论文部分内容阅读
目的构建弓形虫棒状体蛋白（ＲＯＰ１）基因重组质粒并在Ｅ．ｃｏｌｉ中表达。方法用ＲＨ株接种小鼠，收集腹水，纯化速殖子，抽提基因组ＤＮＡ；据ＲＯＰ１基因序列设计合成一对引物，将上、下游引物分别引入ＥｃｏＲＩ，ＢａｍＨＩ酶切位点，用ＰＣＲ技术从ＲＨ株基因组ＤＮＡ中扩增编码ＲＯＰ１的基因片段，插入ｐＢＶ２２０质粒，转化大肠杆菌ＤＨ５α感受态细胞，于氨苄阳性ＬＢ培养平板上筛选阳性克隆，酶切鉴定；经温度诱导在Ｅ．ｃｏｌｉ中表达，ＳＤＳ－ＰＡＧＥ及免疫印迹分析。结果ＲＯＰ１基因体外扩增产物大小与预期值相符，约７５６ｂｐ；构建成功ｐＢＶ２２０－ＲＯＰ１重组质粒；ＳＤＳ－ＰＡＧＥ、免疫印迹显示特异蛋白条带的分子量约４３ｋＤ，表达产量约占菌体蛋白１３．２３％。结论从弓形虫基因组ＤＮＡ中获取ＲＯＰ１基因，并成功构建ｐＢＶ２２０－ＲＯＰ１重组质粒，诱导表达ＲＯＰ１非融合蛋白，为进一步分离纯化、用于对弓形虫侵入机制及免疫特性的研究做好准备。
Objective To construct the recombinant plasmid of Toxoplasma gondii rod-like body protein (ROP1) and express it in E.coli. Expression in E. coli. Methods The mice were inoculated with RH strain, the ascites was collected, the tachyzoites were purified and the genomic DNA was extracted. A pair of primers was designed and synthesized according to the ROP1 gene sequence. The upstream and downstream primers were introduced into EcoRI and BamHI restriction sites respectively. The ROP1 gene fragment was amplified from genomic DNA of RH strain and inserted into pBV220 plasmid. The recombinant plasmid was transformed into E. coli DH5α competent cells. Positive clones were screened on ampicillin-resistant LB culture plates and identified by restriction enzyme digestion. coli expression, SDS-PAGE and Western blot analysis. Results The amplified product of ROP1 gene in vitro was consistent with the expected value of about 756bp. The recombinant plasmid pBV220-ROP1 was constructed successfully. The molecular weight of the specific protein band was about 43kD by SDS-PAGE and the expressed protein was about 13.23 %. Conclusion The ROP1 gene was obtained from the Toxoplasma gondii genomic DNA and the recombinant plasmid pBV220-ROP1 was successfully constructed. The non-fusion protein ROP1 was induced to be further isolated and purified for preparation of the Toxoplasma gondii invasion mechanism and immune characteristics.