 shows the hemagglutination detection results of the bacterium lysate supernatant after OD600 is 0.4, 0.5 and 0.6 after induction, wherein the mark on the far left indicates the OD600 value of each row of samples before induction, and the right side shows the OD600 value of each row of samples Hemagglutination titer.

Figure 6. The HI antibody titer results of the sera to be tested using the 4-unit antigen of PPV-JS or BL-21-VP2, where the text on the left indicates the serum tested in each row, and the text on the right indicates the number of the well.

detailed description

Example 1 Construction of recombinant bacteria expressing VP2 protein

1. Construction of recombinant expression vector

1. Primer Design

Referring to the gene sequence of the PPV-NADL2 strain (KF049424.1), primers PPV-VP2-kpni and PPV-VP2-bamhi were designed using Primerpremier5.0 software to amplify the porcine parvovirus PPV-JS strain (abbreviated as PPV-JS strain , disclosed in CN102965345A, the collection number of the General Microbiology Center of China Microbiological Culture Collection Management Committee is CGMCCNO.6605) the entire sequence of the VP2 protein coding gene.

The nucleotide sequence (SEQ ID NO: 2) of PPV-VP2-kpni is as follows: ggtaccATGAGTGAAAATGTGGAACAAG, the nucleotide sequence (SEQ ID NO: 3) of PPV-VP2-bamhi is as follows: ggatccCTAGTATAATTTTCTTGGTAT, synthesized by Shanghai Yingjun Biological Co., Ltd. Among them, PPV-VP2-kpni carries a KpnⅠ restriction site, and PPV-VP2-bamhi carries a BamhⅠ restriction site.

2. Extraction of Genomic DNA of PPV-JS Strain

(1) Take 450 μl of PPV-JS strain virus sample, add 50 μl of 10% SDS solution, and add 3 μl of proteinase K;

(2) Incubate for 30 minutes in a 56°C water bath with shaking;

(3) Add 500 μl of TRIS-phenol, mix well, after 5 minutes, centrifuge at 4°C, 10000-12000r/min for 10 minutes;

(4) Take the supernatant, add 500 μl of TRIS-phenol and chloroform mixture mixed 1:1 (V/V), and centrifuge at 10000-12000 r/min for 10 minutes after acting for 5 minutes;

(6) Take the supernatant, add 500μl of chloroform, act for 5 minutes, and centrifuge at 10000-12000r/min for 10 minutes;

(7) Take the supernatant, add 2 times the volume of absolute ethanol and 10% (volume percent concentration) sodium acetate aqueous solution;

(8) Place the mixture obtained in step (7) at -20°C for more than 30 minutes;

(9) Take the sample placed at -20°C and centrifuge at 10,000 r/min for 10 minutes at 4°C;

(10) Discard the supernatant, add 75% (volume percent concentration) ethanol aqueous solution to the precipitate, centrifuge at 4°C, 6500r/min for 5 minutes, discard the supernatant; repeat 2 times;

(11) Blot or dry ethanol;

(12) Add 30 μl of ddH ₂ O to dissolve, obtain PPV-JS strain genomic DNA , and store it at -20°C.

3. Amplification and purification of VP2 protein coding gene

(1) Set the reaction system to 50 μL system: 0.5 μL PrimerSTAR High Fidelity Enzyme, 10 μL 5×PSBuffer, 4 μL dNTPs, 1 μL (20 μmol/L) each of primers PPV-VP2-kpni and PPV-VP2-bamhi, 2 μLPPV-JS strain genomic DNA , 28.5 μL ddH ₂ O.

(2) Reaction conditions: 98°C for 2 minutes; 98°C for 15 seconds, 60°C for 30 seconds, 72°C for 2 minutes, 30 cycles; 72°C for 5 minutes. The product was stored at 4°C.

(3) The amplified gene fragments were recovered by 1% agarose gel electrophoresis, and DL2000Marker was used as a control. The results are shown in Figure 1, a specific VP2 protein coding gene band appeared at about 1.7kb. The VP2 protein-encoding gene was recovered using OMEGADNA Gel Extraction Kit. See the manual for specific operations.

The nucleotide sequence of the VP2 protein coding gene of the porcine parvovirus PPV-JS strain is shown in SEQ ID NO:1.

Reagents used in the PCR process were purchased from TAKARA.

4. Construction of expression plasmids

(1) The VP2 protein coding gene was inserted into the pEASY-bluntE1 prokaryotic expression vector (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) to construct the recombinant plasmid p-PPV-VP2. The ligation system is a 5 μL system: 2 μL VP2 protein coding gene gel recovery product, 1 μL pEASY-bluntE1 vector, 2 μL ddH ₂ O. Connect at 24°C for 20min.

(2) The ligation product was added to 100 μL of DH5α competent cells, ice-bathed for 30 minutes, then heat-shocked at 42°C for 90 seconds, and ice-bathed for 5 minutes. Add 1mL of LB medium, incubate at 180rpm on a shaker at 37°C for 1h, take it out and centrifuge at 10,000g for 1min, discard 800μL of supernatant, leave 200μL of supernatant to resuspend the bacteria, spread all on the LB plate with ampicillin (Amp) resistance, and incubate at 37°C overnight.

(3) Randomly pick a single colony on the above LB plate, inoculate it in LB liquid medium with ampicillin resistance, and culture overnight at 37°C.

5. Extraction of Plasmids

Dingguo plasmid mini-extraction kit was used to extract the plasmids in the recombinant bacteria selected in step 4 of this example, and the specific operation steps are shown in the kit instructions.

6. Identification of Recombinant Plasmids

10 μL double enzyme digestion identification system: 0.5 μL BamHI, 0.5 μL KpnⅠ, 1 μL 10×KBuffer, 4 μL plasmid, 4 μL ddH ₂ O, digestion at 37°C for 1 hour. The electrophoresis of the digested product is shown in Figure 2. The plasmid was identified correctly by BamHI and KpnⅠ digestion, and it was a positive recombinant plasmid, named p-PPV-VP2. The recombinant plasmid p-PPV-VP2 was sent to Yingjun Company for sequencing, and the sequencing was correct.

Second construction of recombinant bacteria

1. Transform expression bacteria with recombinant plasmid p-PPV-VP2

(1) Transform the p-PPV-VP2 recombinant plasmid into the competent cells of E. coli BL-21 according to the conventional method, and then spread it on the LB plate containing 50 μg/mL Amp (ampicillin) resistance, and culture at 37°C overnight.

(2) Pick a single colony on the above-mentioned Amp-resistant LB plate, name it as the recombinant strain BL-21-VP2, inoculate it in LB liquid medium containing 50 μg/mL Amp resistance, and culture it with shaking at 37°C and 200rpm overnight.

According to the same method as above, the plasmid pEASY-bluntE1 was transformed into competent cells of BL-21 to obtain the control bacteria BL-21-p.

2. Induced expression of recombinant bacteria and identification by SDS-PAGE

(1) Inoculate BL-21-VP2 and control BL-21-p culture medium at 1:100 (volume ratio) into LB liquid medium containing 50 μg/ml Amp, respectively.

(2) Cultivate at 37°C and 200rpm for 2 hours, and the OD ₆₀₀ is about 0.4 at this time.

(3) Add IPTG with a final concentration of 0.1 mM, shake and culture at 37°C and 200 rpm for 4 hours.

(4) After the culture, take 5ml of BL-21-VP2 and control bacteria BL-21-p respectively, after sonication, centrifuge to separate the supernatant and precipitate of the bacterial lysate, and perform SDS-PAGE electrophoresis. The results are shown in Figure 3, the target band exists in the supernatant of BL-21-VP2 lysate, and the supernatant has high hemagglutination activity, and the HA titer reaches 2 ⁹ There were inclusion bodies of the target protein, the hemagglutination activity was very low, and the HA titer was only 2 ⁵ , which indicated that VP2 protein was solublely expressed in BL-21-VP2, and the expressed VP2 protein had a higher hemagglutination titer. See Example 2 for the detection method of HA titer.

Example 2 Identification of VP2 protein hemagglutination and determination of hemagglutination value (HA)

Preparation of 1.1% guinea pig erythrocyte solution

(1) Draw 1mL of anticoagulant (4% sodium citrate solution) with a syringe; take at least 1 SPF guinea pig, collect about 4mL of blood, mix with 1mL of anticoagulant, then slowly add to a 10mL centrifuge tube and mix well.

(2) Wash guinea pig red blood cells: centrifuge the blood mixture in the 10mL centrifuge tube in step (1) at 1500rpm for 10min, discard the supernatant, take the precipitate, add 5ml of PBS buffer (pH7.2, which contains the following ingredients Aqueous solution: NaCl 137mmol/L, KCl 2.7mmol/L, Na ₂ HPO ₄ 10mmol/L, KH ₂ PO ₄ 2mmol/L, the same below), mix gently, then centrifuge at 1500rpm for 10min, remove the supernatant and precipitate the upper layer of red blood cells with a pipette white blood cell film. After the washing process was repeated twice, 5 mL of PBS buffer (pH 7.2) was added to gently mix the red blood cells, stored at 4°C for later use, and used within 5 days.

(3) 20% guinea pig erythrocyte solution: take the erythrocytes obtained after the treatment in step (2), centrifuge in a conical graduated centrifuge tube at 1500rpm for 10 minutes, discard the supernatant, observe the volume of erythrocytes in the centrifuge tube, add 4 times the volume of PBS buffer solution (pH7.2), mix well to obtain 20% guinea pig erythrocyte solution.

(4) 10% guinea pig erythrocyte solution: take the erythrocytes obtained after the treatment in step (2), centrifuge in a conical graduated centrifuge tube at 1500rpm for 10 minutes, discard the supernatant, observe the volume of erythrocytes in the centrifuge tube, and add 9 times the volume of PBS buffer solution (pH7.2), mix well to obtain 10% guinea pig erythrocyte solution.

(5) 1% guinea pig erythrocyte fluid: take 1mL of 10% guinea pig erythrocyte fluid, add 9mL of PBS buffer (pH7.2), mix well, and obtain 1% guinea pig erythrocyte fluid.

2. Identification of hemagglutination of VP2 protein and determination of hemagglutination (HA) titer

Take the BL-21-VP2 lysate supernatant and precipitate prepared in step 2 in Title 2 of Example 1 to verify the hemagglutination of the VP2 protein prepared by the method of the present invention. The BL-21-VP2 lysate pellet was resuspended with an equal volume of supernatant PBS buffer (pH 7.2) to obtain a VP2 protein inclusion body suspension.

Different samples are detected in each row of 96-well V-type hemagglutination reaction plate, numbered as PBS blank control, PPV positive control, control bacteria BL-21-p negative control, sample well 1 (VP2 protein inclusion body suspension) and sample Well 2 (BL-21-VP2 lysate supernatant). First add 25 μL of PBS buffer (pH7.2) to each well, then add the sample to the first well of each row of wells, add 25 μL VP2 protein inclusion body suspension to sample well 1, add 25 μL BL-21-VP2 to sample well 2 Lysate supernatant, add 25 μL PBS buffer solution (pH7.2) to the PBS blank control, add 25 μL PPV-JS strain virus antigen solution with a hemagglutination value of ²⁹ to the PPV positive control (use ST cells to amplify PPV-JS strain Viral antigen acquisition), add 25 μL of control bacteria BL-21-p whole bacteria ultrasonic lysate before induction to the control bacteria BL-21-p negative control. Dilute each sample by 2 times, then add 25 μL of PBS buffer (pH7.2) to each well, and finally add 25 μL of 1% guinea pig erythrocyte solution to each well, and observe the results after acting at 37°C for 2 hours .

The results are shown in Figure 4. The soluble VP2 protein expressed by BL-21-VP2 has hemagglutination, and the hemagglutination titer is as high as 2 ⁹ , which is similar to the hemagglutination titer of PPV-JS strain virus antigen solution; the hemagglutination effect of VP2 protein inclusion body The valence (HA) is low, only 2 ⁵ .

Embodiment 3 VP2 protein preparation method condition optimization

1. Optimization of the optimum bacterial concentration before the induction of the recombinant strain BL-21-VP2

(1) Inoculate BL-21-VP2 bacterial liquid into LB liquid medium containing 50 μg/ml ampicillin at a volume ratio of 1:100.

(2) Cultivate at 37°C and 200rpm for 2~3h, select the bacterial solution with OD600 (bacterial cell density) of 0.4, 0.5 and 0.6, add IPTG with a final concentration of 0.1mM respectively, and then incubate at 37°C and 200rpm Shaking culture under the conditions for 4 hours to induce expression.

(3) After the cultivation, 5ml of the bacterial liquid was taken for ultrasonication, centrifuged at 12000rpm, and the supernatant of the lysate was taken, and the hemagglutination titer was detected according to the method in Example 2 to determine the expression level of the soluble VP2 protein.

The results are shown in Fig. 5, after induction at OD600 of 0.4, 0.5 and 0.6, the hemagglutination titers of the supernatant of the cell lysate reached 2 ⁹ , 2 ¹⁰ and 2 ¹¹ respectively. That is, when the cell concentration OD600=0.6, the hemagglutination titer of prokaryotic expression of VP2 protein was the highest, reaching 2 ¹¹ .

Example 4 VP2 protein production of 4 units of antigen applied to hemagglutination inhibition test (HI)

1. Prokaryotic expression of VP2 protein to produce 4 units of antigen

Take the PPV-JS strain virus liquid (obtained by ST cell amplification) with a hemagglutination titer of ²⁹ and dilute it with PBS buffer (pH7.2) to 4 times the antigen, that is, carry out the 2-fold ratio of the PPV-JS strain virus liquid Dilute until the hemagglutination titer of the diluent is 2 ² , and obtain 4 units of antigen of PPV-JS. The supernatant of the BL-21-VP2 lysate with a hemagglutination titer of ^2.9 in Example 3 was diluted ² -fold, and the dilution with a hemagglutination titer of 2.2 was used as the 4-unit antigen of BL-21-VP2 .

2. Porcine PPV negative serum and positive serum processing

Take 200 μL of pig PPV negative serum and positive serum after PPV vaccine immunization as follows: inactivate the serum in a water bath at 56°C for 30 minutes, add 400 μL of 25% (mass percent concentration) kaolin suspension, and mix well After leaving at room temperature for 30 minutes, centrifuge at 4000rpm for 10min, take the supernatant, add 200μL of 20% guinea pig erythrocyte solution, vortex and mix well, and incubate at 37°C for 1h; centrifuge at 4000rpm for 10min, absorb the supernatant, and use it as a 1:4 diluted serum sample.

3. Hemagglutination inhibition test

4 unit antigens of BL-21-VP2 and PPV-JS were used to detect the HI antibody titer of treated porcine PPV positive serum to investigate the antigenicity of VP2 protein expressed by BL-21-VP2. At the same time, a negative control of control bacteria BL-21-p (ultrasonic lysate of control bacteria BL-21-p whole bacteria before induction) was set up.

Add 25 μL of PBS buffer solution (pH7.2) to each well of a standard 96-well V-type hemagglutination reaction plate, and test different samples in each row of wells, which are respectively numbered BL-21-VP2 antigen wells and PPV-JS antigen wells And control bacteria BL-21-p negative control. Add 25 μL of the treated serum to be tested in the first well of each row, mix well, take out 25 μL and add it to the second well, and so on, dilute the treated serum to be tested by 2 times until the 12th well, discard 25 μL. In the BL-21-VP2 antigen well, add 25 μL of 4 unit antigen of BL-21-VP2 to each well; in the PPV-JS antigen well, add 25 μL of 4 unit antigen of PPV-JS in each well; the control bacteria BL-21-p is negative In the control, the ultrasonic lysate of the control bacteria BL-21-p whole bacteria before induction was added to each well. After 1 hour at 37°C, add 25 μL of 1% guinea pig erythrocyte solution to each well, shake and mix well, place at 37°C for 2 hours, and observe the results.

The results are shown in Figure 6. It can be seen that the HI antibody titers of the serum to be tested detected with 4 unit antigens of PPV-JS or BL-21-VP2 are the same, both being 2 ⁷ . The strain virus has the same antigenicity, and the antigenic epitope is similar to that of the original virus, which is a characteristic that the prokaryotic expressed VP2 protein in the prior art does not have. Therefore, the VP2 protein prepared by the present invention has the prospect of being applied to a vaccine. The above results show that the VP2 protein prepared by the present invention not only has the same agglutination of guinea pig erythrocyte hemagglutination as the PPV-JS strain virus, but also can react with PPV-specific antibodies in serum to inhibit erythrocyte agglutination.

SEQUENCELISTING

<110>Jiangsu Academy of Agricultural Sciences

<120> Recombinant Vector, Recombinant Bacteria and Application for Expressing Porcine Parvovirus VP2 Protein

<130>20151218

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Claims (8)

1. express the recombinant vectors of PPV VP 2 protein, obtain after it is characterized in that PPV VP 2 protein encoding gene is inserted prokaryotic expression carrier pEASY-bluntE1.

2. express the recombinant vectors of PPV VP 2 protein according to claim 1, it is characterized in that described PPV VP 2 protein encoding gene derives from pig parvoviral PPV-JS strain.

3. expressing the recombinant bacterium of PPV VP 2 protein, is obtain after described for one of claim 1-2 recombinant vectors is imported intestinal bacteria.

4. express the recombinant bacterium of PPV VP 2 protein according to claim 3, it is characterized in that described intestinal bacteria are BL-21.

5. the described recombinant bacterium of one of claim 3-4 is preparing the application in PPV VP 2 protein.

6. apply according to claim 5, it is characterized in that comprising the step that the described recombinant bacterium of induction expresses PPV VP 2 protein.

7. apply according to claim 6, it is characterized in that when recombinant bacterium culture OD600 reaches 0.4-0.6, adopt IPTG abduction delivering PPV VP 2 protein.

8. apply according to claim 7, it is characterized in that the IPTG final concentration be added in recombinant bacterium culture is 0.05-0.2mM.