| Hepatitis E virus is a non-enveloped positive-sense RNA virus belonging to hepatitis E viridae that infects animals and human and causes Hepatitis E. HEV consists of 4 genotypes. Genotype 1 and 2 only infect human while genotype 3 and 4 infect both human and animals. Especially in pigs, HEV has a rather high positive rate, and pigs are important reservoir of HEV. Currently, a lot of swine HEV nucleotide partial sequences are available but there is little description about its complete genome. Complete genome sequence analysis is helpful for a better understanding on the evolutionary relationship between different strains of HEV. Based on this, in this study, we carried out the complete genome sequence analysis of one strain of HEV and prepared the monoclonal antibody against ORF2 coding protein of HEV. The content in detail are as folloing:By Rt-nPCR method, we detected a HEV strain from a pig farm in the suburban area of Wuhan, designated as WH09. Ten pairs of primers were designed according to reported HEV sequence to amplify the complete genome by fragments, and RACE was applied to amplify the two ends. The complete genome sequence of HEV WH09 was obtained and it was 7265bp in size, containing 3 open reading frames, which encode 1707, 674 and 114 amino acids, respectively. It was discovered by genome sequence alignment that the similarity between WH09 strain and genotype 4 was 83.0-86.2%, and that between WH09 and other three genotypes was 73.2-75.2%. The phylogenetic tree based on complete genome sequence showed that swine HEV WH09 strain belonged to genotype 4. It was within a relatively isolated cluster with the human-derived strain HE-JA2 from Japan, and more distant to other genotype 4 strains.2. The preparation of monoclonal antibody against ORF2 coding protein of HEVThe previous studies revealed that the neutralizing epitope of HEV was contained within the 452 - 617 amino acids of the protein encoded by ORF2, which was named sORF2. The protein fragment can induce the generation of specific neutralizing antibodies of HEV and can form virus-like particle. In this study, the primers with certain restriction sites were used to amplify sORF2 gene, and the PCR product was cloned into pMD18-T. After verification by digestion and sequencing, the expression vector pET28a-sORF2 was constructed and the protein was expressed in high efficiency in E.coli. The recombinant soluble protein had a molecular weight of 25kD and existed in a soluble form. The immune activity of expressed protein was validated by Western blot.4-week BALB/c mice were immunized with purifed pET28a-sORF2 protein at a dosage of 100μg/mice. A strengthening immunization was given after 3 times of immunization by conventional methods. Three days later, spleen cells were fused with SP2/0 cells. Then positive hybridoma were screened through indirect ELISA by using sORF2 protein as antigen at the 7th day after fusion. After 3 to 5 times of cloning of the screened positive hybridoma, nine strains that stably secreted monoclonal antibody against HEV sORF2 protein were obtained. |
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