摘要
为构建禽致病性大肠杆菌(APEC)跨膜输出蛋白基因组文库,本研究以APEC O2菌株基因组DNA为模板,采用基因随机片段PCR方法扩增,纯化约250 bp^500 bp的片段构建APEC O2菌株跨膜输出蛋白基因组文库。共获得约1.8×10~5个克隆容量的基因组文库,经氨苄/卡那/阿拉伯糖平板筛选到318个克隆。ORF及Signal P分析显示318个克隆插入片段均含有信号肽序列,其中,78个克隆预测为膜蛋白;启动子分析显示144个克隆含有完整启动子,除去29个能自主表达的克隆,推测其余115个克隆的启动子在实验培养条件下沉默,可能需要特殊条件诱导才能表达;GO功能分析克隆插入片段所对应全长基因的ORF显示,其参与的生物过程主要集中于定位、转运;从分子功能角度分析,其主要集中于催化和结合;从细胞组成角度分析,其主要集中于细胞膜、周间质、外膜等与外壳相关的结构中。本实验将为进一步研究APEC的致病性机理奠定基础。
To construct gene library encoding exported proteins of avian pathogenic Escherichia coli （APEC）, the genes of APEC exported proteins were random amplified by rPCR method, and the 250 bp to 500 bp PCR products were purified and cloned into pMB-Ara-T vector to construct the library. The results showed that the library contained about 1.8×10^5 independent clones, and 318 positive clones were screened by Amp＋kan＋l.0% arabinse plate. ORF and Signal P analysis indicated that the insert fragments had signal peptide sequence and 78 clones were predicted to be the genes for encoding membrane proteins. Promoter analysis showed that 144 clones carried the complete promoter structure, exception of 29 clones were able to independently express, that the rest of promoters in 115 clones were speculated to remain silent under in vitro culturing conditions which need to be specially induced for gene expressions. GO function analysis showed that the protein expressed from these clones involved mainly in localization, transport, catalytic, combination and cell structures, etc. This study established basis for further study the bioprocess and pathogenic mechanism of APEC.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2016年第5期410-412,共3页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金(31272582
30571380)
关键词
大肠杆菌
跨膜输出蛋白基因
基因文库
克隆分析
Escherichia coli
gene encording exported protein
gene library
clone analysis
