弓形虫膜抗原SAG3基因打靶载体的构建及筛选

目的 构建弓形虫膜抗原SAG3基因打靶载体，建立基因敲除突变株，为进一步探讨SAG3功能和弓形虫侵入宿主细胞机制提供可行的研究方法。方法 用基因克隆方法将SAG31．53kb和2．81kb两个同源序列分别插入TUB5／CAT质粒中，构建置换型打靶载体，氯霉素乙酰转移酶(CAT)抗性基因作为筛选标记，采用电转移转染细胞方法进行基因打靶，建立突变型的弓形虫株，采用PCR、酶切分析和Southern Blot杂交方法筛选鉴定。结果 构建了弓形虫RH株5’SAG3-T15135／CAT-3’SAG3置换型载体，获得了敲除SAG3基因的弓形虫突变株，建立基因敲除突变株，获得了阳性的筛选克隆，经鉴定证明基因打靶结果准确。结论 通过构建基因打靶载体，可有目的地敲除弓形虫膜抗原基因，为进一步研究弓形虫侵入机制，探讨弓形虫病防治提供可行的方法。

Construction and screening of a targeting vector for the gene encoding for the surface antigens SAG3 of Toxoplasma gondii

To construct a targeting vector for the gene encoding for the surface antigen SAG3 of Toxoplasma gondii, and to establish a knock C out mutant strain to provide possible method of study for the further investigations on the functions of SAG3 and the mechanism by which the toxoplasma invade into the host cells, two SAG3 homologous sequences of 1.53 kb and 2.81 kb were respectively inserted into plasmid TUB5/CAT in order to construct the displacement targeting vector.The chloramphenicol acetyltransferase (CAT) resistance gene was used as an index of screening.And the selection for the chloramphenicol resistance yielded the desired SAG3 knock out mutant. Analysis of this mutant in vitro was performed with PCR, restriction endonuclease analysis and Southern blot hybridization. It was found that a SAG3 knock out mutant vector with a plasmid encoding the cat gene flanked by SAG3 genomic homologous sequences had been constructed in the present study, and the homologous sequences was found to be stably integrated into the T.gondii genome with the expected targeting fragment obtained.These results demonstrate the utility of the gene targeting approach in the study to investigate the gene function of T.gondii. and the mechanism by which this parasite invades to the host cells.