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当前位置: 首页 > 文章 > 致鹅卵黄性腹膜炎大肠杆菌cya、crp双基因缺失株构建及部分生物学特性分析 中国兽医学报 2013,33 (9) 1358-1363
Position: Home > Articles > Construction and characterization of cya,crpdeletion mutants from Escherichia coli causing goose salpingitis and peritonitis Chinese Journal of Veterinary Science 2013,33 (9) 1358-1363

致鹅卵黄性腹膜炎大肠杆菌cya、crp双基因缺失株构建及部分生物学特性分析

作  者：
王锴;张志强;朱春红;姜露;朱国强
单  位：
扬州大学兽医学院
关键词：
Red重组系统;cya基因突变;cya、crp双基因突变
摘  要：
利用λ噬菌体Red同源重组系统构建致鹅卵黄性腹膜炎大肠杆菌国内分离株G8107、G803、G8120和非致病性大肠杆菌J5的cya、crp基因缺失株,检测所得突变株的部分生物学特性,为大肠杆菌减毒活疫苗的研制提供新思路。首先,以pKD3质粒为模板扩增带有与目的基因(cya和crp)两侧序列同源的氯霉素抗性基因PCR片段;其次将PCR扩增产物电转化入含有pKD46质粒的野生型毒株感受态细胞中,获得目的基因被氯霉素抗性基因同源替换的缺失株;利用携带FLP位点特异性重组酶的温度敏感性质粒pCP20去除上述缺失株中的抗性基因标志,进一步结合PCR扩增和DNA测序,证明基因缺失株的正确构建,本试验成功构建上述各株大肠杆菌的△cya基因缺失株和△cyacrp双基因缺失株。生长试验、酵解试验以及毒力检验试验表明,相对于亲本株,上述缺失株生长速度减慢,分解利用糖的能力较差,对1日龄雏鸡的毒力显著降低,本试验结果初步证实所构建的缺失株可作为减毒疫苗候选株使用。
译  名：
Construction and characterization of cya,crpdeletion mutants from Escherichia coli causing goose salpingitis and peritonitis
作  者：
WANG Kai;ZHANG Zhi-qiang;ZHU Chun-hong;JANG Lu;ZHU Guo-qiang;College of Veterinary Medicine,Yangzhou University;Gansu Agri cultural University;Jiangsu Institute of Poultry Science;
关键词：
Red recombination system;;cya mutant;;cya crp double deletion mutant
摘  要：
In this study,the wild type avian pathogenic Escherichia coli(APEC)isolates G8107,G803,G8120,causing goose salpingitis and peritonitis,and nonpathogenic Escherichia coli(E.coli)J5 were selected as the manipulation prototype for cyaand crpgenes deleting.We constructed the double mutants with theλRed-based recombination system,and then compared some related bio-characteristics among the mutants and the parent strains,aiming a new way of creating effective attenuated live vaccine candidate against APEC.Based on the original sequences of cya,crp genes clusters in the GenBank,we generated PCR products by using primers with the homologies extension of target gene to be deleted and template plasmid pKD3 carrying selectable antibiotic chloramphenicol resistance(cat)gene that is flanked by FRT(FLP recognition target)sites.Then the purified PCR products were electroporated into the wild type isolates containing plasmid pKD46(with an inducible promoter),which was induced under a certain concentration of L-arabinose to encode and express proficientλRed recombinase.By using exogenous DNA fragment,under control of theλRed-based recombination system,the corresponding△cya::cat or△crp::cat deletion mutant was successfully constructed in the flanking homologies,after selection,a helper plasmid pCP20,showing temperature-sensitive and expressing the FLP recombinase,was taken for acting directly on the repeated FRT sites to eliminatethe resistance gene located in the mutant in the second recombination.The mutant obtained was further confirmed by PCR amplification and sequencing.Finally,we successfully obtained the cyaand cyacrpgenes mutants.Compared with the wild type isolate,the mutants,especially the cya,crpdouble mutants,grew slowly during all stages of growth,and showed the inability on catabolism of lactose,maltose,mannitol,and attenuated virulence to 1-day-old chicken.

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