WSSV VAP1 Gene's Optimization, Cloning And Preliminary Study Of The Cultivation Technique Of Simplified Extension For Expression By Constitutive Promoter

White spot syndrome virus (WSSV) is one of the major pathogen of global shrimp farming. Since 1992, a lot of studies have been performed about WSSV and WSS. The whole genome sequence of WSSV provides the basis of the research on the functional gene of the virus and the mechanism of the virus infection. It has been defined that VAP1 is a major envelop protein of WSSV and play an important role in virus entry. This protein is made up of 281 amino acids and no glycation.For obtaining successful gene expression and even with high yields, the gene of VAP1 is reconstructed. It incudes: selecting the special bias of P. pastoris, changing the proportion of A+T and G+C, amending the restriction site of enzymes, analysis of star activity, analysis of intron of P. pastoris.After the reconstruction of the gene of VAP1, the gene was artificially synthesized according to the new sequence. The artificial gene was subcloned into constitutive expression vector pGAPZaA and the recombined plasmid was transformed into DH5a. After the identification of combined plasmids by PCR, EcoR I/Xba I digestion and DNA sequence, the recombined plasmid was transformed into chromosome of Pichia pastoris X-33 strain by electroporation. The X-33-pGAPZαA was identified by PCR.The key point of last part is controlled by the experiment of the cultivation technique of simplified extension in fermentation laboratory...