Cloning Of Hantavirus M1 Segment And Expression In Pichia Pastoris System

Objective: Hantanvirus (HV) is the pathogen of Hemorrhagic Fever with Renal Syndrome (HFRS), which can lead to two dangerous viral diseases: HFRS and Hantavirus Pulmonary Syndrome (HPS). The Glycoprotein (GP) of HV can stimulate host body producing neutralization antibody, GP include G1 and G2. In this study, Gl protein encoded by M1 gene of HV was cloned and expressed in Pichia pastoris , and the expressed product was examined by the mixed serum of the HFRS patients using Western Blot and Dot Blot.Material and Methods: Vero-E6 cell line was used to proliferate HV Z10strain (Hataan virus) and L99 strain (Seoul virus). When the viral virulence tested byIFA reached +++~++++, the total RNA was extracted . The M1 gene was transcribedand amplified by RT-PCR method, the product of which was cloned into pMD18-Tsimple vector and identified by sequencing analysis. The recombinant plasmid wascut by the enzyme, Xba I and Kpn I, and was directionally cloned into the secretaryexpression vector pPICZaA that was cut by the same enzyme. The target geneinserted into the recombinant vector was verified by sequencing analysis. As waslinearized by BstX I enzyme, the recombinant vector was transformed into P.pastoris for integration. The positive recombinant was selected by Zeocin andproliferated for 4 times. The chromosome DNA of the recombinant P. pastoris wasprepared as template and the integrated target gene was examined by PCR. Themethanol was used to induce the recombinant P. pastoris to express the targetprotein, which was identified by Western Blot and Dot Blot.Results: After the RT-PCR products of the M1 gene was cloned into pMD18-T simple vector, the recombinant plasmid pMD18T-Z10M1 and pMD18T-L99M1were sequenced. Compared the sequence with the sequences of Genbank , Z10M1 had 9 nucleotide mutations and L99M1 had 5 nucleotide mutations. The target genes from pMD18T-Z10Ml and pMD18T-L99Ml was cut by the Xba I and the Kpn I and was correctly directional cloned into the secretary expressive vector pPICZaA, named pPICZaA-ZlOMl and pPICZaA-L99Ml respectively. The insertion of target gene was verified by sequence analysis to confirm the open reading frame (ORF) of the recombinant of pPICZaA-ZlOMl and pPICZaA-L99Ml to be correct. The pPICZaA-ZlOMl and pPICZaA-L99Ml was linearized by BstX I and transformed into P. pastoris for integration. The positive recombinant was selected by Zeocin and the chromosome DNA of which was prepared to detect the target gene by PCR. The positive recombinant was named PpX33-Z10Ml and PpX33-L99Ml respectively. The result of Western Blot showed that there were some non-differential bands using the HFRS patient's serum as first antibody. The result of Dot Blot showed that the target protein was expressed in the PpX33-Z10Ml and PpX33-L99Ml strains respectively, and it was still at low level.Conclusions: Using multi-copies target gene for integration will get high level expression of the target protein. This is the genomic HV Ml antigen other than natural HV Ml antigen that will play important role in HFRS diagnosis.