弓形虫GRA8基因真核表达重组质粒的构建、鉴定及测序

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GRA8基因片段，克隆成功pcDNA3．1( )-GRA8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。

Construction, identification and sequence analysis of eukaryotic expression recombinant plasmid containing dense granules protein 8 gene from Toxoplasma gondii in BALB/c mice

Objective To clone the coding dense granules protein 8(GRA8) from Toxoplasma gondii RH isolate for the potential use in the development of DNA vaccination. Methods Amplifing gene fragment coding GRA8 from the genomic DNA of Toxoplasma gondii RH isolate by means of ploymerase chain reaction (PCR), the gene is inserted into cloning vector pUC-19 digesting with restrictive enzymes and linking react ions. The positive colon is screed on LB plates containing ampicilline and IPTG, Xgal identified by blue-white and restrictive enzyme digestion. The inserted GR A8 gene was recombined with pcDNA3.1(+) eukaryotic expression vector by digestion with restrictive enzyme and linking reactions. The positive coloe is screene d o n LB plates containing ampicillin and identified by restrictive enzymes and link ing reactions. Results The GRA8 gene with about 804 base is specifically amplified from genomic DNA of Toxoplasma GRA8/RH and pcDNA3.1(+)-GRA8 recombinant is successfull y constructed. The sequencing results showed that GRA8 gene of isolate RH and R H from genebank shares quite high homology. Conclusion The gene encoding GRA8 is amplified from genomic DNA of Toxoplasma gene isolate GRA8/RH and pcDNA3.1 (+)-GRA8 recombinant is successfully constructed.