鸟分枝杆菌中耐受表面活性物质相关基因的分离及功能鉴定

Isolation and Functional Characterization of Mycobacterium avium Gene Related to Surfactant Stress Tolerance

为筛选鸟分枝杆菌中耐受表面活性物质的基因并探究其相关功能,以认识鸟分枝杆菌适应肺部环境的杀(抑)菌机制,本研究构建鸟分枝杆菌基因组文库,采用生物活性水平筛选策略从基因组文库中筛选耐受十二烷基硫酸钠(sodium dodecyl sulfate,SDS)的克隆子,运用生物信息学分析该克隆子完整的开放阅读框及其编码蛋白的二级结构特点。将克隆子的全基因序列与pGEX-4T-3表达质粒连接后转化大肠杆菌,构建过表达目的蛋白的重组菌。检测重组菌分别在含SDS、H2O2、NaNO2和GSNO条件环境下的存活率。结果表明成功构建滴度为1.18×10^4 cfu/mL的鸟分枝杆菌基因组文库,从文库中筛选到一个耐受SDS的基因MAV-4292。生物信息学分析显示,MAV-4292蛋白是一个无信号肽的跨膜蛋白,二级结构以α-螺旋为主。过表达MAV-4292重组菌在2%SDS、5 mmol/L H2O2、20 mmol/L NaNO2和10 mmol/L GSNO体外条件下的存活率均比空载菌显著提高(p<0.05)。结果表明,MAV-4292是鸟分枝杆菌中一个耐受表面活性物质的基因,它编码的蛋白参与了肺部表面活性物质、活性氧(reactive oxygen species,ROS)及活性氮(reaetive nitrogen species,RNS)的胁迫应答,是鸟分枝杆菌的毒力因子。

To understand the killing(inhibiting)mechanism of Mycobacterium avium(MAV)adapting to lung environment by screening of genes which resistant to surfactant stress in Mycobacterium avium,in this research,the Mycobacterium avium genomic library was constructed and the sodium dodecyl sulfate(SDS)tolerant clones were screened from the genomic library by bioactivity level screening strategy,then the complete ORF of the clone and the secondary structure characteristics of its coding protein were analyzed by bioinformatics.The ORF sequence was linked with the pGEX-4T-3 plasmid and transformed into E.coli to construct a recombinant strain which enabled to overexpress the target protein.The survival rates of recombinant strain were detected under the conditions of SDS,H2O2,NaNO2 and nitroso glutathion(GSNO)respectively.The results showed that a 1.18×10^4 cfu/mL genomic library of Mycobacterium avium was successfully constructed.A SDS tolerant gene named MAV-4292 was screened from the library.Bioinformatics analysis showed that MAV-4292 protein was a transmembrane protein without signal peptide and its secondary structure was mainly alpha-helix.The survival rate of recombinant strain which overexpressed MAV-4292 in vitro was significantly higher than that of the control strain under 2%SDS,5 mmol/L H2O2,20 mmol/L NaNO2 and 10 mmol/L GSNO conditions(p<0.05).MAV-4292 is a novel gene from Mycobacterium avium which resistants to surfactant stress.The protein encoded by MAV-4292 is involved in the stress response of pulmonary surfactant,reactive oxygen and reactive nitrogen,and is an important virulence factor of Mycobacterium avium.