Expression And Phage Display Libraries Construction Of Swine Hepatitis E Virus Capsid Protein Gene

Swine hepatitis E is an important public health problem, it was first described in 1997 in USA by Meng. The consequence study found that not only in developing country but also in developed country ,there was more popular HEV antibody exist in developed country than it was expected. Swine HEV is a non-envelope , single strain and positive RNA virus . Because the swine HEV found in USA is very closed to HEV of human being , US-1 and US-2. So the question if the virus can infect human being came to be in close attention . As there was not a reliable method for swine HEV propagation, the diagnose had to rely on the recombined protein .but almost all the method was not very successful. In 1988-1989, there were 20,000 people that infected hepatitis E in south of Xinjiang. Although the hepatitis E were found in north of China and east of China, the condition of swine hepatitis E was not reported in the northeast China.In this study, with SOE-PCR method, we put five fragement of swine HEV DQ01 together to make a complete virus coat protein gene DQ01ORF2. After we analyzed the antigenic index and hydrophilicity of the coat protein gene with DNAStar protein software, we selected a fragment to do prokaryocyte expression in E.coli. With the complete coat protein gene we developed DNA vaccine to make HEV coat protein antibody which could be used for phage-display peptide libraries biospanning. By inoculated Bal B/C mouse, the serum anti-HEVORF2 protein was made. With two gene phage display system, we digested DQ01ORF2 into 80bp sized fragment to established a DQ01ORF2 gene fragment phage display libraries.The results showed that DQ01 is closed to changchun human being HEV strain. The nucleotide sequence identity between them is 87%, the deduced amino acid sequence identity is 97%. By inserting a fragment into pET32a, we got a soluble protein about 33kD fused with the 109aa Trx·Tag^TM thioredoxin protein, the protein was named DQ01Ag01. Analyzed with SDS-PAGE and western blot, we found that DQ01Ag01 could react to HEV positive serum, and the result was corresponded to the RT-PCR method. On the basis of this, we established ELISA that could be used to diagnose if swine serum was HEV positive. With the whole DQ01ORF2 inserted to plasmid pcDNA3.1, a DNA vaccine was made, named pDQ01ORF2. By transfected pDQ01ORF2 into Vero cells, we demonstrate the DNA vaccine could be expressed in Vero cell. Then the vaccine was inoculated on Bal B/C, and the antibody was collected and titered by ELISA based on DQ01Ag01, the titer of the serum was 1:600. With the established double gene phage display system and the mouse serum, two rounds of biospanning was done and the serum-affinity phage was saved. By indirect ELISA, we demonstrated the phage display libraries could be used for swine HEV DQ01 strain epitope characterization.