表达AAL蛋白重组减毒猪霍乱沙门菌的构建及生物学特性评价
Construction and biological evaluation of recombinant attenuated Salmonella choleraesuis expressing AAL protein
蒋华正 1黄鑫 2赵靖 2杨珊珊 2杨静 2王金泉 3刘光亮1
作者信息
- 1. 新疆农业大学动物医学学院,新疆乌鲁木齐 830000;中国农业科学院兰州兽医研究所动物疫病防控全国重点实验室,甘肃兰州 730046
- 2. 中国农业科学院兰州兽医研究所动物疫病防控全国重点实验室,甘肃兰州 730046
- 3. 新疆农业大学动物医学学院,新疆乌鲁木齐 830000
- 折叠
摘要
为构建表达橙黄网孢盘菌凝集素(AAL)的重组减毒猪霍乱沙门菌,将AAL基因克隆至原核表达载体pTrc-His2A,并通过电穿孔的方法将重组质粒pTrc-AAL转化至减毒猪霍乱沙门菌ΔRposΔslyAΔslrp内,获得了重组减毒沙门菌ΔRposΔslyAΔslrp(pTrc-AAL),利用Western-blot检测AAL蛋白的表达,并对重组菌株的生物学特性进行评价.结果显示,携带AAL基因的重组减毒沙门菌ΔRposΔslyAΔslrp(pTrc-AAL)构建成功并表达AAL蛋白;重组菌株Δ RposΔslyAΔslrp(pTrc-AAL)的生理、生化特性与ΔRposΔslyAΔslrp(pTrc-His2A)菌株相似;与野生株(WT)相比,重组菌株ΔRposΔslyAΔslrp(pTrc-AAL)在对数期的生长速度略慢于野生株,且对抗生素氨苄西林和阿莫西林敏感,而运动性和生化特性与野生株相比无显著变化;LD50结果显示,重组菌株ΔRposΔslyAΔslrp(pTrc-AAL)的LD50较野生株提高了 8.81 ×105倍以上.综上所述,本研究成功构建了表达外源蛋白AAL的重组减毒沙门菌ΔRposΔslyAΔslrp(pTrc-AAL),为深入探究AAL生物学功能和开发靶向M细胞的黏膜疫苗奠定了基础.
Abstract
In order to construct a recombinant attenuated Salmonella choleraesuis expressing the Aleuria aurantia lectin(AAL),the AAL gene was cloned into the prokaryotic expression vector pTrc-His2A.The recombinant plasmid pTrc-AAL was transformed into the attenuated Salmonella cholerae-suis ΔRposΔslyAΔslrp strain by electroporation to obtain recombinant attenuated SalmonellaΔRposΔslyAΔslrp(pTrc-AAL).The expression of AAL protein in the recombinant strain was identified by Western-blot,and the biological characteristics of the strain were evaluated.The results showed that the recombinant attenuated Salmonella ΔRposΔslyAΔslrp(pTrc-AAL)carrying AAL gene was successfully constructed,and AAL protein could be expressed efficiently;the physiological and biochemical charac-teristics of the recombinant strain ΔRposΔslyAΔslrp(pTrc-AAL)were similar with the ΔRposΔslyAΔslrp(pTrc-AAL)strain;Compared with the wild type(WT)strain,the growth rate of the strain ΔRposΔslyAΔslrp(pTrc-AAL)was slower than WT strain in the logarithmic phase.Similarly,the motility and biochemical characteristics of the strain were also not significantly changed compared with the WT strain.However,the strain ΔRposΔslyAΔslrp(pTrc-AAL)was sensitive to the antibiotics ampicillin and amoxicillin.The LD50 results indicated that the LD50of the recombinant strain ΔRposΔslyAΔsslrp(pTrc-AAL)was at least 8.81 X 105 folds higher than that of the WT strain.In conclusion,this study successfully constructed attenuated Salmonella ΔRposΔslyAΔsllrp(pTrc-AAL)expressing the exogenous protein AAL,which provide a foundation for further exploration of the AAL biological function,and development of mucosal vac-cines targeting Mcells.
关键词
橙黄网孢盘菌凝集素(AAL)/猪霍乱沙门菌/生物学特性Key words
Aleuria aurantia lectin(AAL)/Salmonella choleraesuis/biological characterization引用本文复制引用
基金项目
国家自然科学基金区域联合项目(U22A20522)
出版年
2024
国家科技期刊平台
NSTL
万方数据