禽传染性支气管炎病毒S1基因毕赤酵母表达载体的构建

Construction of the Expression Vector of Pichia Pastoris Yeast for S1 Gene of Avian Infectious Bronchitis Virus

本试验根据已公布的IBV株S1基因序列及pPIC9K表达载体序列,去掉由18个氨基酸构成的信号肽后,设计一对IBV S1基因表达片段的PCR引物,利用RT-PCR扩增得到了IBV四川分离株的S1基因片段,将该片段克隆到PMD18-T载体上,通过对所得到的重组质粒进行酶切分析、菌落PCR鉴定,证明得到了含有目的基因片段的阳性重组质粒,测序分析片段长为1566bp,已经成功切除了由18个氨基酸构成的信号肽序列。将该基因亚克隆到毕赤酵母分泌型表达载体pPIC9K的SnaBⅠ和NotⅠ酶切位点,并通过菌落PCR、双酶切鉴定了该重组质粒的正确性,IBV S1基因毕赤酵母表达载体的构建为进一步利用毕赤酵母表达IBVS1蛋白提供了基础材料,并对表达产物的免疫原性和禽传染性支气管炎基因亚单位疫苗及特异性诊断抗原的研究打下了基础。

According to the S1 gene sequence of infectious bronchitis virus （IBV）in GenBank,a pair of specific sequences as primers were designed to amplify the S1 gene of IBV isolated from Sichuan province in China. By reverse transcription polymerase chain reaction （RT-PCR）,S1 gene of IBV was amplified successfully, The PCR products were cloned into pMD18-T vector and sequenced, Sequence analysis indicated that the length of the S1 gene was 1 566 bp, and its signal peptide of 18 amino acids were removed. The S1 gene of IBV was subcloned into the pichia pastoris expression vector pPIC9K. We obtained the recomhinant plasmid successfully by analysis of PCR and Restriction,and the obtained recombinant plasmid was designated as pPIC9K-S1. The construction of pPIC9K-S1 vector was beneficial to study imrnunogenicity of S1 protein expressed in Pichia pastoris ,subunit vaccine and specifical diagnostic antigen of IBV.