沙门氏菌invH基因的重组表达与免疫保护效果研究  

Expression and immunogenicity study of invH gene of Salmonella typhimurium

作　　者：王小龙[1,2] 张伟华 牛钟相[1,2] 

机构地区：[1]山东农业大学动物医学院,山东泰安271018 [2]山东省动物生物技术与疫病防治重点实验室,山东泰安271018 [3]山东华牧天元动物保健品有限公司,山东济南250002

出　　处：《中国兽医科学》2011年第8期799-805,共7页Chinese Veterinary Science

基　　金：国家"十一五"科技支撑计划项目(2006BAD06A11)

摘　　要：在沙门氏菌invH基因重组表达的基础上,研究invH基因表达蛋白的免疫保护功能。采用PCR技术对鼠伤寒沙门氏菌DT104株的入侵基因H进行扩增,将其克隆到原核表达载体pGEX-4T-3中,将重组克隆进行融合蛋白表达、纯化和Western-blot检测,用油乳剂制备融合蛋白疫苗,将该疫苗通过3种不同剂量(40μg/只、60μg/只、80μg/只)免疫注射小鼠进行免疫保护效果测定,同时设油乳剂灭活疫苗组和PBS空白对照组,采用黏附与黏附抑制试验对抗血清的活性进行分析。结果表明,构建的重组克隆成功表达了44ku的融合蛋白GST-invH,且该蛋白具有良好的免疫原性。3种剂量重组蛋白疫苗间的免疫效果差异不显著,均能提高小鼠体内IL-4和IFN-γ的含量,与空白对照组差异显著(P<0.05);油乳剂灭活疫苗组的保护率为90%,3种剂量重组蛋白疫苗组的保护率均在60%以上,虽然重组蛋白疫苗的保护效果不及油乳剂灭活疫苗,但与空白对照组差异均极显著(P<0.01)。体外黏附抑制试验结果显示,抗融合蛋白GST-invH的抗体能抑制沙门氏菌对靶细胞的黏附。表明,GST-invH融合蛋白具有良好的免疫保护性。The objective of the present study was to obtain subunit vaccine of invH gene of Salmonella typhimurium and to evaluate its immunogenicity. Gene of invasionH（invH） from S. typhimurium DT104 amplified by polymerase chain reaction（PCR） was cloned into pGEX-4T-3 getting the recombinant pGEX/ invH,with the invH coexpressed with GST. Mice were injected with three different doses of fusion protein with oil emulsion adjuvant,and two groups were injected with inactivated vaccine and PBS respectively, to evaluate the immune efficacy. The bioactivity of antisera was detected by adherence inhibition assay. Western-blotting analysis indicated that the fusion protein was 44 ku in molecular weight and had immunological activity. We immunized with different doses of the fusion protein invH to mice, challenged with 100-fold LD50 amount of S. typhimurium DT104（4.23〈 107 CFU） ,and found that the survive rate of protein vaccine groups was more than 60% （P〈0. 01）, which are lower than that of inactivated vaccine, but significantly higher than that of control group. And the three groups of protein vaccine all raised levels of mice serum IL-4 and IFN-γ （P〈0.05）. The antiserum of expressed protein can inhibit adherence activity of S. typhimurium DT104 to cell surface by adherence inhibition assay. In conclusion,invH has certain immunogenicity and can offer partial protection against high dose challenge, which provides a theoretical basis for the prevention of S. typhimurium.

关 键 词：沙门氏菌 invH基因 融合表达 免疫保护 黏附抑制 

分 类 号：S852.612[农业科学—基础兽医学]