pORF5质粒蛋白抑制LL37抗菌肽促进沙眼衣原体感染及机制初步研究
发布时间：2018-10-26 12:12
【摘要】：目的：沙眼衣原体（Chlamydia trachomatis，Ct）易造成持续性感染而引发严重的并发症，但其致病机制尚不明确。已有研究表明人源性抗菌肽LL37对衣原体的感染起到一定的抑制作用。pORF5是由Ct质粒基因编码并且分泌到宿主细胞胞浆的分泌性蛋白，与衣原体致病密切相关。本研究拟探讨Ct质粒蛋白pORF5是否通过抑制LL37促进衣原体感染，并初步探讨其分子机制，为Ct致病机制的深入研究提供实验依据。 方法：将已构建好的pGEX-6p-1/pORF5重组质粒转化大肠杆菌XL1-Blue菌株，筛选pORF5重组蛋白表达菌，0.2mM IPTG诱导表达GST-pORF5融合蛋白，融合蛋白经亲和层析纯化及蛋白酶切除GST标签，制备纯化的pORF5蛋白；SDS-PAGE和Western blotting分析和鉴定pORF5蛋白，BCA法测定pORF5蛋白浓度并用鲎试剂对内毒素进行检测；30μg/mL的pORF5和用20μg/mL的LL37单独或者共同孵育衣原体后感染HeLa细胞28h，间接免疫荧光技术检测不同处理组衣原体包涵体的形成数量、大小和形态变化；收集共孵育6h后的HeLa细胞，QPCR技术检测TNF-α的表达情况；衣原体单独或与30μg/mL的pORF5孵育后感染HeLa细胞，收集共孵育6h后的HeLa细胞，QPCR技术检测TNF-α的表达情况；用浓度为10～30μg/mL的pORF5蛋白和浓度为20～50μg/mL的LL37单独或共同刺激HeLa细胞，28h后收集细胞，流式技术检测细胞凋亡情况。 结果： (1) pGEX-6p-1/pORF5成功转化大肠杆菌XL1-Blue菌株，并可高效表达GST-pORF5融合蛋白，融合蛋白经蛋白酶酶切后得到高纯度的pORF5蛋白，纯度约为95%。 (2)当pORF5蛋白浓度为10μg/mL时细胞凋亡率为5.75%，当pORF5蛋白浓度提高到20μg/mL时细胞凋亡率为9.42%，当pORF5蛋白浓度达到30μg/mL时细胞凋亡率为17.52%， pORF5蛋白可诱导HeLa细胞凋亡,且细胞凋亡率随pORF5蛋白浓度的升高而增加，。 (3)间接免疫荧光检测发现pORF5蛋白能降低LL37对衣原体感染的抑制作用，空白对照组衣原体包涵体形成单位(inclusion-forming unit，IFU)为3.80×10~5/mL，20μg/mL LL37处理组IFUs为2.00×10~5/mL，30μg/mL pORF5处理组IFUs为3.00×10~5/mL，20μg/mL LL37和30μg/mL pORF5共同处理组IFUs为3.07×10~5/mL。pORF5蛋白单独处理组和pORF5蛋白与LL37共同处理组IFUs明显高于LL37处理组，差异具有显著性（P0.05）；与空白对照组相比较，，pORF5蛋白单独处理组和pORF5蛋白与LL37共同处理组，IFUs无显著性差异。 (4)实时荧光PCR检测发现，与LL37单独处理组相比较，pORF5蛋白和LL37共同处理组TNF-α的相对表达量增加了7.3倍，pORF5蛋白与与衣原体共同处理组与衣原体单独处理组相比，LL37的相对表达量降低了17%。 结论： (1) pORF5质粒蛋白能明显降低LL37对衣原体感染的抑制作用。 (2) pORF5质粒蛋白可通过上调TNF-α、下调LL37的表达降低LL37的抑制作用，促进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- 伪 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- 伪 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- 伪 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- 伪, down-regulate the expression of LL37 and reduce the inhibitory effect of LL37 on Ct infection.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R374
本文编号：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- 伪 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- 伪 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- 伪 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- 伪, down-regulate the expression of LL37 and reduce the inhibitory effect of LL37 on Ct infection.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R374
【参考文献】
相关期刊论文 前4条
1 李忠玉;汪世平;吴移谋;钟光明;陈玎;;沙眼衣原体质粒蛋白pORF5核酸疫苗抗衣原体生殖道感染免疫保护作用[J];中国科学(C辑:生命科学);2008年09期
2 ZHOU Hui;HUANG QiuLin;LI ZhongYu;WU YiMou;XIE XiaoBing;MA KangKang;CAO WenJuan;ZHOU Zhou;LU ChunXue;ZHONG GuangMing;;pORF5 plasmid protein of Chlamydia trachomatis induces MAPK-mediated pro-inflammatory cytokines via TLR2 activation in THP-1 cells[J];Science China(Life Sciences);2013年05期
3 刘超;袁红艳;焦淑凡;余磊;常雅萍;;hCAP-18/LL-37基因转染对RAW264.7细胞活化功能的影响[J];中国生物制品学杂志;2011年02期
4 李忠玉;汪世平;吴移谋;朱翠明;何卓;;沙眼衣原体质粒蛋白pORF5原核表达及免疫原性鉴定[J];中国现代医学杂志;2009年04期
本文编号：2295677
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2295677.html
最近更新
教材专著
