解脲支原体Ⅰ型MB抗原主蛋白氮端基因的克隆及原核表达

Cloning and Expression of the N-terminal Segment of URAI MB in E.coli

目的 从新鲜培养的解脲支原体 ( URA) 型培养液获得其 MB抗原主蛋白 N端 1 /3保守区的基因 ,并体外表达 ,获得具有抗原活性的原核表达蛋白 ,为研制广谱、低廉及方便准确的 URA感染的诊断及治疗方法提供物质基础。方法 从新鲜培养的 URA标准株 型培养液提取 DNA模板 ,采用特异引物及聚合酶链方法获得其 MB抗原主蛋白 N端 1 /3保守区的基因 ,经克隆入测序载体验证 DNA大小及序列后 ,将 MB抗原主蛋白 N端 1 /3保守区的基因克隆入高效原核表达载体 ,并在大肠杆菌中表达 ,初步以Western- blot测定表达产物的抗原性即与感染者阳性血清的反应活性。结果 以笔者设计的特异引物 ,能得到预期大小的 PCR产物 ,并能插入预定的克隆载体中测序 ,所得基因的序列符合目的基因的序列 ,其插入原核表达载体能获得表达 ,且表达产物具有抗原活性 ,能与感染患者的血清发生抗原抗体反应。结论 所得到的 MB抗原主蛋白 N端 1 /3保守区片段的基因 ,与 GENE BANK检索序列一致。在大肠杆菌中表达此片段所获得的表达产物 ,有抗原活性 ,有望用于

Objective To acquired the N terminal conserved segment of MB antigen DNA,expressed its DNA in E.coli ,and prepared for developing a novel method for detection and vaccination of URA.Methods First the N terminal conserved segment of MB antigen DNA was ascertained by comparing the MB sequence of URAI throrgh BLAST2.0 on NCBI,then by using specific primers,the subjective DNA segment was acquired by PCR from culture URAI.After that it was cloned into vector of pGEM 3zf(-)and sequenced.At last it was inserted into an expression vector pBV220 and expressed in E.coli .The antigen activity of expressed product was detected by Western blot.Results The desired DNA of N terminal conserved segment of MB antigen could be acquired by PCR using special primer from culture of URAI,the acquired DNA fragment could be cloned into vector of pGEM 3zf(-)and sequenced.It could also be cloned into an expression vector and expressed actively in E.coli .Conclution The N terminal conserved segment of MB antigen DNA can be acquired and expressed actively in E.coli .That may provide a cheap,convenience method for detection and vaccination study of URA.