弓形虫RH株表面抗原P30基因重组质粒的构建及鉴定

目的：构建弓形虫RH株表面抗原P30基因的重组表达质粒，为下一步免疫与诊断研究制备高纯度P30重组蛋白奠定基础。方法：自弓形虫RH株感染小鼠腹水中收集速殖子，用酚／氯仿法提取基因组DNA，用PCR法扩增编码P30的基因片段(经电泳切带纯化)，定向插入到PQE-30表达质粒中，转化入TG1宿主菌，在含有青霉素的SOB平板上筛选阳性重组子，采用酶切、PCR扩增和DNA序列分析等方法对插入片段进行鉴定。结果：阳性重组质粒PQE30-P30经Sal HindⅢ双酶切下的插片及PCR扩增产物均与原插入的P30基因片段大小一致为976bp，通过序列测定证实插入片段为编码P30的基因片段。结论：成功构建弓形虫表面抗原P30基因的重组质粒PQE30-P30。

Construction and identification of recombinant plasmids of P30 surface antigen of Toxoplasma gondii

Objective To construct recombinant plasmids DMA encoding P30 surface antigen from Toxoplasma gondii and identify the cloning gene, as a base for developing pure P30 recombinant protein and further study of immunology and diagnosis. Methods Tachyzoites were collected from ascites of mice infected with RH strain of Toxoplasma gondii, from which the genomic DNA was extracted by means of phenol/chloroform. Fragments of gene coding P30 antigen were amplified by polymerase chain reaction (PCR) using primers designed according to the sequence of published P30 gene. The purified P30 gene fragments were inserted into PQE30 by double digestion with SaiI+ HindIII and ligation with T4 ligase, then transformed into E. coli TG1. Positive clones were screened and identified by endonulease digestion PCR amplification and sequence determination analysis. Results Double digestion and PCR amplification of the recombinant plas-mid PQE30-P30 has showed that the size of the inser-ted fragment is 976 bp and in accordance with the expected one. Sequence determination analysis hasdemonstrated that the cloned gene is exactly the gene fragment encoding P30 antigen. Conclusions The expression plasmids containing P30 gene from Toxoplasma gondii were constructed successfully.