甘肃省乳房炎奶牛乳源肠球菌的分离、药敏试验及毒力基因原核表达载体的构建

doi:10.16431/j.cnki.1671-7236.2020.10.039

摘要/Abstract

摘要： 为分离临床型奶牛乳房炎中的肠球菌，检测其耐药性和携带毒力基因的情况，本试验采集了甘肃省东、中和西部3个地区41头临床型乳房炎奶牛的奶样93份，并构建了肠球菌毒力基因的原核表达载体。试验使用选择性培养基分离纯化肠球菌，16S rRNA和生化试验结合的方法鉴定所分离菌株的种；选取16种抗菌药进行药敏试验；常规PCR方法检测11种毒力基因的携带情况，最后对具有免疫原性的毒力基因进行原核表达载体的构建。鉴定结果显示，93份乳样中18株为肠球菌，并分为9种。药敏结果显示，分离株多数为多重耐药菌（multiple resistant bacteria，MDR），占88.89%，未发现耐万古霉素菌株（vancomycin-resistant enterococcus，VRE），万古霉素敏感率94.12%，所有分离株至少对一种抗菌药耐药。PCR检测结果表明，11种毒力基因均有检出，cob基因检出率最高（44.44%），efaA、hyl、ccf和esp基因检出率分别为33.33%、27.78%、27.78%和22.22%，Asa1、cylA、EF3314和gelE基因检出率均为16.67%，Ace和cylM基因检出最低（11.11%）；毒力基因组合因菌种不同而存在差异，选择具有免疫原性的Ace和gelE基因成功构建出原核表达载体pET32a-Ace和pET32a-gelE。本试验为后续绘制3个地区奶牛乳房炎流行病学区域谱以及制备相应抗体和亚单位疫苗提供了基础数据及生物材料。

关键词: 奶牛乳房炎; 肠球菌; 耐药性; 毒力因子; 原核表达

Abstract: The aim of this study was to isolate Enterococcus in clinical dairy cow mastitis,detect its drug resistance and virulence genes,a total of 93 milk samples were collected from 41 dairy cosw with clinical mastitis in eastern,central and western regions of Gansu province,and then construct a prokaryotic expression vector for virulence genes.This experiment used selective medium to isolate and purify bacteria.16S rRNA and biochemical experiments combined method to identify the isolated strains.16 antibiotics were selected for drug sensitivity test,and conventional PCR method was used to detect the carrying of 11 virulence genes.Finally,the detected virulence genes with immunogenicity were selected for the construction of prokaryotic expression vectors.The separation and identification results showed that 18 strains of Enterococcus were isolated and identified from the 93 milk samples,which were divided into 9 species.Drug susceptibility results showed that most of the isolates were multiple resistant to bacteria,accounting for 88.89%.No vancomycin-resistant Enterococcus was found,vancomycin sensitivity rate was 94.12%,and all isolates were resistant to at least one antibiotic.Virulence gene test results demonstrated that 11 virulence genes were detected,the detection rate of cob gene (44.44%) was the highest,the detection rates of efaA,hyl,ccf and esp genes were 33.33%,27.78%,27.78% and 22.22% respectively,the detection rates of Asa1,cylA,EF3314 and gelE genes were all 16.67%,while Ace and cylM genes had the lowest detection rate (11.11%).The virulence genes combination was different due to different strains.Ace and gelE genes with immunogenicity were selected and the prokaryotic expression vector pET32a-Ace and pET32a-gelE were successfully constructed.The results provided basic data and biological materials for subsequent mapping of regional epidemiology of cow mastitis in three regions and preparation of corresponding antibodies and subunit vaccines.

Key words: dairy cow mastitis; Enterococcus; drug resistance; virulence factor; prokaryotic expression

中图分类号:

S857.2⁺6

申玉龙, 门倩云, 陈婷婷, 李宗帅, 李海江, 杨洋, 张勇, 赵兴绪. 甘肃省乳房炎奶牛乳源肠球菌的分离、药敏试验及毒力基因原核表达载体的构建[J]. 中国畜牧兽医, 2020, 47(10): 3389-3400.

SHEN Yulong, MEN Qianyun, CHEN Tingting, LI Zongshuai, LI Haijiang, YANG Yang, ZHANG Yong, ZHAO Xingxu. Isolation,Drug Sensitivity Test and Construction of Prokaryotic Expression Vector of Virulence Genes of Enterococcus from Dairy Cow with Mastitis in Gansu Province[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(10): 3389-3400.

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