副猪嗜血杆菌5型galE基因缺失株HPS-YA-008ΔgalE的构建及鉴定

Construction and characterizations of a galE gene-deleted strain of Haemophilus parasuis serotype 5

为构建副猪嗜血杆菌5型galE基因缺失株,本研究通过重叠PCR构建了galE基因的上、下游同源臂,然后将其连接到pRE112载体而构建了重组自杀性质粒pRE112ΔgalE;使重组质粒pRE112ΔgalE转化大肠杆菌X7213,与副猪嗜血杆菌接合转移,应用两步法筛选galE基因缺失株HPS-YA-008ΔgalE,用PCR方法验证galE基因的缺失突变。在此基础上进一步研究galE基因缺失株HPS-YA-008ΔgalE的生长特性、药物敏感性、对保育猪的毒力等生物学特性。结果,副猪嗜血杆菌血清5型galE基因缺失株HPS-YA-008ΔgalE构建成功,且缺失株的毒力已经被致弱,对药物的敏感性增加。本试验的研究结果为研制更加安全的副猪嗜血杆菌弱毒疫苗提供了实验材料。

In order to develop a safer vaccine strain for immunization,a galE-deleted strain of Haemophilus parasuis was constructed.The galE gene＇s upstream and downstream as homologous arms were cloned,and ligated by recombinant PCR.The recombinant plasmid pRE112ΔgalE was obtained by ligating the recombinant PCR products into the suicide vector pRE112.The recombinant plasmid pRE112ΔgalE was transformed into E.coli X7213 and conjugal transfer occured when the transformants were mixed with HPS on TSA plates.The galE-deleted strain HPS-YA-008ΔgalE was selected by the two-step method and identified by PCR.The growth properties,drug sensitivity and virulence in nursery pigs of the galE-deleted strain HPS-YA-008ΔgalE were further characterized.The results showed that the galE-deleted mutant strain HPS-YA-008ΔgalE was successfully constructed.The mutant had lower virulence and was sensitive to drugs.