【摘要】：百日咳是由百日咳鮑特氏菌（以下簡稱百日咳桿菌）引起的一種呼吸道傳染病，在嬰幼兒當(dāng)中較為常見。百日咳桿菌致病機(jī)理包括粘附、侵襲、產(chǎn)毒等一系列的過程。絲狀血凝素（Filamentous hemagglutmin）、粘附素（Pertactin)、百日咳氣管定植因子（Tracheal colonization factor; TcfA）等諸多膜表面蛋白參與到細(xì)菌的粘附過程。百日咳氣管定植因子是百日咳桿菌膜表面蛋白，相關(guān)文獻(xiàn)報(bào)道，推測該蛋白可能是百日咳桿菌的粘附分子，但對其粘附功能尚未進(jìn)行系統(tǒng)的驗(yàn)證。因此本研究采用DNA重組技術(shù)，在大腸桿菌（BL21；DE3）內(nèi)表達(dá)TcfA蛋白，并純化重組TcfA蛋白。然后通過重組表達(dá)菌的體外細(xì)胞粘附實(shí)驗(yàn)、重組蛋白免疫熒光實(shí)驗(yàn)、競爭抑制實(shí)驗(yàn)證明TcfA蛋白的黏附作用。并在此基礎(chǔ)上，就TcfA蛋白第317-319位RGD三聯(lián)肽的功能進(jìn)行驗(yàn)證。同時(shí)通過構(gòu)建FHA-PRN-基因缺失株，探究TcfA蛋白在粘附過程中的重要性，并嘗試找出TcfA蛋白與經(jīng)典模型細(xì)胞HeLa相互作用的受體。 通過基因重組技術(shù)成功的表達(dá)了TcfA蛋白，并純化出純度達(dá)85%的重組蛋白，免疫印跡實(shí)驗(yàn)結(jié)果顯示，重組蛋白能與疫苗生產(chǎn)菌株（CS菌）全菌體血清發(fā)生抗原抗體反應(yīng)，說明重組蛋白具有良好的免疫反應(yīng)性和抗原特異性；玻片凝集實(shí)驗(yàn)和V管凝集實(shí)驗(yàn)證明TcfA蛋白表達(dá)于大腸桿菌膜表面；熒光粘附試驗(yàn)證實(shí)表達(dá)有TcfA的大腸桿菌與含有空載體的大腸桿菌對經(jīng)典模式細(xì)胞的粘附有明顯的差異，CFU實(shí)驗(yàn)定量證實(shí)兩者之間具有統(tǒng)計(jì)學(xué)差異。粘附ELISA實(shí)驗(yàn)從另一技術(shù)方法反映了兩者之間具有統(tǒng)計(jì)學(xué)差異。重組蛋白的免疫熒光實(shí)驗(yàn)間接反映了TcfA蛋白與細(xì)胞受體的相互作用，重組蛋白的競爭抑制實(shí)驗(yàn)表明TcfA蛋白能競爭抑制重組TcfA大腸桿菌的粘附，并表現(xiàn)出蛋白濃度的依賴性。TcfA小鼠多抗封閉實(shí)驗(yàn)結(jié)果顯示，封閉了重組大腸桿菌膜表面的TcfA蛋白，細(xì)菌的粘附率出現(xiàn)明顯的下降，說明了TcfA蛋白的粘附的特異性（依賴性）。含RGD的短肽與含RGE的短肽的競爭抑制試驗(yàn)表明，TcfA蛋白可能通過其氨基酸位點(diǎn)上的RGD模序發(fā)揮作用。慶大霉素胞外殺傷實(shí)驗(yàn)是檢測病原菌是否具有入侵宿主細(xì)胞能力的有效方法之一，該方法主要是檢測粘附分子是否介導(dǎo)了病原菌的入侵。實(shí)驗(yàn)結(jié)果顯示，TcfA蛋白并不介導(dǎo)重組大腸桿菌入侵HeLa細(xì)胞。FHA、PRN基因缺失株與抗TcfA小鼠多克隆抗體作用后，粘附力相比于FHA、PRN基因缺失株和CS野生株有較明顯的下降，說明TcfA蛋白作為百日咳桿菌膜表面的一個(gè)粘附因子，，在細(xì)菌的粘附過程中發(fā)揮著重要的作用。 GST pull down實(shí)驗(yàn)初步證明Myosin-9蛋白可能是TcfA蛋白與HeLa細(xì)胞相互作用的受體，但需要進(jìn)一步驗(yàn)證。
[Abstract]:Pertussis is a respiratory infectious disease caused by Bordetella pertussis (hereinafter referred to as pertussis bacillus). The pathogenic mechanism of pertussis bacillus includes a series of processes, such as adhesion, invasion, toxin production and so on. Many membrane surface proteins such as fibroin (Filamentous hemagglutmin), adhesin (Pertactin), pertussis tracheobronchial colonization factor (Tracheal colonization factor; TcfA) and so on are involved in the bacterial adhesion process. Pertussis trachea colonization factor is a pertussis bacillus membrane surface protein. It is speculated that this protein may be the adhesion molecule of pertussis bacillus, but its adhesion function has not been systematically verified. Therefore, TcfA protein was expressed in Escherichia coli (BL21;DE3) by DNA recombination technique, and the recombinant TcfA protein was purified. Then the adhesion of TcfA protein was proved by cell adhesion assay in vitro, immunofluorescence assay and competitive inhibition test. On this basis, the function of RGD tripeptide at position 317-319 of TcfA protein was verified. At the same time, by constructing the FHA-PRN- gene deletion strain, the importance of TcfA protein in the adhesion process was explored, and the receptor of HeLa interaction between TcfA protein and classical model cells was found. TcfA protein was successfully expressed by gene recombination technique, and the recombinant protein was purified with 85% purity. The results of Western blot showed that the recombinant protein could react with the whole cell serum of vaccine producing strain (CS). The results showed that the recombinant protein had good immunoreactivity and antigen specificity. Glass slide agglutination assay and V tube agglutination assay showed that TcfA protein was expressed on the surface of Escherichia coli membrane. Fluorescence adhesion test confirmed that there was significant difference between E. coli expressing TcfA and Escherichia coli containing empty vector on classical model cell adhesion. Quantitative CFU experiment confirmed that there was a statistical difference between them. Adhesion to ELISA test from another technical method reflects a statistical difference between the two. The immunofluorescence assay of the recombinant protein indirectly reflected the interaction between the TcfA protein and the cell receptor. The competitive inhibition test of the recombinant protein showed that the TcfA protein could compete to inhibit the adhesion of the recombinant TcfA Escherichia coli. The results of TcfA mouse polyclonal blocking test showed that the adhesion rate of the bacteria to TcfA protein on the surface of recombinant Escherichia coli was significantly decreased, indicating the specificity (dependence) of the adhesion of TcfA protein. The competitive inhibition tests of short peptides containing RGD and short peptides containing RGE showed that TcfA proteins may function through RGD motifs at their amino acid sites. The extracellular killing test of gentamicin is one of the effective methods to detect whether the pathogen has the ability to invade the host cells, and the main method is to detect whether the adhesion molecule mediates the invasion of the pathogen. The results showed that TcfA protein did not mediate the invasion of HeLa cells by recombinant Escherichia coli. The adhesion of FHA,PRN gene deletion strain to polyclonal antibody against TcfA mice was significantly lower than that of FHA,PRN gene deletion strain and CS wild strain. As an adhesion factor of pertussis bacillus membrane, TcfA protein plays an important role in the process of bacterial adhesion. GST pull down assay showed that Myosin-9 protein may be the receptor of the interaction between TcfA protein and HeLa cells, but it needs further verification.
【學(xué)位授予單位】：中國食品藥品檢定研究院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R392

【共引文獻(xiàn)】

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1 柯兵兵;徐穎華;侯啟明;馬霄;;百日咳鮑特菌黏附分子的研究進(jìn)展[J];中國生物制品學(xué)雜志;2013年11期

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2 韓雙;疏綿狀嗜熱絲胞菌脂肪酶基因lgy的表達(dá)及分子改造[D];山東農(nóng)業(yè)大學(xué);2013年

3 錢微;兔波氏桿菌免疫原性及致病性研究[D];南京農(nóng)業(yè)大學(xué);2013年

4 范蕊;家蠶腸道短小芽孢桿菌SW41脂肪酶基因的克隆、表達(dá)及酶活性分析[D];西南大學(xué);2014年

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