Detection Of The Swine HEV In Jiangsu Province And The Fusion Protein Of ORF2Immune Protective Experiments Of SD Rats

Hepaitits E (HE) is a zoonotic disease, it is caused by hepatitis E virus.HE occurred on breakout epidemic stage in Asia and Africa with poor sanitation condition,whereas on sporadic stage in developed countries,China is on of the HE-epidemic country of the world. Various animals are susceptible to HE.Swine is one of the most important intermediate hosts and play an important role in interspecies transmission.So the study of swine hepatitis E virus is of great significance for the prevention and treatment of HE.This study is used RT-nPCR detection of HEV RN A of pig feces and bile samples of a local pig farm in central region of Jiangsu was conducted. The positive PCR products were cloned and sequenced. Through investigate the HEV infection in swine of central region of Jiangsu, the popular features of swine hepatitis E virus was expbred.Ruselts showed that totally4of90pigs’ bile samples were positive for HEV RNA, positive rate is4.44%. In the140pigs’feces samples, only the35feces samples of three or four mouths pigs wrer positive for HEV RNA. The other feces samples of pigs were ne gative for HEV RNA. The sequence analysis showed that the identity at nucleotide level was88.0%-100.0%among them. They shared78.0-86.0%,81.3%～82.7%,78.7～84.0%and86.0%～98.7%nucleotide sequence identity with HEV genotype Ⅰ、Ⅱ、ⅢandⅣ, respectively, in the region (nt6317-6466). So we concluded that the hepatitis E virus in swine was widespread and it belonged to genotype Ⅳ.After that the test used the Protean software to analyze the potential antigentic sites of ORF2of Swine Hepatitis E Virus, and selected four pieces of antigen concentration area(398-460aa,290-405aa,398-619aa and377-604aa)was named BE,BH,BW and BZ and amplified the target gene by RT-PCR.Then we transferred the target gene into pGEX-6p-1expression vectors and induced its expression in E. coli BL21. Results showed that only two sets of recombinant plasmids pGEX-BH and pGEX-BW successfully expressed the39ku and48ku fusion protein, respectively. While the other two groups of recombinant plasmids pGEX-BE and pGEX-BZ cannot express the protein. The Western blotting analysis showed that the48ku recombinant protein has a good antigenicity with positive SHEV serum after purified the recombinant protein. It proved that the natural immune epitope is located in between398to619aa.This result laid a foundation of screening of antigenic epitope of structural protein of HEV and study on sero logical diagnosis of swine HEV.According to the related literature reports SD rats can be used as animal model of hepatitis e virus infection. Three SD rats were vaccinated with three doses (200μg/doses) purified GST-BW protein in Freud’s adjuvant under a schedule of0d,14d and28d.Specific antibodies can be detected on third week in two SD rats, the other one SD rat can be detected on fourth week. Three times after immunization with HEV feces suspension in the poison attack experiment in SD rats. Three SD rats in control group presented typical acute hepatitis E manifestation:increased seral amino transferase (ALT) and continuous virus excretion in stool in two weeks. In contrast, the ALT of SD rats in vaccinated group continued to be normal, stool virus had not been detected in two SD rats, and presented only a short duration in another one. One vaccinated rat serum with antibody titer125600was first incubated with HEV for neutralization, then the mixture were used to challenge two monkeys, the results showed that two rats in control group continued to excrete virus for more than two weeks and presented obvious ALT increase. Both two rats challenged with antibody neutralized virus had not detected virus in stool and ALT continued to be normal. These results suggested that the prokaryotic expression recombinant protein GST-BW have good immunogenicity and immunoprotectivity, and it provides the related fundamental research to prevent the spread of hepatitis virus, and should be a candidate for an effective epitope-based vaccines.