单增李斯特菌溶血素LLS/LLO缺失株的构建及小鼠免疫保护性研究

Construction of the listeriolysin LLS/LLO deletion strains of Listeria monocytogenes and their immune protection in mice

旨在探究单增李斯特菌溶血素S/O(LLS/LLO)对单增李斯特菌(LM)的部分生物学特性影响及单增李斯特菌溶血素S/O基因缺失株的免疫原性。利用同源重组技术在本实验室已成功构建并保存的LM90SB2Δlls菌株基础上构建LM90SB2Δlls-Δllo,并绘制LM90SB2和LM90SB2Δlls-Δllo的生长曲线,测定半数致死量(LD_(50))、组织载菌量(同时采用灌服和腹腔注射方式)等,进行小鼠免疫保护性研究,分析溶血素基因llsB/hly缺失对LM的生长特性以及毒力的影响。结果:成功构建LM90SB2Δlls-Δllo缺失株,且能在体外稳定遗传。测定生长曲线,LM90SB2的生长活性略高于LM90SB2Δlls-Δllo;LM90SB2的LD_(50)为3.65×10^(7.23)CFU,LM90SB2Δlls-Δllo的LD_(50)大于2.7×10^(9)CFU;通过灌服及腹腔注射,与LM90SB2相比,LM90SB2Δlls-Δllo在小鼠脑、肝、脾中的载菌量均明显减少,差异极显著(P<0.01)。抗体含量除第21天亲本株略高于缺失株,其余天数缺失株均略高于亲本株;通过小鼠免疫保护率结果分析,2株菌免疫后小鼠存活率均为75%,对照组小鼠存活率为30%。本研究表明,llsB/hly基因缺失对LM90SB2的毒力有明显影响,但并不会影响LM的免疫原性,缺失株LM90SB2Δlls-Δllo对亲本株LM90SB2的侵袭具有良好的免疫保护效果。

This study was to explore the effect of listeriolysin S/O (LLS/LLO) on some biological characteristics of Listeria monocytogenes(LM) and to understand the immunogenicity of the S/O gene deletion strain of L.monocytogenes.The homologous recombination technology was used to construct LM90SB2Δlls-Δllo based on the LM90SB2Δlls strain that had been successfully constructed and stored in our laboratory.Then,the growth curve of LM90SB2 and LM90SB2Δlls-Δllo was drawn;and some of their biological characteristics,including their LD_(50)and the bacterial load of them in the brain,liver and spleen,were determined.Next,immune protection was performed on mice to analyze the influence of the lls B/hly gene on the growth characteristics and virulence of LM.The results were as follows:An LM90SB2Δlls-Δllo deletion strain was successfully constructed,and it could be inherited stably in vitro.The growth curve showed that the growth activity of LM90SB2 was slightly higher than that of LM90SB2Δlls-Δllo,The LD_(50)of LM90SB2 was 3.65×10^(7.23) CFU,but there was an LD_(50)value greater than 2.7×10^(9) CFU for LM90SB2Δlls-Δllo.Compared with LM90SB2 by oral administration and intraperitoneal injection,the bacterial load of LM90SB2Δlls-Δllo in the brain,liver and spleen of the mice was significantly lower (P<0.01).In terms of antibody content,that of the parent strain on the 21st day of the experiment was slightly higher than that of the deletion strain,but on all the other days,the content of the deletion strain was slightly higher than that of the parent strain.The results of the immune protection in the tested mice showed that the survival rate of mice vaccinated by the two strains was 75%,and that of the control mice was 30%.The present research indicated that the lls B/hly gene was able to directly or indirectly regulate,to a certain extent,some of the biological characteristics of LM90SB2,that the deletion of the lls B/hly gene did not affect the immunogenicity of LM,that the deletion strain LM90SB2Δlls-Δllo had a good immune protection against the invasion of the parent strain LM90SB2;however,whether the strain could be used as a vaccine candidate strain would require further experimental research.