淋病奈瑟菌外膜蛋白Porin B通过抑制CD4^(+)T细胞增殖调控机体免疫应答的分析

Analysis of Neisseria gonorrhoeae outer membrane protein Porin B regulates the immune response by inhibiting the proliferation of CD4^(+)T-cells

目的:探究淋病奈瑟菌(NG)外膜蛋白Porin B能否通过抑制CD4^(+)T细胞增殖来调控机体免疫应答。方法:从GenBank中获取NG菌株的Porin B基因的DNA序列,以NG菌株FA1090的DNA为模板,扩增目Porin B基因,并采用1.5%琼脂糖凝胶电泳对Porin B基因进行检测。将得到的PCR产物用Bam HⅠ和Eco RⅠ双酶切后克隆到酶切的载体pET30a中得到重组质pET30a-Porin B,并采用1.5%琼脂糖凝胶电泳对pET30a-Porin B进行检测。从C57BL/6小鼠的脾脏和淋巴结中分离CD4^(+)T细胞,分为对照组、低剂量pET30a-Porin B(LPB)组、中剂量pET30a-PorinB(MPB)组和高剂量PET30a-PorinB(HPB)组,对照组不做任何处理,LPB组、MPB组和HPB组分别与低、中、高剂量的pET30a-Porin B共培养。采用流式细胞术检测各组CD4^(+)T细胞增殖及凋亡情况并进行比较分析。结果:将PCR扩增产物进行1.5%琼脂糖凝胶电泳测定,在1.0 kb处得到一条清晰的条带,表明扩增出了正确的Porin B基因;将pET30a-Porin B进行1.5%琼脂糖凝胶电泳测定,出现1.0 kb的目的片段和5.4 kb的载体片段,说明重组质粒pET30a-Porin B构建成功。与对照组比较,LPB组、MPB组和HPB组的CD4^(+)T细胞增殖指数均显著下降,且随着重组质粒pET30a-Porin B的剂量增加,CD4^(+)T细胞增殖指数下降显著;与对照组比较,LPB组、MPB组和HPB组的CD4^(+)T细胞凋亡比例均显著增加,且随着重组质粒pET30a-Porin B的剂量增加,CD4^(+)T细胞凋亡比例增加更显著。结论:Porin B能够抑制CD4^(+)T细胞的增殖并促进其凋亡,且呈剂量依赖性。这表明Porin B能够通过抑制CD4^(+)T细胞增殖来调控机体免疫应答。后期还需大量研究及临床试验来证明Porin B的临床效果,为临床NG疫苗的研究提供基础临床数据。

Objective To investigate whether the outer membrane protein Porin B of Neisseria gonorrhoeae can regulate the immune response by inhibiting the proliferation of CD4^(+)T cells.Method The DNA sequence of the Porin B gene of NG strains was obtained from GenBank,and the DNA of NG strain FA1090 was used as a template to amplify the Porin B gene,and the Porin B gene was detected by 1.5%agarose gel electrophoresis.The PCR product was double digested with Bam HⅠand Eco RⅠand cloned into the digested vector pET30a to obtain the recombinant plasmid pET30a-Porin B.The pET30a-Porin B was detected by 1.5%agarose gel electrophoresis.CD4^(+)T cells were isolated from spleens and lymph nodes of C57BL/6 mice and divided into control,LPB,MPB and HPB groups.The control group was not treated in any way,and the LPB,MPB and HPB groups were co-cultured with low,medium and high doses of pET30a-Porin B,respectively.Flow cytometry was used to detect the proliferation and apoptosis of CD4^(+)T cells in each group and comparatively analysed.Results The PCR amplification products were subjected to 1.5%agarose gel electrophoresis,and a clear band at 1.0 kb was obtained,indicating that the correct Porin B gene was amplified.pET30a-Porin B was subjected to 1.5%agarose gel electrophoresis,and 1.0 kb of the target fragment and 5.4 kb of vector fragment appeared,which indicated that the recombinant plasmid pET30a-Porin B was successfully constructed.Porin B was successfully constructed.The proliferation index of CD4^(+)T cells in LPB,MPB and HPB groups were significantly decreased compared with the control group,and with the increase of the dose of recombinant plasmid pET30a-Porin B,the proliferation index of CD4^(+)T cells was decreased about significantly.The apoptotic proportion of CD4^(+)T cells in LPB,MPB and HPB groups were significantly increased compared with the control group,and with the increase of the dose of recombinant plasmid pET30a-Porin B dose increased,the increase in the proportion of CD4^(+)T cell apoptosis was more significant.Conclusion Porin B can inhibit the proliferation of CD4^(+)T cells and promote their apoptosis in a dose-dependent manner.This indicates that Porin B can regulate the body′s immune response by inhibiting CD4^(+)T cell proliferation.A large amount of research and clinical trials are needed in the later stage to demonstrate the clinical efficacy of Porin B and provide basic clinical data for the study of NG vaccines in clinical practice.