CN116284434A - Variant pseudorabies virus gC-gD epitope concatemer and application thereof - Google Patents
Variant pseudorabies virus gC-gD epitope concatemer and application thereof Download PDFInfo
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Abstract
The invention provides a variant pseudorabies virus (Pseudorabies virus, PRV) gC-gD epitope concatemer and application thereof. The amino acid sequence of the variant pseudorabies virus gC-gD expression concatemer is shown as SEQ ID NO.1, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2. The invention establishes an indirect ELISA detection method for PRV neutralizing antibodies with strong specificity, high sensitivity and good repeatability by utilizing the variant pseudorabies virus gC-gD epitope concatemer, can completely meet the serological diagnosis requirements of PRV wild virus epidemic pig farms and PRV wild virus purification pig farm pig groups, and lays a foundation for the development of novel PRV polypeptide vaccines.
Description
Technical Field
The invention belongs to the technical field of livestock and veterinary, and particularly relates to a pseudorabies virus gC-gD epitope concatemer and application thereof.
Background
Porcine pseudorabies virus (Pseudorabies virus, PRV) is a porcine neurogenic herpesvirus. Although pigs are a natural host of PRV, a variety of mammals, including ruminants, carnivores, and rodents, can also be infected. It is an important nervous system pathogen in livestock and animals infected with PRV may die from central nervous system diseases. PRV infection poses a serious threat to the pig industry and attenuated live or inactivated vaccines are commonly used to control disease. Since 2011, the pig farm vaccinated with Bartha-K61 in China reappears pseudorabies caused by PRV gene mutation, and the first inventor of the invention separates PRV variant strain FJ-2012 from the pig farm, and discovers that the strain has a certain degree of mutation. Many studies have shown that antibodies produced by PRV classical strain vaccines fail to completely protect against infection by virulent PRV variant strains.
The PRV genome is linear double-stranded DNA with the size of 150kb, the G+C content is up to 75%, and the PRV genome contains 72 reading frames and totally encodes 70-100 proteins, and the gC and gD proteins are closely related to the immune response of a host in the currently known encoded glycoprotein 11. gC. The gD glycoprotein is envelope protein, is mainly distributed on the envelope surface of virus particles, has high conservation, can induce organisms to generate high-level neutralizing antibodies, can protect mice or pigs from PRV infection, has good immunogenicity and reactivities, and is the preferred protein for constructing recombinant DNA vaccine and vaccine immunodetection. Multi-epitope subunit vaccines are a vaccine development technique that has developed in recent years. The epitope vaccine is a vaccine prepared based on antigen epitopes, has strong pertinence to induced immune response, is nontoxic and relatively stable, is easy to produce, store and use, and is a direction of vaccine development in the future compared with the traditional vaccine. Therefore, new genes based on gC and gD sequences can be designed as antigens of vaccine candidates, or gC and gD epitope fragments capable of stimulating the body to generate protective humoral immune response can be selected as diagnostic antigens for the detection of PRV antibodies and the development of epitope vaccines.
According to the method, epitope genes gC and gD on PRV variant strains are connected in series for the first time, recombined onto a pET-28a (+) vector, pET-28a (+) -gC and gD prokaryotic recombinant expression plasmids are constructed, and transformed into BL21 (DE 3) for induced expression, so that PRV novel strain gC-gD epitope tandem recombinant proteins are obtained, an indirect ELISA detection method for a pig serum PRV neutralizing antibody is established, and a foundation is laid for developing a PRV multi-epitope vaccine.
Disclosure of Invention
The invention aims to provide a variant pseudorabies virus gC-gD epitope concatemer and application thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention firstly provides a variant pseudorabies virus gC-gD epitope concatemer, wherein the amino acid sequence of the variant pseudorabies virus gC-gD epitope concatemer is shown as SEQ ID NO. 1.
The invention further provides a gene for coding the variant pseudorabies virus gC-gD epitope concatemer, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2.
The invention also provides application of the variant pseudorabies virus gC-gD epitope concatemer in preparation of PRV diagnostic kit and vaccine.
The invention has the advantages that:
the invention takes a variant pseudorabies virus (Pseudorabies virus, PRV) as a research and development basis, obtains the gene sequences of the major antigen proteins gC and gD of the variant PRV epitope through screening and cloning sequencing, artificially synthesizes the cDNA serial sequence of the gene sequences after optimizing sequence codons, is connected to a prokaryotic expression vector pET-28a (+) multiple cloning site, is transformed to BL21 (DE 3) competent cells, and is induced to express by IPTG. The result of plasmid sequencing and enzyme digestion shows that the construction of gC-gD antigen epitope tandem gene recombinant expression plasmid is successful, and the SDS-PAGE and Westren blot result shows that the gC-gD antigen epitope tandem recombinant protein is successfully induced and expressed. Therefore, the invention initiates the in vitro tandem expression of the main antigen protein gC and gD antigen epitope gene sequences of the PRV variant strain FJ-2012, establishes the gC and gD antigen epitope cDNA tandem sequences, obtains the tandem recombinant protein for induced expression and the corresponding amino acid sequences, and lays a foundation for PRV epitope screening, PRV multi-epitope vaccine and diagnostic reagent research.
Drawings
Fig. 1: PCR amplification result of PRV virus FJ-2012 strain gC and gD epitope gene. M,2K Marker;1,2,3, performing gC epitope gene PCR amplification (405 bp) by taking FJ-2012 strain nucleic acid as a template; 4,5,6, gD epitope gene PCR amplification (318 bp) was performed using FJ-2012 strain nucleic acid as template.
Fig. 2: nucleotide comparison before and after PRV virus gC-gD epitope gene tandem optimization.
Fig. 3: and (5) PCR verification and enzyme digestion identification results. Wherein A: m,2K Marker;1,2,3,4, PCR verification with plasmid as template; b: m1,2K Marker; m2,8K Marker;5, PCR verification by taking the plasmid as a template; 6, double enzyme digestion identification.
Fig. 4: sequencing peak shape of gC-gD-PET28a (+) recombinant plasmid.
Fig. 5: and (3) comparing the sequencing result of the gC-gD-PET28a (+) recombinant plasmid with the optimized sequence.
Fig. 6: and (5) inducible expression and identification of the recombinant protein. A, M: protein Maker; NC, bacterial liquid precipitation is not induced; 2,3,4 are bacterial liquid precipitations when IPTG is used to induce gC-gD-pET-28a (+) -BL21 (DE 3) for 2h,4h and 6 h; b, M: protein Maker;4, serially expressing recombinant proteins by the purified gC-gD epitope; NC, control group did not induce mycoprotein.
Detailed Description
Example 1
1 Experimental materials and methods
1.1 reagent Material
PBST（pH7.4，0.01 M），FlyCut® EcoRI，FlyCut ® NcoI，EasyPure ® Quick Gel Extraction Kit，EasyPure® Plasmid MiniPrep Kit (EM101)，Agarose，GelStain，BL21(DE3) Chemically Competent Cell，ProteinRuler ® II (12-120 kDa), IPTG, his tag monoclonal antibody, horseradish peroxidase-labeled goat anti-pig antibody and the like are all purchased from Beijing full gold Co.
1.2 Amplification of gC and gD epitope genes
Primers for PCR amplification of gC and gD epitope genes are designed according to a PRV reference strain Becker sequence (JF 79721 9.1.1) registered in GenBank, PCR amplification is carried out by taking the nucleic acid of a novel newly isolated pseudorabies virus FJ-2012 strain as a template, and amplified products are sent to a biological engineering Co., ltd for sequencing, so that the gC and gD epitope gene sequences of the FJ-2012 strain are obtained. Primers used for PCR were synthesized by the engineering Co., ltd., of the biological engineering (Shanghai), namely:
gC-F：5’- TACTTTGACGAGCCCCCGCG-3’，
gC-R：5’- GAAGGCGGCGTCCACCG’；
gD-F：5’- GATCGTCTGCTGAATGAAGC -3’，
gD-R：5’- CAGGGCAACCATAAAATCTGT -3’。
1.3 Construction of gC-gD recombinant expression vector
According to FJ-2012 strain gC and gD antigen epitope gene sequence, gC-gD tandem coding sequence is designed, and codon optimization is carried out, gC-gD tandem gene fragment is synthesized by Fuzhou Shangya biotechnology Co Ltd, and the fragment is inserted between NcoI and EcoRI cleavage sites on pET-28a+ carrier, so as to construct recombinant plasmid gC-gD-PET28a (+). The recombinant plasmid was transferred into BL21 (DE 3) competent cells, the plasmid was extracted according to the instructions of the plasmid extraction kit, PCR and double cleavage (NcoI, ecoRI) verification were performed using the plasmid as a template, and the recombinant plasmid was sent to the engineering Co.Ltd for sequencing. Primers used for PCR were synthesized by the engineering Co., ltd., of the biological engineering (Shanghai), namely:
F：5’- CCCAATCCATGGCCTATTTTGATGAACCGCCGCGT -3’，
R：5’- CCCAATGAATTCGGCAGGGCAACCATAAAATCTGTCAG -3’。
1.4 Inducible expression of gC-gD epitope tandem recombinant protein
The correct monoclonal colonies were verified by double digestion and PCR, and were cultured overnight (200 rpm,37 ℃) with LB liquid medium containing 50. Mu.g/mL kanamycin. Inoculating into fresh LB liquid culture medium (containing kanamycin 50 [ mu ] g/mL) at a ratio of 1vol%, and culturing at 37deg.C under shaking (200 rpm) to OD 600 At 0.6, IPTG was added to a final concentration of 0.5mmol/L and the expression of the target protein was induced at 37 ℃. Respectively taking 500 mu L culture solution in IPTG induction for 2h,4h and 6h, centrifuging for 5min at 3000 Xg, collecting bacterial precipitate, adding a proper amount of 1X SDS Loading buffer heavy suspension bacterial precipitate, boiling in boiling water bath for 10min, centrifuging for 10min at 12000 Xg, taking 8 mu L supernatant, and carrying out Western blot detection on the expression of target protein by using 12% SDS-PAGE and anti-His tag antibody. The recombinant target protein was purified by using a Ni-NTA purification column, and the recombinant protein concentration after renaturation was measured by BCA method.
1.5 Establishment of indirect ELISA antibody detection method
According to an array titration method, gC-gD epitope tandem recombinant proteins with different concentrations are added into an ELISA plate at the concentration of 5 mg/L, 3mg/L, 1 mg/L and 0.5 mg/L of 100 mu L per hole, the ELISA plate is coated for 12 hours at 4 ℃, then blocked by PBST containing 2% BSA, 100 mu L per hole is blocked for 1 hour at 37 ℃, blocking liquid is discarded, after PBST is washed for 3 times, pig serum with different dilution ratios is added according to 100 mu L per hole, and incubation is carried out for 60 minutes at 37 ℃; after PBST is washed for 3 times, adding 1:2000 diluted horseradish peroxidase-labeled antibody (HRP) at a rate of 100 [ mu ] L/hole, and incubating at 37 ℃ for 60min; PBST is washed 3 times, TMB substrate chromogenic solution is added according to 100 mu L/hole, and the mixture is incubated for 15min at room temperature and then is led throughReading OD of each hole by enzyme labeling instrument 650 Numerical values, and the optimal antigen (gC-gD epitope tandem recombinant protein) coating concentration and the optimal serum dilution ratio are determined through analysis. And determining a critical value determined by the result of the PRV negative serum. The stability of the process was verified by both the inter-batch and intra-batch repeat tests.
1.6 detection of clinical porcine serum antibodies
According to the indirect ELISA antibody detection method established by the gC-gD fusion protein obtained by the invention, PRV neutralizing antibodies are detected on clinical pig sample serum.
1.6.1 Comparison with gB antibody detection
The method provided by the invention is used for detecting 90 pig serum randomly extracted from a plurality of pig farms, simultaneously detecting the gB antibody by using an IDEXX pig pseudorabies virus gB antibody detection kit (competition ELISA method), and carrying out comparative analysis on the detection result.
1.6.2 PRV antibody detection contrast in different pig farms
60 parts of pig serum from PRV wild virus positive pig farms and 60 parts of pig farms from PRV complete purification are detected by the method established by the invention, and the detection results are compared and analyzed.
2. Results
2.1 Construction of epitope tandem expression recombinant vector
According to the sequencing result of PCR amplification of FJ-2012 strain gC and gD antigen epitope gene (figure 1,SEQ ID NO.3,SEQ ID NO.4), gC-gD tandem coding sequence is designed, and codon optimization (shown in figure 2 and SEQ ID NO. 2) is carried out, so as to construct gC-gD-PET28a (+) recombinant plasmid, after T1 competent cells are transformed, cloning is selected, bacteria are shaken and plasmids are extracted, PCR amplification is carried out by using the extracted plasmids as templates, and agarose gel electrophoresis observation is carried out. As a result, as shown in FIG. 3, a distinct target band was seen, consistent with the expected band (723 bp) size. The extracted plasmid was digested with NcoI and EcoRI, and the digested product was run out to visualize the expected bands. The extracted plasmid was sent for sequencing, the sequencing result showed (FIG. 4) that the peak shape was single, indicating that the sequencing result was good, and the comparison was 100% with the expected gC-gD fragment (FIG. 5), indicating that the construction of the gC-gD-pET28a (+) recombinant plasmid was successful.
2.2 Inducible expression and verification of gC-gD recombinant protein
The resulting recombinant plasmid gC-gD-pET-28a (+) was transferred into BL21 (DE 3), after induction with IPTG for 2h,4h,6h, bacterial pellet was collected and lysed, SDS-PAGE gel showed a distinct recombinant protein band at about 27kDa (FIG. 6A), and WB experiments also showed a specific band at about 27kDa (FIG. 6B). The PRV variant strain FJ-2012 strain gC-gD antigen epitope recombinant protein constructed by the invention is expressed in BL21 (DE 3), and the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1.
2.3 Indirect ELISA method for gC-gD recombinant protein
2.3.1 Antigen coating optimal concentration and serum optimal dilution
The array titration method test results show that: when the antigen coating concentration is 3mg/L and the sample serum is diluted according to the ratio of 1:50, the positive sample serum OD 650 The value is 0.801, and the serum OD of the negative sample is larger 650 The value was 0.359, and the ratio of the two (positive sample serum OD 650 Serum OD with negative sample 650 I.e. P/N value) is also 2.23 at maximum. Therefore, the optimal antigen coating concentration of the indirect ELISA antibody detection method established by the invention is 3mg/L, and the optimal serum dilution is 1:50.
TABLE 1 gC-gD recombinant protein coating optimal concentration and serum optimal dilution (OD 650 Value of
2.3.2 Determination of the threshold of an Indirect ELISA method
With reference to the above method steps, 20 parts of pig serum with PRV antibody as negative (namely PRV-gB and PRV-gE antibodies as negative) are detected, and the OD of the sample serum is obtained 650 Mean value (Mean) of 0.339, variance (SD) of 0.03, variationThe difference coefficient was 8.8%. According to mean+3×sd=0.429 and mean+2×sd=0.399, the decision criteria of the indirect ELISA method for detecting PRV neutralizing antibodies by using gC-gD recombinant proteins of the present invention were initially determined: sample to be tested OD 650 A value of 0.429 or more is determined to be positive, and a value of 0.399 or less is determined to be negative, and a value between the values is determined to be suspicious.
TABLE 2 negative threshold of gC-gD recombinant protein indirect ELISA method
2.3.3 Specificity verification
The method established by the invention is adopted to detect positive pig serum (gB and gE antibodies are positive), and the result shows that the method is negative to the detection results of PRRSV, PEDV, PCV-2, CSFV and PPV positive serum, and only PRV antibody positive serum is positive, so that the indirect ELISA antibody detection method established by the invention has good specificity and has no cross reaction to other pathogenic antibodies.
TABLE 3 results of specificity test of gC-gD recombinant protein by indirect ELISA method
2.3.4 Repeatability test
By adopting the method established by the research, the repeated detection results in the sample batch are shown as follows: OD was tested repeatedly for each sample 650 The variance of the values is between 0.006 and 0.013, and the variation coefficient is between 1.1 and 2.4 percent; the results of the batch-to-batch repeated detection show that: OD was tested repeatedly for each sample 650 The variance of the values is between 0.009 and 0.026, and the variation coefficient is between 3.0% and 4.0%. Therefore, the indirect ELISA antibody detection method established by the invention is stable and applicable and has good repeatability.
TABLE 4 results of reproducibility of the indirect ELISA method for gC-gD recombinant protein
2.3.5 Sensitivity test
PRV antibody positive porcine serum (positive for both gB and gE antibodies) 2 N The detection result of the method shows that: after 16 times dilution, the positive serum is subjected to dilution detection according to a ratio of 1:50, namely positive can still be detected after 800 times dilution, which shows that the indirect ELISA antibody detection method established by the invention has higher sensitivity.
TABLE 5 sensitivity test (OD) of indirect ELISA method for gC-gD recombinant protein 650 Value of
2.4 Preliminary application and compliance rate
The indirect ELISA antibody detection method and the commercial antibody detection kit established by the invention are used for detecting 90 clinical pig serum, and the result shows that: the method provided by the invention has the advantages that the positive rate of the PRV neutralizing antibody is 71.11 percent (64/90), the negative rate is 9.24 percent (17/90), and the suspicious rate is 4.89 percent (9/90); the commercial kit detects the PRV gB antibody with the positive rate of 74.44 percent (67/90), the negative rate of 8.7 percent (70/90) and the suspicious rate of 3.8 percent. Dividing the number of the detection results of the two methods which are consistent by the total number to obtain the overall coincidence rate of the two methods of 96.67%. The results show that the indirect ELISA antibody detection method established by the invention is basically consistent with the detection results of the commercial antibody detection kit, and the research invention can be used for clinical detection and provides a new method for PRV antibody epidemiological research and vaccine immune effect monitoring.
TABLE 6 comparative test results of 90 pig serum from clinical samples
2.5 PRV antibody detection results in different pig farms
60 parts of pig serum from PRV wild-toxin positive pig farms and 60 parts of pig serum from PRV wild-toxin purified pig farms are detected by the method and the commercial antibody detection kit, and the gE gene deletion vaccine of classical strain of pseudorabies is immunized in both pig farms, so that the positive rate of neutralizing antibodies in the PRV wild-toxin positive pig farms is 95% (57/60), the positive rate of neutralizing antibodies in the PRV wild-toxin completely purified pig farms is only 80% (48/60), and the positive rate of gB antibodies in the PRV wild-toxin completely purified pig farms is 96.67% (58/60) by the commercial kit. According to the detection results of PRV antibodies in different pig farms, the positive rate of neutralizing antibodies in the PRV wild toxin completely purified pig farms is obviously lower than that in PRV wild toxin positive pig farms, and is also obviously lower than that of detecting gB antibodies by using a commercial antibody detection kit, and the fact that antibodies generated by immunization of a classical PRV strain gE gene deletion vaccine and gC-gD epitope fusion proteins of variant FJ-2012 cannot play a complete immune reaction is indirectly reflected. Therefore, the method of the invention has important clinical guidance significance for detecting PRV wild virus positive PRV antibody condition of pig farm and presuming the PRV wild virus capability of completely purifying PRV variant strain infection of pig farm; and the gC-gD epitope fusion protein constructed by the invention can be used for preparing polypeptide seedlings, and can effectively resist PRV variant strain infection.
TABLE 7 PRV antibody detection results for different pig farms
In conclusion, the research constructs a tandem expression vector containing PRV gC and gD epitopes, converts an escherichia coli expression system to perform induction expression, and obtains gC-gD epitope tandem recombinant protein with good expression characteristics and immunocompetence, thereby laying a certain foundation for the development of PRV novel vaccines. The invention uses the escherichia coli to express PRV gC-gD epitope tandem recombinant protein in vitro, has simple production process, and the expressed recombinant protein has good immunoreactivity and antigenicity and good application prospect. In order to elucidate the mechanism of PRV gC-gD immunization of piglets to reduce viral transmission, cell immunity-related studies are required to be carried out in future researches.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (3)
1. A variant pseudorabies virus gC-gD epitope concatemer, characterized in that: the amino acid sequence of the variant pseudorabies virus gC-gD expression concatemer is shown as SEQ ID NO. 1.
2. A gene encoding the variant pseudorabies virus gC-gD epitope concatemer of claim 1, characterized by: the nucleotide sequence of the gene is shown as SEQ ID NO. 2.
3. The use of the variant pseudorabies virus gC-gD epitope concatemers according to claim 1 in the preparation of PRV diagnostic kits and vaccines.
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