弓形虫P30蛋白抗原表位的克隆表达及纯化鉴定

目的构建表达P30蛋白抗原表位的重组质粒及工程菌,获得纯化的P30蛋白抗原表位,用于检测弓形虫特异性抗体。方法采用PCR技术,设计一对引物(P30P3、P30P4),从弓形虫基因组DNA中扩增出编码P30蛋白抗原表位的基因片段,与载体pET28a(+)连接,重组质粒转化大肠杆菌BL21(DE3),经IPTG诱导后进行SDSPAGE分析。用离子交换纯化表达的P30抗原表位,用ELISA鉴定其抗原性。结果表达的P30蛋白抗原表位以包涵体形式存在,纯化的P30蛋白抗原表位片段有较好的抗原性,可用于检测弓形虫抗体。结论已成功构建了高表达P30蛋白的工程菌,为研制弓形虫抗体检测试剂或疫苗奠定了基础。

Cloning and Expression of Antigenic Epitope of Toxoplasma P30 Protein and Purification and Identification of Expressed Product

Objective To construct the recombinant plasmid and engineering bacteria for expression of antigenic epitope of Toxoplasma P30 protein and use the expressed product for detection of Toxoplasma specific antibody.Methods Design a pair of primers and amplify the gene fragment encoding antigenic epitope of Toxoplasma P30 protein from the genomic DNA of Toxoplasma by PCR then insert into vector pET28a( ).Transform the contructed recombinant plasmid to E.coli BL21(DE3) for expression under induction of IPTG.Identify the expressed product by SDS-PAGE,purify by ion exchange chromatography and analyze for antigenicity by ELISA.Results The expressed product existed in a form of inclusion body and contained about 30% of total somatic protein.It showed good antigenicity and could be used for detection of Toxoplasma antibody.Conclusion The engineering bacteria for high expression of antigenic epitope of Toxoplasma P30 protein was successfully constructed.It laid a foundation of development of Toxoplasma antibody detection kit or Toxoplasma vaccine.