Studies On Epidemic Investigation Of Hepatitis E And Eukaryotic Expression Of Virus ORF2 Gene And Immunology Effects

Hepatitis E virus (HEV), which is responsible for epidemic and sporadic hepatitis E in a variety of animals including huaman, has a genome of single-stranded, polyadenylated RNA of positive polarity which encodes three open reading frames (ORFs). IgG anti-HEV were firstly confirmed by dot-blot hybridization (Dot-bot) method in pet dogs in this study. Moreover, eukaryotic expression vector concluding capsid gene of swine HEV were successfully constructed, and then expressed in COS-7 cell. Immune reaction induced by these proteins were then systemic studied in BaLB/c mice.Objective: Eukaryotic expression vectors containing the HEV ORF2 gene named PCDQL and PCDPL were constructed and expressed in COS-7 cells. In order to produce immune defense ability in BalB/c mice, those vectors were intramuscular injected comply with saturation sucrose solution.Methods: The dog HEV antibodies were detected by double antigen sandwich ELISA (DS-ELISA) method , and the existence of false positive was further detected by Dot-bot method. The full length and the C terminal sequence of ORF2 gene were amplified by RT-PCR, and the PCR products were then subcloned into pcDNA3.1(+) vector. The recombinant plasmids named PCDQL and PCDPL were transfected into COS-7 cells.The expression of these plasmids were determed used immuocytochemistry and Western-Blot methods. IgG of Anti-HEV wsa detected by ELISA method 2 weeks later of primary immunization.Results: The results showed that total positive rate of dog was about 12.0%, the maximum OD value of that was 2.5, and the minimum value was 0.01, the critical value was 0.13 and the HEV is epidemic in degree in the Jiangsu-Zhejiang region, and may play an important role in the spread of HEV; The results showed the PCDQL in the construction of eukaryotic expression plasmid had well immune response induced by the body, the immune after 4 weeks about 90% of the mice produced anti-HEV antibodies, recombinant plasmid PCDPL also can produced immune response induced by the body, the immune after 4 weeks about 25% of mice produced anti-HEV antibodies, the experimental group was significantly higher than the control group (P <0.05). Conclusions:(1)The results of dot-blot hybridization technology validated HEV in blood serum of pet dogs, and provided the theory reference.(2) The ORF2 gene and the C terminal sequence in swine HE were clone successfully.(3) Recombinant expression vectors of PCDQL and PCDPL were successfully constructed and expressed in eukaryotic cells and can induced the anti-HEV antibodies.