| 致倦库蚊天蚕素CecB2基因的原核表达及蛋白纯化 |
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| 引用本文： | 王吉平,张堤,张春林,赵文静,张晶,翟素珍.致倦库蚊天蚕素CecB2基因的原核表达及蛋白纯化[J].动物医学进展,2016(11):31-35. |
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| 作者姓名： | 王吉平 张堤 张春林 赵文静 张晶 翟素珍 |
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| 作者单位： | 贵州医科大学生物学教研室,贵州贵阳,550025 |
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| 基金项目： | 贵州省科技厅项目(黔科合 LH 字[2015]7363号) |
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| 摘 要： | 构建致倦库蚊(贵阳株)天蚕素B2(CecB2)基因原核表达载体,并原核表达获得重组蛋白。定向克隆CecB2成熟肽序列至原核表达载体pET32a(+)上,将成功构建的pET32a-CecB2重组表达质粒转化大肠埃希菌Rosetta中经IPTG诱导表达,对IPTG诱导浓度和诱导时间优化后表达所得目的蛋白采用镍离子亲和层析纯化,SDS-PAGE和Western blot检测鉴定表达蛋白。结果表明,成功构建原核表达载体pET32a-CecB2,IPTG浓度和时间优化结果为IPTG浓度为0.05mmol/L,诱导时间为3h。SDS-PAGE检测获得大小约25ku的可溶性纯化蛋白,Western blot鉴定纯化蛋白可与鼠抗His-tag单克隆抗体发生抗原抗体结合反应。说明所构建原核表达载体pET32a-CecB2能在大肠埃希菌中可溶表达,为进一步研究其生物学功能奠定了基础。
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| 关 键 词： | 致倦库蚊 天蚕素基因 原核表达 蛋白纯化 |
Prokaryotic Expression and Protein Purification of Cecropin CecB2 Gene of Culex quinquefasciatus |
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| Abstract: | To construct the prokaryotic expression vector of cecropin CecB2 gene of Culex quinquefasciatus for prokaryotic expression recombinant protein.The mature peptide of CecB2 was inserted into the multiple cloning sites of the expression vector pET32a to construct recombinant plasmid firstly,then transformed it into E.coli Rosetta for expression by IPTG induction.The expression conditions were optimized by investi-gating the effects of concentration of IPTG and induction time.SDS-PAGE and Western blot were used to detect and indentify the recombinant protein respectively.The results showed that prokaryotic expression vector pET32a-CecB2 was constructed successfully and optimum induction condition of fusion protein were as follows:IPTG concentration 0.05 mmol/L,induction time 3 h.The recombinant protein was about 25 ku and identified to react with monoclonal antibody 6× His tag by Western blot.This suggested that the re-combinant plasmid was constructed successfully and can be expressed as a soluble fusion protein in E.coli Rosetta.It laid the necessary theoretical basic for further study of biological funtions of cecropin of Culex quinquefasciatus in future. |
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| Keywords: | Culex quinquefasciatus cecropin gene prokaryotic expression protein purification |
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