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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 construct the galE gene deletion mutant of H. parasuis, we constructed the upstream and downstream homology arms of galE gene by overlapping PCR and ligated it to pRE112 vector to construct recombinant suicide plasmid pRE112ΔgalE. pRE112ΔgalE was transformed into Escherichia coli X7213 to conjugate with Haemophilus parasuis. The galE gene deletion mutant HPS-YA-008ΔgalE was screened by two-step method. The deletion mutation of galE gene was verified by PCR. On this basis, the biological characteristics of galE gene deletion strain HPS-YA-008ΔgalE, such as the growth characteristics, drug sensitivity and virulence of piglets, were further studied. As a result, H. parasuis serotype 5 galE gene deletion strain HPS-YA-008ΔgalE was successfully constructed, and the virulence of the deletion strain was already weakened and the sensitivity to the drug was increased. The results of this study provide experimental materials for the development of safer Haemophilus parasuis vaccine.