弓形虫膜抗原SAG3基因打靶载体构建方法探讨

目的 探讨构建弓形虫膜抗原SAG3基因打靶载体的方法，为建立基因敲除弓形虫基因突变株打下基础。方法 用PCR方法扩增弓形虫SAG3基因同源序列，将所获得的1．53kb和2．81kb序列连接克隆插入TUB5／CAT质粒中，构建置换型打靶载体，采用PCR扩增、酶切分析鉴定。结果 将SAG3基因中1．53kb和2．81kb两个同源序列分别插入TUB5／CAT质粒中，构建了弓形虫RH株5’SAG3-TUB5／CAT-3’SAG3置换型载体，经鉴定所获得的打靶载体结构准确。结论 建立了弓形虫膜抗原SAG3基因打靶载体，并获得了弓形虫RH株SAG3基因置换型打靶载体，为分析弓形虫基因功能提供了可行的方法。

Construction of A Knockout Vector for Surface Antigen SAG3 Gene of Toxoplasma gondii

Objective To construct a SAG3 knockout vector, which can be used for establishing a mutant model of Toxoplasma gondii.Methods To amplify two fragments,1 53kb and 2 81kb,of the SAG3 gene by polymerase chain reaction(PCR) and inserted them in front and after the bacterial chloramphenicol acetyltransferase (cat) gene of the cloning vector, respectively.The recombinant plasmid was screened by PCR and restriction analysis. Results A SAG3 gene targeting vector of T.gondii was constructed with a TUB5/CAT plasmid encoding the bacterial chloramphenicol acetyltransferase (cat) gene. Analysis of the vector with PCR and enzyme restriction showed that both gene fragments were integrated into the vector and an expected targeting fragment was obtained. Conclusions A SAG3 gene knockout vector was constructed. The results demonstrate the possibility of using gene knockout approach in the study of T.gondii gene function and provide a further selectable method for investigating the mechanism of the invasion of host cell.