Genetic Evolution Analysis Of PEDV Epidemic Strains In Guizhou Province And Study On Tandem Expression Of Antigenic Epitope

Porcine epidemic diarrhea virus is one of the main pathogens that cause diarrhoeal diseases in pig herds at different growth stages.PEDV is a positive-sense,single-stranded RNA-encapsulated virus with a single stranded,the S gene is its main virulence gene,insertion,deletion,and mutation of the S gene may lead to changes in the virulence of PEDV,which is the main basis for PEDV typing.The ORF3 gene is associated with the virulence of PEDV and encodes 224 amino acids.ORF3 gene can be used to distinguish vaccine strains from wild virus strains,and is a valuable tool for epidemiological investigation of PEDV.In order to understand the prevalence of PEDV and the genetic variation of PEDV epidemic strains in Guizhou Province,and to explore the immune effect of the tandem expression protein of the main antigen epitope of the S gene.In this experiment,Specific primers and probes were designed for the conserved region of PEDV S gene,and a Taq Man RT-q PCR method was established after optimizing the conditions.Subsequently,this study used this method to retrospectively detect PEDV in diarrhea samples submitted for clinical examination by Guizhou pig farms from2017 to February 2023,and to clone and analyze the genetic evolution of S and ORF3 genes in some PEDV positive samples.At the same time,this study predicted and analyzed the main antigen epitopes of PEDV S gene,designed and constructed a tandem expression plasmid with the main antigen epitopes,and studied the immunogenicity of the purified recombinant protein.The specific method results are as follows:1、Establishment and Application of Fluorescence Quantitative RT-PCR Method for Porcine Epidemic Diarrhea VirusIn order to establish a more convenient,specific,and sensitive Taq Man RT-q PCR method and use this method to detect the prevalence of PEDV in Guizhou Province.In this study,primers and probes were designed for the conserved region of PEDV S gene,and standard plasmids were constructed through RT-PCR amplification and cloning of target fragments.After optimizing a series of conditions,a Taq Man RT-q PCR detection method was established.The results show that,the optimal reaction procedure for this method was reverse transcription at 50℃15 min;95℃30 s;95℃5 s,56℃30 s,A total of 40 cycles.This method has a good linear relationship within the range of 1×10⁷～1×10²copies/μL standard,with a correlation coefficient of 0.995,and high reliability.This method is highly specific and does not have cross reactions with viruses such as Po RV,TGEV,and PDCo V.The minimum detection limit of this method can reach 1.0 copies/μL,which is 100 times more sensitive than ordinary RT-PCR reaction detection methods.Within the range of 1×10⁵～1×10²copies/μL standard,the intra assay and inter assay repeatability coefficient of variation is less than 1.5%,with good repeatability and stability.Using this method,119 clinical samples from pig farms in different regions of Guizhou Province from 2017 to February 2023 were retrospectively tested for PEDV.The results showed that the positive rate of PEDV was 76.92%（92/119）,and the largest number of diarrhea samples were submitted for examination from January to April each year,with a positive rate of 84.85%（56/66）.The above results,in Guizhou Province,indicate that PED mainly occurs in spring.This study not only enriched the epidemiological data of PEDV in Guizhou Province,but also laid a material foundation for analyzing the genetic evolution of PEDV S and ORF3 genes in Guizhou Province.2、Cloning and Genetic Evolution Analysis of S and ORF3 Genes from 30Positive SamplesIn order to understand the genetic variation characteristics of PEDV epidemic strains in Guizhou Province,this study designed specific amplification primers for PEDV ORF3 and S genes,and selected 30 positive samples with high nucleic acid concentration for amplification,cloning,and sequencing of S and ORF3 genes.The obtained sequences were analyzed for genetic evolution,and the major antigenic epitopes of S protein B lymphocytes and T lymphocytes were predicted.The results show that:The S gene size of 30 samples ranged from 4146 to 4170 bp,encoding 1382 to1390 aa.The ORF3 gene size was 675 bp,encoding 224 aa.Compared with CV777,the S gene of 30 samples had 52 identical amino acid mutations,mainly in the S1-NTD region;There are 6 identical amino acid mutations in the ORF3 gene,which are relatively conservative.The amino acid sequence homology between the S gene of 30 samples and the S gene of CV777 was 93.2%to 95.0%,and the amino acid sequence homology between the ORF3 gene and the ORF3 gene of CV777was 94.2%to 96.4%.Based on the PEDV S gene,30 samples are prevalent in both GⅠand GⅡtypes,with the main epidemic type being GⅡ,of which 4 share belong to GⅠb subtype;8 share belong to the GⅡa subtype and are closely related to the vaccine strain XJ-DB2;18 share belong to the GⅡb subtype and are closely related to vaccine strains AJ1102 and AJ1102-R.Based on the ORF3 gene,all 30 samples belong to the GⅡsubtype,of which 15 belong to the GⅡb subtype,and are closely related to the vaccine strain XJ-DB2;Fifteen strains belong to the GⅡa subtype and are closely related to vaccine strains AJ1102 and AJ1102-R.Compared with CV777strain,based on PEDV S gene typing,N¹¹⁶⁷D,T¹¹⁹⁸D,G¹³⁶⁸C,and A¹³⁸⁵V can be used as molecular markers for the GⅡa subtype,T¹¹⁹⁸D,F¹²¹²Y,S¹²²⁰G,P¹²⁷⁰S,and Y¹¹⁹⁹deletions can be used as molecular markers for the GⅡb subtype,and K⁵⁸⁹N,I⁹⁶⁸L,Y⁷⁶⁶F,Y⁷⁷¹F,N¹³¹¹D,C¹³⁷⁰F,F¹³⁷¹S,A¹³⁸⁵D,and G⁷⁰deletions can be used as molecular markers for the GⅡc subtype;Based on ORF3 gene typing,L²⁵S and I⁷⁰V can be used as molecular markers for GⅡa subtype,and F⁸⁰V and H¹⁸²Q can be used as molecular markers for distinguishing GⅡb subtype.Compared with the S gene of CV777 strain,there were 3 deletions and 2insertions in the S protein glycosylation sites of 26 GⅡPEDV strains,with a significant difference between the two;Mutations in the four neutralizing antigen epitopes of the S gene of 30 samples were concentrated in the COE region,with7～11 amino acid mutations.It is predicted that there are mainly 9 B lymphocyte epitopes and 5 T lymphocyte epitopes in PEDV S protein.The above results indicate that the 30 samples cloned in this study are all mutant strains,compared with classical vaccine strains,there are significant differences in homology,antigenic epitopes,and N-linked glycosylation sites,with a low degree of matching.Therefore,the low immune effect of using classical PED vaccine in Guizhou Province may be related to the low matching degree of S protein between PEDV strains and classical vaccine strains in Guizhou Province.3、Study on tandem expression of antigenic epitopes of PEDV S proteinsIn order to explore the immune effects of proteins expressed in tandem with the major antigenic epitopes of PEDV S gene.In this study,Linker was used to connect the major antigen epitopes of PEDV S gene B and T lymphocytes,optimize the connection sequence,and send it to a biological company to synthesize and construct are combinant plasmid p ET32a（+）-BD1-S.After identification,the recombinant plasmid was transformed into BL21（DE3）receptor cells for induction,expression,and purification.The purified multi-epitope fusion expression protein was used to immunize female Balb/c mice aged 6 to 8 weeks,and its immune effects were studied.The results showed that the optimal induction time for p ET32a（+）-BD1-S recombinant plasmid was 3 hours,and the optimal IPTG induction concentration was 0.1 mmol/L;SDS-PAGE and Western blot analysis showed that the multi-epitope fusion protein was mainly expressed in the form of inclusion bodies,with a size of 51.8 KDa.Studies on the immune effects of recombinant proteins on mice have shown that the immune antibody titer of the multi-epitope protein group is 1:10⁵,it’s 10³times higher than the attenuated vaccine group.Compared with the control group,the levels of IL-6 and IL-10secretion in the multi-epitope protein group increased,but the difference in IL-6was not significant,and the difference in IL-10 was significant（P<0.05）;The levels of IL-6 and IL-10 secretion in the vaccine group were significantly higher than those in the control group;IL-12 and INF-γin multi-epitope proteomics and vaccine groups,the secretion level was significantly higher than that of the control group and the multi-epitope protein group INF-γSecretion levels were higher than in the vaccine group.The results showed that the multi-epitope fusion protein can stimulate the body of mice to produce better B and T cell immune responses.Compared with the vaccine group,the multi-epitope fusion protein has a poor effect on stimulating the body to produce B-cell immune responses,but can stimulate the body to produce better T-cell immune responses.Western blot identification and indirect immunofluorescence experiments showed that immune reactions can occur between multi-epitope fusion proteins and whole virus antibodies,as well as between multi-epitope fusion protein antibodies and PEDV pathogens.The above results indicate that the multi-epitope fusion protein prepared in this study can stimulate mice to produce a high degree of humoral and B,T cell immune responses,has immunogenicity.At the same time,multiple epitope fusion proteins and PEDV whole virus antibodies,as well as multiple epitope binding protein antibodies and PEDV pathogens can undergo immune reactions.In summary,the multi-epitope fusion protein prepared in this study has good antigenicity.The study of PEDV multi epitope fusion protein in this paper lays a foundation for the exploratory research of PEDV gene engineering epitope vaccine.