布鲁氏菌毒力缺失疫苗株的构建

Construction of Virulence Deleted Vaccine Strain of Brucella

本研究在S19弱毒菌株的基础上通过一系列手段获得布鲁氏菌Bp26蛋白、Bmp18蛋白双缺失株,待遗传学鉴定合格后用于小鼠实验。将120只小鼠分为实验组、S19对照组和空白对照组3组,将双基因缺失株和S19弱毒株培养24 h后离心收集菌体,使用PBS清洗重悬后进行细菌计数,实验组小鼠腹腔接种检验合格的双基因缺失株5×10~5 CFU的剂量,病菌对照组腹腔接种S19弱毒菌株5×10~5 CFU的剂量,空白对照组腹腔注射同等体积的PBS溶液,观察分析Bp26蛋白做抗原的免疫原性、双基因缺失菌株的毒力情况和免疫应答情况。最终构建出能够区分自然免疫和人工免疫并具低毒性的布鲁氏弱毒菌株。双基因缺失株具有遗传稳定性,采用Bp26蛋白做抗原对3组小鼠的免疫原性测定,表明实验组40只小鼠均未表现出免疫原性,S19对照组40只小鼠中有28只表现出了免疫原性,占70%,空白对照组40只小鼠均未表现出免疫原性,双基因缺失株与原S19弱毒株的免疫应答基本一致,且更为安全。

This study obtained Brucella Bp26 protein and Bmp 18 double-deleted strain through a series of means based on S19 attenuated strain, which was used for the genetic test of mice after the qualification of genetic identification. 120 mice were divided into three groups： the experimental group, S19 control group and the blank control group. The double gene deletion strain and S19 attenuated strain were collected after 24 h culture by centrifugation, and then we took bacteria counting after the clearance and resuspension of PBS. Mice in experimental group were intraperitoneally inoculated with double gene deletion strain of 5×10^5 CFU dosage, and those in S 19 control group and blank control group were intraperitoneally inoculated with S 19 attenuated strain of 5×10^5 CFU dosage and PBS solution of the same volume, respectively, from which we observed and analyzed the immunogenicity of Bp26 protein as antigen and the virulence and immune response of double gene deletion strain. Finally, we constructed a Brucella attenuated strain which could distinguish between natural and artificial immune and was of low toxicity. Double gene deletion strains were genetically stable. Immunogenicity of three groups of mice were measured by Bp26 protein antigen, of which the results showed that all 40 mice in the experimental group did not show immunogenicity, 28 of the 40 mice in S19 control group showed immunogenicity, accounting for 70%, and the 40 mice in the blank control group all did not show immunogenicity, and the immune response of double gene deletion strain was basically consistent with that of the original S 19 attenuated strain, and it was safer.