弓形虫复合抗原基因P30-ROP2体外扩增、克隆及真核表达重组质粒的构建

目的　构建弓形虫RH株 pcDNA3.1 P30 ROP2 真核表达重组质粒,为进一步表达及 DNA疫苗的研制作准备。　方法　用PCR技术从弓形虫RH分离株的基因组DNA中扩增编码 P30基因片段和棒状体蛋白(ROP2)的基因片段,重组入 pUC18克隆载体,然后将 pUC18 P30 ROP2中的 P30 ROP2 外源基因片段经酶切、连接等反应,亚克隆入pcDNA3.1真核表达载体,再经含氨苄青霉素的LB培养基筛选、酶切及PCR鉴定。　结果　从弓形虫RH株基因组中扩增出特异的 P30、ROP2 片段,克隆成功 pUC18 P30 ROP2 重组质粒;经亚克隆、筛选鉴定获得了 pcDNA3. 1 P30 ROP2重组表达质粒。　结论　成功构建了弓形虫 pUC18 P30 ROP2重组克隆质粒,亚克隆成功 pcDNA3.1 P30 ROP2真核表达重组质粒,为下一步DNA疫苗的研究奠定了基础。

IN VITRO AMPLIFICATION, CLONING OF TOXOPLASMA GONDII MULTI-ANTIGENIC GENE ENCODING P30 AND RHOPTRY PROTEIN 2 AND CONSTRUCTION OF ITS EUKARYOTIC EXPRESSION PLASMID

Objective To construct a recombinant eukaryotic expression plasmid encoding Toxoplasma gondii multi antigen P30 and rhoptry protein 2(ROP2) and prepare for further study. Methods Amplifying gene fragment encoding P30 and ROP2 from the genomic DNA of T. gondii RH strain by means of PCR, the gene was inserted into cloning vector pUC18. Then the inserted P30 ROP2 gene fragment was subcloned to pcDNA3.1 eukaryotic expression vector by digesting with restrictive enzymes and ligating reactions. The positive clone was screened on LB plate containing ampicillin and identified by restrictive enzyme digestion and PCR amplification. Results The specific gene fragments P30 and ROP2 were amplified from genomic DNA of T. gondii RH strain; The cloning and eukaryotic expression recombinant plasmid were correctly constructed. Conclusion The recombinant plasmid pUC18 P30 ROP2 and pcDNA3.1 P30 ROP2 were successfully constructed.This would pave the way for further study of DNA vaccination of T. gondii .