本文選題：晶體蛋白 + 結(jié)核分枝桿菌��； 參考：《廣西醫(yī)科大學(xué)》2012年碩士論文

【摘要】：結(jié)核分枝桿菌(Mycobacterium tuberculosis, MTB)目前世界上最具致病性的人類病原體。全球目前有30%的人口被MTB感染,其中有90%的感染者以臨床上無癥狀的形式存在,即以潛伏狀態(tài)存在。結(jié)核桿菌晶體蛋白(Alpha-Crystallin, Acr)是結(jié)核分枝桿菌潛伏感染期機(jī)體免疫反應(yīng)的重要靶抗原,對MTB進(jìn)入休眠狀態(tài)及MTB休眠菌在體內(nèi)的存活起著重要的作用。本研究選擇MTB休眠期重要的保護(hù)性抗原a-crystallin(Acr),從大容量人源噬菌體抗體庫中篩選特異的抗MTB Acr蛋白的人源抗體。構(gòu)建IgA真核表達(dá)載體,在中國倉鼠卵巢細(xì)胞中表達(dá)抗結(jié)核桿菌的人源IgA抗體,為進(jìn)一步研究這些抗體對于結(jié)核桿菌感染的預(yù)防和治療作用,探索抗晶體蛋白防治結(jié)核桿菌感染的可行性和可能的作用機(jī)理鑒定基礎(chǔ)。 1結(jié)核分枝桿菌Acr蛋白表達(dá)載體的構(gòu)建及Acr的表達(dá)純化 以MTB H37Rv基因組DNA為模板,通過PCR方法對Acr蛋白的基因進(jìn)行擴(kuò)增,以pCold為載體構(gòu)建重組質(zhì)粒,再轉(zhuǎn)化到表達(dá)宿主菌BL21(DE3)中,以IPTG誘導(dǎo)表達(dá)。經(jīng)SDS-PAGE和Western blotting分析并純化該表達(dá)產(chǎn)物。用超濾的方式將純化后蛋白質(zhì)溶液的buffer替換為PBS緩沖液。構(gòu)建了具有正確基因序列的Acr蛋白重組表達(dá)質(zhì)粒,重組蛋白在大腸桿菌BL21(DE3)中表達(dá)。分別用6×his的單克隆抗體(mAb)和抗Acr蛋白mAb對表達(dá)產(chǎn)物進(jìn)行Western blotting分析,結(jié)果顯示在相對分子量約19kDa處均有特異性條帶,與預(yù)計(jì)大小相吻合,表明成功表達(dá)目的蛋白。低溫誘導(dǎo)培養(yǎng),Acr蛋白獲得了可溶性表達(dá)。純化、超濾后蛋白純度達(dá)90%,濃度達(dá)0.8mg/ml 2結(jié)核分枝桿菌Acr蛋白人源抗體的篩選 以MTB Acr蛋白包被免疫管,通過對噬菌體抗體庫進(jìn)行4輪“吸附-洗脫-擴(kuò)增”的過程,從大容量抗體庫中篩選特異性(?)(?)MTB Acr蛋白的抗體,并對可變區(qū)序列進(jìn)行了測序分析。將特異性的噬菌體抗體感染HB2151菌,經(jīng)I PTG誘導(dǎo)表達(dá),制備了抗MTB Acr蛋白的可溶性單鏈抗體；對其序列和抗原結(jié)合活性進(jìn)行分析鑒定。經(jīng)過4輪篩選,獲得了43個與MTB Acr蛋白結(jié)合的陽性克隆,29個特異結(jié)合的克�。粶y序分析有26不同的可變區(qū)片段；通過可溶性scFv表達(dá)篩選到14株特異性結(jié)合Acr蛋白的可溶性scFv抗體克隆；經(jīng)過基因測序,分析了可變區(qū)基因的亞群。成功制備了可溶性抗體。Westren blotting分析證實(shí)篩選的人源抗體能與天然蛋白結(jié)合。 3結(jié)核分枝桿菌Acr蛋白人源IgA抗體真核表達(dá)載體的構(gòu)建 通過融合PCR將重鏈可變區(qū)基因與本室構(gòu)建的IgA2恒定區(qū)的序列拼接,輕鏈可變區(qū)基因與本室構(gòu)建的k鏈恒定區(qū)基因連接,完成了IgA抗體基因的構(gòu)建。將IgA抗體克隆到真核表達(dá)載體pEF-dhfr-Neo中,構(gòu)建了pEF-dhfr-IGH,pEF-dhfr-IGK IgA真核表達(dá)載體。 4結(jié)核分枝桿菌Acr人源IgA抗體在真核細(xì)胞中的表達(dá) 用構(gòu)建的pEF-dhfr-IGH和pEF-dhfr-IGK表達(dá)質(zhì)粒共轉(zhuǎn)染CHO/dhfr-細(xì)胞。72小時后,用鼠抗人的工gA(a)和鼠抗人的kappa鏈mAb和結(jié)核分枝桿菌的Acr蛋白包被酶聯(lián)板,ELISA檢測細(xì)胞上清IgA抗體的表達(dá)。 綜上所述：本實(shí)驗(yàn)表達(dá)出MTB Acr蛋白,通過對大容量噬菌體抗體庫的篩選,篩選到14株特異性結(jié)合MTB Acr蛋白的可溶性抗體。通過western blotting實(shí)驗(yàn)驗(yàn)證了可溶性抗體與天然蛋白的結(jié)合活性。構(gòu)建了抗MTB Acr蛋白的IgA真核表達(dá)載體,通過轉(zhuǎn)染中國倉鼠卵巢細(xì)胞,獲得了工gA抗體,免疫檢測結(jié)果顯示該重組全分子人源IgA抗體具有特異性識別結(jié)核桿菌Acr蛋白的能力。
[Abstract]:Mycobacterium tuberculosis (MTB) is currently the most pathogenic human pathogen in the world. 30% of the world's population is currently infected by MTB, of which 90% of the infected people exist in the form of asymptomatic in clinical, that is, the latent state. Mycobacterium tuberculosis (Alpha-Crystallin, Acr) is the potential of Mycobacterium tuberculosis. The important target antigen of the immune response in the stage of volt infection plays an important role in the entry of MTB into dormancy and the survival of MTB dormant bacteria in the body. This study selected the important protective antigen A-crystallin (Acr) of the MTB dormancy period, and screened the human antibody against the specific anti MTB Acr protein from the large human phage antibody library. The expression vector expressed IgA antibody against Mycobacterium tuberculosis in Chinese hamster ovary cells, in order to further study the preventive and therapeutic effects of these antibodies on Mycobacterium tuberculosis infection, and to explore the feasibility and possible mechanism of anti tuberculous bacillus to prevent Mycobacterium tuberculosis infection.
Construction of Mycobacterium tuberculosis Acr protein expression vector 1 and expression and purification of Acr
The MTB H37Rv genome DNA was used as a template to amplify the gene of Acr protein by PCR method, construct the recombinant plasmid with pCold as the carrier, and then convert it into the expression host strain BL21 (DE3) and induce expression in IPTG. The expression product was analyzed and purified by SDS-PAGE and Western blotting. The purified protein solution was replaced by ultrafiltration. The recombinant expression plasmid of Acr protein with correct gene sequence was constructed, and the recombinant protein was expressed in Escherichia coli BL21 (DE3). The expression products were analyzed with 6 x his monoclonal antibody (mAb) and anti Acr protein mAb, respectively. The results showed that there were specific bands at the relative molecular weight around 19kDa. The results showed that the target protein was expressed successfully. The soluble expression of Acr protein was obtained by low temperature induction culture. The purity of protein was 90% after ultrafiltration, and the concentration was up to 0.8mg/ml
Screening of human antibody for 2 Mycobacterium tuberculosis Acr protein
MTB Acr protein was coated with the immune tube, and the antibodies of specific (?) MTB Acr protein were screened from the large capacity antibody library by the 4 rounds of "adsorption elution amplification" of the phage antibody library, and the sequence of the variable region was sequenced. The specific phagocytic antibody was infected with HB2151 bacteria, induced by I PTG, and the anti MTB was prepared. Acr protein soluble single chain antibody; analysis and identification of its sequence and antigen binding activity. After 4 rounds of screening, 43 positive clones combined with MTB Acr protein were obtained, 29 specific clones were cloned, and 26 different variable region fragments were sequenced, and 14 specific binding Acr proteins were screened by soluble scFv expression. The soluble scFv antibody clones were cloned, and the subpopulations of the variable region genes were analyzed by gene sequencing. The soluble antibody.Westren blotting analysis was successfully prepared to confirm that the screened human antibody could be combined with the natural protein.
Construction of eukaryotic expression vector for human IgA antibody of 3 Mycobacterium tuberculosis Acr protein
By splicing the gene of the heavy chain variable region with the sequence of the IgA2 constant region constructed in this room by fusion of PCR, the light chain variable region gene was connected with the K chain constant region gene of the room, and the construction of the IgA antibody gene was completed. The IgA antibody was cloned into the eukaryotic expression vector pEF-dhfr-Neo, and the pEF-dhfr-IGH and pEF-dhfr-IGK IgA eukaryotic expression vector was constructed.
Expression of human IgA antibody against Mycobacterium tuberculosis Acr 4 in eukaryotic cells
After transfecting CHO/dhfr- cells with the constructed pEF-dhfr-IGH and pEF-dhfr-IGK plasmids for.72 hours, the mouse anti human gA (a) and the mouse anti human kappa chain mAb and the Acr protein of Mycobacterium tuberculosis were used to detect the expression of the IgA antibody in the cell supernatant.
To sum up: the MTB Acr protein was expressed in this experiment. By screening the large capacity phage antibody library, 14 soluble antibodies with specific binding of MTB Acr protein were screened. The binding activity of soluble antibody and natural protein was verified by Western blotting experiment. The IgA eukaryotic expression vector of anti MTB Acr protein was constructed and transfected through transfection. The Chinese hamster ovary cells obtained the gA antibody. The results of immunoassay showed that the recombinant human IgA antibody had the ability to identify the Acr protein of Mycobacterium tuberculosis.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392.1

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