Feline calicivirus VP1 recombinant protein and application thereof

Abstract

The invention discloses a feline calicivirus VP1 recombinant protein and application thereof. The amino acid sequence of the feline calicivirus VP1 recombinant protein is shown as SEQ ID No.1 in a sequence table. The neutralizing titers of the feline calicivirus positive serum prepared by adopting the recombinant protein as an antigen and different gene groups are all up to 1: 4096 times or above, the requirements of ELISA and IFA tests can be met, the feline calicivirus positive serum obtained after immunization of the prepared recombinant protein is good in purity, free of bacteria, free of mycoplasma and exogenous virus pollution, high in specificity and good in clinical application prospect. And the strain can react with strains of different evolution groups well, and has good broad spectrum. Positive serum prepared from the recombinant protein prepared by the invention can be used for quality control of different gene group cat calicivirus vaccines, such as specificity test, exogenous virus test, diagnostic detection and the like.

Description

Technical Field

[0001] The invention belongs to the fields of biotechnology and animal virology, and in particular relates to a feline calicivirus VP1 recombinant protein and an application thereof. Background Art

[0002] Feline calicivirus belongs to the family Calicivirdae and the genus Vesivirus. FCV is a single-stranded positive-strand non-enveloped RNA virus. Feline calicivirus (FCV) is one of the main pathogens that cause respiratory diseases in cats. The FCV genome is about 7.7kb in size and contains 3 open reading frames. ORF2 encodes the structural protein VP1, including 6 regions from A to F. Changes in nucleotides in regions C, D, and E can affect the reactogenicity of FCV. Regions C and E are highly variable regions. The variability of region E is the reason why most FCV strains have low cross-protection from each other.

[0003] The replication mechanism of FCV also increases the mutation rate of FCV. In the early stage, the clinical symptoms of FCV infection were increased eye and nasal discharge, fever, oral ulcers, nasal congestion and other respiratory symptoms. With environmental pressure, FCV strains of two major evolutionary groups, GⅠ and GⅡ, appeared. In recent years, FCV has become prevalent throughout the country, and strains of both GⅠ and GⅡ have been reported.

[0004] There are no reports or patents in the published articles and patents on the method of preparing positive serum that has good cross-reactivity with feline calicivirus of the two major evolutionary groups GⅠ and GⅡ. Summary of the invention

[0005] The present invention discloses an amino acid sequence of a recombinant protein of an antigenic dominant region of the VP1 gene of a feline calicivirus covering two major evolutionary groups GⅠ and GⅡ, as well as a method for preparing the recombinant protein and its application in preparing positive serum with good cross-reactivity. The positive serum prepared using the recombinant protein of the present invention has high neutralization titer, good purity, strong specificity, and good cross-reactivity. The prepared serum meets the test requirements of exogenous virus testing, specific testing, and the like, and lays a foundation for the development of related detection and prevention products in the later stage, and has important practical application value for veterinary biological product companies.

[0006] 1. Technical issues to be resolved

[0007] Feline calicivirus is an RNA virus that is easily mutated and has genetic diversity, which results in poor cross-reactivity of positive serum. In order to obtain feline calicivirus-specific positive serum with good cross-reactivity and meet the technical requirements of feline calicivirus vaccine quality control, diagnosis and prevention, the present invention provides a series of recombinant proteins covering the antigenic dominant regions of the VP1 gene of feline calicivirus of the two major evolutionary groups GⅠ and GⅡ, and prepares a subunit vaccine on this basis, and then immunizes rabbits to obtain feline calicivirus-specific positive serum.

[0008] (II) Technical solution

[0009] The first aspect of the present invention provides a tandem recombinant protein of the VP1 gene IVP1-IIVP1 of feline calicivirus covering two major evolutionary groups GⅠ and GⅡ, and its sequence is the amino acid sequence shown in SEQ ID NO.1.

[0010] In the present invention, the recombinant protein is also called fusion protein or recombinant fusion protein, which is the expression product obtained by DNA recombination technology after the recombination of the antigen dominant regions of the VP1 gene of the two major evolutionary groups GⅠ and GⅡ. Specifically, the molecular weight of the target protein expressed by the E. coli expression system is about 80KD, and it is highly expressed in the precipitate after the bacterial cell is broken.

[0011] VP1 protein is the main immunogenic antigen of feline calicivirus, which can induce and activate the body's immune system to produce immune responses and induce host cells to produce neutralizing antibodies. Therefore, fusing the main antigenic regions of VP1 proteins from the two major evolutionary groups GⅠ and GⅡ together for expression can not only increase the titer of neutralizing antibodies, but also enable antibodies to produce better cross-reactions with strains from both the two major evolutionary groups GⅠ and GⅡ.

[0012] In some embodiments of the present invention, preferably, the recombinant protein consists of the amino acid sequence shown in SEQ ID NO.1.

[0013] The second aspect of the present invention provides a gene encoding the recombinant protein of the first aspect of the present invention, which comprises the nucleotide sequence shown in SEQ ID NO.2.

[0014] This gene sequence is to express the recombinant protein in E. coli, and the codons are optimized according to the preference of E. coli for codons. Different species use different frequencies of synonymous codons, and this codon preference has an impact on the translation process. If an mRNA has many clustered rare codons, this will have a negative impact on the movement speed of the ribosome and greatly reduce the protein expression level. Here, the gene sequence is codon optimized for E. coli expression.

[0015] The present invention firstly provides a feline calicivirus VP1 recombinant protein.

[0016] The present invention further provides the use of the feline calicivirus VP1 recombinant protein in preparing feline calicivirus-specific positive serum.

[0017] The present invention further provides a method for preparing feline calicivirus-specific positive serum, using rabbits or guinea pigs as immunized animals.

[0018] Preferably, the feline calicivirus VP1 recombinant protein is used in sequence to perform basic immunization on the immunized animal; more preferably, the feline calicivirus VP1 recombinant protein has an antigen content of 800 μg / ml during basic immunization.

[0019] More preferably, the basic immunization is performed twice, with an interval of 7 to 14 days. The dose for each immunization is 2 ml per animal. The immunization method is intramuscular or subcutaneous injection, and the immunization site is the leg muscle or the subcutaneous part of the back.

[0020] Preferably, the immunized animal is a rabbit or a guinea pig.

[0021] After the basic immunization, booster immunization is also included. As a preferred embodiment, the booster immunization specifically includes: booster immunization of the immunized animal with feline calicivirus VP1 recombinant protein; more preferably, the recombinant protein antigen content is 500 μg / ml.

[0022] More preferably, the booster immunization is performed twice, with an interval of 7 to 14 days. The booster immunization has a dose of 2 ml per animal each time; the immunization method is intramuscular or subcutaneous injection; and the immunization site is the leg muscle or the back subcutaneous.

[0023] According to common knowledge in the art, after the immunization, there are also steps of collecting serum and measuring titer, wherein the time for collecting serum is generally determined according to the interval between immunizations and is not further limited here.

[0024] In some embodiments of the present invention, generally 14 to 21 days after the last immunization, the blood of the immunized animal is collected aseptically, the serum is separated, and the serum neutralizing antibody titer is measured. If the titer is above 1:4096, feline calicivirus-positive serum is obtained.

[0025] After the serum of the present invention passes the purity, mycoplasma and exogenous virus tests, it is packaged, labeled with the serum name, specification, date, etc., and stored below -20°C. It can be used for identification testing, exogenous virus testing and diagnosis.

[0026] Those skilled in the art can combine the preferred solutions of the present invention to obtain the preferred embodiments of the present invention.

[0027] (III) Beneficial effects

[0028] (1) The present invention selects the tandem recombinant proteins of the dominant regions of the feline calicivirus VP1 gene antigen located in the two major evolutionary groups GⅠ and GⅡ as antigens, and immunizes rabbits or guinea pigs to prepare positive serum. The titer of the prepared feline calicivirus-specific positive serum is as high as 1:4096 times, which is higher than the titer of the serum obtained by directly immunizing cats, and can meet the requirements of feline calicivirus diagnostic technology and vaccine quality control.

[0029] (2) In the present invention, the immunized animals are rabbits or guinea pigs, which are standard experimental animals, thus reducing costs and avoiding the difficulty in obtaining experimental animals that meet the requirements and the risk of contamination by other exogenous viruses caused by homologous animals when preparing positive serum using cats.

[0030] (3) The feline calicivirus-specific positive serum prepared by the present invention uses immunogenic proteins expressed by Escherichia coli, and the antigen preparation process is safe, the antigen content is controllable, and the preparation cost is low, thereby avoiding the risk of virus shedding caused by using feline calicivirus to prepare antigens and the disadvantage of high preparation cost.

[0031] (4) The immune titer is as high as 1:4096 times, which is higher than the titer of serum obtained by direct immunization of cats, and can meet the requirements of feline calicivirus diagnostic technology and vaccine quality control.

[0032] (5) The feline calicivirus-positive serum prepared by the present invention has good purity, is sterile, free of mycoplasma, and free of exogenous virus contamination, has strong specificity, and can react well with strains of different evolutionary groups, and has good broad spectrum.

[0033] (6) The positive serum prepared by the present invention can be used for feline calicivirus vaccine quality control such as specificity testing, identification testing, exogenous virus testing, and diagnostic testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The SDS-PAGE results for the purification of FCV-IVP1-IIVP1-1 recombinant protein;

[0035] Figure 2 It is the result of positive IFA reactivity identification and titer detection of FHV rabbit. DETAILED DESCRIPTION

[0036] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0037] Example 1. Expression and identification of feline calicivirus recombinant protein

[0038] 1. Expression and preparation of recombinant proteins

[0039] According to the genetic evolution analysis of some FCV viruses of the two major evolutionary groups GⅠ and GⅡ, the DNAstar software Protean was used to analyze the epitope-related information of the VPI proteins of the FCV viruses of the two major evolutionary groups GⅠ and GⅡ, mainly including antigenicity analysis (Antigenicity), accessibility analysis (Accessibility), hydrophilicity analysis (Hydrophilioity), primary bond flexibility (Flexibility) and antigenic epitope positioning (Determinant). The possible antigenic advantage region was preliminarily predicted to be aa398~aa524. The predicted nucleotide fragments were concatenated with linkers, and the concatenated nucleotide sequences were optimized, designed and artificially synthesized according to the codons preferred by Escherichia coli. According to the correlation between virulence sites and pathogenicity, the corresponding amino acid sites in the recombinant protein were replaced with the corresponding sites of the highly pathogenic strains. The tandem nucleotide sequences were optimized and designed and artificially synthesized according to the codons preferred by Escherichia coli. The nucleotide sequence and amino acid sequence of the recombinant protein with replaced amino acid sites were SEQ ID No. 2 and SEQ ID No. 1, respectively, recorded as FCV-IVP1-IIVP1-1. The nucleotide sequence and amino acid sequence of the recombinant protein without replacing the virulence-related amino acid sites were SEQ ID No. 4 and SEQ ID No. 3, respectively, recorded as FCV-IVP1-IIVP1-2.

[0040] For the protein gene fragment synthesized in vitro, EcorⅠ and SacⅠ were used to connect with the pET-32a(+) expression vector treated in the same way, and the obtained recombinant plasmid was transformed into E. coli BL21(DE3) competent cells to screen positive clones. The positive recombinant plasmids identified as positive were transformed into E. coli BL21(DE3) competent cells, and the positive single colonies containing the specific protein expression plasmids were picked and inoculated into LB medium containing ampicillin. After shaking at 37℃ overnight, the positive recombinant bacteria were screened, and the recombinant bacteria expressing FCV-IVP1-IIVP1-1 were named pET32-IVP1-IIVP1-1, and the recombinant bacteria expressing FCV-IVP1-IIVP1-2 were named pET32-IVP1-IIVP1-2.

[0041] 2. Expression results identification

[0042] The results of SDS-PAGE expression identification of FCV recombinant protein showed that compared with the control before induction, the expression products of FCV-IVP1-IIVP1-1 and FCV-IVP1-IIVP1-2 were mainly insoluble after IPTG induction. The optimal induction expression conditions of recombinant protein were: pET32-IVP1-IIVP1-1 and pET32-IVP1-IIVP1-2 were induced at 37°C for 4 hours, and the bacteria and supernatant were harvested. The size of the recombinant protein was estimated to be about 80 kDa by scanning analysis of SDS-PAGE gel stained with Coomassie Brilliant Blue. Figure 1 .

[0043] 3. Purification results and specificity identification

[0044] The expressed feline calicivirus recombinant protein was purified according to the protein purification method of the BeyoGoldTM His-tag Purification Resin kit. The collected samples were concentrated by ultrafiltration (ultrafiltration tube), and the purity was >90%.

[0045] Example 2: Preparation method of feline calicivirus-positive serum and verification of its effect

[0046] 1. Preparation of Feline Calicivirus Subunit Vaccine

[0047] 1) The concentrations of the expressed and purified feline calicivirus recombinant protein FCV-IVP1-IIVP1-1 or FCV-IVP1-IIVP1-2 were adjusted to 1000 μg / ml and 1600 μg / ml, respectively.

[0048] 2) Preparation of adjuvant:

[0049] Weigh palmitic acid: hornbene: Tween 80: Span 85 in a weight ratio of 15:15:7:63, heat and melt in a constant temperature magnetic stirring water bath (80°C), and stir evenly to obtain the oil phase. Then weigh chitosan according to the weight percentage of 0.1%, dissolve it in citric acid buffer (10mmol / L, pH 6.5), and heat it in a 80°C water bath to obtain the water phase. Under magnetic stirring, add the oil phase to the hot water phase (oil phase: water phase = 1:10). After stirring evenly, use a high shear instrument to shear at a speed of 12000r / min for 5 minutes to obtain a coarsely dispersed adjuvant. Then use an ultrasonic crusher to crush it at an amplitude of 300W for 20 minutes to obtain a primary emulsified adjuvant.

[0050] 3) Subunit vaccine preparation: FCV-IVP1-IIVP1-1 or FCV-IVP1-IIVP1-2 antigen liquid was mixed with colostrum adjuvant in a volume ratio of 1:1, sheared at a speed of 12000 r / min for 1 minute, and ice-water bathed for 10 minutes to solidify to obtain FCV-IVP1-IIVP1-1 or FCV-IVP1-IIVP1-2 feline calicivirus recombinant protein subunit vaccine. The content of FCV-IVP1-IIVP1-1 or FCV-IVP1-IIVP1-2 feline calicivirus recombinant protein antigen in the vaccine was 500 μg / ml and 800 μg / ml, respectively.

[0051] 2. Preparation of feline calicivirus-positive serum.

[0052] 1) Basic immunization: Each rabbit was subcutaneously injected with 2 ml of feline calicivirus recombinant protein FCV-IVP1-IIVP1-1 (800 μg / ml) or FCV-IVP1-IIVP1-2 (800 μg / ml). After 7 to 14 days, the same dose and method were used for re-immunization.

[0053] 2) Reinforced immunization: After the basic immunization, each rabbit was subcutaneously injected with 2 ml of feline calicivirus subunit vaccine FCV-IVP1-IIVP1-1 (500 μg / ml) or FCV-IVP1-IIVP1-2 (500 μg / ml) every 14 days for two immunizations.

[0054] 3. Collect serum.

[0055] 14 days after the last immunization, blood was collected from the immunized animals aseptically, and serum was separated to obtain positive serum prepared with FCV-IVP1-IIVP1-1 or FCV-IVP1-IIVP1-2 as antigen.

[0056] Serum testing: The positive serum samples prepared above were subjected to sterility testing, mycoplasma testing, exogenous virus testing, neutralization titer determination, and specificity testing. Specificity testing is mainly aimed at common cat pathogens such as feline panleukopenia virus and feline calicivirus. The results showed that the prepared positive sera were all light yellow clear liquids, without obvious hemolysis or foreign matter; sterile, mycoplasma-free, and free of exogenous virus contamination; no feline panleukopenia virus or feline calicivirus antibodies were detected, and the specificity was good. The qualified sera were mixed, packaged, labeled, and the serum name, specification, date, etc. were indicated, and stored below -20°C.

[0057] 4. According to the method specified in the current "Chinese Veterinary Pharmacopoeia", the neutralization test is carried out to determine the serum neutralization 200TCID 50Neutralization titer of feline calicivirus. The results showed that the neutralization antibody titer of the positive serum produced after immunization with FCV-IVP1-IIVP1-1 as the antigen and FCV (genogroup I (G(A)I)) and FCV (genogroup II (G(A)II)) could reach 1:4096; the neutralization antibody titer of the positive serum produced after immunization with FCV-IVP1-IIVP1-1 as the antigen and FCV (genogroup I (G(A)I)) and FCV (genogroup II (G(A)II)) were 1:512 and 1:1024, respectively.

[0058] Table 1 Neutralizing antibody titer of positive serum

[0059]

[0060] 5. Positive serum ELISA titer detection

[0061] The purified FCV CAP protein was diluted to 4 μg / ml with coating solution, added to the ELISA enzyme labeling plate at 100 μL / well, and coated at 4°C overnight; the liquid in the wells was discarded, washed 5 times with PBST, and blocked with 50 g / L skim milk at 37°C for 1 hour; the liquid in the wells was discarded, washed 5 times with PBST, and the positive serum was diluted twice with PBS, i.e. 1:200-1:102400, 100 μL / well, incubated at 37°C for 1 hour; the liquid in the wells was discarded, washed 5 times with PBST, and HRP goat anti-rabbit secondary antibody diluted 1:10000 was added, and incubated at 37°C for 1 hour; the liquid in the wells was discarded, washed 5 times with PBST, TMB was added for color development, 100 μL / well, and reacted at 37°C in the dark for 15 minutes, and 50 μL of stop solution was added to each well, and OD was measured immediately 450nm Reading of values; when positive serum OD 450nm ≥0.7 was considered positive, and negative serum OD 450nm When the value is ≤0.3, the test is considered to be successful, and if positive, the highest antibody dilution factor corresponding to the positive result is taken as the antibody titer.

[0062] The indirect ELISA method was used to determine the titer of the anti-FCV positive serum of immunized rabbits. The results showed that the ELISA titers of the two polyclonal antibodies were different. Among them, the serum antibody titer of FCV-IVP1-IIVP1-1 inactivated virus was higher, not less than 1:32000; the serum antibody titer of FCV-IVP1-IIVP1-2 was lower, at 1:16000 (Table 2).

[0063] Table 2 Positive serum ELISA test results (OD 450nm )

[0064]

[0065] 6. Identification of positive serum IFA reactivity

[0066] FCV(G(A)I) and FCV(G(A)II) were used to infect F81 cells, respectively. After 24 hours, the culture supernatant was discarded, 100 μl of V(methanol)∶V(acetone)=1∶1 fixative was added to each well, fixed at -20℃ for 20 min, and the fixative was discarded; 100 μl of 50 g / L skim milk was added to each well, and the cells were blocked at room temperature for 1 hour; the blocking solution was discarded, polyclonal antibody was diluted with PBS at 1∶200-16400, 100 μL was added to each well, incubated at 37℃ for 2 hours, and washed 3 times with PBST; protected from light, 100 μL of FITC goat anti-rabbit antibody (1∶500 diluted with 50 g / L PBS) was added to each well, incubated at 37℃ for 30 min, and washed 3 times with PBST; the experimental results were observed under a fluorescence microscope. The results showed that the rabbit anti-FCV positive serum was diluted in two-fold ratio and incubated with FCV-infected cell samples, and the IFA reactivity of each polyclonal antibody was identified. The results showed that the IFA reaction titer of FCV-IVP1-IIVP1-1 antibody could reach 1:1000, and an ideal fluorescent signal could be obtained when it was diluted 1:500; the reactivity of FCV-IVP1-IIVP1-2 antibody with FCV was still weak at a dilution of 1:200 ( Figure 2 ). The above results show that FCV-IVP1-IIVP1-1 antibody has good IFA reactivity, sensitive reaction, high titer and strong specificity. However, the preparation process of whole virus antigen is complicated, the antigen composition is not single, the positive serum is highly non-specific, and the cross-reactivity with strains of different gene groups is poor.

[0067] In this study, the polyclonal antibody prepared by using a recombinant protein of the advantageous regions of the VPI protein of FCV viruses from different evolutionary groups as an antigen has the advantages of being easy to prepare, simple in composition, and producing antibodies with high titer, good specificity, and good cross-reactivity with strains from different evolutionary groups.

[0068] In addition, compared with monoclonal antibodies, this method has a short cycle for preparing positive serum, low cost, high success rate, and low experimental difficulty.

[0069] Example 3: Application of positive serum in detection of feline calicivirus exogenous virus

[0070] The positive serum with a neutralization titer of 1:4096 prepared by FCV-IVP1-IIVP1-1 in Example 2 of the present invention was selected for the exogenous virus test of feline calicivirus (the virus content was 10 9.0 TCID 50 / ml, 10 9.5 TCID 50 / ml). According to the current method of the third appendix of the Chinese Pharmacopoeia, 1ml of diluted virus solution was taken to neutralize with positive serum, and then the exogenous virus test was performed. Results: All samples did not show incomplete neutralization, and the positive serum did not cause cytotoxicity.

Claims

1. Feline calicivirus recombinant protein, characterized in that It is a recombinant protein of the antigenic dominant regions of the VP1 gene of feline calicivirus of different gene groups in series, and its amino acid sequence is SEQ ID No.

1.

2. The feline calicivirus recombinant protein according to claim 1, characterized in that The sequence of the gene encoding the feline calicivirus recombinant protein is SEQ ID No.

2.

3. Application of the feline calicivirus recombinant protein according to claim 1 or 2 in the preparation of feline calicivirus high titer positive serum.

4. A method for preparing a high-titer positive serum for feline calicivirus, comprising using a feline calicivirus recombinant protein as an antigen to perform basic immunization on an immune animal; collecting blood from the immune animal and preparing serum to obtain a feline calicivirus-positive serum; the immune animal is a rabbit or a guinea pig; the feline calicivirus recombinant protein is the feline calicivirus recombinant protein according to claim 1 or 2.

5. The method according to claim 4, characterized in that: The basic immunization is performed twice, each ml of the vaccine contains 800 μg of antigen protein, and the immunization dose is 2 ml / time.

6. The method according to claim 4, characterized in that: After the basic immunization, two booster immunizations are included. The booster immunizations specifically include: using a feline calicivirus recombinant protein subunit vaccine, each milliliter of the vaccine containing 500 μg of antigen protein, to boost the immunized animals; each immunization dose is 2 ml / time; the interval between two immunizations is 7 to 14 days.

7. The method according to claim 6, characterized in that: The preparation method of the feline calicivirus subunit vaccine is as follows: the expressed and purified feline calicivirus recombinant protein according to claim 1 or 2 is mixed with an adjuvant.

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