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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 预览 被引量:4

IN VITRO AMPLIFICATION, CLONING OF TOXOPLASMA GONDII MULTI-ANTIGENIC GENE ENCODING P30 AND RHOPTRY PROTEIN 2 AND CONSTRUCTION OF ITS EUKARYOTIC EXPRESSION PLASMID
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摘要 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. 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 .
作者 蒋华 何深一 周怀瑜 丛华 古钦民 李瑛 赵群力 JIANG Hua, HE Shen yi, ZHOU Huai yu, CONG Hua, GU Qin min, LI Ying, ZHAO Qun li (Department of Pathogenic Biology, Shandong University, Jinan 250012, China)
出处 《中国寄生虫病防治杂志》 CSCD 2005年第1期 1-4,共4页 Chinese Journal of Parasitic Disease Control
基金 国家自然科学基金,教育部留学回国人员科研启动基金
关键词 弓形虫 P30 棒状体蛋白2 克隆 基因重组 Toxoplasma gondii P30 rhoptry protein 2(ROP2) cloning gene recombinant
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