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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 obtain the E9 esterase gene from Culex pipiens larvae by molecular biology and to construct the expression vector PET-32a E9 to express and purify the soluble protein in Escherichia coli. Methods The E9 esterase gene was amplified from the larvae of Culex pipiens pallens by RT-PCR. The target gene was ligated with PET-32a vector through gene ligation reaction. The recombinant plasmids were sequenced to construct PET-32a and the target gene Expression plasmid, induced by isopropylthiogalactoside (IPTG), purified by Ni2 + column and purified by Western-blot. Results The E9 esterase gene of Culex pipiens pallens larvae was successfully obtained. The gene mutation rate was 0 and the expression of the gene was double digested (BamHⅠ and NcoⅠ). The target gene was expressed and induced by IPTG to obtain the HIS tagged fusion protein E9, the relative molecular mass of 80.6 × 103, confirmed by Western-blot analysis of antigenicity. Conclusion E9 esterase gene can be efficiently expressed in vitro by genetic engineering, providing a basis for its function, screening of inhibitors and environmental management of pesticide contamination.