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

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ΔRposΔslyAΔslrp(pTrc-AAL)的生理、生化特性与ΔRposΔslyAΔslrp(p Trc-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8.81×10^(5)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。

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 Salmonella ARposAslyAAslrp(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 characteristics of the recombinant strain ΔRposΔslyAΔslrp(pTrc-AAL)were similar with theΔRposΔslyAΔslrp(pTrc-AAL)strain;Compared with the wild type(WT)strain,the growth rate of the strainΔRposΔslyAΔslrp(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 LDso results indicated that the LDso of the recombinant strainΔRposΔslyAΔslrp(pTrc-AAL)was at least 8.81 ×10^(5) folds higher than that of the WT strain.In conclusion,this study successfully constructed attenuated SalmonellaΔRposΔslyAΔslrp(pTrc-AAL)expressing the exogenous protein AAL,which provide a foundation for further exploration of the AAL biological function,and development of mucosal vaccines targeting Mcells.