Predominant B cell antigen epitope of PEDV S protein RBD, chimeric gene, recombinant strain and preparation method and application thereof

Abstract

The present invention discloses a dominant B cell antigen epitope of the receptor binding domain (RBD) of the PEDV spike (S) protein, a chimeric gene thereof, a recombinant strain and a preparation method thereof, and an application thereof in a multi-linked and multivalent vaccine of ETEC and PEDV. The F4-F18-PEDV-S recombinant vaccine strain prepared by the present invention can simultaneously express the F4 and F18 pili of the porcine ETEC, as well as the dominant neutralizing B cell antigen epitope of the RBD of the PEDV S protein. The pili of the extracted recombinant vaccine strain are emulsified with Freund's adjuvant to immunize mice, and high levels of anti-FaeG, anti-FedF and anti-PEDV S protein antibodies can be simultaneously induced in the mice, and the serum of the immune mice can inhibit the adhesion of the F4+ ETEC and F18+ ETEC standard strains to the small intestinal cells of piglets in vitro; the neutralization titer to the PEDV2b strain (HSW) reaches 1:80. The recombinant vaccine strain is expected to be developed into a multi-linked and multivalent vaccine for preventing diarrhea in piglets caused by mixed infection of ETEC and PEDV, and has great application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a dominant B cell antigen epitope of a PEDV S protein RBD, a chimeric gene thereof, a recombinant strain, and a preparation method and application thereof. Background Art

[0002] my country is the world's largest pig farming and pork consumption country. The pig farming industry plays a pivotal role in my country's animal husbandry, agriculture and national economy. The survival rate of piglets is one of the key factors affecting the economic benefits of pig farming. Piglet diarrhea includes neonatal piglet diarrhea and weaned piglet diarrhea. Neonatal piglet diarrhea mainly occurs within 1 week of birth; weaned piglet diarrhea usually occurs within 3-12 days after weaning. Due to the high infection rate and mortality rate of piglet diarrhea in piglets, it has caused huge and continuous economic losses to the global pig industry.

[0003] There are many factors that cause diarrhea in piglets, among which pathogenic microorganisms are the main cause. The pathogens that cause diarrhea in piglets mainly include bacteria, viruses and parasites. Among these pathogens, PEDV is the most important viral diarrhea pathogen, and its proportion in viral diarrhea can be as high as 50%. Porcine epidemic diarrhea (PED) is a highly contact enteric infectious disease caused by PEDV, and the clinical symptoms are mainly diarrhea, vomiting and dehydration. PED is most fatal to newborn piglets, with an morbidity rate of up to 100% and a mortality rate of more than 80%. PEDV is an enveloped, linear, non-segmented single-stranded positive-strand RNA virus. Its full genome is 28kb long, with a cap structure at the 5' end and a Poly (A) tail at the 3' end. From 5' to 3', it is 5'UTR-PP1a / 1b-S-ORF3-EMN-3'UTR. The S protein of the virus shell is the most basic and important functional protein of PEDV. It has a high degree of genetic diversity and plays a key role in mediating the virus' entry into host cells, inducing neutralizing antibodies and viral virulence. The S protein can be divided into the S1 region that binds to the receptor and the S2 region that promotes membrane fusion. The RBD domain located in the S1 region is an important target for the development of drugs, antibodies, and vaccines.

[0004] ETEC is the main bacterial pathogen that causes diarrhea in piglets, among which pili and enterotoxin are important virulence factors in the pathogenesis of ETEC. ETEC from pigs can express F4, F5, F6, F18 and F41 pili, among which F4 and F18 pili are the most common. Fimbriae promote ETEC adhesion and colonization to the intestinal mucosa of piglets, and then secrete enterotoxins to cause water and electrolyte metabolism disorders in piglets, and finally lead to diarrhea.

[0005] Vaccines are currently the most effective measure to prevent diarrhea in piglets caused by PEDV and ETEC infections. However, due to the continuous emergence of PEDV variants, the expression of multiple virulence factors by ETEC and the heterogeneity of virulence factors, and mixed bacterial and viral infections, current vaccines cannot provide good protection. Therefore, it is urgent to apply new strategies and technologies to develop more efficient and broad-spectrum multi-valent new vaccines to meet these challenges.

[0006] Multiepitope fusion antigen (MEFA) technology is a new structural vaccine construction technology and platform based on the combination of computer biology and structural biology. This technology integrates neutralizing B cell antigen epitopes from different target antigens into one antigen using computer biology and structural biology techniques, and simulates the immunogenicity and natural spatial structural conformation of the antigen epitope itself to the maximum extent, aiming to construct a new multi-linked and multivalent vaccine with broad protective power. When selecting a skeleton protein, an antigen that is non-toxic, highly immunogenic, has multiple separated continuous antigen epitopes, and has a stable secondary structure is usually used as the skeleton protein for MEFA construction. The B cell antigen epitopes of other pre-constructed MEFA heterogeneous virulence factors are predicted using relevant software, and then the dominant neutralizing B cell antigen epitopes are selected to replace the antigen epitopes with weak immunogenicity and exposed on the surface of the skeleton protein, respectively, to ensure that the constructed multi-epitope fusion antigen protein containing multiple pathogens or multiple virulence factors has a similar and stable structure to the structural protein, and the inserted epitopes are still located on the surface of the MEFA protein, which can induce the production of specific antibodies against each epitope. The emergence of MEFA technology provides an innovative technology platform for constructing new multi-linked and multivalent epitope structure vaccines. Summary of the invention

[0007] Purpose of the invention: The technical problem to be solved by the present invention is to provide a dominant B cell antigen epitope from the RBD region of the S protein of PEDV.

[0008] The technical problem that the present invention also solves is to provide a gene encoding the dominant B cell antigen epitope of the PEDV S protein RBD. The technical problem that the present invention also solves is to provide a F18-PEDV-S chimeric pili or a pili protein expressed by a F4-F18-PEDV-S recombinant strain and a gene encoding the F18-PEDV-S chimeric pili.

[0009] The technical problem that the present invention also solves is to provide an expression cassette, a recombinant vector or a recombinant strain containing the gene or the chimeric gene.

[0010] The technical problem that the present invention also aims to solve is to provide a method for preparing F18-PEDV-S chimeric pili or pili protein expressed by F4-F18-PEDV-S recombinant strains and a method for constructing the recombinant vector or recombinant strain.

[0011] The technical problem that the present invention also aims to solve is to provide the B cell antigen epitope, the gene of the B cell antigen epitope, the F18-PEDV-S chimeric pili or the pili protein expressed by the F4-F18-PEDV-S recombinant strain, the chimeric gene, the expression cassette, the recombinant vector or the recombinant strain for use in preparing antibodies against F4 and / or F18 pili and PEDV or in preparing multi-valent vaccines or antibodies.

[0012] Based on technical platforms such as MEFA technology, efficient expression technology of bacterial fimbriae adhesins in vitro, and surface presentation of target antigen B cell antigen epitopes, a new type of highly efficient, broad-spectrum, multi-linked and multivalent vaccine with extensive protection against diarrhea in piglets caused by ETEC and PEDV is developed. One vaccine can provide multiple protections, effectively improve the survival rate and feed utilization rate of piglets, and thus safeguard the healthy growth of piglets. The main structural subunit FedA of the F18 fimbriae is inserted into the RBD region of the PEDV S protein described in the present invention, and the F18 fimbriae are used as a carrier to realize its functional presentation on the surface of the non-pathogenic Escherichia coli PEC2021-8 host bacteria that express F4 fimbriae.

[0013] Technical solution: In order to solve the above technical problems, the present invention provides a dominant B cell antigen epitope of PEDV S protein RBD, the amino acid sequence of the antigen epitope is shown in SEQ ID NO.1, specifically GYVSKSQDSNC.

[0014] The present invention also provides a gene encoding the dominant B cell antigen epitope of the PEDV S protein RBD, the nucleotide sequence of which is shown in SEQ ID NO.2, specifically GGTTACGTGTCTAAATCACAGGACAGTAATTGC.

[0015] The present invention also provides F18-PEDV-S chimeric pili or pilus protein expressed by F4-F18-PEDV-S recombinant strain, wherein the amino acid sequence of the F18-PEDV-S chimeric pili is shown in SEQ ID NO.3, and the pilus protein expressed by the F4-F18-PEDV-S recombinant strain contains a mixture of F18-PEDV-S chimeric pili and F4 pili.

[0016] The present invention also provides a chimeric gene encoding the F18-PEDV-S chimeric pili, and the nucleotide sequence of the chimeric gene is shown in SEQ ID NO.4.

[0017] The present invention also provides an expression cassette, a recombinant vector or a recombinant strain, which contains the gene or the chimeric gene.

[0018] Among them, the recombinant vector is obtained by introducing the chimeric gene encoding the F18-PEDV-S chimeric pili into the pBR322 plasmid; the recombinant strain is obtained by introducing the recombinant vector into a host bacterium that does not express F4 pili or a non-pathogenic host bacterium PEC2021-8 that expresses F4 pili. Preferably, the host bacterium includes Escherichia coli.

[0019] Specifically, the chimeric pilus expressing the dominant B cell antigen epitope of the PEDV S protein RBD of the present invention uses the FedA protein, the main structural subunit of the F18+ETEC pilus adhesin, as the skeleton protein, and replaces the weak B cell immunogenic antigen epitope of the F18 pilus adhesin with the dominant B cell antigen epitope of the PEDV S protein RBD to construct the FedA-PEDV-S chimeric protein and perform structural prediction, wherein the FedA amino acid sequence is shown in SEQ ID NO.5, and the FedA gene sequence is shown in SEQ ID NO.6. The amino acid sequence of the FedA-PEDV-S chimeric protein is shown in SEQ ID NO.7, and the FedA-PEDV-S chimeric gene sequence is shown in SEQ ID NO.8.

[0020] Among them, the construction method of the recombinant vector or the recombinant strain, the construction method of the recombinant vector comprises the following steps: introducing the gene or the chimeric gene into an expression vector, preferably, the gene includes a chimeric gene encoding the F18-PEDV-S chimeric pili, and the vector includes the pBR322 plasmid; the construction method of the recombinant strain comprises the following steps: introducing the recombinant vector into a host bacteria that does not express F4 pili or a non-pathogenic host strain PEC2021-8 that expresses F4 pili, preferably, the host bacteria is Escherichia coli.

[0021] Among them, the preparation method of the F18-PEDV-S chimeric pili or the pilus protein expressed by the F4-F18-PEDV-S recombinant strain comprises the following steps: introducing the chimeric gene encoding the F18-PEDV-S chimera into a plasmid to obtain a recombinant vector, and then introducing the recombinant vector into a host bacterium that does not express F4 pili or a non-pathogenic host bacterium PEC2021-8 that expresses F4 pili to obtain an F18-PEDV-S recombinant strain or a F4-F18-PEDV-S recombinant strain, inoculating the cultured seed liquid into LB liquid culture medium, adding Amp+, culturing and centrifuging to collect the bacteria, extracting and purifying to obtain the F18-PEDV-S chimeric pili or the pilus protein expressed by the F4-F18-PEDV-S recombinant strain.

[0022] The present invention also provides the use of the B cell antigen epitope, the gene of the B cell antigen epitope, the F18-PEDV-S chimeric pili or the pili protein expressed by the F4-F18-PEDV-S recombinant strain, the chimeric gene, the expression cassette, the recombinant vector or the recombinant strain in preparing antibodies against F4 and / or F18 pili and PEDV or preparing a multi-valent vaccine.

[0023] The present invention also provides a multi-link multivalent vaccine of bacterial pathogen ETEC and viral pathogen PEDV for preventing piglet diarrhea, wherein the vaccine comprises the recombinant strain.

[0024] The multi-link multivalent vaccine of the present invention is used to prevent the bacterial pathogen ETEC and the viral pathogen PEDV of piglet diarrhea.

[0025] The present invention also provides a neutralizing antibody against F4, F18 pili and PEDV. The neutralizing antibody is obtained by immunizing an animal with pili proteins expressed by the F4-F18-PEDV-S recombinant strain.

[0026] Specifically, the present invention also includes the use of a recombinant plasmid or a recombinant strain in the preparation of a vaccine, wherein the recombinant expression plasmid is constructed by inserting the pili chimeric gene F18-PEDV-S into the expression vector pBR322. The recombinant strain includes the recombinant vector introduced into the non-pathogenic Escherichia coli PEC2021-8 strain.

[0027] The present invention also includes a multi-link and multi-valent piglet diarrhea vaccine, which contains the vaccine that can effectively prevent piglet diarrhea caused by F4+ETEC, F18+ETEC and PEDV virus infection.

[0028] The pilus protein of the F4-F18-PEDV-S recombinant strain extracted by the present invention can induce high levels of IgG antibodies against FaeG (the main structural subunit of F4 pilus), FedF (the adhesion structural subunit of F18 pilus) and PEDV-S1 (S protein S1 domain) in BALB / c mice, and can simultaneously induce Th1 and Th2 cellular immune responses. The pilus protein extracted from the recombinant strain can induce neutralizing antibodies against PEDV, and the neutralization titer is as high as 1:80.

[0029] Given that the F4-F18-PEDV-S recombinant strain has a good vaccine immune effect against ETEC and PEDV infections in piglets, it can be used as a candidate vaccine strain for preventing and controlling piglet diarrhea caused by ETEC and PEDV.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the F4-F18-PEDV-S recombinant vaccine strain constructed by the present invention can simultaneously express the F4 and F18 pili that are dominantly expressed by porcine ETEC, as well as the dominant neutralizing B cell antigen epitopes in the RBD region of the S protein of PEDV, and can induce high levels of humoral immunity and cellular immunity against these three antigens after immunization. The present invention innovatively develops and prepares a multi-link multivalent vaccine that simultaneously targets the bacterial pathogen ETEC and the viral pathogen PEDV that cause diarrhea in piglets, achieving multiple protections with one vaccine, and fundamentally solving and overcoming the problem of piglet diarrhea that has been plaguing the pig farming industry. This vaccine is the first multi-link multivalent vaccine that simultaneously prevents bacterial and viral infections in pigs, and it provides an important strategy and technical platform for the development of new multi-link multivalent vaccines for mixed bacterial and viral infections in livestock and poultry. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the construction of F18-PEDV-S chimeric pili, where E represents the dominant neutralizing B cell antigen epitope in the RBD region of the PEDV S protein.

[0032] Figure 2 This is the electrophoresis diagram of PCR amplification and identification of F18-PEDV-S chimeric gene. Lane M is 2KPlusⅡDNAMarker; Lane 1 is fedA-PEDV-S chimeric gene; Lane 2 is F18-2 gene fragment; Lane 3 is F18-3 gene fragment; Lane 4 is the complete F18-PEDV-S chimeric gene.

[0033] Figure 3 Identification of the F4-F18-PEDV-S recombinant strain. 1: The reaction result of the PEC2021-8 / pBR322 recombinant strain culture with PEDV positive serum, negative control; 2: The agglutination result of the BL21 / pET28α-PEDV-S-recombinant strain culture with PEDV positive serum, negative; 3: The agglutination result of the PEC2021-8 / F4-F18-PEDV-S recombinant strain culture with PEDV positive serum, positive.

[0034] Figure 4 SDS-PAGE and Western blot images of the extracted pilus proteins of the F4-F18-PEDV-S recombinant strain.

[0035] Figure 5 ELISA was used to detect the levels of anti-FaeG, anti-FedF and anti-PEDV-S1 IgG antibodies in the serum of mice immunized with the pilus proteins extracted from the F4-F18-PEDV-S recombinant strain.

[0036] Figure 6ELISA was used to detect the expression levels of inflammatory cytokines IL-4, IL-6, IFN-γ and TNF-α in the spleen cells of immunized mice.

[0037] Figure 7 The inhibitory effect of immune mouse serum on the adhesion of F4+ETEC and F18+ETEC strains to piglet small intestinal epithelial cells IPEC-J2.

[0038] Figure 8 It is the neutralizing antibody titer of immune mouse serum against PEDV type 2b (HSW) virus. DETAILED DESCRIPTION

[0039] The embodiments of the present invention will be clearly and completely described below in conjunction with examples. It should be pointed out that the examples described below are only used to illustrate some examples of the present invention and should not be regarded as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the reagents or instruments used are not specified by the manufacturer, they are regarded as conventional products that can be purchased commercially. In addition to the specific methods, equipment, and materials used in the examples, according to the technical personnel in the art's grasp of the prior art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the examples of the present invention can also be used to realize the invention.

[0040] Example 1 Construction and identification of F4-F18-PEDV-S recombinant vaccine strain

[0041] 1. Construction of pBR322-F18-PEDV-S recombinant expression plasmid

[0042] (1) Construction and structure prediction of FedA-PEDV-S multi-epitope fusion protein

[0043] The B cell antigen epitope prediction website (http: / / tools.immuneepitope.org / bcell / ) was used to predict the B cell antigen epitopes of FedA, the main structural subunit of the dominant F18 fimbriae adhesin expressed by porcine ETEC, and the RBD region of the S protein of PEDV. Figure 1The diagram shows the construction of the FedA-PEDV-S fusion protein in the embodiment of the present invention. FedA (amino acid sequence as shown in SEQ ID NO.5, gene sequence as shown in SEQ ID NO.6), the main structural subunit of F18 pili, is used as the skeleton protein, and the dominant B cell antigen epitope in the RBD region of the S protein of PEDV (amino acid sequence as shown in SEQ ID NO.1, gene sequence as shown in SEQ ID NO.2) replaces the B cell antigen epitope with weak immunogenicity in FedA and exposed on the protein surface, constructs the FedA-PEDV-S multi-epitope fusion protein (amino acid sequence as shown in SEQ ID NO.7, gene sequence as shown in SEQ ID NO.8), and then uses the Phyre2 portal website for modeling, predicting and analyzing the biological structure, and scoring according to parameters such as credibility, linear coverage and homology percentage (>90%) in the established protein model library, and the highest score is used as the final FedA-PEDV-S protein model. The results showed that the insertion of exogenous B cell antigen epitopes would not change the native conformation of the framework protein FedA, and the inserted epitopes were located on the surface of the FedA-PEDV-S multi-epitope fusion protein.

[0044] (2) PCR amplification of F18-PEDV-S chimeric gene

[0045] The FedA-PEDV-S chimeric gene (see sequence listing SEQ ID NO.8) was synthesized by Nanjing Qingke Biotechnology Co., Ltd., and the FedA-PEDV-S chimeric gene and F18+ETEC (O139:H1) genomic DNA (GeneBank accession number CP080237.1) were used as templates, and primers P1 (SEQ ID NO.9) and P4 (SEQ ID NO.12) were used for PCR amplification of the FedA-PEDV-S chimeric gene, respectively; the F18+ETEC (O139:H1) genomic DNA was used as a template, and primers P3 (SEQ ID NO.11) and primers P6 (SEQ ID NO.14) were used to PCR amplify the F18-2 gene fragment; the F18+ETEC (O139:H1) genomic DNA was used as a template, and primers P2 (SEQ ID NO.10) and primers P5 (SEQ ID NO.13) were used to amplify the F18-3 gene fragment. The PCR product was recovered and purified using a DNA gel recovery and purification kit (Beijing Tiangen Biochemical Technology Co., Ltd., catalog number: W0220). The recovered and purified PCR products of the FedA-PEDV-S chimeric gene, F18-2 and F18-3 genes were mixed again as amplification templates, and the F18-PEDV-S chimeric gene (SEQ ID NO.4) was amplified using SOE-PCR with primers P1 (SEQ ID NO.9) and P2 (SEQ ID NO.10). The reaction amplification system is shown in Table 1, and the PCR reaction conditions are: 95°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 5 min, 30 cycles, 72°C×10 min, and termination at 4°C. The P1 (SEQ ID NO.9) and P2 (SEQ ID NO.10) primers carry XbaI-HF and SalI-HF restriction sites, respectively. The results are shown in Table 1. Figure 2 As shown, the sizes of the fedA-PEDV-S chimeric gene, F18-2 gene, F18-3 gene and F18-PEDV-S chimeric gene were approximately 500 bp, 2000 bp, 3000 bp and 5500 bp, respectively, which were consistent with the expected results.

[0046] Table 1 PCR amplification system

[0047]

[0048]

[0049] (3) Construction of pBR322-F18-PEDV-S recombinant plasmid

[0050] The F18-PEDV-S chimeric gene PCR product was double-digested with XbaI-HF (NEB, Catalog No.: R3138) and Sal I-HF (NEB, Catalog No.: R3138) endonucleases in a 37°C water bath for 2 h;

[0051] The pBR322 vector was double-digested with NheI-HF and Sal I-HF endonucleases in a 37°C water bath for 3 hours. The double-digestion reaction system is shown in Table 2. After the digestion product was purified and recovered, it was connected overnight at 16°C using T4 DNA ligase. The ligation product was transformed into DH5α competent cells. Then it was added to the LB liquid culture medium and shaken for 1 hour. An appropriate amount of the culture product was coated on the Amp+ LB solid culture medium and cultured overnight in a 37°C bacterial constant temperature incubator for resistance screening. The next day, several single colonies were picked and the positive clones after PCR identification were sent to the company for sequencing. Finally, the clone construction was confirmed to be correct by DNA sequencing, and the recombinant plasmid was named pBR322-F18-PEDV-S.

[0052] Table 2 The restriction enzyme digestion system

[0053]

[0054] 2. Construction and identification of the F4-F18-PEDV-S recombinant vaccine strain

[0055] (1) Construction of F4-F18-PEDV-S recombinant strain

[0056] The pBR322-F18-PEDV-S recombinant expression plasmid identified by DNA sequencing was electrotransformed into non-pathogenic Escherichia coli (Escherichia coli PEC2021-8) and deposited in the China Center for Type Culture Collection on January 26, 2024, with a deposit number of CCTCC NO: M2024224, and the deposit address is Wuhan, China. Then it was spread on LB solid medium with ampicillin (Amp+) and cultured overnight in a 37°C bacterial constant temperature incubator. The next day, several single colonies were picked and correctly identified by PCR using faeG-F (SEQ ID NO.15) faeG-R (SEQ ID NO.16) primers, and fedF-F (SEQ ID NO.17) fedF-R (SEQ IDNO.18) primers, and the F4-F18-PEDV-S recombinant strain was obtained. Transfer a single positive clone to 6 mL of LB medium containing Amp+ and culture overnight in a shaker at 37°C. The next day, inoculate 5 mL of overnight cultured seed solution into 500 mL of fresh autoclaved LB medium at a ratio of 1:100, and add 500 μL (30 μg / mL) of Amp+. When the bacterial solution grows to the stationary phase, centrifuge at 4500 prm for 5 min, discard the supernatant, resuspend in 0.01 M PBS (PH=7.4), and wash twice with the same parameters to prepare the bacterial suspension required for the agglutination test. The agglutination reaction results are as follows: Figure 3 As shown, PEC2021 / pBR322 recombinant strain

[0057] (pBR322 plasmid was transferred into PEC2021-8 host bacteria) and BL21 / pET28α-PEDV-S1-recombinant strain (pET28α-PEDV-S1 plasmid was transferred into BL21 host bacteria. Primers S1-F (SEQ ID NO.19) and S1-R (SEQ ID NO.20) were used to amplify the gene encoding the S1 subunit of the PEDV S protein, and the two restriction sites of BamHI and SalI were used to connect to the expression vector pET28α(+) to construct the pET28α-PEDV-S1 recombinant expression plasmid.) could not agglutinate with PEDV positive serum, while the F4-F18-PEDV-S recombinant strain could significantly agglutinate with PEDV positive serum, indicating that the F4-F18-PEDV-S recombinant strain could functionally express the F4 and F18 pili of porcine ETEC at the same time, and display the dominant B cell antigen epitopes of the PEDV S protein.

[0058] (2) Extraction of fimbriae proteins from the F4-F18-PEDV-S recombinant strain

[0059] The seed solution of the overnight culture of the F4-F18-PEDV-S recombinant strain was inoculated into 500mL LB liquid medium sterilized by autoclave at a ratio of 1:100, and 500μL Amp+ was added. After shaking and culturing at 37℃ for about 16h, the bacteria were collected by centrifugation at 4℃, 12000rpm×10min. An appropriate amount of 0.01M PBS (PH=7.4) was added to the collected bacteria, mixed evenly, and frozen at -70℃. The next day, the pilus protein was extracted and purified by heat extraction, and dialyzed and protein quantified. The protein quantification results showed that the concentration of pilus protein was 1.36mg / mL.

[0060] (3) Western blot identification of the extracted pilus proteins of the F4-F18-PEDV-S recombinant strain

[0061] After 12% SDS-PAGE electrophoresis of the extracted pilus protein samples of the recombinant strain, the target protein was transferred to the PVDF membrane using a semi-dry transfer apparatus at 25V, 1.0A, 30min. The transferred PVDF membrane was placed in 10% skim milk prepared in PBST (PBS + 0.5‰ Tween 20), and the membrane was slowly shaken on a shaker at room temperature for 3h and then transferred to 4℃ for overnight blocking. Anti-FaeG polyclonal antibody and anti-FedF polyclonal antibody (Duan et al., 2020, A multivalent vaccine candidate targeting enterotoxigenic Escherichia coli fimbriae for broadlyprotecting against porcine post-weaning diarrhea) and anti-PEDV S1 polyclonal antibody (Aimejie Technology, Catalog No.: ASM-PVV31302) were used as primary antibodies, diluted with 5% skim milk powder (1:8000), incubated slowly on a shaker at room temperature for 1.5 hours, and washed 4 times with PBST. The secondary antibody goat anti-mouse IgG-HRP (ABclonal, Catalog No.: AS014) was diluted with PBST (1:15000) and placed on a shaker for incubation with slow shaking for 1 hour. PBST was washed 4 times, 5 minutes / time, and the PVDF membrane was developed using the ECL luminescence method. The results are as follows Figure 4 As shown, the pili of the recombinant strain can be recognized by anti-FaeG polyclonal antibodies, anti-FedF polyclonal antibodies and anti-PEDV S1 polyclonal antibodies at the same time, which once again proves that the F4-F18-PEDV-S recombinant strain can functionally express the F4 and F18 pili of porcine ETEC at the same time, and present the dominant B cell antigen epitopes expressing the PEDV S protein. Example 2 Study on the immunogenicity of the F4-F18-PEDV-S recombinant strain

[0062] (1) Mouse immunization

[0063] Twelve 6-8 week old SPF BALB / c mice were randomly divided into two groups, 6 mice in each group. In the first group, the extracted pilus protein (1.36 mg / mL) of the F4-F18-PEDV-S recombinant strain was fully emulsified with complete Freund's adjuvant at a ratio of 1:1 (volume ratio) and then immunized subcutaneously (antigen 50 μg / mouse). After the first immunization, booster immunization was performed every two weeks for a total of three immunizations, and incomplete Freund's adjuvant was used for the subsequent second and third immunizations. The second group of mice injected with only 0.01M PBS (PH=7.4) (100 μL / mouse) served as the control group. 14 days after the last immunization, the mice were painlessly killed by cervical vertebrae, blood was collected from the eyeballs to collect serum, and spleen cells were collected by sterile dissection. The separated serum was used for subsequent detection of IgG antibody levels of the corresponding antigens in mouse serum, and spleen cells were used to detect the levels of related inflammatory cytokines induced and secreted by mice.

[0064] (2) ELISA to detect antibody levels in immunized mice

[0065] The ELISA method was used to detect the IgG antibody titers against FaeG, FedF and PEDV S1 proteins in the serum of mice immunized in step (1). The FaeG recombinant protein and FedF recombinant protein expressed and purified by the prokaryotic expression system of this laboratory (Duan et al., 2020, A multivalent vaccine candidate targeting enterotoxigenic Escherichia coli fimbriae for broadly protecting against porcine post-weaningdiarrhea) and PEDV-S1 recombinant protein (obtained by expression and purification using BL21 / pET28α-PEDV-S1 recombinant strain) were used as coating antigens (500 ng / well) to coat a 96-well ELISA plate, incubated in a 37°C incubator for 1 hour, and then transferred to 4°C overnight. The next day, the ELISA plate was taken out to room temperature, and after rewarming, the ELISA plate was washed 3 times with PBST buffer and patted dry. Add 200 μL of 10% skim milk diluted with PBST buffer to each well, block in a 37°C incubator for 1 hour, wash 3 times with PBST buffer and pat dry, dilute mouse serum with PBST at a ratio of 1:200, 1:400, and 1:800 to 1:12800, add 100 μL / well to the ELISA plate, and incubate in a 37°C incubator for 1.5 hours. Wash again with PBST 3 times and pat dry, dilute goat anti-mouse IgG-HRP (ABclonal, catalog number: AS014) secondary antibody with PBST at 1:5000, add 100 μL / well to the ELISA plate, and incubate in a 37°C incubator for 1 hour. Wash 3 times with PBST buffer and pat dry to remove residual liquid, add 200 μL of TMB substrate colorimetric solution to each well, incubate at room temperature in the dark for 30 minutes, and read the OD of each well on the ELISA reader. 650nm The result is Figure 5 The serum of mice immunized with the pilus protein of the F4-F18-PEDV-S recombinant strain showed high titers of anti-FaeG, anti-FedF and anti-PEDV S1 IgG antibodies, while these three antibodies were not detected in the PBS control group, indicating that the pilus protein of the F4-F18-PEDV-S recombinant strain has good immunogenicity.

[0066] (3) Expression levels of related inflammatory cytokines in mouse spleen cells

[0067] The mice in step (1) were painlessly killed by cervical vertebrae and then immersed in 75% alcohol for disinfection for 5 minutes. They were quickly dissected aseptically in a clean bench and chopped with tissue scissors. The filtered spleen cells were fully ground and filtered in a 200-mesh copper mesh using a rubber piston of a syringe. The filtered spleen cells were centrifuged at 1000 rpm for 10 minutes, the supernatant was discarded, and a sufficient amount of 1× red blood cell lysis buffer was added to the cell pellet to lyse the red blood cells at room temperature for 10 minutes. The cells were centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded to keep the cell pellet. The cells were washed three times with 0.01 M PBS (PH = 7.4), and then fully resuspended in RPMI 1640 medium containing 1% penicillin-streptomycin + 0.1% FBS, and the concentration of the cell suspension was adjusted to 2.5×10 5 cells / mL. The cells were 5×10 5 The cells were inoculated at a density of 10 cells / well in a 6-well cell culture plate and cultured in a constant temperature incubator at 37°C and 5% CO2. 5 μg / mL of the pilus protein of the recombinant strain F4-F18-PEDV-S extracted in Example 1 was added to each well to stimulate spleen cells. After 72 hours, the spleen cell supernatant was collected and the concentrations of IL-4 (Xinbosheng Company, Catalog No.: EMC003), IL-6 (Xinbosheng Company, Catalog No.: EMC004), IFN-γ (Xinbosheng Company, Catalog No.: EMC101g) and TNF-α (Xinbosheng Company, Catalog No.: EMC102a) in the spleen cell supernatant were detected according to the instructions of the ELISA kit. The results are as follows. Figure 6 The F4-F18-PEDV-S chimeric fimbriae protein immunization group induced significantly higher Th1 (IFN-γ and TNF-α) immune responses and Th2 (IL-4 and IL-6) concentrations in the spleen cells of immunized mice than those in the PBS control group, indicating that the F4-F18-PEDV-S chimeric fimbriae can simultaneously induce Th1 and Th2 cell immune responses.

[0068] (4) Bacterial adhesion inhibition test

[0069] Piglet epithelial cells IPEC-J2 were cultured in DMEM medium with 10% fetal bovine serum (FBS) and subcultured for 2-3 generations. Then the cells were cultured at 2.0×10 5 The cells were inoculated into a 24-well cell culture plate at a concentration of 100 cells / well and were used when the cells grew to a monolayer.

[0070] The culture was continued until the logarithmic growth phase (OD 600nm =0.4-0.6) of F4ac+ETEC and F18ac+ETEC standard strains (purchased from China Veterinary Culture Collection Center) were diluted with sterile 0.01M PBS (PH=7.4) and the bacterial concentration was adjusted to 1.0×10 7 CFU / mL. Take 150μL (1.5×10 6CFU / mL) bacterial solution was mixed with 30 μL of serum collected from immunized mice in step (1), and supplemented with DMEM medium to a total volume of 600 μL. The bacteria and serum were then gently mixed and evenly placed on a horizontal shaker for incubation at room temperature for 30 minutes. The incubated bacterial solution and serum mixture was added to IPEC-J2 cells and placed in a 37°C constant temperature incubator for incubation for 1 hour. Then, the bacteria that were not attached to the cells were gently washed away with sterile 0.01M PBS (PH=7.4), 200 μL of trypsin was added to each well to fully digest the cells from the cell plate, and then 0.01M PBS (PH=7.4) was added to dilute the cells in a volume ratio of 1:10, and the cells were spread on an antibiotic-free LB solid culture medium. After inverted overnight in a 37°C constant temperature incubator, single colonies were counted. The results are as follows Figure 7 As shown, the mouse serum in the PBS group had no inhibitory effect on the adhesion of F4+ETEC and F18+ETEC standard strains, while the mouse serum in the group immunized with the chimeric fimbriae protein extracted from the F4-F18-PEDV-S recombinant strain was able to significantly inhibit the adhesion of F4+ETEC and F18+ETEC standard strains to piglet epithelial cells IPEC-J2 in vitro, indicating that the fimbriae immunity of the F4-F18-PEDV-S recombinant strain can simultaneously induce neutralizing antibodies against F4 and F18 fimbriae.

[0071] (5) Virus neutralization test

[0072] First, the Vero cell suspension (ATCC) was cultured at 1×10 5 The cells were inoculated into 96-well cell culture plates and placed in a 37°C constant temperature incubator until the cell viability reached 80%. The serum samples of the immunized mice in step (1) were inactivated in a 56°C water bath for 30 min, and the serum antibodies were diluted with cell maintenance medium at a volume ratio of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128 and 1:256. 50 μL of the diluted serum sample was mixed with 100 TCID 50 / mL PEDV 2b (HSW) strain (Yangzhou Youbang Biological Pharmaceutical Co., Ltd.) was incubated in a 37°C CO2 constant temperature incubator for 1 hour. The Vero cell culture supernatant in the 96-well cell plate was discarded, and after washing twice with cell maintenance solution, the incubated serum and virus mixture was added to the Vero cells in the 96-well plate, 100 μL / well, and 4 wells were inoculated for each dilution. At the same time, 4 wells of normal cell control and 4 wells of 100 TCID 50 / mL PEDV 2b (HSW) virus was used as a control and placed in a 37℃ CO2 constant temperature incubator for adsorption for 1 hour. After 1 hour, the supernatant was discarded and 100μL serum-free DMEM medium (containing 8μg / mL trypsin) was added to continue culturing, and the cell lesions were observed in the next three days. Figure 8As shown, compared with the control group, the neutralizing antibody titer of PEDV in the mouse serum of the group immunized with the pili of the extracted F4-F18-PEDV-S recombinant strain reached 1:80, indicating that the F4-F18-PEDV-S recombinant strain has good immunogenicity and can be used as a candidate vaccine strain for PEDV.

Claims

1. The dominant B cell antigen epitope peptide of the S protein RBD of PEDV, characterized in that: The amino acid sequence of the antigen epitope peptide is shown in SEQ ID NO.

1.

2. A gene encoding the dominant B cell antigen epitope peptide of the PEDV S protein RBD according to claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. F18-PEDV-S chimeric pili, characterized in that The amino acid sequence of the F18-PEDV-S chimeric pilus is shown in SEQ ID NO.

3.

4. A chimeric gene encoding the F18-PEDV-S chimeric pilus according to claim 3, characterized in that: The nucleotide sequence of the chimeric gene is shown in SEQ ID NO.

4.

5. The pilus protein expressed by the F4-F18-PEDV-S recombinant strain is characterized in that: The pilus protein expressed by the F4-F18-PEDV-S recombinant strain contains a mixture of the F18-PEDV-S chimeric pilus and the F4 pilus according to claim 3.

6. An expression cassette, characterized in that It contains the gene according to claim 2 or the chimeric gene according to claim 4.

7. A recombinant vector, characterized in that: It contains the gene according to claim 2 or the chimeric gene according to claim 4.

8. A recombinant strain, characterized in that It contains the gene according to claim 2 or the chimeric gene according to claim 4.

9. The recombinant vector according to claim 7, characterized in that The recombinant vector is obtained by introducing the chimeric gene of the F18-PEDV-S chimeric pili described in claim 4 into the pBR322 plasmid.

10. The recombinant strain according to claim 8, characterized in that The recombinant strain is obtained by introducing the recombinant vector according to claim 7 into a host bacterium that does not express F4 pili or a non-pathogenic host bacterium that expresses F4 pili.

11. The recombinant strain according to claim 10, characterized in that The host bacteria include Escherichia coli.

12. The method for constructing a recombinant vector according to claim 7, characterized in that: The method for constructing the recombinant vector comprises the following steps: introducing the gene described in claim 2 or the chimeric gene described in claim 4 into an expression vector.

13. The method for constructing the recombinant vector according to claim 12, characterized in that: The expression vector includes the pBR322 plasmid.

14. The method for constructing a recombinant strain according to claim 8 or 10, characterized in that: The method for constructing the recombinant strain comprises the following steps: introducing the recombinant vector described in claim 7 into a host strain that does not express F4 pili or a non-pathogenic host strain that expresses F4 pili.

15. The method for constructing a recombinant strain according to claim 14, characterized in that: The host bacteria is Escherichia coli.

16. The method for preparing the F18-PEDV-S chimeric pili according to claim 3, wherein: The method comprises the following steps: introducing the chimeric gene of the F18-PEDV-S chimeric pili according to claim 4 into a plasmid to obtain a recombinant vector, then introducing the recombinant vector into a host bacterium that does not express F4 pili to obtain an F18-PEDV-S recombinant strain, inoculating the cultured seed liquid into an LB liquid culture medium, adding ampicillin, culturing and centrifuging to collect the bacterial cells, extracting and purifying to obtain the F18-PEDV-S chimeric pili.

17. The method for preparing the pilus protein expressed by the F4-F18-PEDV-S recombinant strain according to claim 5, wherein: The method comprises the following steps: introducing the chimeric gene of the F18-PEDV-S chimeric pili according to claim 4 into a plasmid to obtain a recombinant vector, and then introducing the recombinant vector into a non-pathogenic host bacterium expressing F4 pili to obtain an F4-F18-PEDV-S recombinant strain, inoculating the cultured seed liquid into an LB liquid culture medium, adding ampicillin, culturing and centrifuging to collect the bacterial cells, and extracting and purifying to obtain the pili protein expressed by the F4-F18-PEDV-S recombinant strain.

18. Use of the B cell antigen epitope peptide according to claim 1 in preparing antibodies against F4 and / or F18 pili or in preparing a combined multivalent vaccine against ETEC and PEDV.

19. Use of the gene of the B cell antigen epitope peptide according to claim 2 in preparing antibodies against F4 and / or F18 pili or in preparing a combined multivalent vaccine against ETEC and PEDV.

20. Use of the F18-PEDV-S chimeric pili according to claim 3 in preparing antibodies against F18 pili or preparing a combined multivalent vaccine against ETEC and PEDV.

21. Use of the chimeric gene according to claim 4 in preparing antibodies against F18 pili or preparing multivalent vaccines against ETEC and PEDV.

22. Use of the pilus protein expressed by the F4-F18-PEDV-S recombinant strain according to claim 5 in preparing antibodies against F4 and / or F18 pili or in preparing a combined multivalent vaccine against ETEC and PEDV.

23. Use of the expression cassette of claim 6 in preparing antibodies against F4 and / or F18 pili or in preparing a combined multivalent vaccine against ETEC and PEDV.

24. Use of the recombinant vector of claim 7 in preparing antibodies against F4 and / or F18 pili or in preparing a combined multivalent vaccine against ETEC and PEDV.

25. Use of the recombinant strain according to claim 8 in preparing antibodies against F4 and / or F18 pili or in preparing a combined multivalent vaccine against ETEC and PEDV.

26. A multivalent vaccine against ETEC and PEDV, characterized in that: The vaccine comprises the recombinant strain according to claim 8.

Images