Cloning And Expression Of Hepatitis E Virus ORF2 Gene

Hepatitis E is recognized as an acute infectious disease spread by the faecal-oral route, and frequently provokes epidemic outbreaks in many developing countries in Asia, Africa, Central America and so on. The genome of virus is around 7.5 kb of positive (that is, mIRNA) sense, single-stranded PNT, with short 5'- noncoding region, 3'-noncoding region, and coding region containing three separate open reading frames (ORFs). ORF2 is located at the 3'end, extends l98Obp, and encodes for structural proteins. In our study, the fragment of 492bp cDNA of hepatitis E virus (HEy) amplified from Xinjiang epidemic strain (CH1.1) was inserted into an expression vector pGEX-4T-3 and transformed into Escherichia coli TO-i. The recombinant fusion protein with glutathione S-transferase (GST) was used as an immunodiagnostic target in an enzyme imtnunoassay (EIA). 1. Amplification of REV gene designated By reverse transcription-nested polymerase chain reaction (RT-nPCR) method, the nucleotide of HEV ORF2 was amplified. The product extending 492bp was named REV ORF2 cDNA. 2. Construction of the recombinant containing the target gene and the vector pGEX-4T-3 for the expression of fusion protein REV QRF2 cDNA was digested with BamH I and EcoR I, and ligated with vector pGEX-4T-3 which was also digested with the same enzymes. The recombinant 4 was transformed into Eseherichia coil TG-1. The piasmid DNA was extracted by the alkaline lysis procedure. Then the recombinant was identified with enzyme digestion and PCR. The fragment was analyzed on 1% agarose gel electrophoresis and visualized by ultraviolet instrument. An about 500bp strip was demonstrated, and confinned by sequencing. The recombinant pGEX-T-HEV has been successfully constructed, and HEV nucleotide of which was 92.9% homologous with that of the Burma strain of HEV (ET1.l). 3. Expression of pGEX-T-HEV in TGT Induced with IPTG, the recombinant was expressed in TG-1. An about 46KD additional protein band was demonstrated by SDS-AGE. The result of western blot analysis show that the fusion protein reacted with anti-HEV positive sera 1:100 diluted. 4. The preliminary application of the recombinant protein in ETA The recombinant fusion protein was used as an immunodiagnostic target in ETA. 97 sera isolated from the patients with hepatitis E were tested for anti-HEV. The result showed that the consistent rate with Genelabs ant i-HEV ETA kit was 96. 9%. Among sera of 6 hepatitis A, 11 hepatitis B, 12 hepatitis C, 8 hepatitis D and 10 hepatitis G, one serum of hepatitis D and one serum of hepatitis G were positive for anti-HEV, whereas others were negative. Conclusions 1. The cloning recombinant of HEV ORF2 cDNA was successfully constructed. 2. An about 46KD fusion protein has been expressed by the recombinant of pGEX-T-HEV in TG-1. 3. The recombinant fusion protein was preliminary applied in anti-HEV ETA, and the result suggested that the consistent rate was high when compared with Genelabs anti-HEV ETA Kit.