棉铃虫乙醇脱氢酶5的原核表达和活性分析及其抗血清的制备

Prokaryotic Expression,Activity Analysis and Antibody Preparation of Alcohol Dehydrogenase 5 from Helicoverpa armigera

本课题组前期基于酵母单杂交技术筛选出响应2-十三烷酮诱导CYP6B6过表达的6个调控因子,乙醇脱氢酶5(HaADH5)是其中之一,乙醇脱氢酶是一种代谢乙醇的重要酶类,在哺乳动物中的研究很多,但在昆虫中的研究很少.为了进一步探究HaADH5是否参与P450 CYP6B6的过表达,进而参与棉铃虫对包括杀虫剂在内的外界有毒物质的代谢.本实验将pET32a-HaADH5转化至大肠杆菌Transetta中进行诱导表达,Western blot进一步验证融合蛋白HisHaADH5的表达,通过镍柱纯化获得融合蛋白His-HaADH5,且测定了酶活.用蛋白免疫法制备了鼠抗His-HaADH5的血清,Western blot分析了该抗血清的免疫特异性,继而用此抗体检测了HaADH5在不同组织中的表达.活性分析表明,在NAD+的存在下,融合蛋白可以对不同醇类和甲醛进行脱氢,尤其对异戊醇的脱氢活性最高,活性为1333 U/mg.制备的抗血清效价为1∶409600.Western blot结果表明,该抗血清既能与融合蛋白His-HaADH5结合,也能与棉铃虫体内的HaADH5结合,还发现HaADH5在脂肪体中的表达量最高.原核表达的融合蛋白能代谢异戊醇,制备的抗血清具有较好的免疫特性,这些结果为HaADH5蛋白水平的鉴定以及功能研究提供了重要的检测工具,也有助于我们更好地探索棉铃虫乙醇脱氢酶5的功能.

On the basis of H.armigera CYP6 B6 overexpression response to 2-tridecanone,six regulators in response to 2-tridecan-one were selected by yeast one-hybrid,and ethanol dehydrogenation of Helicoverpa armigera(HaADH5)is one of them.Alcohol dehydrogenase is an important ethanol metabolic enzyme and has been studied mainly in mammals,and a little in insects.We aim to study whether alcohol dehydrogenase 5 is involved in the overexpression of P450 CYP6 B6,and further participates in the metabolism of external toxic substances including insecticides of H.armigera,these results will lay a foundation for effective control of H.armigera.In this study,pET32 a-HaADH5 was transformed into E.coli Transetta for induction expression,the fusion protein His-HaADH5 was further detected by Western-blot,purified using Ni-NTA,and its enzyme activity was measured.The antiserum of H.armigera His-HaADH5 was prepared by protein immunoassay.The titer of the polyclonal antibody was detected by enzyme-linked immune sorbent assay(ELISA).Western blot was used to analyze the immimu-specifity of prepared antiserum,and then used antiserum to detect the expression of HaADH5 in different tissues of H.armigera.Activity analysis showed that the fusion protein could catalyze the dehydrogenation of different alcohols and formaldehyde in the presence of NAD+condition.However,it had the highest metabolic capacity for isoamyl alcohol and its activity was 1333 U/mg.The titer of the polyclonal antibody,which was detected by enzymelinked immune sorbent assay(ELISA),was 1∶409600.Immimu-specifity of polyclonal antibody showed that the prepared antiserum could not only bind to the fusion protein His-HaADH5,but also to HaADH5 in H.armigera,and then used it to detect the expression level of HaADH5 in different tissues of H.armigera,the results showed that the expression level of HaADH5 was highest in the fat body.The fusion protein expressed by prokaryote can metabolize isoamyl alcohol,and the prepared mouse anti-His-HaADH5 antiserum has better immune properties.These results will provide an important detection tool for the identification of HaADH5 protein.These results will help us better investigate the function of HaADH5 of H.armigera.