肺孢子虫（菌）p55抗原嵌合基因的克隆及表达产物分析

目的构建肺孢子虫（菌）p55蛋白嵌合基因的原核表达载体,分析、鉴定及纯化表达产物。方法自NCBI网站的蛋白数据库中获取p55抗原及4个变异体信息,运用生物信息学方法预测可能的抗原表位,设计包含多个可能抗原表位的多肽,根据遗传中心法则及大肠杆菌密码子偏好性进行密码子优化,将氨基酸序列转化为核苷酸序列,将人工合成的嵌合基因（CAG）片段连接到质粒pGEX-6p-1（含GST标签）上,构建原核表达载体pGEX-6p-1/CAG,酶切、测序鉴定。pGEX-6p-1/CAG转化大肠杆菌,异丙基-β-D硫代半乳糖苷（IPTG）诱导表达重组融合蛋白CAG-GST。表达产物用SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）及Western-blotting分析鉴定。结果双酶切及测序结果显示重组质粒pGEX-6p-1/CAG构建成功。SDS-PAGE显示重组融合蛋白相对分子量约为69 000。Western-blotting结果显示,表达的融合蛋白为CAG-GST。结论成功构建表达p55蛋白嵌合基因的原核表达载体pGEX-6p-1/CAG,建立表达重组蛋白的原核表达系统。

Cloning of chimeric gene of p55 antigen from Pneumocystis and analysis on the expression Product

To construct the prokaryotic expression vector of chimeric gene of p55 antigen from Pneumocystis and to analyze and identify the expression product,the possible epitopes of p55 protein and its variants were forecasted by the bioinformatics technology and a polypeptide containing multiple possible epitopes was designed.The amino acid sequences of the polypeptide were converted to the nucleotide sequences according to the genetic center ruler and the codon preference of E.coli to make the codon optimization.The chimeric gene（CAG） fragment was synthesized and inserted into the plasmid pGEX-6p-1（containing glutathione S-transferase;GST） to construct the recombinant plasmid.The expression of the recombinant protein in E.coli was induced by IPTG.The recombinant protein was analysized and identified by SDS-polyacrylamide gel electrophoresis and western-blotting.The recombinant plasmid pGEX-6p-1/CAG was constructed successfully.The relative molecular weight of the recombinant protein was about 69 000.The result of Western-blotting showed the recombinant protein was recognized by anti-GST.Tt＇s suggested that the prokaryotic expression vector pGEX-6p-1/CAG was constructed successfully and the prokaryotic expression system was established.