肺炎衣原体Cpn0147相互作用分子的筛选及初步鉴定

发布时间：2017-12-27 08:06

本文关键词：肺炎衣原体Cpn0147相互作用分子的筛选及初步鉴定　出处：《河北北方学院》2016年硕士论文　论文类型：学位论文

【摘要】：肺炎衣原体(Chlamydia pneumonia,Cpn)是一类严格活细胞内寄生的、具有独特生活周期的微生物。虽然肺炎衣原体的致病机制尚不清楚,但包涵体作为肺炎衣原体生物合成和代谢的重要场所,与肺炎衣原体的致病性密切相关。包涵体膜蛋白(inclusion protein,Inc)是包涵体的重要组成成分,是衣原体与宿主进行一系列信息和物质交换的基础,在肺炎衣原体的一系列生命活动中扮演重要角色。Cpn0147是已确定的肺炎衣原体包涵体膜蛋白之一,具有较强的免疫原性。本研究采用酵母双杂交技术对Cpn0147相互作用的蛋白质进行初步筛选,并采用免疫共沉淀和亚细胞共定位技术对筛选出的Cpn0147相互作用的蛋白质进一步验证。首先检测p GBKT7-Cpn0147诱饵质粒对酵母菌株AH109和Y187有无自激活和毒性作用。检测无自激活和毒性作用后,将诱饵质粒p GBKT7-Cpn0147转化的酵母菌株AH109与含有He La细胞c DNA文库的p GADT7转化的酵母菌株Y187进行酵母双杂交,当酵母结合子(三叶草或米奇结构)形成后,将该菌液涂布于腺嘌呤、组氨酸、亮氨酸、色氨酸营养缺陷型平板(SD/-Ade/-His/-Leu/-Trp)进行初步筛选,收集经过三次筛选后的阳性菌落进行蓝白斑筛选,蓝斑菌落(阳性)进行PCR验证。选取PCR阳性的酵母菌落提取质粒转化大肠杆菌XL1-Blue后再提取质粒,然后分别转化酵母菌Y187后与p GBKT7-Cpn0147转化的AH109酵母菌进行回交实验,回交实验阳性的质粒进行碱基序列测定,测序结果在Genebank中进行BLAST检索比对确定其相互作用蛋白基因。酵母双杂交实验结果表明,Cpn0147与He La细胞c DNA文库中环腺苷酸应答原件结合蛋白3(CREB3)发生相互作用。为进一步验证Cpn0147与CREB3的相互作用,采用了亚细胞共定位技术与免疫共沉淀技术。运用脂质体转染试剂Lipo2000将pc DNA3.1+/His/Myc-Cpn0147和pc DNA3.1+/Flag-CREB3共转染至He La细胞。转染48 h后,一部分细胞经固定、封闭,然后加入兔抗c-Myc抗体、鼠抗Flag抗体,再经Alexa Fluor 488标记羊抗鼠Ig G抗体、Cy3标记羊抗兔Ig G抗体染色,激光共聚焦显微镜下观察。结果显示,c-Myc-Cpn0147呈现红色荧光,Flag-CREB3呈现绿色荧光,当二者有重叠时,呈现黄色荧光,初步证明Cpn0147可与重组CREB3结合;另一部分共转染的He La细胞裂解后离心,取上清经抗c-Myc agarose翻转吸附共沉淀后,进行SDS-PAGE电泳,转膜后进行封闭,再用兔抗c-Myc抗体和鼠抗Flag抗体进行孵育,洗膜后加入HRP标记羊抗兔Ig G、HRP标记羊抗鼠Ig G进行孵育。经ECL检测在分子量为16KD、45KD处显带,与c-Myc-Cpn0147、Flag-CREB3重组蛋白分子量相符,表明共转染的Cpn0147与CREB3结合。为进一步确认Cpn0147与内质网上CREB3的相互作用,同时进行下列实验:(1)将爬片24 h的He La细胞固定、封闭,再经鼠抗Calnexin抗体、兔抗CREB3抗体共同孵育,最后经Alexa Fluor 488标记羊抗小鼠Ig G抗体和Cy3标记羊抗兔Ig G抗体染色,激光共聚焦显微镜下观察;(2)用pc DNA3.1+/His/Myc-Cpn0147重组质粒转染He La细胞,转染48 h后,用鼠抗c-Myc抗体和兔抗CREB3抗体,已及对应的Alexa Fluor 488标记羊抗鼠Ig G抗体和Cy3标记羊抗兔Ig G抗体进行染色,激光共聚焦显微镜下观察;(3)用pc DNA3.1+/His/Myc质粒作为对照重复(2)。结果显示:当绿色荧光的Calnexin与红色荧光的CREB3重叠时,呈现黄色荧光;当绿色荧光的c-Myc-Cpn0147与内质网上红色荧光的CREB3重叠时,也有黄色荧光出现。证明Cpn0147可与内质网上的CREB3结合。本研究发现了肺炎衣原体包涵体膜蛋白Cpn0147可以与He La细胞CREB3相互作用,为进一步研究肺炎衣原体与宿主相互作用的分子机制打下基础。
[Abstract]:Chlamydia pneumonia (Cpn) is a kind of microorganism which is strictly living cell parasitized and has a unique life cycle. Although the pathogenesis of Chlamydia pneumoniae is not yet clear, inclusion bodies play an important role in the biosynthesis and metabolism of Chlamydia pneumoniae, and are closely related to the pathogenicity of Chlamydia pneumoniae. Inclusion protein (Inc) is an important component of inclusion bodies. It is a basis for chlamydia and host to carry out a series of information and material exchange. It plays an important role in a series of life activities of Chlamydia pneumoniae. Cpn0147 is one of the identified proteins of the Chlamydia pneumoniae inclusion body membrane protein, which has strong immunogenicity. In this study, yeast two hybrid technology was used to screen the proteins interacting with Cpn0147. Further, the Cpn0147 interaction proteins identified by immunoprecipitation and subcellular collocation were further verified. First, the P GBKT7-Cpn0147 decoy plasmid has no self activation and toxicity to yeast strain AH109 and Y187. Detection of no self activation and toxicity, the yeast strain Y187 bait plasmid P GBKT7-Cpn0147 transformed yeast strains AH109 and He containing La cell C DNA library P GADT7 transformed yeast two hybrid yeast, when sub (clover or Mickey structure) after the formation of the liquid coating on adenine and histidine, leucine, tryptophan auxotrophic plate (SD/-Ade/-His/-Leu/-Trp) were collected after preliminary screening, three positive colonies were screened by blue white screening, blue spot colony (positive) PCR verification. Selection of PCR positive yeast colonies extraction plasmid was transformed into E.coli XL1-Blue after extraction of plasmid AH109, then transformed into yeast yeast after Y187 conversion and P GBKT7-Cpn0147 by Backcross experiments, plasmid backcross experiment were nucleotide sequencing, sequencing results were compared to determine the retrieval of BLAST interacting protein gene in Genebank. The results of yeast two hybrid experiment showed that Cpn0147 and He La cell C DNA library interacted with cyclic adenylate responsive element binding protein 3 (CREB3). In order to further verify the interaction between Cpn0147 and CREB3, the technique of subcellular co localization and immunoprecipitation was used. PC DNA3.1+/His/Myc-Cpn0147 and PC DNA3.1+/Flag-CREB3 were transfected into He La cells by using liposome transfection reagent Lipo2000. After transfection for 48 h, a part of cells were fixed and sealed, then added anti c-Myc antibody and anti Flag antibody. Then Alexa Fluor 488 was used to mark Goat anti mouse Ig G antibody, Cy3 labeled Goat anti rabbit Ig G antibody staining, and observed under confocal laser scanning microscope. The results showed that c-Myc-Cpn0147 showed red fluorescence, Flag-CREB3 showed green fluorescence when the two overlap, showed yellow fluorescence showed that Cpn0147 can be combined with recombinant CREB3; the other part of the He co transfected La cell lysis after centrifugation, the supernatant after anti c-Myc agarose turn adsorption coprecipitation, SDS-PAGE electrophoresis, transfer after the film was closed, anti c-Myc antibody and anti Flag antibody in rabbits and rats were incubated after washing the membrane with HRP labeled Goat anti rabbit Ig G and HRP labeled Goat anti mouse Ig were incubated with G. The molecular weight of 16KD and 45KD was detected by ECL, which was consistent with the molecular weight of recombinant protein of c-Myc-Cpn0147 and Flag-CREB3, indicating that the co transfected Cpn0147 was combined with CREB3. To further confirm the interaction between Cpn0147 and ER CREB3, and carried out the following experiments: (1) He La will climb 24 h cell sheet is fixed, closed, and then by mouse anti Calnexin antibody, Rabbit anti CREB3 antibody were incubated with Alexa, finally Fluor 488 labeled Goat anti mouse Ig antibody and Cy3 conjugated goat G Ig anti rabbit G antibody staining, confocal laser scanning microscope; (2) using PC DNA3.1+/His/Myc-Cpn0147 recombinant plasmid was transfected into He La cells 48 h after transfection, using mouse anti c-Myc antibody and Rabbit anti CREB3 antibody, and the corresponding Alexa Fluor 488 labeled Goat anti mouse Ig antibody and G Cy3 antibody labeled goat G anti rabbit Ig were observed under laser confocal microscope; (3) using PC DNA3.1+/His/Myc plasmid as control repeat (2). The results showed that yellow fluorescence appeared when the green fluorescence Calnexin overlapped with the red fluorescence CREB3, and yellow fluorescence appeared when the green fluorescence c-Myc-Cpn0147 overlapped with the red fluorescence CREB3 of endoplasmic reticulum. It has been proved that Cpn0147 can be combined with the CREB3 of the endoplasmic reticulum. This study found that Chlamydia pneumoniae inclusion body membrane protein Cpn0147 can interact with He La cell CREB3, and lay the foundation for further studying the molecular mechanism of Chlamydia pneumoniae and host interaction.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R374

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