pORF5質(zhì)粒蛋白抑制LL37抗菌肽促進沙眼衣原體感染及機制初步研究
發(fā)布時間：2018-10-26 12:12
【摘要】：目的：沙眼衣原體（Chlamydia trachomatis，Ct）易造成持續(xù)性感染而引發(fā)嚴重的并發(fā)癥，但其致病機制尚不明確。已有研究表明人源性抗菌肽LL37對衣原體的感染起到一定的抑制作用。pORF5是由Ct質(zhì)�；蚓幋a并且分泌到宿主細胞胞漿的分泌性蛋白，與衣原體致病密切相關(guān)。本研究擬探討Ct質(zhì)粒蛋白pORF5是否通過抑制LL37促進衣原體感染，并初步探討其分子機制，為Ct致病機制的深入研究提供實驗依據(jù)。 方法：將已構(gòu)建好的pGEX-6p-1/pORF5重組質(zhì)粒轉(zhuǎn)化大腸桿菌XL1-Blue菌株，篩選pORF5重組蛋白表達菌，0.2mM IPTG誘導(dǎo)表達GST-pORF5融合蛋白，融合蛋白經(jīng)親和層析純化及蛋白酶切除GST標簽，制備純化的pORF5蛋白；SDS-PAGE和Western blotting分析和鑒定pORF5蛋白，BCA法測定pORF5蛋白濃度并用鱟試劑對內(nèi)毒素進行檢測；30μg/mL的pORF5和用20μg/mL的LL37單獨或者共同孵育衣原體后感染HeLa細胞28h，間接免疫熒光技術(shù)檢測不同處理組衣原體包涵體的形成數(shù)量、大小和形態(tài)變化；收集共孵育6h后的HeLa細胞，QPCR技術(shù)檢測TNF-α的表達情況；衣原體單獨或與30μg/mL的pORF5孵育后感染HeLa細胞，收集共孵育6h后的HeLa細胞，QPCR技術(shù)檢測TNF-α的表達情況；用濃度為10～30μg/mL的pORF5蛋白和濃度為20～50μg/mL的LL37單獨或共同刺激HeLa細胞，28h后收集細胞，流式技術(shù)檢測細胞凋亡情況。 結(jié)果： (1) pGEX-6p-1/pORF5成功轉(zhuǎn)化大腸桿菌XL1-Blue菌株，并可高效表達GST-pORF5融合蛋白，融合蛋白經(jīng)蛋白酶酶切后得到高純度的pORF5蛋白，純度約為95%。 (2)當pORF5蛋白濃度為10μg/mL時細胞凋亡率為5.75%，當pORF5蛋白濃度提高到20μg/mL時細胞凋亡率為9.42%，當pORF5蛋白濃度達到30μg/mL時細胞凋亡率為17.52%， pORF5蛋白可誘導(dǎo)HeLa細胞凋亡,且細胞凋亡率隨pORF5蛋白濃度的升高而增加，。 (3)間接免疫熒光檢測發(fā)現(xiàn)pORF5蛋白能降低LL37對衣原體感染的抑制作用，空白對照組衣原體包涵體形成單位(inclusion-forming unit，IFU)為3.80×10~5/mL，20μg/mL LL37處理組IFUs為2.00×10~5/mL，30μg/mL pORF5處理組IFUs為3.00×10~5/mL，20μg/mL LL37和30μg/mL pORF5共同處理組IFUs為3.07×10~5/mL。pORF5蛋白單獨處理組和pORF5蛋白與LL37共同處理組IFUs明顯高于LL37處理組，差異具有顯著性（P0.05）；與空白對照組相比較，，pORF5蛋白單獨處理組和pORF5蛋白與LL37共同處理組，IFUs無顯著性差異。 (4)實時熒光PCR檢測發(fā)現(xiàn)，與LL37單獨處理組相比較，pORF5蛋白和LL37共同處理組TNF-α的相對表達量增加了7.3倍，pORF5蛋白與與衣原體共同處理組與衣原體單獨處理組相比，LL37的相對表達量降低了17%。 結(jié)論： (1) pORF5質(zhì)粒蛋白能明顯降低LL37對衣原體感染的抑制作用。 (2) pORF5質(zhì)粒蛋白可通過上調(diào)TNF-α、下調(diào)LL37的表達降低LL37的抑制作用，促進Ct感染。
[Abstract]:Objective: chlamydia trachomatis (Chlamydia trachomatis,Ct) is easy to cause persistent infection and cause serious complications, but its pathogenesis is unclear. Human antimicrobial peptide LL37 has been shown to inhibit chlamydia infection. PORF5 is a secreted protein encoded by Ct plasmid gene and secreted into the cytoplasm of host cells, which is closely related to the pathogenesis of chlamydia. The purpose of this study was to investigate whether Ct plasmid pORF5 could promote Chlamydia chlamydia infection by inhibiting LL37, and to explore the molecular mechanism of Chlamydia chlamydia infection, and to provide experimental evidence for the further study of the pathogenesis of Ct. Methods: the constructed pGEX-6p-1/pORF5 recombinant plasmid was transformed into Escherichia coli XL1-Blue strain to screen pORF5 recombinant protein expression bacteria, and 0.2mM IPTG induced expression of GST-pORF5 fusion protein. The fusion protein was purified by affinity chromatography and the GST label was removed by protease to prepare the purified pORF5 protein. PORF5 protein was analyzed and identified by SDS-PAGE and Western blotting. The concentration of pORF5 protein was determined by BCA method and endotoxin was detected by Limulus lysate reagent. HeLa cells were infected with 30 渭 g/mL pORF5 and 20 渭 g/mL LL37 for 28 h. The number, size and morphology of chlamydia inclusion bodies in different treatment groups were detected by indirect immunofluorescence technique. The expression of TNF- 偽 was detected by QPCR, chlamydia was incubated with 30 渭 g/mL pORF5 alone or with 30 渭 g/mL pORF5, HeLa cells were collected after 6 h incubation, and TNF- 偽 expression was detected by QPCR technique. HeLa cells were stimulated with pORF5 protein of 10 ~ 30 渭 g/mL and LL37 of 20 ~ 50 渭 g/mL, respectively. After 28 hours, the cells were collected and the apoptosis was detected by flow cytometry. Results: (1) pGEX-6p-1/pORF5 was successfully transformed into Escherichia coli XL1-Blue strain, and GST-pORF5 fusion protein was expressed efficiently. The fusion protein was digested by protease to obtain high purity pORF5 protein, the purity of which was about 95%. (2) the apoptotic rate was 5.75 when the concentration of pORF5 protein was 10 渭 g/mL, 9.42 when the concentration of pORF5 protein increased to 20 渭 g/mL, and 17.52 when the concentration of pORF5 protein reached 30 渭 g/mL. PORF5 protein could induce apoptosis of HeLa cells, and the apoptosis rate increased with the increase of pORF5 protein concentration. (3) indirect immunofluorescence assay showed that pORF5 protein could reduce the inhibitory effect of LL37 on chlamydia infection. In blank control group, the IFUs of chlamydia inclusion body formation unit (inclusion-forming unit,IFU) was 3.80 脳 10 ~ (5) / mL ~ (-1) and that of 20 渭 g/mL LL37 treatment group was 2.00 脳 10 ~ (5) / mL 路mL ~ (-1). The IFUs of 30 渭 g/mL pORF5 treatment group was 3.00 脳 10 ~ (-5) / mL ~ (-1) 20 渭 g/mL LL37 and the IFUs of 30 渭 g/mL pORF5 co-treatment group was 3.07 脳 10~5/mL.pORF5 protein treatment group, and the IFUs of pORF5 and LL37 co-treatment group was significantly higher than that of LL37 treatment group. The difference was significant (P0.05). Compared with the control group, there was no significant difference in IFUs between pORF5 protein treatment group and pORF5 protein co-treatment group and LL37 co-treatment group. (4) compared with LL37 alone, the relative expression of TNF- 偽 in pORF5 protein and LL37 co-treated group was increased by 7.3 times, and pORF5 protein was compared with chlamydia cotreatment group and chlamydia alone treatment group. The relative expression of LL37 decreased by 17%. Conclusion: (1) pORF5 plasmid protein can significantly reduce the inhibitory effect of LL37 on chlamydia infection. (2) pORF5 plasmid protein could up-regulate TNF- 偽, down-regulate the expression of LL37 and reduce the inhibitory effect of LL37 on Ct infection.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R374
本文編號：2295677
[Abstract]:Objective: chlamydia trachomatis (Chlamydia trachomatis,Ct) is easy to cause persistent infection and cause serious complications, but its pathogenesis is unclear. Human antimicrobial peptide LL37 has been shown to inhibit chlamydia infection. PORF5 is a secreted protein encoded by Ct plasmid gene and secreted into the cytoplasm of host cells, which is closely related to the pathogenesis of chlamydia. The purpose of this study was to investigate whether Ct plasmid pORF5 could promote Chlamydia chlamydia infection by inhibiting LL37, and to explore the molecular mechanism of Chlamydia chlamydia infection, and to provide experimental evidence for the further study of the pathogenesis of Ct. Methods: the constructed pGEX-6p-1/pORF5 recombinant plasmid was transformed into Escherichia coli XL1-Blue strain to screen pORF5 recombinant protein expression bacteria, and 0.2mM IPTG induced expression of GST-pORF5 fusion protein. The fusion protein was purified by affinity chromatography and the GST label was removed by protease to prepare the purified pORF5 protein. PORF5 protein was analyzed and identified by SDS-PAGE and Western blotting. The concentration of pORF5 protein was determined by BCA method and endotoxin was detected by Limulus lysate reagent. HeLa cells were infected with 30 渭 g/mL pORF5 and 20 渭 g/mL LL37 for 28 h. The number, size and morphology of chlamydia inclusion bodies in different treatment groups were detected by indirect immunofluorescence technique. The expression of TNF- 偽 was detected by QPCR, chlamydia was incubated with 30 渭 g/mL pORF5 alone or with 30 渭 g/mL pORF5, HeLa cells were collected after 6 h incubation, and TNF- 偽 expression was detected by QPCR technique. HeLa cells were stimulated with pORF5 protein of 10 ~ 30 渭 g/mL and LL37 of 20 ~ 50 渭 g/mL, respectively. After 28 hours, the cells were collected and the apoptosis was detected by flow cytometry. Results: (1) pGEX-6p-1/pORF5 was successfully transformed into Escherichia coli XL1-Blue strain, and GST-pORF5 fusion protein was expressed efficiently. The fusion protein was digested by protease to obtain high purity pORF5 protein, the purity of which was about 95%. (2) the apoptotic rate was 5.75 when the concentration of pORF5 protein was 10 渭 g/mL, 9.42 when the concentration of pORF5 protein increased to 20 渭 g/mL, and 17.52 when the concentration of pORF5 protein reached 30 渭 g/mL. PORF5 protein could induce apoptosis of HeLa cells, and the apoptosis rate increased with the increase of pORF5 protein concentration. (3) indirect immunofluorescence assay showed that pORF5 protein could reduce the inhibitory effect of LL37 on chlamydia infection. In blank control group, the IFUs of chlamydia inclusion body formation unit (inclusion-forming unit,IFU) was 3.80 脳 10 ~ (5) / mL ~ (-1) and that of 20 渭 g/mL LL37 treatment group was 2.00 脳 10 ~ (5) / mL 路mL ~ (-1). The IFUs of 30 渭 g/mL pORF5 treatment group was 3.00 脳 10 ~ (-5) / mL ~ (-1) 20 渭 g/mL LL37 and the IFUs of 30 渭 g/mL pORF5 co-treatment group was 3.07 脳 10~5/mL.pORF5 protein treatment group, and the IFUs of pORF5 and LL37 co-treatment group was significantly higher than that of LL37 treatment group. The difference was significant (P0.05). Compared with the control group, there was no significant difference in IFUs between pORF5 protein treatment group and pORF5 protein co-treatment group and LL37 co-treatment group. (4) compared with LL37 alone, the relative expression of TNF- 偽 in pORF5 protein and LL37 co-treated group was increased by 7.3 times, and pORF5 protein was compared with chlamydia cotreatment group and chlamydia alone treatment group. The relative expression of LL37 decreased by 17%. Conclusion: (1) pORF5 plasmid protein can significantly reduce the inhibitory effect of LL37 on chlamydia infection. (2) pORF5 plasmid protein could up-regulate TNF- 偽, down-regulate the expression of LL37 and reduce the inhibitory effect of LL37 on Ct infection.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R374
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相關(guān)期刊論文 前4條
1 李忠玉;汪世平;吳移謀;鐘光明;陳玎;;沙眼衣原體質(zhì)粒蛋白pORF5核酸疫苗抗衣原體生殖道感染免疫保護作用[J];中國科學(xué)(C輯:生命科學(xué));2008年09期
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3 劉超;袁紅艷;焦淑凡;余磊;常雅萍;;hCAP-18/LL-37基因轉(zhuǎn)染對RAW264.7細胞活化功能的影響[J];中國生物制品學(xué)雜志;2011年02期
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