结核杆菌ESAT-6抗原及Flt3配体双表达核酸疫苗的构建与体外表达
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R378.911

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江苏省教育厅自然科学基金资助项目(05KJB310077);江苏省现代病原重点实验室开放课题(8853)


Construction and in vitro expression of a bicistronic DNA vaccine carrying ESAT-6 antigen of Mycobacterium tuberculosis and Flt3 ligand genes
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    摘要:

    目的:构建可同时表达flt3 配体(flt3-ligand,FL)和结核杆菌6kD早期分泌蛋白(ESAT-6)的重组pIRES质粒,并在大鼠肾小球系膜细胞(GMC)中表达,为进一步研究结核杆菌DNA疫苗提供实验基础。方法:采用PCR方法将FL和ESAT-6基因分别定向克隆入真核双表达载体pIRES。在酶切分析及序列测定后,用脂质体转染至GMC细胞,Western blot 鉴定其体外表达。结果:核酸序列测定证实重组质粒构建正确,该重组质粒在体外GMC细胞中能表达FL和ESAT-6两种蛋白。结论: 成功构建了结核杆菌FL和ESAT-6双顺反子真核表达质粒,并在体外实现了共表达。

    Abstract:

    Objective:To construct bicistronic DNA vaccines containing extra-cellular fragment of Flt3 ligand(FL) genes and early secreted antigen(ESAT-6) of Mycobacterium tuberculosis and to express them in glomerular mesangial cells(GMC). Methods:FL and ESAT-6 genes were cloned into pIRES, a bicistronic vector, using polymerase chain reaction(PCR). After being screened and identified, the vector was transfected into GMC cells in order to examine FL and ESAT-6 and their expression levels. Results:The recombinant vector was confirmed by sequencing and the expression of FL and ESAT-6 genes in GMC cells were detected by western blot. Conclusion:The recombinant pIRES-FL-ESAT6 bicistronic vector could be successfully constructed and expressed in vitro.

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徐 娟,陈 霞.结核杆菌ESAT-6抗原及Flt3配体双表达核酸疫苗的构建与体外表达[J].南京医科大学学报(自然科学版),2007,(1):23-26

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  • 收稿日期:2006-08-28
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  • 在线发布日期: 2007-01-15
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