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刘世国, 左丽, 王娇. DEN-2分离株E基因部分序列原核蛋白表达[J]. 中国公共卫生, 2009, 25(6): 660-662. DOI: 10.11847/zgggws2009-25-06-10
引用本文: 刘世国, 左丽, 王娇. DEN-2分离株E基因部分序列原核蛋白表达[J]. 中国公共卫生, 2009, 25(6): 660-662. DOI: 10.11847/zgggws2009-25-06-10
LIU Shi-guo, ZUO Li, WANG Jiao. Prokaryotic expression of E partial nucleotide sequence of clinical isolated strain of dengue virus type 2[J]. Chinese Journal of Public Health, 2009, 25(6): 660-662. DOI: 10.11847/zgggws2009-25-06-10
Citation: LIU Shi-guo, ZUO Li, WANG Jiao. Prokaryotic expression of E partial nucleotide sequence of clinical isolated strain of dengue virus type 2[J]. Chinese Journal of Public Health, 2009, 25(6): 660-662. DOI: 10.11847/zgggws2009-25-06-10

DEN-2分离株E基因部分序列原核蛋白表达

Prokaryotic expression of E partial nucleotide sequence of clinical isolated strain of dengue virus type 2

  • 摘要: 目的 通过构建登革2型病毒(DEN-2)临床分离株(B株)E基因区1-476bp的原核表达载体,进行原核表达,为DEN-2E蛋白功能的研究提供基础依据。方法 将DEN-2B株E基因区部分序列克隆入原核表达载体pET28a(+),命名为pET28a(+)-Eb;经酶切、PCR及测序鉴定后转化BL21(DE3)菌株,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,通过十二烷基硫酸钠-聚丙烯胺凝胶电泳(SDS-PAGE)、蛋白印迹(Western blot)鉴定表达蛋白;对表达蛋白进行纯化,并滴定对C6/36细胞的细胞半数致死量(TCID50)。结果 成功构建了pET28a(+)-Eb原核表达重组质粒;SDS-PAGE分析表明,E基因区部分序列获得高效表达,其相对分子量约为23kDa,表达量约占菌体总蛋白的29%;Western blot表明,该目的蛋白可与DEN-2鼠单克隆抗体结合。用Ni柱亲和层析法纯化原核表达蛋白,纯度达90%。DEN-2B株E基因部分序列原核表达蛋白对C6/36细胞的TCID50为10-5.31。结论 pET28a(+)-Eb可在BL21(DE3)菌株中高效表达;DEN-2B株E基因部分序列原核表达蛋白对C6/36细胞有明显的细胞毒作用。

     

    Abstract: Objective To construct prokaryotic expression vector of Egene partial sequence of Bstra in dengue virus type 2 for proka ryotic expression for further study of dengue virus.Methods Egene partial sequence of B strain dengue virus type 2 was amplified by RT-PCR,and then in serted into prokaryotic vector pET28a(+)and transformed to E.coli BL21 cells.The Egene partial sequence was expressed with IPTG induction.Results The expressed products was identified by SDS-PAGE and Western B lot and purified.Mean while,the cyto to xicity of the purified protein on C6/36 was detected.The exper miental results showed that prokaryotic expression recom binant vectors pET28a(+)-Eb was successfully constructed.SDS-PAGE assay suggested that the recom binant proteins with a relative molecular weight of 23KD a could be highly expressed in BL21 and the yields were 29% of total bacterial proteins.there sult of Western B lot indicated that the expression products could specifically react with monoantibody aga inst dengue virus type.Vitrocy to toxic expermients suggested that target proteins had relatively cyto toxic function for C6/36.Conclusion pET28a(+)-Eb with Egene partial sequence can be highly expressed in BL21.The antigenicity of target prote in provide a potential source for fur ther developing dengue virus diagnosis reagent.Cyto toxic expermients suggest that target proteins had relatively cyto toxic function for C6/36.

     

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