弓形虫ROP1基因真核表达重组质粒DNA免疫小鼠的研究Ⅰ.pcDNA3-ROP1真核表达重组质粒的构建

STUDIES ON DNA VACCINATION WITH RECOMBINANT PLASMID pcDNA3 CONTAINING ROP1 GENE FROM TOXOPLASMA GONDII IN BALB/C MICE Ⅰ.CONSTRUCTING EUKARYOTIC EXPRESSION RECOMBINANT PLASMID pcDNA3 ROP1

目的构建弓形虫ＺＳ２株ｐｃＤＮＡ３－ＲＯＰ１真核表达重组质粒，为进一步表达及ＤＮＡ免疫做准备。方法用ＰＣＲ技术从弓形虫ＺＳ２分离株的基因组ＤＮＡ中扩增编码棒状体蛋白１（ＲＯＰ１）的基因片段，重组入ｐＵＣ１８克隆载体。将ｐＵＣ１８－ＲＯＰ１中的ＲＯＰ１外源基因片段经酶切、连接等反应，亚克隆入ｐｃＤＮＡ３真核表达载体，再经含氨苄ＬＢ培养基筛选，酶切、ＰＣＲ鉴定。结果从ＺＳ２株基因组ＤＮＡ中扩增出特异的ＲＯＰ１基因片段，克隆成功ｐＵＣ１８－ＲＯＰ１；经亚克隆，筛选鉴定获得了ｐｃＤＮＡ－ＲＯＰ１重组质粒。结论构建成功弓形虫ｐＵＣ１８－ＲＯＰ１重组质粒，亚克隆成功ｐｃＤＮＡ－ＲＯＰ１重组质粒。

Aim Cloning the gene coding rhoptry protein 1(ROP1)from Toxoplasma gondii ZS2 isolate to prepare for expression and DNA vaccinating with the recombinant plasmid,pcDNA3 ROP1.Methods Amplifying gene fragment coding ROP1 from the genomic DNA of T.gondii ZS2 isolate by means of polymerase chain reaction (PCR),the gene was inserted into cloning vector,pUC18.The inserted ROP1 gene was recombined with pcDNA3 eukaryotic expression vector by digesting with restrictive enzymes and linking reactions.The positive colon was screed on LB plates containing ampicillin and identified by restrictive enzyme digestion and PCR amplification.Results The specific gene fragment ROP1 was amplified from genomic DNA of ZS2 T.gondii islate;Constructed pUC18 ROP1 and pcDNA3 ROP1 were constructed successfully and the further research will be carried out.