丹毒丝菌SpaA基因免疫保护区的克隆及其在毕赤酵母中的表达

Cloning surface protective antigen A gene from Erysipelothrix rhusiopathiae and its expression in Pichia pastoris

【目的】以毕赤酵母Pichia pastoris X-33为宿主表达猪丹毒丝菌Erysipelothrix rhusiopathiae Spa A基因氨基端的免疫保护区蛋白.【方法】以采集于广东某猪场的猪丹毒丝菌为模板,根据NCBI中已发表的Spa基因c DNA序列设计1对引物,通过PCR扩增得到Spa A-N,并将其连接到表达载体p PICZαC上,得到重组表达质粒p PICZαC-Spa A-N;用Sac I酶将重组表达质粒p PICZαC-Spa A-N线性化后电转化入毕赤酵母X-33,经含博来霉素ZencinTM抗性的YPDS平板筛选和PCR鉴定的阳性转化子,用含不同浓度博来霉素抗性的YPDS平板筛选出高拷贝子并进行甲醇诱导培养,分别于诱导48、72、96 h后离心收集上清液,立即做SDS-PAGE,并进行SDS-PAGE及Western-blot试验.【结果和结论】成功克隆并表达了Spa A-N基因,构建了重组表达质粒p PICZαC-Spa A-N,并以毕赤酵母X-33为宿主成功表达了猪丹毒丝菌Spa A基因氨基端的免疫保护区蛋白.丹毒丝菌Spa A-N作为免疫保护区在酵母宿主中得以成功表达.

【Objective】Immunization protection area protein,which lies in the N-terminal protective domain of surface protective antigen A( Spa A) of Erysipelothrix rhusiopathiae,was expressed in Pichia pastoris X-33.【Method】Using the swine erysipelas,which was isolated from a pig farm in Guangdong as template,a pair of primers was designed according to the c DNA sequence of Spa gene from NCBI. The amino terminal sequence was achieved by PCR amplification,after being inserted into the expression vector p PICZαC to get the recombinant plasmid of p PICZαC-Spa A-N. Recombinant plasmid of p PICZαCSpa A-N by Sac I enzyme was linearized,and electro transformed it into P. pastoris X-33. The positive transformant,screened by YPDS tablet with ZencinTMand identified by PCR at different concentrations of ZencinTM,achieved the high number of copies which were induced and cultured by methanol. Supernatant was collected by centrifugation at 48,72,96 h respectively after induction,and SDS-PAGE and Western-blot tests were carried out.【Result and conclusion】The Spa A gene was successfully cloned and expressed,and the recombinant plasmid of p PICZαC-Spa A-N was constructed. The immunization protection area protein,which lies in Spa A gene amino terminal of swine E. rhusiopathiae in P. pastoris X-33,was successfully expressed. Spa A gene of swine E. rhusiopathiae has been successfully expressed asim-munization protection area protein in yeast host,which will lay a foundation for the development and the mass production of subunit vaccine of swine E. rhusiopathiae.