摘要
为了提高绵羊肺腺瘤病毒受体透明质酸酶-2(Hyal-2)在大肠杆菌BL21(DE3)中的表达量,试验对含pGEX-4T-1-Hyal-2重组质粒的BL21(DE3)重组菌原核表达条件进行了优化。结果表明:诱导剂IPTG浓度、作用时间、温度对目的蛋白的表达影响很大,在37℃条件下加入不同终浓度的IPTG(0.2,0.4,0.6,0.8,1.0 mmol/L)对重组菌诱导4 h,在终浓度为0.4 mmol/L时目的蛋白的表达量最高;加入终浓度为0.5 mmol/L的IPTG,在37℃条件下诱导,在不同时间点(2,4,6,8小时)取样,在作用时间为2 h,37℃条件下表达的目的蛋白主要以包涵体形式存在,当温度降低时可溶性形式明显增加。通过对诱导条件的优化和摸索发现,绵羊肺腺瘤病毒受体Hyal-2原核表达在诱导剂IPTG浓度为0.4 mmol/L、作用时间为2 h、温度为37℃时目的蛋白的表达量最高。
To improve the expression level of the jaagsiekte sheep retrovirus receptor hyaluronidase - 2 （ Hyal - 2 ） in E. coli BL21 （ DE3 ）, the prokaryotic expression conditions were optimized for the recombinant bacteria E. coil BL21 （ DE3 ） which containing recombinant plasmid pGEX - 4T - 1 - Hyal - 2. The results showed that the expression of target protein was affected greatly by the inducer IPTG concentration, reac- tion time and temperature. When the recombinant bacteria was induced for 4 hours with different final concentrations of IPTG （0.2,0.4,0.6, 0.8,1.0 mmol/L） under the condition of 37℃ , the highest expression level for the target protein was found with a final concentration of 0.4 mmol/L of IPTG. The recombinant bacteria was induced at different time points （2 h, 4 h, 6 h, and 8 h） at 37 qC for samplirg with a fi- nal concentration of 0.5 mmol/L of IPTG, it was found that the expressed target protein existed mainly in the form of inclusion bodies at 37℃ for 2 h of action time, and the soluble form was significantly increased when the temperature decreased. The results indicate that the highest expression level of the target protein for prokaryotic expression of the jaagsiekte sheep retrovirus receptor Hyal - 2 is found under the following conditions ： a concentration of 0.4 mmol/L of inducer IPTG, 2 hours reaction time, temperature at 37℃, by means of optimization and exploration of inducing conditions.
出处
《黑龙江畜牧兽医》
CAS
北大核心
2014年第3期25-28,共4页
Heilongjiang Animal Science And veterinary Medicine
基金
国家自然科学基金项目(31101788
31260593)
关键词
绵羊肺腺瘤病毒
受体
透明质酸酶-2
原核表达
诱导条件优化
Jaagsiekte sheep retrovirus
receptor
hyaluronidase - 2
prokaryotie expression
optimization of inducing conditions
