本文關(guān)鍵詞：肺炎衣原體Cpn0147相互作用分子的篩選及初步鑒定　出處：《河北北方學(xué)院》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 肺炎衣原體 包涵體膜蛋白 Cpn0147 CREB3 酵母雙雜交 免疫共沉淀 亞細(xì)胞共定位技術(shù)

【摘要】：肺炎衣原體(Chlamydia pneumonia,Cpn)是一類嚴(yán)格活細(xì)胞內(nèi)寄生的、具有獨(dú)特生活周期的微生物。雖然肺炎衣原體的致病機(jī)制尚不清楚,但包涵體作為肺炎衣原體生物合成和代謝的重要場(chǎng)所,與肺炎衣原體的致病性密切相關(guān)。包涵體膜蛋白(inclusion protein,Inc)是包涵體的重要組成成分,是衣原體與宿主進(jìn)行一系列信息和物質(zhì)交換的基礎(chǔ),在肺炎衣原體的一系列生命活動(dòng)中扮演重要角色。Cpn0147是已確定的肺炎衣原體包涵體膜蛋白之一,具有較強(qiáng)的免疫原性。本研究采用酵母雙雜交技術(shù)對(duì)Cpn0147相互作用的蛋白質(zhì)進(jìn)行初步篩選,并采用免疫共沉淀和亞細(xì)胞共定位技術(shù)對(duì)篩選出的Cpn0147相互作用的蛋白質(zhì)進(jìn)一步驗(yàn)證。首先檢測(cè)p GBKT7-Cpn0147誘餌質(zhì)粒對(duì)酵母菌株AH109和Y187有無自激活和毒性作用。檢測(cè)無自激活和毒性作用后,將誘餌質(zhì)粒p GBKT7-Cpn0147轉(zhuǎn)化的酵母菌株AH109與含有He La細(xì)胞c DNA文庫的p GADT7轉(zhuǎn)化的酵母菌株Y187進(jìn)行酵母雙雜交,當(dāng)酵母結(jié)合子(三葉草或米奇結(jié)構(gòu))形成后,將該菌液涂布于腺嘌呤、組氨酸、亮氨酸、色氨酸營(yíng)養(yǎng)缺陷型平板(SD/-Ade/-His/-Leu/-Trp)進(jìn)行初步篩選,收集經(jīng)過三次篩選后的陽性菌落進(jìn)行藍(lán)白斑篩選,藍(lán)斑菌落(陽性)進(jìn)行PCR驗(yàn)證。選取PCR陽性的酵母菌落提取質(zhì)粒轉(zhuǎn)化大腸桿菌XL1-Blue后再提取質(zhì)粒,然后分別轉(zhuǎn)化酵母菌Y187后與p GBKT7-Cpn0147轉(zhuǎn)化的AH109酵母菌進(jìn)行回交實(shí)驗(yàn),回交實(shí)驗(yàn)陽性的質(zhì)粒進(jìn)行堿基序列測(cè)定,測(cè)序結(jié)果在Genebank中進(jìn)行BLAST檢索比對(duì)確定其相互作用蛋白基因。酵母雙雜交實(shí)驗(yàn)結(jié)果表明,Cpn0147與He La細(xì)胞c DNA文庫中環(huán)腺苷酸應(yīng)答原件結(jié)合蛋白3(CREB3)發(fā)生相互作用。為進(jìn)一步驗(yàn)證Cpn0147與CREB3的相互作用,采用了亞細(xì)胞共定位技術(shù)與免疫共沉淀技術(shù)。運(yùn)用脂質(zhì)體轉(zhuǎn)染試劑Lipo2000將pc DNA3.1+/His/Myc-Cpn0147和pc DNA3.1+/Flag-CREB3共轉(zhuǎn)染至He La細(xì)胞。轉(zhuǎn)染48 h后,一部分細(xì)胞經(jīng)固定、封閉,然后加入兔抗c-Myc抗體、鼠抗Flag抗體,再經(jīng)Alexa Fluor 488標(biāo)記羊抗鼠Ig G抗體、Cy3標(biāo)記羊抗兔Ig G抗體染色,激光共聚焦顯微鏡下觀察。結(jié)果顯示,c-Myc-Cpn0147呈現(xiàn)紅色熒光,Flag-CREB3呈現(xiàn)綠色熒光,當(dāng)二者有重疊時(shí),呈現(xiàn)黃色熒光,初步證明Cpn0147可與重組CREB3結(jié)合;另一部分共轉(zhuǎn)染的He La細(xì)胞裂解后離心,取上清經(jīng)抗c-Myc agarose翻轉(zhuǎn)吸附共沉淀后,進(jìn)行SDS-PAGE電泳,轉(zhuǎn)膜后進(jìn)行封閉,再用兔抗c-Myc抗體和鼠抗Flag抗體進(jìn)行孵育,洗膜后加入HRP標(biāo)記羊抗兔Ig G、HRP標(biāo)記羊抗鼠Ig G進(jìn)行孵育。經(jīng)ECL檢測(cè)在分子量為16KD、45KD處顯帶,與c-Myc-Cpn0147、Flag-CREB3重組蛋白分子量相符,表明共轉(zhuǎn)染的Cpn0147與CREB3結(jié)合。為進(jìn)一步確認(rèn)Cpn0147與內(nèi)質(zhì)網(wǎng)上CREB3的相互作用,同時(shí)進(jìn)行下列實(shí)驗(yàn):(1)將爬片24 h的He La細(xì)胞固定、封閉,再經(jīng)鼠抗Calnexin抗體、兔抗CREB3抗體共同孵育,最后經(jīng)Alexa Fluor 488標(biāo)記羊抗小鼠Ig G抗體和Cy3標(biāo)記羊抗兔Ig G抗體染色,激光共聚焦顯微鏡下觀察;(2)用pc DNA3.1+/His/Myc-Cpn0147重組質(zhì)粒轉(zhuǎn)染He La細(xì)胞,轉(zhuǎn)染48 h后,用鼠抗c-Myc抗體和兔抗CREB3抗體,已及對(duì)應(yīng)的Alexa Fluor 488標(biāo)記羊抗鼠Ig G抗體和Cy3標(biāo)記羊抗兔Ig G抗體進(jìn)行染色,激光共聚焦顯微鏡下觀察;(3)用pc DNA3.1+/His/Myc質(zhì)粒作為對(duì)照重復(fù)(2)。結(jié)果顯示:當(dāng)綠色熒光的Calnexin與紅色熒光的CREB3重疊時(shí),呈現(xiàn)黃色熒光;當(dāng)綠色熒光的c-Myc-Cpn0147與內(nèi)質(zhì)網(wǎng)上紅色熒光的CREB3重疊時(shí),也有黃色熒光出現(xiàn)。證明Cpn0147可與內(nèi)質(zhì)網(wǎng)上的CREB3結(jié)合。本研究發(fā)現(xiàn)了肺炎衣原體包涵體膜蛋白Cpn0147可以與He La細(xì)胞CREB3相互作用,為進(jìn)一步研究肺炎衣原體與宿主相互作用的分子機(jī)制打下基礎(chǔ)。
[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.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R374

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 苗小艷;孔繁斗;石敏;Bos JL;趙春艷;;酵母雙雜交系統(tǒng)篩選與RapGAP相互作用的蛋白質(zhì)[J];中國(guó)微生態(tài)學(xué)雜志;2015年06期

2 賈棟;張彬;馬瑞燕;唐貴剛;趙捷;王文華;周琳;;免疫共沉淀結(jié)合基因芯片對(duì)m_3G帽子結(jié)構(gòu)RNA的分離鑒定[J];中國(guó)生物工程雜志;2014年02期

3 沈瑤瑤;嚴(yán)慶豐;;蛋白質(zhì)相互作用研究進(jìn)展[J];生命科學(xué);2013年03期

4 代國(guó)知;馬忠夏;周安文;陳虹亮;吳移謀;;肺炎嗜衣原體Cpn0147重組蛋白的免疫學(xué)活性研究[J];國(guó)際檢驗(yàn)醫(yī)學(xué)雜志;2012年14期

5 付曉達(dá);高美華;;人環(huán)指蛋白11與核因子κB調(diào)控蛋白腫瘤壞死因子α誘導(dǎo)蛋白3相互作用蛋白1相互作用的研究[J];醫(yī)學(xué)研究生學(xué)報(bào);2012年06期

6 童偉;周淑芬;賈天軍;;肺炎衣原體感染兒童血清中抗Cpn0147抗體的檢測(cè)[J];臨床檢驗(yàn)雜志;2012年04期

7 陳謀通;劉建軍;;蛋白質(zhì)相互作用的研究方法[J];生物技術(shù)通報(bào);2009年01期

，

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