弓形虫P~(30)的克隆表达及其对巨噬细胞凋亡的影响

目的　获得能表达弓形虫P3 0 蛋白的小鼠巨噬细胞克隆并观察内源性表达的P3 0 蛋白对小鼠巨噬细胞凋亡的影响。方法　通过PCR扩增获得P3 0 基因片段 ,定向克隆到真核表达载体pcDNA3.1/Hygro(- )中 ,利用酶切、DNA序列分析鉴定阳性克隆 ,采用脂质体将重组质粒转染到RAW 2 6 4 .7巨噬细胞中 ,通过Hygromycin筛选和PCR、免疫组化鉴定 ,用流式细胞术DNA倍体分析计算稳定转染和瞬时转染P3 0 基因的巨噬细胞的凋亡率。结果　 1.PCR、酶切、连接的产物经电泳鉴定 ,均与预期设计相符合 ,DNA序列分析发现重组质粒中的目的基因序列与文献报道相符。2 .PCR和免疫组化鉴定发现转染P3 0 重组质粒的巨噬细胞能稳定地复制质粒并表达弓形虫P3 0 蛋白。 3.P3 0 稳定转染的细胞凋亡率均在 2 %左右 ,不同细胞之间没有区别。 4 .瞬时转染空载体和P3 0 重组质粒的巨噬细胞其凋亡率均在 6 %左右 ,没有区别。结论　 1.获得了含P3 0 基因的重组质粒以及能稳定表达弓形虫P3 0 蛋白的小鼠巨噬细胞克隆。2 .无论是稳定转染还是瞬时转染 ,均不能引起巨噬细胞的凋亡 ,说明内源性表达的弓形虫P3 0 蛋白对小鼠巨噬细胞的凋亡没有影响。

Cloning and expression of the gene encoding for the major surface antigen P30 of Toxoplasma gondii and the effects of P30 on the apptosis of murine macrophages

To obtain mouse macrophage cell clone steadily expressing the major surface antigen P 30 of Toxoplasma gondii and to explore the effects of P 30 on the apoptosis of murine macrophages,the gene encoding P 30 was amplified by PCR,using the primer designed for the DNA sequences of P 30 gene and introduced into eukaryotic expression vector pcDNA3.1/Hygro(-) by using EcoRI and Xho I,and then transformed into E.coli Top10.The positive clones were identified by restriction enzyme digestion and DNA sequence analysis,and the correct recombinant plasmid was transfected to murine macrophage cell line RAW264.7 and screened with Hygromycin.The Hygromycin-resistant cell clones were identified by PCR and immunohistochemical analysis.DNA propidiumiodide (PI) staining flow cytometric assay was used to detect the percentages of apoptosis of RAW264.7 cells transfected with P 30 gene.The experimental results showed that the PCR products and the cleavage and link reaction were just the same as those expected,and the sequence of the inserted fragments in the recombinant plasmid were also the same as reported.The recombinant plasmid with P 30 gene could replicate and express correctly in the transfected RAW264.7 cells,and the rates of apoptosis of RAW264.7 cells,steadily and transiently transfected with P 30 gene recombinant plasmid were around 2% and 6% respectively,without any differences from which transfected with or without vacant vector pcDNA3.1.In conclusion,the recombinant plasmid containing the gene encoding for the major surface antigen P 30 of Toxoplasma gondii has been obtained in the present study.Both the stead or the transient transfection of this gene are unable to induce apoptosis of the transfected cells,indicating no influence of the endogenous expression of P 30 antigen on the apoptosis of murine macrophages.