朱砂叶螨几丁质酶TecCht1基因的原核表达与纯化

Prokaryotic expression and purification of chitinase gene TecCht1 from Tetranychus cinnabarinus

【目的】朱砂叶螨是世界范围广泛分布的重要经济害螨,害螨防治十分困难。而几丁质酶是昆虫体内几丁质代谢中分解几丁质的关键酶,在更替新旧表皮和降解围食膜中起着重要的作用,几丁质酶是潜在的螨害生物防治靶标。本研究对朱砂叶螨几丁质酶TecCht1进行原核表达和纯化。【方法】克隆朱砂叶螨TecCht1基因的催化结构域,连接到pColdⅡ载体上,构建pColdⅡ/TecCht1重组表达载体,在大肠杆菌中进行高效表达,采用镍柱纯化目的蛋白,并进行Western Blot检测。【结果】TecCht1在pColdⅡ载体上成功高水平表达,确定最佳诱导表达时间为24h。对TecCht1进行大量表达和纯化,经Western Blot验证,获得纯度较高的目的蛋白。【结论】实现TecCht1体外高效原核表达,获得大量的TecCht1重组蛋白。

【Objective】The carmine spider mite,Tetranychus cinnabarinus(Boisduval),is an important agricultural worldwide pest,and it is an increasing challenge to controlling mites.Chitinase is a key enzyme in insect chitin metabolism,and plays a critical role in molting and degrading peritrophic membranes.Chitinase can be potential target for pest biological control.This study is to express and purify the mite chitinase TecCht1in E.Coli.【Methods】The catalytic domain of TecCht1was cloned and ligated to pColdII vector,and then the pColdⅡ/TecCht1recombinant expression vector was constructed.TecCht1was highly expressed by IPTG induction,and then was purified using Ni-NTA column.The purified protein was verified by Western Blot.【Results】The TecCht1protein was successfully expressed in pColdⅡ,and the optimal induction time was 24h.High quality TecCht1protein was obtained,which were verified by Western Blot.【Conclusion】In the study,TecCht1was highly expressed by IPTG induction and purified TecCht1protein was obtained.