Cry2A杀虫蛋白在大肠杆菌中的表达及纯化

Expression andpurification of Bacillus thuringiensis Cry2A insecticidal protein in E.coli

通过PCR方法从克隆载体上扩增转基因水稻中的抗虫基因cry2A,经限制性内切酶EcoRI和BamHI双酶切定向插入到原核表达载体pET-30a(+)中,成功构建了蛋白表达载体pET-30a(+)/Cry2A,并转入大肠杆菌BL21(DE3)中进行诱导表达。通过对其表达条件进行优化,发现在IPTG浓度为0.4mmol/L、诱导时间为6h、诱导温度为30℃的表达条件下,目的蛋白表达量最高,大部分以包涵体形式表达。将包涵体裂解并用Ni-NTA亲和柱纯化,所得纯化蛋白在复性缓冲液中复性,最终得到有活性的高纯度目的蛋白。

The insect-resisting cry2A gene of transgenic rice from the cloning vector was amplified by polymerase chain reaction（PCR）,and then the PCR products of cry2A gene were inserted into the prokaryotic expression vector pET-30a（ ＋ ）via digestion of both restriction endonucleases EcoRI and Bam HI, thus successfully constructing the recombinant expression plasmid pET-30a （ ＋ ）/Cry2A. The recombinant vector was introduced into E. coli BL21 （DE3）. Through optimization of the induced ex- pression conditions it was found that at 0.4 mmol/L IPTG and 30 ~C for 6 h the target protein was highly expressed in E. coli BL21 mostly as inclusion bodies. The inclusion bodies could be dissolved and purified by Ni-NTA affinity column, then the purified protein was renatured in renaturation buff- er solution and finally the high-purity active protein was obtained.