弓形虫微线体蛋白1部分基因原核表达重组质粒pWR450-1-MIC1的构建、鉴定及测序

目的：构建弓形虫ZS2分离株pWR450-1-MIC1原核表达重组质粒，为进一步表达及免疫做准备。方法：用PCGENE软件分析MIC1基因可能的TB抗原表位，自行设计引物，用聚合酶链反应（PCR）技术从弓形虫ZS2分离株的基因组DNA中扩增编码微线体蛋白1（MCC1）的基因片段，经酶切，连接，重组入pWR450-1原核表达载体，再经含氨苄培养基筛选，酶切，PCR鉴定和测序。结果：从ZS2分离株基因组DNA中扩增出特异的MIC1基因片段，克隆成功pWR450-1－MIC1重组质粒，测序表明MIC1这部分基因与RH株相应碱基序列完全一致。高度保守，为下一步表达及免疫奠定基础。

Constructing Protoaryotic Expression Recombinant Plasmid pWR450-1-MIC1

Aim:To clone the coding microneme 1(MIC1) protein from Toxoplasma gondii ZS2 to prepare for expression and vaccinating. Methods Amplifing gene fragment coding microneme 1(MIC1) protein from the genomic DNA of Toxoplasma.gondii ZS2 by means ploymerase chain reaction (PCR),The gene was inserted to cloning expression vector pWR450-1by digesting with restrictive enzymes and linking reactions.The positive colon was screed on LB plates containing ampicillin and identified by restrictive enzyme digestion and PCR amplification and sequence analysis.Results:The partial microneme 1gene with about 483 base was specifically amplified from genomic DNA of Toxoplasma gene isolate MIC1/ZS2 and pWR450-1MIC1 recombinant was successfully constructed. Using softwares to analyze the microneme 1 gene structure and RH gene sequencing ,The microneme 1 gene of isolate ZS2 and RH shared quite high homology. Conclusions:The gene encoding microneme 1 was amplified from genomic DNA of Toxoplasma gene isolate MIC1/ZS2 and pWR450-1MIC1 recombinant was successfully constructed.