| Hepatitis E virus(HEV)is a single-stranded positive-stranded RNA virus that is divided into 8 genotypes,of which genotype 4 is mainly reported in China.HEV-infected swine generally have no obvious clinical symptoms,but genotypes 3and genotypes 4 can break through the species barrier,causing abortion and death in human,and have important public health significance in developing countries.The HEV genome contains ORF1,ORF2,and ORF3,and ORF2 encodes for capsid proteins and plays a key role in virion assembrage and viral adsorption of host cells,and it is the primary target antigen for neutralizing antibodies.The development of porcine virus-like particles(VLP)vaccine based on ORF2 is of great practical significance for the prevention and control of the harm of the disease to the pig industry and the spread between humans and swine.In this study,556 porcine serum samples and 846 porcine fecal samples were detected by using PCR,and the viral nucleic acid positive rates were 0.9% and 9.1%,respectively.Subsequently,the ORF2 function prediction and analysis of genotype 4swine Hepatitis E virus(sHEV)was carried out by using molecular biology information method to determine the protective antigen coding region,and the results showed that there were 2 stable proteins encoded by ORF2,namely,p496 protein containing 496 amino acids and p476 protein containing 476 amino acids,both of which belong to hydrophilic proteins without transmembrane region,and their secondary structure is mainly irregular curling.The p496 protein had 11 T-cell epitopes and 21 B-cell epitopes,and the p476 protein had 14 T-cell epitopes and 18B-cell epitopes.The dominant peptides 128-623 aa and 140-615 aa corresponding to p496 and p476,together with full-length capsid protein p674,were selected for eukaryotic expression,the difference between the three proteins to form VLP was compared.Specifically,through PCR amplification,digestion,ligation,transformation and other steps,the insect cell expression vectors p Fast Bac-HTB-sHEV-ORF2(674/496/476)corresponding to ORF2(674),ORF2(496)and ORF2(476)were constructed,the recombinant plasmids were transformed into DH10Bac-competent cells containing baculovirus shuttle vectors to obtain the recombinant shuttle plasmid Bacmid-sHEV-ORF2(674/496/476),followed by using Cellfectin Reagent II.mediated transfection of sf9 cells with these plasmids.Upon harvesting and purification of recombinant proteins,protein hybridization technology(Western blotting)was employed to detect the recombinant proteins.As a result,there existed protein bands that could be recognized by His-tagged monoclonal antibody and porcine hepatitis E virus antibody-positive sera,and transmission electron microscopy revealed the formation of VLP from p496 and p476.In order to construct an immunogenic sHEV vaccine candidate,the immunogenicity of the p674,p496 and p476 recombinant proteins was initially evaluated through mouse trials.The established indirect ELISA method based on the recombinant proteins was used to detect the serum specific antibody level of mice.Consequently,the lowest effective dose of p674 that ilicited the body’s immune response is 10 μg/pc,p496 and p476 2μg/pc,the positive rate of antibodies in p496 and p476 groups was more than 85% when the injection dose was 10 μg/pc,and all mice produce antibodies when the injection dose was 20 μg/pc,indicating that the recombinant proteins and the derived VLPs induced an obvious antibody response in the body.In summary,compared to p674,p496 and p476-derived VLPs of sHEV showed good immunogenicity,which laid a foundation for the further development of swine hepatitis E vaccine candidates. |
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