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目的 构建弓形虫RH株 pcDNA3 .1( + ) GRA8真核表达重组质粒 ,为进一步DNA免疫做准备。方法 用聚合酶链反应 (PCR)从弓形虫RH株的基因组DNA中扩增编码致密颗粒蛋白(GRA8)的基因片段 ,纯化后重组入 pUC 19克隆载体 ,再经含IPTG ,XGal氨苄培养基蓝白筛选 ,挑选白色克隆酶切 ,低溶点琼脂糖纯化 ,回收目的基因亚克隆入pcDNA3 .1( + ) ,经氨苄培养基过夜培养挑选 6个克隆提纯酶切 ,PCR鉴定和测序。结果 从RH株基因组DNA中扩增出特异的GRA 8基因片段 ,克隆成功pcDNA3 .1( + ) GRA 8重组质粒。测序表明GRA8这部分基因与GENEBANK相应基因序列完全一致。结论 本结果为研究抗弓形虫核酸疫苗打下基础
Objective To construct the eukaryotic expression plasmid pcDNA3. 1 (+) GRA8 of Toxoplasma gondii RH strain for preparation of further DNA immunization. Methods The gene fragment encoding compact granule protein (GRA8) was amplified from the genomic DNA of Toxoplasma gondii RH strain by polymerase chain reaction (PCR). The fragment was purified and inserted into pUC 19 cloning vector. The recombinant plasmids were cloned into pUC 19 vector containing IPTG, White selection, selection of white cloning enzyme digestion, low melting point agarose purification, the target gene was subcloned into pcDNA3 .1 (+), cultured overnight with ampicillin culture selected six clones purified digestion, PCR identification and sequencing. Results The specific GRA 8 gene fragment was amplified from the genomic DNA of RH strain and the recombinant plasmid pcDNA3. 1 (+) GRA 8 was successfully cloned. Sequencing showed that this part of GRA8 gene and GENEBANK corresponding gene sequence exactly the same. Conclusion The results laid the foundation for the study of anti-Toxoplasma nucleic acid vaccine