本文選題：沙門(mén)菌 + 質(zhì)粒　； 參考：《蘇州大學(xué)》2012年碩士論文

【摘要】：目的： 構(gòu)建沙門(mén)菌質(zhì)粒毒力基因(Salmonella plasmid virulence gene B, spvB)突變株,通過(guò)上皮細(xì)胞感染模型,研究該基因?qū)?xì)胞自噬的影響,為探討沙門(mén)菌質(zhì)粒毒力基因spvB致病機(jī)制及減毒活疫苗的制備提供理論和實(shí)驗(yàn)依據(jù),并為后續(xù)利用小鼠感染模型進(jìn)一步研究該基因的功能提供工具和手段。 方法： 一.沙門(mén)菌質(zhì)粒毒力基因spvB突變株的構(gòu)建 1.根據(jù)GeneBank序列數(shù)據(jù)庫(kù)中沙門(mén)菌質(zhì)粒毒力基因spvB序列,用Primer Premier5.0設(shè)計(jì)PCR特異性引物,以鼠傷寒沙門(mén)菌標(biāo)準(zhǔn)株SR-11基因組為模板,擴(kuò)增獲得spvB基因缺陷性核苷酸片段后連接至自殺質(zhì)粒PGMB151。將改建的自殺載體導(dǎo)入含有spvB基因的鼠傷寒沙門(mén)菌SR-11野生株中進(jìn)行同源重組,用PCR方法挑選穩(wěn)定傳代的突變株并進(jìn)行基因測(cè)序鑒定。 2.根據(jù)spvB基因序列設(shè)計(jì)引物,以鼠傷寒沙門(mén)菌標(biāo)準(zhǔn)株SR-11基因組為模板,通過(guò)PCR擴(kuò)增獲得含spvB基因編碼區(qū)域的全長(zhǎng)DNA,并與表達(dá)載體pBAD/gⅢ定向連接,轉(zhuǎn)化至大腸桿菌TOP10中篩選陽(yáng)性克隆質(zhì)粒,經(jīng)序列分析后將陽(yáng)性重組載體電轉(zhuǎn)化至spvB基因突變株,作為spvB基因突變回補(bǔ)株,用PCR、核酸測(cè)序及蛋白免疫印跡(Western blot, WB)鑒定。 二.沙門(mén)菌質(zhì)粒毒力基因spvB對(duì)上皮細(xì)胞自噬的影響 1.以攜帶spvB的鼠傷寒沙門(mén)菌標(biāo)準(zhǔn)株SR-11,敲除spvB的鼠傷寒沙門(mén)菌突變株(SR-11-ΔspvB)及其回補(bǔ)株(SR-11-c-spvB),與穩(wěn)定轉(zhuǎn)染了帶有紅色和綠色熒光蛋白標(biāo)記微管相關(guān)蛋白1輕鏈3(microtubule-associated protein1light chain3, LC3)(mRFP-GFP-LC3)的HeLa細(xì)胞體外共培養(yǎng),制作細(xì)胞感染模型。將對(duì)數(shù)生長(zhǎng)期細(xì)菌按感染復(fù)數(shù)(multiplicity of infection, MOI)100:1感染細(xì)胞,在細(xì)菌和細(xì)胞共培養(yǎng)1h后吸除上清,此時(shí)定為“0”點(diǎn)。隨后加入含100μg/ml阿米卡星(Amikacin, AMK)培養(yǎng)基作用2h以殺滅胞外菌,最后將AMK濃度降至10μg/ml以抑制從感染細(xì)胞中釋放至培養(yǎng)基中的細(xì)菌生長(zhǎng)。分別在共培養(yǎng)后1h、3h和5h收集細(xì)胞,用共聚焦激光掃描顯微鏡(Confocal laser scanning microscope, CLSM)觀察胞內(nèi)LC3-Ⅱ黃色點(diǎn)狀結(jié)構(gòu)。 2.以攜帶spvB的鼠傷寒沙門(mén)菌標(biāo)準(zhǔn)株SR-11,敲除spvB的鼠傷寒沙門(mén)菌突變株(SR-11-ΔspvB)及其回補(bǔ)株(SR-11-c-spvB)與HeLa細(xì)胞體外共培養(yǎng),制作細(xì)胞感染模型。以對(duì)數(shù)生長(zhǎng)期細(xì)菌按感染復(fù)數(shù)(multiplicity of infection, MOI)100:1感染細(xì)胞,在細(xì)菌和細(xì)胞共培養(yǎng)1h后吸除上清,此時(shí)定為“0”點(diǎn),隨后加入含100μg/ml阿米卡星(Amikacin, AMK)培養(yǎng)基作用2h以殺滅胞外菌,最后將AMK濃度降至10μg/ml以抑制從感染細(xì)胞中釋放至培養(yǎng)基中的細(xì)菌生長(zhǎng)。分別在共培養(yǎng)后1h、3h和5h收集細(xì)胞,用蛋白免疫印跡法(Western blot, WB)檢測(cè)自噬相關(guān)蛋白Beclin-1和p62蛋白的表達(dá)情況,并用平板菌落計(jì)數(shù)法檢測(cè)胞內(nèi)集落形成單位(colony forming unit, CFU)。 結(jié)果： 一.沙門(mén)菌質(zhì)粒毒力基因spvB突變株的構(gòu)建 1.經(jīng)PCR和基因測(cè)序證實(shí),成功獲得了spvB基因缺陷的鼠傷寒沙門(mén)菌突變株SR-11-AspvB。 2.經(jīng)PCR、基因測(cè)序和WB證實(shí),成功獲得了spvB基因突變回補(bǔ)株SR-11-c-spvB。 二.沙門(mén)菌質(zhì)粒毒力基因spvB對(duì)上皮細(xì)胞自噬的影響 1.CLSM檢測(cè)自噬相關(guān)蛋白LC3-Ⅱ結(jié)果顯示,細(xì)菌和細(xì)胞共培養(yǎng)1h后,SR-11和回補(bǔ)株SR-11-c-spvB感染組細(xì)胞內(nèi)LC3-II的黃色熒光點(diǎn)狀聚集數(shù)明顯低于SR-11-ΔspvB感染組(P0.05)；3h和5h時(shí)三組黃色熒光點(diǎn)狀聚集數(shù)未見(jiàn)明顯差異(P0.05)。在細(xì)菌和細(xì)胞共培養(yǎng)的各時(shí)間點(diǎn),SR-11和回補(bǔ)株SR-11-c-spvB感染組細(xì)胞內(nèi)LC3-II的黃色熒光點(diǎn)狀聚集數(shù)無(wú)明顯差異(P0.05)。 2.WB檢測(cè)結(jié)果顯示,在細(xì)菌和細(xì)胞共培養(yǎng)1h后,SR-11和SR-11-c-spvB感染組細(xì)胞自噬相關(guān)蛋白Beclin-1表達(dá)低于SR-11-ΔspvB感染組(P0.05),3h和5h無(wú)明顯變化(P0.05)。在細(xì)菌和細(xì)胞共培養(yǎng)1h后,SR-11和SR-11-c-spvB感染組細(xì)胞自噬相關(guān)蛋白p62表達(dá)高于SR-11-AspvB感染組(P0.05),3h和5h無(wú)明顯差異(P0.05)。CFU結(jié)果顯示,在細(xì)菌和細(xì)胞共培養(yǎng)1h后,各感染組間胞內(nèi)活菌數(shù)無(wú)明顯差異(P0.05)；共培養(yǎng)3h和5h后,SR-11和SR-11-c-spvB感染組胞內(nèi)活菌數(shù)高于SR-11-ΔspvB感染組(3h,P0.05；5h,P0.01)；在細(xì)菌和細(xì)胞共培養(yǎng)的各時(shí)間點(diǎn),SR-11和回補(bǔ)株SR-11-c-spvB感染組胞內(nèi)活菌數(shù)無(wú)明顯差異(P0.05)。 結(jié)論： 1.運(yùn)用自殺質(zhì)粒載體成功構(gòu)建了鼠傷寒沙門(mén)菌質(zhì)粒毒力基因spvB突變株SR-11-ΔspvB;運(yùn)用原核表達(dá)載體pBAD/gⅢ成功構(gòu)建了回補(bǔ)株SR-11-c-spvB,為進(jìn)一步研究該質(zhì)�；虻墓δ芎蜏p毒疫苗的制備提供了工具和手段。 2.沙門(mén)菌質(zhì)粒毒力基因spvB對(duì)上皮細(xì)胞的自噬有抑制作用。在細(xì)菌和細(xì)胞共培養(yǎng)1h后,鼠傷寒沙門(mén)菌標(biāo)準(zhǔn)株SR-11和回補(bǔ)株SR-11感染組細(xì)胞內(nèi)自噬相關(guān)蛋白LC3-Ⅱ聚集量和自噬相關(guān)蛋白Beclin-1表達(dá)量均低于突變株SR-11-ΔspvB感染組；自噬相關(guān)蛋白p62表達(dá)量則高于突變株SR-11-ΔspvB感染組。在細(xì)菌和細(xì)胞共培養(yǎng)3h和5h后,SR-11和回補(bǔ)株SR-11-C-spvB感染組胞內(nèi)活菌數(shù)明顯高于突變株SR-11-ΔspvB感染組。以上結(jié)果說(shuō)明,毒力基因spvB對(duì)發(fā)生在細(xì)菌細(xì)胞共作用早期的自噬有抑制作用,以利于攜帶該基因的宿主菌在細(xì)胞內(nèi)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)錛，

本文編號(hào)：2000918