E9基因的克隆及蛋白的原核表达和纯化

Cloning of E9 gene and expression of its protein

目的通过分子生物学的手段从库蚊幼虫中获得E9酯酶基因,构建表达载体PET-32a E9,实现在大肠杆菌中高效表达可溶性蛋白并将其纯化。方法通过RT-PCR技术从库蚊幼虫中特异性扩增E9酯酶基因,将目的基因通过基因连接反应与PET-32a载体连接,重组质粒进行基因序列测序,构建PET-32a和目的基因的高表达质粒,异丙基-B-D-硫代半乳糖(isopropylthiogalactoside,IPTG)诱导表达,Ni2+柱纯化,Western-blot法检测纯化的目的蛋白。结果成功获得库蚊幼虫E9酯酶基因,基因测序显示基因突变率为0,表达质粒构建双酶切(BamHⅠ和NcoⅠ)可见目的基因的条带,经IPTG诱导表达,获得带HIS标签的融合蛋白E9,相对分子质量为80.6×103,经Western-blot分析证实抗原性正确。结论 E9酯酶基因可以通过基因工程手段获得体外高效表达,为其功能的研究、抑制剂的筛选以及农药污染的环境治理提供基础。

Objective To clone the gene of E9 from Culex pipiens, recombinant with PET - 32a vector and realize the expression of its protein in vitro. Methods Used of RT - PCR techniques,the E9 gene was cloned from the larva of Culex pipiens by species-specific primers, and the objective gene was bonded to PET - 32a vector through gene cou- pled action, recombinant plasmids were sequencing, and the gene was cloned into PET -32a vector, then the recombi- nant plasmids were induced by isopropyhhiogalactoside （IPTG） to express the proteins. The proteins were purified by Ni-eolumn and detected by using Western-blot test. Results The E9 gene was successfully cloned from Culex pip- lens, adding the enzyme site. No gene mutations were detected in the gene after sequencing. The expression plasmids were cut by the two enzyme （ BamH I and Nco I ）, the target band was seen on the results of electrophoresis. The ex- pression plasmids were induced by IPTG and got the purified HIS fusion protein E9, which relative molecular weight was 80.6 x 103. The results of Western-blot test confirmed that the antigenicity of the protein was precise. Conclusion E9 proteins can be expressed in a high efficiency in vitro using genetic engineering , so it provides a good basis for further research on its function, inhibitor screening and pesticide pollution environment management.