Expression Research Of Avain Infectious Bronchitis Virus S1 Gene In Pichia Pastoris

A pair of primers were designed according to isolated IBV's S1 gene sequences which were accepted by Genbank and the sequence of Pichia pastoris expression vector pPIC9K's Multiple Cloning Site(MCS). Then the S1 gene was amplified by RT-PCR, The amplified products were cloned and sequenced. The results showed that the nucleotide sequence length of S1 gene was 1566bp and the deduced amino acid sequence length is 521aa,and the sequence included stop codon in 3' terminal but didn't include signal peptide in 5' terminal. The S1 fragment was digested by SnaB Ⅰ and Not Ⅰ, then ligated with the yeast secreted expressing vector pPIC9K which was also digested by SnaB Ⅰ and Not Ⅰ ,the results of PCR and enzyme digestion of plasmid proved that recombination vector was obtained (named pPIC9K-S1).The expressing plasmid pPIC9K-S1 was linearized with Bgl Ⅱ, then it was transformed into Pichia pastoris GS115 by electroporation. After the electroporatic transformants were selected by PCR and Methanol-utilization-plus,the positive recombination yeast was obtained (named GS115/ pPIC9K-S1 His~+Mut~s). GS115/ pPIC9K-S1 His~+Mut~s was induced with 1% methanol at 28℃ for target protein expression. IBV S1 protein was analysised by SDS-PAGE and Western blotting assay.The results showed that IBV S1 gene was expressed successfully in Pichia pastoris and the product was 76KD which could act with the positive serum of IBV. We optimized the inoculated dosage,the concentration of methanol and the induced time which could affect the expressing level of IBV S1 protein.The results showed that the target protein was up to the highest yield after 3 days induced by 1% methanol when inoculated dosage was 2%.The amount of target protein shares 12.5% of the total protein which consist in the supernatant fluid of yeast culture medium. The expressed S1 protein may act as diagnostic antigen of IB and genetic vaccine basic meterial.