| Hepatitis E (Hepatitis E, HE), caused by Hepatitis E virus (Hepatitis E Virus, HEV), is an acute viral hepatitis. The disease is transmitted primarily by the fecal-oral route though contaminated water and causes sporadic and epidemic forms of acute hepatitis. The mortality rate is generally low, ranging from 0.5% to 1%, but it can be as high as 15% to 25% among pregnant women.HEV is a non enveloped, single-stranded, positive sense RNA virus of 27-34nm in diameter. The genome HEV has approximately 7.5 kb in length, and contains three open reading frames (ORFs), named ORF1, ORF2 and ORF3, and short 5'and 3'untranslated regions. ORF2 encodes the putative capsid protein, which is rich in essential epitope(s). Studies reveal that anti-HEV antibodies are present in numerous animal species, including pigs, cows, sheep, chickens, boar, rats, deer and cats. Geographically Hepatitis E is epidemic in many developing countries of Asia, Africa, and North America, and sporadic cases of acute hepatitis have also been reported in some industrialized countries, including the United States, England, and France. Generally speaking, large-scale epidemics and sporadic cases of Hepatitis E occur in developing countries, and HEV infection in industrialized countries of nonendemicity is sporadic. In China HEV prevalence rate is 17.2%. Although the distribution is uneven in provinces and cities, HEV infection rate in rural areas is higher than the city. Based on phylogenetic analysis, HEV sequences worldwide are classified into four major genotypes, but according to the analysis of the gene sequence of HEV isolates at the size of nucleotide and amino acid homology and the phylogenetic trees, at present, in the world HEV is classified into eight genotypes.Nearly two decades, Reyes and other researchers obtained the gene cloning of human HEV by molecular cloning technologies, and formally the Hepatitis and its related virus were named Hepatitis E and Hepatitis E virus. For Hepatitis E and swine Hepatitis E, so far there is no specific treatment, no specific passive and active immunization for the prevention. The key of controlling hepatitis E is mainly to cut off the route of transmission in the comprehensive preventive measures. Related researches mainly focus on genotype analysis and diagnosis of Hepatitis E virus. Based on the HEV genome research, to date, for different genotypes, researchers have established many corresponding diagnostic methods to detect anti-HEV antibodies and HEV RNA. That is, using genetic engineering re-establishment of the antigen to establish antibody diagnostic methods, and using reverse transcription - polymerase chain reaction (RT - PCR) to establish the method of genetic diagnosis.In this study we separated and identified swine Hepatitis E virus by Nested PCR. According to registered AJ272108 sequence and other related sequences in GenBank, and referring the literatures of Meng and other researchers, two pairs of primers were designed by Oligo6 software, then using Nested PCR technology to amplify swine Hepatitis E virus which was isolated from the laboratory and analysising the sequence. The results showed that using Nested primers to expand the fragment, and sequence analysis showed that the gene sequence belonged to genotypesⅣ, and with genotypesⅣbetween 85.6﹪and 94.8﹪in nucleotide identity, between 99.1% and 100% in the amino acid identity. But keeping the minimum with genotypesⅢin nucleotide identity, and keeping the minimum with genotypesⅡin amino acid identity.So far, because HEV mature cell culture model has not yet been established, at present by genetic engineering technology, using the recombinant protein or synthetic peptide as the antigen to detect HEV specific antibody and as immunogen to prepare monoclonal antibody and immune serum. Through comparison of different detection methods for HEV antibody, Mast and other researchers found that the encoded proteins in HEV ORF2 region as known antigens to detect unknown antibodies have better sensitivity. In addition, many studies have confirmed that there are many epitopes but complex structure on ORF2-encoded capsid protein, and in hydrophobic domain of C-terminal 2/3 part there are multiple immunodominant B-cell epitope(s), including the antigen binding site to induce neutralizing antibody. In the present, we used the detected and isolated swine hepatitis E virus by Nested PCR in the first chapter to research the recombinant protein (p223) consisting of amino-acid residues 395 to 617 in the C terminus of ORF2 protein. Applying the specific primer to amplify the corresponding gene fragment, and in order to construct the recombinant plasmid, we inserted the gene into the expression vector pGEX-4T-1, named pGEX-HEV, then transformed into E.coli BL21 (DE3), and induced expression of fusion protein GST-ORF2 by 0.2mmol/L IPTG. By SDS-PAGE analysis it showed that the gene fragment with molecular weight of 50kDa obtained fusion expression, and Western blot analysis showed that the recombinant protein reacted with SHEV serum-positive and had good reactionogenicity. So we used the purified recombinant protein to establish indirect ELISA method, by the square test the results determined that the optimal concentration of coated antigen was 2.08μg/mL, optimal dilution of Goat Anti-mouse IgG was 1:3000, the best dilution ratio of positive serum was 1:80 and the best time was 10 min for reacting with the OPD. In addition, we selected 2% BSA as the best sealant and the sealed time was 120 min, the best time was 60 min for reacting with HRP labeled goat anti-mouse IgG. Goose paramyxovirus, Gosling plague virus and Swine vesicular disease virus as the antigen-coated had no cross-reaction with the positive serum of swine Hepatitis E virus, so it showed that the method had good specificity. Using the established methods to detect specific antibodies of swine Hepatitis E virus, so we laid the foundation for further more in-depth researching the epitope(s) of HEV, at the same time, we provided technical support for the seroepidemiological survey of swine hepatitis E virus.In this study we used real-time quantitative PCR method to detect swine hepatitis E virus at the level of cDNA. Using the identified sequence of swine HEV in the first chapter, and according to registered AJ272108 sequence and other related sequences in GenBank, a pair of primers and the probe were designed and synthesized for the conservative region of HEV ORF2 fragment, and a real-time fluorescence PCR assay was established by the TaqMan probe. The standard curve was established though using the positive plasmid as template, in which the linear range of Ct value was from 14.89 to 27.02, and correlation coefficient was 0.996. The real-time fluorescence quantitative PCR assay with the efficient amplification and the wide linear range laid the foundation for rapidly detecting the absolute quantitative analysis of swine Hepatitis E virus. In this study, by genetic engineering technology using the recombinant protein as the antigen to detect HEV-specific antibodies and applying TaqMan probe-based real-time fluorescence quantitative PCR assay to detect the absolute content of HEV in the environment and clinical samples. The two methods provid a convenient for laboratory diagnosis, at the same time provid technical support for swine Hepatitis E seroepidemiological survey and detection of the pathogens, and then lay the technical foundation for the research of human Hepatitis E. |
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