携带Lipocalin 2基因重组减毒沙门菌的构建及其对分枝杆菌生长影响的研究

Construction of recombinant attenuated Salmonella typhimurium carrying the lipocalin 2 gene and its effect on the growth of mycobacteria

摘要:

目的：探讨携带脂质运载蛋白2（lipocalin 2，Lcn2）的减毒沙门菌对分枝杆菌生长的影响。方法：以巨噬细胞RAW264.7提取RNA逆转录为cDNA为模版，采用PCR法扩增Lcn2基因，定向克隆到质粒pBudCE4.1-orim的多克隆位点中，构建重组质粒 pBudCE4.1-orim-Lcn2；将重组质粒转染RAW264.7细胞，用RT-qPCR法检测Lcn2的mRNA 相对表达量，并以空白载体为对照组；将重组质粒转入减毒沙门菌SL7207得到重组菌株 SL7207-pBudCE4.1-orim-Lcn2；重组菌感染RAW264.7细胞，Western blot检测Lcn2的蛋白表达，RT-qPCR法检测Lcn2的mRNA的相对表达量，并以空载菌组为对照；7H10平板上通过菌落计数观察重组菌对感染 RAW264.7 细胞的卡介苗（Bacille calmette-guerin，BCG）生存的影响，以空载菌组为对照。用重组菌灌胃小鼠，检测脾组织Lcn2和血清IFN-γ水平，以空载菌和生理盐水为对照组。结果：PCR扩增出Lcn2基因；重组质粒经双酶切及基因测序鉴定正确。重组菌感染 RAW264.7细胞Lcn2的蛋白表达量较对照组增加（P=0.000）。RT-qPCR 法检测重组菌、重组质粒Lcn2 mRNA表达较对照组明显升高（P=0.000，F=5.281；P=0.000，F=22570）。7H10平板上重组菌组BCG数量与空载菌组相比明显减少（P=0.000，F=11.41）。重组菌组与空载对照菌组相比小鼠脾组织 Lcn2表达升高（P=0.000，F=4.449），重组菌组与生理盐水组血清IFN-γ升高（P=0.004，F=15.27）。结论：成功构建了携带Lcn2基因的重组减毒沙门菌。重组菌感染RAW264.7细胞后Lcn2的表达增加，对感染RAW264.7的BCG存活起到一定的抑制作用。重组菌灌胃后小鼠脾组织Lcn2表达增加，血清IFN-γ含量增加，小鼠免疫状态有利于结核病转归。该研究为进一步研究脂质运载蛋白和结核分枝杆菌感染的关系及分子机制打下基础。

Abstract:

Objective：To construct attenuated Salmonella typhimurium carrying the lipoprotein 2（Lcn2） gene and to investigate its effect on the growth of mycobacteria. Methods：RNA was extracted from macrophage RAW264.7 cells and reverse transcribed into cDNA. The Lcn2 gene was amplified by PCR using the cDNA as a template and inserted into the multiple cloning sites of the plasmid pBudCE4.1-orim to construct the recombinant plasmid pBudCE4.1-orim-Lcn2. The recombinant plasmid was trans－fected into RAW264.7 cells. The mRNA expression of Lcn2 was measured by RT-qPCR，with the empty plasmid for a control group. Then the recombinant plasmid was transfected into the attenuated Salmonella typhimurium SL7207 to obtain the recom－binant strain SL7207-pBudCE4.1-orim-Lcn2. RAW264.7 cells were infected with the recombinant strain. The protein expres－sion of the Lcn2 gene was measured by Western blot，and its mRNA expression was measured by RT-qPCR，with the empty strain for a control group. Bacterial colonies were counted on the 7H10 plate to observe the effect of the recombinant strain on the survival of bacille Calmette-Guérin（BCG） in RAW264.7 cells，with the empty strain for a control group. Mice were intragastrically given the re－combinant strain to measure the Lcn2 expression in the spleen and the interferon gamma（IFN-γ） level in serum，with the empty strain and normal saline for control groups. Results：The Lcn2 gene was amplified by PCR，and the recombinant plasmid was identified by double-enzyme digestion and DNA sequencing. The protein expression of Lcn2 in RAW264.7 cells infected with the recombinant strain was significantly increased compared with the control group（P=0.000）. The RT-qPCR showed that the mRNA expression of Lcn2 in the recombinant strain group and the recombinant plasmid group was significantly higher than that in the control groups（F=5.281，P=0.000；F=22.570，P=0.000）. The colony number of BCG on the 7H10 plate in the recombinant strain group was significantly reduced compared with the control group（F=11.41，P=0.000）. The expression of Lcn2 in mouse spleen tissues and the level of IFN-γ in serum were significantly higher in the recombinant strain group than in the control groups（F=4.449，P=0.000；F=15.27，P=0.004）. Conclusion：Recombinant attenuated Salmonella typhimurium carrying the Lcn2 gene was successfully constructed. The expression of Lcn2 increased in RAW264.7 cells infected with the recombinant strain，which inhibited the survival of BCG in the cells. The expres－sion of Lcn2 in mouse spleen tissues and the IFN-γ level in serum were elevated after the mice were intragastrically given the recom－binant strain，so that the immune state of mice was beneficial to anti-tuberculosis outcome. This study lays a foundation for further study of the relationship between lipid carrier proteins and Mycobacterium tuberculosis infection and its molecular mechanism.