摘要
目的优化高表达禽流感病毒NS1蛋白的实验方法。方法将基因工程菌接种LB培养基,设定IPTG终浓度为0.3mmol/L,诱导温度为37℃,诱导表达6.0h,SDS-PAGE分析融合蛋白表达情况,确定最佳诱导时间。以0.1mmol/L为梯度,设置不同IPTG诱导浓度,37℃诱导4.0h,分析融合蛋白表达,确定最佳IPTG诱导浓度。设定IPTG至终浓度为0.6mmol/L,分别于24℃、28℃、32℃、37℃和42℃下诱导表达4.0h,分析融合蛋白表达,确定最适诱导温度。采用最优表达条件,超声裂菌后SDS-PAGE分析融合蛋白,Western blotting对融合蛋白进行鉴定。结果当培养温度为37℃,IPTG浓度为0.3mmol/L时,融合蛋白GST-NS1在IPTG诱导5.0h时的表达量最高,达28.5%;当IPTG诱导浓度为0.6mmol/L,在37℃诱导表达4.0h时,融合蛋白的表达量可达27.9%。选用优化的表达条件,IPTG 0.6mmol/L,37℃诱导表达4.0h,融合蛋白的表达量最高可达33.2%。进一步分析显示,融合蛋白大部分以可溶形式表达,其表达量可占菌体总蛋白的25.1%。经SDS-PAGE电泳、电转移后,用小鼠抗GST单克隆抗体进行免疫印迹分析,出现一条阳性反应带。结论通过实验优化了工程菌诱导表达的最佳IPTG浓度、最适时间和培养温度,实现了融合蛋白的可溶性高表达。
Objective To optimize the culture conditions for the high expression of NS1 protein. Methods Engineering bacteria were inoculated into LB medium and incubated with shaking. The IPTG concentration and culture temperature were set for 0.3mmol/L and 37℃ respectively,then the duration of induction were optimized. Inductions were initiated in turn at 0.5h intervals from 0.5h to 6.0h. The expression of fusion protein were assessed by SDS-PAGE using 12% acrylamide. The culture temperature and the duration of induction were set for 37℃ and 4.0h respectively, then the amount of IPTG were added from 0. lmmol/L to 1.0mmol/L final concentration, and the expression of fusion protein were assessed by SDS-PAGE. The IPTG concentration and the duration of induction were set for 0.6mmoFL and 4.0h respectively,then the optimal temperature was investigated by testing 24℃, 28℃, 32℃, 37℃ ,42℃. The culture conditions were optimized,and bacteria were pelleted by centrifugation and disrupted by sonication,then the expression of fusion protein were assessed by SDS-PAGE, and identified using Western blotting. Results The expression level of GST-NS1 fusion protein was 28.5% by using the induce temperature at 37℃, the IPTG concentration of 0.3mmol/L for 5.0h induction. The expression amount of fusion protein was also 27.9% ,when the IPTG concentration was 0.6mmol/L and the induce temperature was 37℃ for 4.0h induction and the expression level was 33.2%. The recombinant fusion protein GST-NS1 was expressed in a soluble form by SDS-PAGE. Analysis and the expression amount of soluble GST-NS1 was 25.1% using 0.6mmol/L IPTG concentration and 37% for 4.0h induction. Separated proteins on SDS-PAGE gel were transferred electrophoretically onto PVDF membrane, then the membrane incubated with anti-GST mouse monoclonal antibody,finally the expected band was shown. Conclusion Conditions for induction were optimized resulting in high expression of fusion protein GST-NS1 in soluble form.
出处
《中国热带医学》
CAS
2012年第1期1-5,共5页
China Tropical Medicine
基金
东莞市高等院校科研机构科技计划重点项目经费资助(No.201108101007)
