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根据已发表的弓形虫主要表面抗原Ｐ３０基因序列，自行设计合成了一对引物，分别在其正义链和反义链的５′末端加上了ＢａｍＨⅠ和ＥｃｏＲⅠ的酶切位点，利用ＰＣＲ技术获取Ｐ３０抗原基因整个开放阅读框架的碱基序列。ＰＣＲ产物经ＢａｍＨⅠ和ＥｃｏＲⅠ双酶切后，与经同样两种内切酶切割的质粒载体ｐＢｌｕｅｓｃｒｉｐｔＳＫ连接构成重组质粒ｐＢｌｕｅｓｃｒｉｐｔＳＫ－Ｐ３０，转化大肠杆菌ＤＨ５α。通过遗传标记筛选、ＰＣＲ扩增及酶切分析对重组子进行了鉴定，为进一步选择真核系统或原核系统体外表达该抗原打下了必要的基础。
A pair of primers was designed and synthesized according to published P30 gene sequence of Toxoplasma gondii major surface antigen. BamHI and EcoRI restriction sites were added to the 5 ’ends of the sense and antisense strands, respectively. The PCR products were obtained The base sequence of the entire open reading frame of the P30 antigen gene. After digested by BamHⅠand EcoRⅠ, the PCR product was ligated with the plasmid vector pBluescriptSK cut by the same two kinds of endonucleases to construct the recombinant plasmid pBluescriptSK-P30 and transformed into E. coli DH5α. The recombinants were identified by genetic marker screening, PCR amplification and enzyme digestion analysis, laying the necessary foundation for the further selection of eukaryotic or prokaryotic expression of the antigen in vitro.