产肠毒素大肠杆菌F4ac黏附素新基因sfm缺失株的构建及相关功能验证

发布时间：2018-05-29 16:47

本文选题：sfm基因 + Red同源重组系统　；参考：《中国预防兽医学报》2017年12期

【摘要】：为研究产肠毒素大肠杆菌菌株F4ac(F4ac~+ETEC)菌毛粘附素sfm基因的功能,本研究利用Red同源重组方法对F4ac+ETEC的sfm基因进行敲除,构建缺失株F4acΔsfm。再将sfm基因克隆到表达载体pACYC184中,获得重组质粒pACYC184-sfm,并转化至F4acΔsfm得到回补株F4acΔsfm/psfm。对ETEC野生菌、缺失株和回补株的生长曲线进行了测定和比较,结果显示sfm的缺失和回补对F4ac~+ETEC的生长没有影响;生物被膜定性和定量试验结果一致:均表明sfm在F4ac~+ETEC生物被膜形成的过程中作用不明显;血凝试验和甘露糖敏感性血凝试验结果显示3种类型的菌株均能够与2%猪血红细胞发生凝集反应,且该凝集现象能被D-甘露糖抑制。本研究为进一步研究sfm菌毛粘附素功能奠定了基础。
[Abstract]:In order to study the function of sfm gene of Enterotoxigenic Escherichia coli strain F4ac-F4ac-ETEC, the sfm gene of F4ac ETEC was knockout by Red homologous recombination method, and the missing strain F4ac 螖 sfm was constructed. The sfm gene was cloned into the expression vector pACYC184, and the recombinant plasmid pACYC184-sfm was obtained, and then transformed into F4ac 螖 sfm to obtain the complementary strain F4ac 螖 sfm / psfm. The growth curves of wild bacteria, missing plants and supplementary plants of ETEC were measured and compared. The results showed that the absence and compensation of sfm had no effect on the growth of F4ac- ETEC. The qualitative and quantitative results of biofilm showed that sfm did not play a significant role in the formation of F4ac- ETEC biofilm. The results of hemagglutination test and mannose sensitive hemagglutination test showed that all three strains could agglutinate with 2% porcine red blood cells, and the agglutination could be inhibited by Dmannose. This study laid a foundation for further study of the function of sfm pili adhesion hormone.
【作者单位】：扬州大学兽医学院;江苏省动物重要疫病与人兽共患病防控协同创新中心;江苏省家禽科学研究所;国药集团扬州威克生物工程有限公司;
【基金】：科技部畜禽重大疫病防控与高效安全养殖综合技术研发重点专项(2016YFD0500900);科技部转基因生物新品种培育重大专项(2014ZX08006-001B) 国家自然科学基金(31672579、31270171、31072136、31502075、30771603) 江苏省自然科学基金(BK20150442) 江苏高校优势学科建设工程资助项目
【分类号】：S852.61

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