重组大肠杆菌表达BAFF及其受体的优化研究

Optimization of Recombinant Escherichia Coli Strain Expressing sBAFF and BAFF Receptor Derivative

构建游离态BAFF及其受体突变体的表达载体,采用大肠杆菌系统进行表达,并优化温度、诱导物浓度对蛋白表达的影响。分别提取K562细胞系mRNA和小鼠脾脏组织mRNA,进行逆转录,后经PCR扩增,获取BAFF和BAFF受体的基因片段,采用单因素实验,选取不同温度及诱导物浓度梯度诱导表达,探索重组蛋白表达的最适条件,提高目标蛋白表达量及可溶性蛋白含量。结果表明BAFF受体突变体在37℃,1 mmol/L IPTG诱导条件下表达量可达到25%左右,且可溶性蛋白含量均高于60%;可溶性BAFF的最适诱导条件为28℃,1 mmol/L IPTG,表明温度可显著提高其可溶性蛋白的含量。BAFF及其受体突变体的克隆和表达,为BAFF及其受体生物学功能的深入研究奠定了坚实的基础。

Two recombinant expression plasmids were constructed to express the soluble BAFF and CDR2 domain of BAFF receptor TACI in E. coli expression system, and then expression parameters including temperature, IPTG concentration were optimized to improve expression and yield of soluble expression of target proteins. The genes encoding soluble BAFF and CDR2 domain of BAFF receptor TACI were amplified respectively from K562 cell line and murine spleen tissue, and expression plasmids were constructed. The recombinant plasmids were transformed into BL21 （DE3）, induced by IPTG for expression and both sBAFF and CDR2 were expressed. Through single factor experiment,temperature and IPTG concentration were optimized to explore the optimum conditions for recombinant protein expression. The results showed that under the conditions of 37 ~C, 1 mol/L IPTG, the expression of CDR2 reached about 25% of total cellular proteins, and soluble protein content was more than 60% ;for sBAFF, the optimal induction conditions is 28 ~C, 1 mol/L IPTG. Low temperature fermentation can increase the production of soluble target proteins. The construc- tion, expression of sBAFF and its receptor derivative lay a solid foundation for further in-depth investigation of biological function of BAFF and its receptor.