沙门菌质粒毒力基因spvB突变株的构建及其对上皮细胞自噬的影响

发布时间：2018-06-09 21:38

  本文选题：沙门菌 + 质粒　； 参考：《苏州大学》2012年硕士论文

【摘要】：目的： 构建沙门菌质粒毒力基因(Salmonella plasmid virulence gene B, spvB)突变株,通过上皮细胞感染模型,研究该基因对细胞自噬的影响,为探讨沙门菌质粒毒力基因spvB致病机制及减毒活疫苗的制备提供理论和实验依据,并为后续利用小鼠感染模型进一步研究该基因的功能提供工具和手段。 方法： 一.沙门菌质粒毒力基因spvB突变株的构建 1.根据GeneBank序列数据库中沙门菌质粒毒力基因spvB序列,用Primer Premier5.0设计PCR特异性引物,以鼠伤寒沙门菌标准株SR-11基因组为模板,扩增获得spvB基因缺陷性核苷酸片段后连接至自杀质粒PGMB151。将改建的自杀载体导入含有spvB基因的鼠伤寒沙门菌SR-11野生株中进行同源重组,用PCR方法挑选稳定传代的突变株并进行基因测序鉴定。 2.根据spvB基因序列设计引物,以鼠伤寒沙门菌标准株SR-11基因组为模板,通过PCR扩增获得含spvB基因编码区域的全长DNA,并与表达载体pBAD/gⅢ定向连接,转化至大肠杆菌TOP10中筛选阳性克隆质粒,经序列分析后将阳性重组载体电转化至spvB基因突变株,作为spvB基因突变回补株,用PCR、核酸测序及蛋白免疫印迹(Western blot, WB)鉴定。 二.沙门菌质粒毒力基因spvB对上皮细胞自噬的影响 1.以携带spvB的鼠伤寒沙门菌标准株SR-11,敲除spvB的鼠伤寒沙门菌突变株(SR-11-ΔspvB)及其回补株(SR-11-c-spvB),与稳定转染了带有红色和绿色荧光蛋白标记微管相关蛋白1轻链3(microtubule-associated protein1light chain3, LC3)(mRFP-GFP-LC3)的HeLa细胞体外共培养,制作细胞感染模型。将对数生长期细菌按感染复数(multiplicity of infection, MOI)100:1感染细胞,在细菌和细胞共培养1h后吸除上清,此时定为“0”点。随后加入含100μg/ml阿米卡星(Amikacin, AMK)培养基作用2h以杀灭胞外菌,最后将AMK浓度降至10μg/ml以抑制从感染细胞中释放至培养基中的细菌生长。分别在共培养后1h、3h和5h收集细胞,用共聚焦激光扫描显微镜(Confocal laser scanning microscope, CLSM)观察胞内LC3-Ⅱ黄色点状结构。 2.以携带spvB的鼠伤寒沙门菌标准株SR-11,敲除spvB的鼠伤寒沙门菌突变株(SR-11-ΔspvB)及其回补株(SR-11-c-spvB)与HeLa细胞体外共培养,制作细胞感染模型。以对数生长期细菌按感染复数(multiplicity of infection, MOI)100:1感染细胞,在细菌和细胞共培养1h后吸除上清,此时定为“0”点,随后加入含100μg/ml阿米卡星(Amikacin, AMK)培养基作用2h以杀灭胞外菌,最后将AMK浓度降至10μg/ml以抑制从感染细胞中释放至培养基中的细菌生长。分别在共培养后1h、3h和5h收集细胞,用蛋白免疫印迹法(Western blot, WB)检测自噬相关蛋白Beclin-1和p62蛋白的表达情况,并用平板菌落计数法检测胞内集落形成单位(colony forming unit, CFU)。 结果： 一.沙门菌质粒毒力基因spvB突变株的构建 1.经PCR和基因测序证实,成功获得了spvB基因缺陷的鼠伤寒沙门菌突变株SR-11-AspvB。 2.经PCR、基因测序和WB证实,成功获得了spvB基因突变回补株SR-11-c-spvB。 二.沙门菌质粒毒力基因spvB对上皮细胞自噬的影响 1.CLSM检测自噬相关蛋白LC3-Ⅱ结果显示,细菌和细胞共培养1h后,SR-11和回补株SR-11-c-spvB感染组细胞内LC3-II的黄色荧光点状聚集数明显低于SR-11-ΔspvB感染组(P0.05)；3h和5h时三组黄色荧光点状聚集数未见明显差异(P0.05)。在细菌和细胞共培养的各时间点,SR-11和回补株SR-11-c-spvB感染组细胞内LC3-II的黄色荧光点状聚集数无明显差异(P0.05)。 2.WB检测结果显示,在细菌和细胞共培养1h后,SR-11和SR-11-c-spvB感染组细胞自噬相关蛋白Beclin-1表达低于SR-11-ΔspvB感染组(P0.05),3h和5h无明显变化(P0.05)。在细菌和细胞共培养1h后,SR-11和SR-11-c-spvB感染组细胞自噬相关蛋白p62表达高于SR-11-AspvB感染组(P0.05),3h和5h无明显差异(P0.05)。CFU结果显示,在细菌和细胞共培养1h后,各感染组间胞内活菌数无明显差异(P0.05)；共培养3h和5h后,SR-11和SR-11-c-spvB感染组胞内活菌数高于SR-11-ΔspvB感染组(3h,P0.05；5h,P0.01)；在细菌和细胞共培养的各时间点,SR-11和回补株SR-11-c-spvB感染组胞内活菌数无明显差异(P0.05)。 结论： 1.运用自杀质粒载体成功构建了鼠伤寒沙门菌质粒毒力基因spvB突变株SR-11-ΔspvB;运用原核表达载体pBAD/gⅢ成功构建了回补株SR-11-c-spvB,为进一步研究该质粒基因的功能和减毒疫苗的制备提供了工具和手段。 2.沙门菌质粒毒力基因spvB对上皮细胞的自噬有抑制作用。在细菌和细胞共培养1h后,鼠伤寒沙门菌标准株SR-11和回补株SR-11感染组细胞内自噬相关蛋白LC3-Ⅱ聚集量和自噬相关蛋白Beclin-1表达量均低于突变株SR-11-ΔspvB感染组；自噬相关蛋白p62表达量则高于突变株SR-11-ΔspvB感染组。在细菌和细胞共培养3h和5h后,SR-11和回补株SR-11-C-spvB感染组胞内活菌数明显高于突变株SR-11-ΔspvB感染组。以上结果说明,毒力基因spvB对发生在细菌细胞共作用早期的自噬有抑制作用,以利于携带该基因的宿主菌在细胞内的存活。
[Abstract]:Purpose :

The effect of the gene on the autophagy of the cells was investigated by constructing the Salmonella plasmid virulence gene B ( spvB ) mutant strain , which provided a theoretical and experimental basis for the study of the pathogenic mechanism of the plasmid virulence gene spvB and the preparation of attenuated live vaccine , and provided tools and means for further study on the function of the gene for the subsequent use of the mouse infection model .

Method :

1 . Construction of the spvB mutant strain of the plasmid virulence gene of Salmonella

1 . According to the spvB sequence of Salmonella typhimurium plasmid in GeneBank sequence database , a PCR - specific primer was designed by Primer Premier5.0 , and then ligated to the suicide plasmid PGMB151 with the strain SR - 11 of Salmonella typhimurium as a template . The modified suicide vector was introduced into the wild strain of Salmonella typhimurium SR - 11 containing spvB gene .

2 . According to the spvB gene sequence design primer , the full - length DNA of the coding region containing spvB gene was obtained by PCR amplification , and the whole - length DNA containing spvB gene coding region was amplified by PCR . The positive clone plasmid was transformed into E . coli TOP10 . After sequence analysis , the positive recombinant vector was transformed into spvB gene mutation strain , which was identified by PCR , nucleic acid sequencing and Western blot ( WB ) .

Effect of Salmonella typhimurium plasmid virulence gene spvB on autophagy of epithelial cells

1 . In vitro co - culture of Salmonella typhimurium mutant ( SR - 11 - 螖spvB ) with spvB and its return strain ( SR - 11 - c - spvB ) were carried out .

2 . In vitro co - culture of Salmonella typhimurium mutant ( SR - 11 - 螖spvB ) with spvB and its return line ( SR - 11 - c - spvB ) and HeLa cells were carried out in vitro to produce a model of cell infection . The cells were harvested at 1 h , 3 h and 5 h after co - culture . The expression of Beclin - 1 and p62 proteins were detected by Western blot and WB . The colony forming unit ( CFU ) was detected by plate colony counting method .

Results :

1 . Construction of the spvB mutant strain of the plasmid virulence gene of Salmonella

1 . The Salmonella typhimurium mutant SR - 11 - AspvB with spvB gene deficiency was successfully obtained by PCR and gene sequencing .

2 . After PCR , gene sequencing and WB confirmation , the spvB gene mutation was successfully obtained in SR - 11 - c - spvB .

Effect of Salmonella typhimurium plasmid virulence gene spvB on autophagy of epithelial cells

1 . The results showed that LC3 - II in SR - 11 and SR - 11 - c - spvB cells were significantly lower in SR - 11 and SR - 11 - c - spvB infection group than SR - 11 - 螖spvB infection group ( P0.05 ) .
There was no significant difference in the number of yellow fluorescent spots at 3h and 5h ( P0.05 ) . There was no significant difference in the number of yellow fluorescence spots in SR - 11 and SR - 11 - c - spvB infection group ( P0.05 ) at each time point of co - culture of bacteria and cells .

2 . The results showed that the expression of autophagy - related protein Beclin - 1 in SR - 11 and SR - 11 - c - spvB infection group was lower than SR - 11 - 螖spvB infection group ( P0.05 ) .
The number of viable bacteria in SR - 11 and SR - 11 - c - spvB infection group was higher than SR - 11 - 螖spvB infection group ( 3h , P0.05 ) .
5h,P0.01)锛，

本文编号：2000918