BVDV E2 recombinant protein as well as preparation method and application thereof

Abstract

The invention provides a BVDV E2 recombinant protein and a preparation method and application thereof, and relates to the technical field of biological antibody preparation and biological detection.The BVDV E2 recombinant protein is obtained from Genebank, the antigen protein gene is expressed and purified and then serves as an antigen to immunize a mouse, and a mouse anti-BVDV antibody is obtained and applied to double-antibody sandwich detection. And detecting the bovine viral diarrhea virus. The invention has the beneficial effects of good stability and high sensitivity.

Description

Technical Field

[0001] The present invention provides BVDV E2 recombinant protein and a preparation method and application thereof, and relates to the technical field of biological antibody preparation and biological detection. Background Art

[0002] Bovine viral diarrhea virus (BVDV) can infect cattle and cause viral diarrhea / mucosal disease (BVD / MD), so it is also called bovine viral diarrhea virus-mucosal disease virus. The virus attacks the respiratory, digestive and reproductive systems of infected animals, and clinical manifestations include fever, diarrhea, poor appetite, mental depression, erosion of the digestive tract mucosa, large amounts of oral and nasal secretions, and stillbirth or miscarriage in pregnant cows. Therefore, it causes significant economic losses to the cattle breeding industry.

[0003] Bovine viral diarrhea virus is widely distributed in the world, especially in countries with developed cattle farming, such as the United States, Japan, Australia and New Zealand. At present, there is no effective treatment for bovine viral diarrhea virus in the world, so countries mainly adopt effective detection and group purification to control the spread of the virus in cattle farms, which puts forward relatively high requirements on the throughput, accuracy and cost of the detection method. At present, the main detection methods for this virus are serum neutralization test (SNT), immunohistochemistry (IHC), antigen ELISA, PCR, in vitro virus separation and the newly developed CRISPR, which have problems such as low throughput, poor specificity and high cost. Summary of the invention

[0004] The present invention provides a BVDV E2 recombinant protein and a preparation method and application thereof, which solve the technical problems of poor sensitivity, low throughput and high cost in some in vitro detection methods in the prior art.

[0005] The present invention is achieved in that the amino acid sequence of the BVDV E2 recombinant protein is shown in SEQ ID NO:4.

[0006] As further preferred, the gene encoding the BVDV E2 recombinant protein is as shown in SEQ ID NO:3.

[0007] The preparation method of BVDV E2 recombinant protein comprises the following steps: the coding gene of BVDV E2 recombinant protein is recombined with pET-28b plasmid vector to obtain a recombinant plasmid, the recombinant plasmid is transformed into BL21 competent cells, and IPTG is induced to express, the coding gene of BVDV E2 recombinant protein is shown in SEQ ID NO:3.

[0008] As a further preferred embodiment, the method specifically comprises the following steps: (1) obtaining the BVDV E2 protein gene sequence, amplifying and digesting the BVDV E2 protein gene sequence, and connecting the digested BVDV E2 protein gene sequence with the pET-28b plasmid; (2) The pET-28b plasmid obtained in step (1) was transformed into BL21 competent cells, and the BVDV E2 protein was expressed using BL21 Escherichia coli. The BVDV E2 protein was denatured and dissolved with urea solution, and then purified by Ni-NTA chromatography to obtain the BVDV E2 recombinant protein.

[0009] As further preferred, in step (1), the primers used for amplification include an upstream primer and a downstream primer, the sequence of the upstream primer is shown in SEQ ID NO:1, and the sequence of the downstream primer is shown in SEQ ID NO:2.

[0010] As a further preference, in step (2), the expression conditions of BVDV E2 protein are: 0.5-1 mM IPTG, 30-37° C., and induction for 5-7 h.

[0011] As a further preference, in step (2), the concentration of the urea solution is 8 mol / L.

[0012] Application of BVDV E2 recombinant protein, wherein the BVDV E2 recombinant protein is used for producing bovine viral diarrhea virus antibodies.

[0013] As a further preferred embodiment, the production step is specifically as follows: the BVDV E2 recombinant protein is completely emulsified with Freund's adjuvant and then immunized with mice, the spleen is removed and submerged, PEG1450 is used to induce fusion of Sp2 / 0 cells and mouse spleen cells to obtain a monoclonal cell line-hybridoma cell line producing anti-E2 protein, and then subcloned and expanded for culture, the positive cell line is cultured and inoculated into the peritoneal cavity of BALB / c mice to obtain a monoclonal antibody, and the encoding gene of the BVDV E2 recombinant protein is shown in SEQ ID NO: 3.

[0014] As a further preference, the bovine viral diarrhea virus antibody is used to prepare a double antibody sandwich Elisa kit for detecting bovine viral diarrhea virus.

[0015] The BVDV E2 recombinant gene provided by the invention can be used to prepare a bovine viral diarrhea virus monoclonal antibody, and the monoclonal antibody can be used to prepare a double antibody sandwich Elisa kit for detecting the bovine viral diarrhea virus, and has good sensitivity and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1It is the agarose electrophoresis diagram of the PCR amplification result of BVDV E2 gene, M is DL 10000 DNA Marker, and lane 1 is the PCR amplification product of E2 gene; Figure 2 This is a schematic diagram of the electrophoresis of three repetitions of the recombinant E2 protein expression experiment induced by different IPTG concentrations. Figure 2 a, Figure 2 b and Figure 2 c are the electrophoretic images of three repeated experiments; Figure 3 This is a schematic diagram of electrophoresis of three repetitions of the experiment of inducing recombinant E2 protein expression at different temperatures. Figure 3 a, Figure 3 b and Figure 3 c are the electrophoretic images of three repeated experiments; Figure 4 This is a schematic diagram of electrophoresis of three repetitions of the experiment of inducing recombinant E2 protein expression at different induction times. Figure 4 a, Figure 4 b and Figure 4 c are the electrophoretic images of three repeated experiments; Figure 5 This is the SDS-PAGE electrophoresis of pET28b-E2 recombinant protein purification, M is Blue plus protein Marker, lane 1 is inclusion body, lane 2 is flow-through, lane 3 is wash, and lane 4 is elution; Figure 6 The hydrophobicity analysis map of E2 recombinant protein was analyzed by DNAMAN software; Figure 7 The hydrophilicity analysis map of E2 recombinant protein was analyzed by DNAMAN software; Figure 8 This is the purified SDS-PAGE electrophoresis of the anti-E2 recombinant protein paired antibody. M is Blue plus protein Marker. Lane 1 is the eluted sample of antibody 1, and lane 2 is the eluted sample of antibody 2. DETAILED DESCRIPTION

[0017] To further illustrate the technical means and effects adopted by the present invention, the present invention is further described below in conjunction with the examples and drawings. Obviously, the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The raw materials, reagents, and devices used in the examples can be obtained from conventional commercial sources or prior art methods unless otherwise specified, and the experiments or test methods are all conventional methods in the art. Example

[0018] 1. Gene amplification and construction of expression vector The gene sequence of E2 protein was obtained from Genebank. The sequence number of E2 protein gene is ABP57735.1. DNAStar software was used to analyze the hydrophilic region, antigenic determinant region and surface display probability of E2 gene, the main antigenic domain was selected, and specific primers of E2 protein gene were designed. Specific primers include upstream primers and downstream primers. The sequence information of the upstream primer is shown in SEQ ID NO:1, specifically: 5'-GCCATATGGACCTGGACTGCAAGC-3'; the sequence information of the downstream primer is shown in SEQ ID NO:2, specifically: 5'-GCCTCGAGTTAAGATGCTTTCTGTTCAG-3'. The conditions for PCR amplification of E2 protein gene are: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 2 min, 55℃ annealing for 1 min, 72℃ extension for 2 min, for a total of 30 cycles; 72℃ extension for another 10 min. See Figure 1 , the size of the E2 gene was compared by agarose gel electrophoresis to see if it was consistent with the design, and the PCR amplification product was recovered using a gel recovery kit. The purified PCR product and pET-28b plasmid vector were double-digested with EcoRI and BamHI, respectively, and the double-digested E2 gene and pET-28b plasmid vector were connected by T4 DNA ligase, and transformed into competent Escherichia coli BL21 (DE3), coated on LB solid plates containing 50 mg / L kanamycin and 50 mg / L chloramphenicol, and the plates were inverted in a 37°C constant temperature incubator for overnight culture for 16 h, and single colonies were picked for PCR identification. The sequence of the E2 gene after double digestion is shown in SEQ ID NO:3.

[0019] 2. Expression and purification of recombinant proteins The pET28b-E2 plasmid successfully verified in step (I) was transformed into BL21 (DE3) competent cells, inoculated into LB liquid culture medium at a ratio of 1:100, and cultured in a shaking incubator at 37°C until OD600 (absorbance at 600nm on a spectrophotometer) was 0.4-0.6, and then expressed with IPTG (isopropyl-β-D-thiogalactopyranoside) at a certain temperature. In order to select the best expression conditions for the recombinant protein, repeated expression experiments were performed with different IPTG concentrations, different induction temperatures, and different induction times. Figure 2 The IPTG concentrations were 0.2 mM, 0.5 mM, and 1 mM, respectively. The induction temperature was 37°C and the induction time was 6 hr. The recombinant E2 protein expression experiment was repeated three times. Figure 2 a, Figure 2 b and Figure 2 c, The results of each repetition showed that the best induction effect was obtained when 1mM IPTG was used and induced at 37℃ for 6 hours. Figure 3 The induction temperature was 18℃, 25℃, and 37℃, the IPTG concentration was 1mM, and the induction time was 6 hr. The recombinant E2 protein expression experiment was repeated three times. Figure 3 a, Figure 3 b and Figure 3 c, The results of each repetition showed that the best induction effect was obtained when 1 mM IPTG was used and induced at 37°C for 6 hr. Figure 4 The induction time was set to 2 hr, 4 hr, and 6 hr, the IPTG concentration was 1 mM, and the induction temperature was 37 °C. The recombinant E2 protein expression experiment was repeated three times. Figure 4 a, Figure 4 b and Figure 4 c, The results of each repetition showed that the best induction effect was obtained under the conditions of 1 mM IPTG and 6 hours of induction at 37°C.

[0020] The optimal induction conditions were selected: 1 mM IPTG, induction at 37°C for 6 hr, and then centrifugation at 10,000 g for 20 min to collect the bacteria, discard the supernatant, resuspend the bacteria in 1×TE (0.01 M Tris+0.001 M EDTA) and break the bacteria with an ultrasonic disruptor. Gel running showed that the E2 protein was expressed in the form of inclusion bodies in Escherichia coli, and the inclusion bodies were washed three times with detergent, denatured and dissolved with 8 mol / L urea, and impurities such as cell debris were removed by centrifugation. The E2 recombinant protein was purified with Ni-NTA filler. The buffers used in the purification process included equilibration buffer, washing buffer and elution buffer. Equilibration buffer: 10 mM Tris, 100 mM NaCl, 10 mM imidazole, 8 M urea, pH 7.4; wash buffer: 10 mM Tris, 100 mM NaCl, 50 mM imidazole, 8 M urea, pH 7.4; elution buffer: 10 mM Tris, 100 mM NaCl, 400 mM imidazole, 8 M urea, pH 7.4. The eluted E2 protein was pumped into PBS at a flow rate of 1 ml / min for renaturation while stirring. After renaturation, it was identified by 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide) gel electrophoresis. The results are shown in Figure 5 The protein purified by Ni-NTA filler was dialyzed and renatured in PBS solutions containing 6, 4, 2, 1, and 0 mol / L urea, and the renatured protein was the desired BVDV E2 protein. The amino acid sequence information of the BVDV E2 protein is shown in SEQ ID NO:4.

[0021] The purity of the target protein was 91.4% as determined by SDS-PAGE, and the concentration of the target protein was 3.25 mg / ml as determined by the Coomassie Brilliant Blue method. Figure 6 and Figure 7 ,The hydrophobicity and hydrophilicity of the BVDV E2 protein obtained in the previous steps were analyzed by DNAMAN software, and it can be seen that the optimized protein has better hydrophilicity and solubility.

[0022] (III) Immunization of BALB / c mice with recombinant E2 protein and preparation of hybridoma cells E2 recombinant protein: Freund's complete adjuvant = 1:1 emulsified, 0.1 mg each was injected subcutaneously into 6 6-week-old female BALA / c mice for the first immunization; 2 weeks later, Freund's complete adjuvant was replaced with Freund's incomplete adjuvant at the same dose, and immunization was performed once every 2 weeks for a total of 4 times.

[0023] On the 4th day after the 4th immunization, the eyeballs of the mice were removed and blood was collected, which was centrifuged for detection. The titer of the obtained mouse serum was detected by indirect ELISA, and the detection process included the following steps: ① Antigen coating: E2 protein was used as antigen, diluted to 5 μg / ml with phosphate buffer, and 100 μL / well was added to a polystyrene 96-well reaction plate. It was placed at 4°C overnight.

[0024] ② Washing: On the second day, pour out the liquid in the concave wells and wash three times with washing solution PBST (i.e., PBS solution with Tween added).

[0025] ③ Blocking: Add 100 μL / well blocking solution, leave at room temperature for 0.5 h, then wash 3 times with washing solution PBS-T.

[0026] ④ Add the sample to be tested: dilute the immunized mouse serum in multiples and add 100 μL / well to the 96-well plate. Make two replicates for each sample and use PBS as a negative control. After sealing, incubate in a 37°C incubator for 1 hour.

[0027] ⑤ Washing: After incubation, wash three times with washing buffer PBST.

[0028] ⑥ Add enzyme-labeled anti-antibody: rabbit anti-mouse IgG-HRP, dilute to 5μg / ml with phosphate buffer, 100μL / well, seal, and incubate in a 37℃ constant temperature incubator for 1h.

[0029] ⑦ Washing: Wash 3 times with washing buffer PBST.

[0030] ⑧ Color development: Add 100 μL / well of freshly prepared substrate solution and place in the dark at room temperature for 10 min to display blue color.

[0031] ⑨ Stop reaction and compare color: add 50 μL / well stop solution. The color turns yellow; use an enzyme-labeled instrument to measure the absorbance of each well at 450nm. The maximum dilution of the positive reaction is the titer of the sample to be tested.

[0032] The test results are shown in Tables 1 and 2, where ABCDEFGH are the longitudinal numbers of the 96-well plate, and 1-12 are the transverse numbers of the 96-well plate.

[0033] Table 1 Results of antibody titer test for mice No. 1-3

[0034] Table 2 Results of antibody titer test for mice No. 4-6

[0035] As can be seen from Tables 1 and 2, the titers of mice No. 2 and No. 3 were the highest at 1:32768 and 1:65536, respectively, so their spleens were taken to prepare hybridoma cells, and the spleens of mice No. 2 and No. 3 were taken and mixed with SP2 / 0 myeloma cells at a ratio of 1:10, and PEG-1450 was used to promote cell fusion. The fusion conditions were as follows: SP2 / 0 cells and immunized mouse spleen cells were resuspended with 1640 incomplete culture medium, counted with a hemacytometer, and then mixed in a 50 ml sterile centrifuge tube at a ratio of spleen cells: SP2 / 0 cells = 1:3. After thorough mixing, 1640 incomplete culture medium was added to 40 ml, and the mixture was centrifuged at a speed of 1,500 rpm for 5 minutes. After centrifugation, the supernatant and the culture medium remaining on the tube wall were discarded, the mixed precipitate of SP2 / 0 cells and immunized mouse cells was gently tapped to loosen it, and the bottom of the centrifuge tube was immersed in 37°C warm water. Take out 1 ml of fusion agent PEG1450 that has been incubated at 37°C from the incubator, and evenly drip it into the cell mixed pellet within 60 seconds, while rotating the centrifuge tube. Incubate for 45 seconds, take out 1640 incomplete culture medium preheated in the 37°C incubator, take 2 ml, and evenly drip it into the pellet for 120 seconds, while rotating the centrifuge tube, and then slowly drip the remaining 45 ml of culture medium. After gently inverting to mix, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and resuspend with 100 ml of HAT complete culture medium solution.

[0036] The fused cells were screened for monoclonal clones using a 96-well plate, and the fused cells were positively screened using the indirect ELISA method. The screening process was as follows: ① Use a pipette to evenly spread the resuspended cells into a 96-well plate at 200 μL / well. After spreading, place the plate in a 5% CO2, 37°C incubator for culture. After 7 days of culture, replace half of the culture medium in the well with HAT complete culture medium and further culture the cells.

[0037] ②Cell subcloning: Using the limiting dilution method, the cultured cells were diluted at 0.8 cells / well and evenly coated on a 96-well plate. The 96-well plate was then placed in a 5% CO2 37°C cell culture incubator for culture. After observation for about 5 days, the wells containing only single colony cells were selected for supernatant detection. If the single colony wells reached more than 50% and the result of the selected single colony well detection was 100% positive, the cell was considered to be a monoclonal cell line. Four cell lines that could express high-titer antibodies (the selection method was the same as the above-mentioned indirect ELISA method) were selected for use. They were verified by double antibody sandwich ELISA to ensure that they could become antibody pairs. Finally, two monoclonal cell lines that could produce anti-E2 proteins - hybridoma cell lines were obtained. Monoclonal antibody 1 came from B3 hybridoma cells, and monoclonal antibody 2 came from E5 hybridoma cells.

[0038] 4. Ascites or antibody preparation Select female BALB / c mice aged 6-10 weeks, expand the culture of the two selected hybridoma cell lines and inoculate them into the peritoneal cavity of 10 BALB / c mice respectively. The injection volume for each mouse is 1 ml 5×10 6 hybridoma cells. About 7 days after the injection of hybridoma cells, extract the ascites from the mouse peritoneal cavity, centrifuge at 10,000 r / min for 20 min, and take the supernatant. Dilute the ascites supernatant 3-5 times with PBS, filter with a 0.45μm filter membrane, and purify with Protein A. The purification conditions are: ① Dilute the ascites supernatant 3-5 times with PBS and filter it with a 0.45μm filter membrane.

[0039] ② Use 10mM PBS, pH7.4 equilibration buffer to equilibrate the protein A (Novo A Diamond filler from Boglon) chromatography column at a flow rate of 2ml / min. After equilibration, use 10mM PBS, pH7.4 washing buffer to rinse the chromatography column. After 3-5 CV, use elution buffer: 0.1M glycine, pH3.0, neutralization buffer to elute the chromatography column, collect the elution peak, and use 1M Tris-HCL, pH8.5 to neutralize the eluate. The pH after neutralization is 7.4. High-purity monoclonal antibodies 1 and 2 against E2 protein were purified. The purification results are shown in Figure 8 The relevant cell lines are stored in the company by the applicant. The purity of monoclonal antibody 1 and monoclonal antibody 2 is 98%.

[0040] (V) Double Antibody Sandwich Elisa Method Development 100 μL of anti-E2 protein monoclonal antibody 1 (diluted to 3 μg / mL) diluted with 50 mmol / L phosphate buffer was added to each well of a 96-well plate and incubated overnight at 4°C. Blocked with 150 μL 3% BSA for 45 min and washed four times with PBS-Tween. BVDV serum was diluted in PBS at pH 7.0 (containing 0.05% Tween 20) at 0.5-200 μg / ml, and 50 μL was added to each well. Incubated at 37°C for 1 h, washed four times with PBS-Tween, and 100 μL of HRP-labeled anti-E2 protein monoclonal antibody 2 (diluted to 1 μg / mL) was added to each well and incubated in a 37°C incubator for 45 min. After incubation, the cells were washed four times with PBS-Tween. 100 μL TMB substrate was added, and the reaction was terminated by adding 2 mol / L sulfuric acid after incubation at room temperature in the dark for 10 min. The absorbance at 450 nm was measured by a microplate reader.

[0041] (VI) Confirmation of detection limit of double antibody sandwich Elisa The E2 protein with known concentration was diluted in a gradient (50-0.01 µg / mL) and tested by double antibody sandwich ELISA to determine the calibration curve. At the same time, the cutoff value of this detection method was determined according to the OD450 value of the negative sample (P / N>2.5). The data are shown in Table 3.

[0042] Table 3. Statistical table of calibration curves of BVDV double antibody sandwich ELISA

[0043] As can be seen from Table 3, the experimental results show that the lower limit of detection in this experiment is 1:4096 dilution, that is, 0.012 µg / mL. At this time, the OD450 (the absorbance value of the microplate reader at 450 nm) detection result is 0.226, which is 2.6 times the background well OD450 detection of 0.087.

[0044] 7. Clinical sample testing 50 BVDV-1 serum positive specimens and 30 BVDV serum negative specimens were purchased from the China Veterinary Drug Administration, and the samples were determined by q-PCR. The double antibody sandwich ELISA kit of this embodiment was used to detect it, and compared with the known results, and the test was repeated three times to detect all positive sera. The test results are shown in Tables 4, 5 and 6.

[0045] Table 4 Double antibody sandwich ELISA kit first test results

[0046] Table 5 The second test results of the double antibody sandwich ELISA kit

[0047] Table 6 The third test results of the double antibody sandwich ELISA kit

[0048] As shown in Tables 4, 5 and 6, all 50 positive sera were positive, and 1 of the 30 negative sera was positive. The sensitivity was 100% and the specificity was 98.75%. The results of the three repeated experiments were consistent, with good stability and no false positives.

[0049] The BVDV E2 recombinant gene provided by the invention can be used to prepare a bovine viral diarrhea virus monoclonal antibody, and the monoclonal antibody can be used to prepare a double antibody sandwich Elisa kit for detecting the bovine viral diarrhea virus, and has good sensitivity and stability.

Claims

1. BVDV E2 recombinant protein, characterized in that The amino acid sequence of the BVDV E2 recombinant protein is shown in SEQ ID NO:

4.

2. The BVDV E2 recombinant protein according to claim 1, characterized in that The gene encoding the BVDV E2 recombinant protein is shown in SEQ ID NO:

3.

3. The method for preparing the BVDV E2 recombinant protein according to claim 2, characterized in that: The coding gene of the BVDV E2 recombinant protein is recombined with the pET-28b plasmid vector to obtain a recombinant plasmid, and the recombinant plasmid is transformed into BL21 competent cells and induced to express by IPTG.

4. The preparation method according to claim 3, characterized in that: The specific steps include: (1) obtaining the BVDV E2 protein gene sequence, amplifying and digesting the BVDV E2 protein gene sequence, and connecting the digested BVDV E2 protein gene sequence with the pET-28b plasmid; (2) The pET-28b plasmid obtained in step (1) was transformed into BL21 competent cells, and the BVDV E2 protein was expressed using BL21 Escherichia coli. The BVDV E2 protein was denatured and dissolved with urea solution, and then purified by Ni-NTA chromatography to obtain the BVDVE2 recombinant protein.

5. The preparation method according to claim 4, characterized in that: In step (1), the primers used for amplification include an upstream primer and a downstream primer, the sequence of the upstream primer is shown in SEQ ID NO:1, and the sequence of the downstream primer is shown in SEQ ID NO:

2.

6. The preparation method according to claim 4, characterized in that: In step (2), the expression conditions of BVDV E2 protein are: 0.5-1 mM IPTG, 30-37°C, and induction for 5-7h.

7. The preparation method according to claim 6, characterized in that: In step (2), the concentration of the urea solution is 8 mol / L.

8. The use of the BVDV E2 recombinant protein according to claim 1, characterized in that: The BVDV E2 recombinant protein is used for producing bovine viral diarrhea virus antibodies.

9. The use according to claim 8, characterized in that: The specific production steps are: immunize mice after the BVDVE2 recombinant protein described in claim 1 is completely emulsified with Freund's adjuvant, remove the spleen and submerge it, use PEG1450 to induce the fusion of Sp2 / 0 cells and mouse spleen cells to obtain a monoclonal cell line-hybridoma cell line that produces anti-E2 protein, and then subclone and expand the culture to obtain a positive cell line and inoculate it into the abdominal cavity of BALB / c mice to obtain a monoclonal antibody. The encoding gene of the BVDV E2 recombinant protein is shown in SEQ ID NO:

3.

10. The use according to claim 8, characterized in that: The bovine viral diarrhea virus antibody is used to prepare a double antibody sandwich Elisa kit for detecting bovine viral diarrhea virus.

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