Cloning Of Hantavirus S Segment And Expression In Pichia Pastoris System

Hemorrhagic fever with renal syndrome(HFRS) is a dangerous emerging viral disease, whose pathogen is Hantanvirus. And the Hantaan virus(HTNV) and the Seoul virus(SEOV) are two main pathogen types in our country, whose representative strains are Z10 strain and L99 strain respectively. The main structural protein, nucleoprotein (NP) encoded by S gene, is the main part of nucleocapsid. The antigenicity and immunogenicity of NP is strong and highly conservative. The antibody of patients special for NP is produced early and the antibody titer is high. Therefore NP could be regarded as an ideal antigen for diagnosis. The expression system of Pichia pastoris is a new one for heterologous gene, which is as controllable as prokaryotic cells and is capable of many of the posttranslational modifications performed by higher eukaryotic cells. Target genes inserted into chromosome were more stable and the conditions for fermentations were simple, as was fitted to high-density expression. It was high expression efficiency and easy to purify, providing great convinences for industrialization.The total RNA were prepared from Vero-E6 cells infected by Z10 strain of Hataan virus and L99 strain of Seoul virus. The RT-PCR products, the whole S gene, was cloned into pGEM-T-Easy vector. After identified by enzymes cutting and sequence analysis, the insertion of target gene was verified. Then the heterologous gene in the recombinant of pGEM-T-Easy-Z10 and pGEM-T-Easy-L99 was inserted into secretary expressive vector pPICZa of Pichia pastoris. After identified by enzymes cutting and sequence analysis, open reading frame (ORF) of the recombinant of pPICZa-Z10-S and pPICZa-L99-S could be proved to be correct. And the configution of the targetprotein was not changed by the analysis of 3D-PASSM server software. After linearized by Sac I enzyme, the recombinant plasmid was inserted into Pichia pastoris to integrate. It was confirmed that S gene segment were inserted into Pichia pastoris chromosome by PCR, named Z10-S/X-33 strain and L99-S/X-33 strain respectively. After induction by 0.5% methanol, the result of SDS-PAGE showed that the target protein encoded by S gene was in the supernatant. The molecular weight of target protein was the same as the predicted. Moreover, the result of Dot blot reveals that the expressive protein can be recognized by HFRS patient's serum. The cloning and expression of Hantanvirus S gene laid primary basis on further reaserches.