本文選題：梅毒螺旋體 + 單克隆抗體��； 參考：《南華大學(xué)》2007年碩士論文

【摘要】： 目的:將本室構(gòu)建的含梅毒螺旋體(Treponema pallidum,Tp)外膜蛋白Tp0453的優(yōu)勢(shì)表位(28～288aa)基因的重組表達(dá)體在E.coli M15中進(jìn)行誘導(dǎo)表達(dá),純化表達(dá)產(chǎn)物并進(jìn)行抗原性分析。應(yīng)用雜交瘤技術(shù),將SP2/0細(xì)胞與經(jīng)重組蛋白Tp0453免疫的小鼠脾細(xì)胞進(jìn)行融合,篩選抗重組蛋白Tp0453的單克隆抗體(mAb) ,并進(jìn)行鑒定和純化。用此mAb檢測(cè)Tp感染疑似患者的分泌物標(biāo)本,由此確定該mAb的應(yīng)用價(jià)值,并為Tp感染診斷的研究奠定基礎(chǔ)。 方法:(1)使用鎳親和層析法純化重組蛋白Tp0453。SDS-PAGE和Western- blot鑒定表達(dá)產(chǎn)物。(2)以復(fù)性后純化的Tp0453重組蛋白免疫BALB/c小鼠,將免疫小鼠的脾細(xì)胞與SP2/0細(xì)胞融合,用間接酶聯(lián)免疫吸附試驗(yàn)(ELISA)篩選分泌特異性抗體的雜交瘤細(xì)胞。(3)體內(nèi)誘生腹水法制備抗體,用硫酸銨沉淀法對(duì)腹水中的mAb進(jìn)行純化,ELISA法檢測(cè)抗體效價(jià),mAb亞類測(cè)定試劑盒鑒定mAb的類別,通過(guò)細(xì)胞涂片染色鏡檢計(jì)數(shù)雜交瘤細(xì)胞株染色體數(shù)目。(4)間接ELISA和間接熒光免疫試驗(yàn)(IIF)檢測(cè)臨床標(biāo)本及mAb的特異性,并進(jìn)行統(tǒng)計(jì)學(xué)分析。 結(jié)果:重組目的基因表達(dá)菌在IPTG的誘導(dǎo)下,表達(dá)了相對(duì)分子量(Mr)約為32kDa的目的蛋白,目的蛋白在菌體細(xì)胞內(nèi)主要以包涵體形式存在;經(jīng)Ni-NTA親和層析法純化獲得了純度在95%以上的重組蛋白;Western-blot檢測(cè)其能與Anti-His mAb發(fā)生特異性反應(yīng);應(yīng)用純化復(fù)性后的重組Tp0453免疫8周齡的BALB/c小鼠,經(jīng)過(guò)雜交瘤技術(shù)進(jìn)行細(xì)胞融合;應(yīng)用ELISA法篩選出了4株分泌抗Tp0453重組蛋白mAb的雜交瘤細(xì)胞株,分別命名為2F8、8B9、9C6和15B2;Western-blot結(jié)果顯示mAb均能與重組Tp0453很好的結(jié)合。用腹水誘生法大量制備mAb;ELISA檢測(cè)4株雜交瘤細(xì)胞誘生腹水中的mAb效價(jià)分別為1:25 000、1:8 000、1:50 000和1:10 000;根據(jù)mAb親和常數(shù)公式計(jì)算4株雜交瘤細(xì)胞分泌的mAb親和常數(shù)分別為4.12×107M-1,2.73×108M-1,4.71×108M-1和3.64×107M-1;經(jīng)Mouse Monoclonal Antibody Isotyping Kit測(cè)定,4株雜交瘤細(xì)胞分泌的mAb,2F8為IgG2b,其它3株均為IgG1。4株mAb腹水經(jīng)SDS-PAGE均顯示出一條分子量約為50kDa的重鏈條帶和一條分子量為26kDa的輕鏈條帶。4株雜交瘤細(xì)胞染色體數(shù)在90～108范圍內(nèi)變化。IIF和間接ELISA對(duì)85份臨床標(biāo)本進(jìn)行檢測(cè)結(jié)果表明自制mAb能與天然抗原結(jié)合,并與鍍銀染色的檢測(cè)結(jié)果基本符合。 結(jié)論: (1)篩選出4株穩(wěn)定分泌抗Tp0453 mAb的雜交瘤細(xì)胞株,經(jīng)鑒定4株雜交瘤細(xì)胞分泌的mAb均能識(shí)別重組Tp0453。 (2)獲得的抗重組Tp0453的mAb均為IgG類抗體,且能識(shí)別Tp0453抗原的天然表位,具有較好的特異性。 (3)自制mAb與鍍銀染色的檢測(cè)結(jié)果比較具有較好的一致性,有望應(yīng)用于Tp抗原的檢測(cè)。
[Abstract]:Aim: to induce the recombinant expression of the outer membrane protein Tp0453 containing Treponema pallidum Tp1 in our laboratory into E.coli M15, and to purify the expressed product and analyze its antigenicity.SP2/0 cells were fused with murine spleen cells immunized with recombinant protein Tp0453 by hybridoma technique. Monoclonal antibodies against recombinant protein Tp0453 were screened, identified and purified.The mAb was used to detect the secretions of suspected patients with TP infection. The application value of the mAb was determined and the foundation for the diagnosis of TP infection was established.Methods the recombinant protein Tp0453.SDS-PAGE and Western blot were purified by nickel affinity chromatography. The purified recombinant Tp0453 protein was used to immunize BALB/c mice, and the spleen cells of the immunized mice were fused with SP2/0 cells.Indirect enzyme-linked immunosorbent assay (Elisa) was used to screen hybridoma cells secreting specific antibodies.MAb in ascites was purified by ammonium sulfate precipitation method. Elisa was used to detect antibody titer and mAb subclass test kit was used to identify the type of mAb.Indirect ELISA and indirect fluorescence immunoassay (IIFI) were used to detect the specificity of clinical specimens and mAb.Results: under the induction of IPTG, the recombinant target gene expressed the target protein of 32kDa, and the target protein existed mainly in the form of inclusion body.The recombinant protein with purity of more than 95% was purified by Ni-NTA affinity chromatography and its specific reaction with Anti-His mAb was detected by Western-blot. The purified recombinant Tp0453 was used to immunize the 8-week-old BALB/c mice, and the cells were fused by hybridoma technique.Four hybridoma cell lines secreting mAb against Tp0453 recombinant protein were screened by ELISA method and named as 2F8B9C6 and 15B2 Western-blot, respectively. The results showed that mAb could bind well with recombinant Tp0453.The mAb titers in ascites of four hybridoma cell lines were measured by Elisa with the method of ascites induction. The mAb affinity constants of the four hybridoma cells were calculated by mAb affinity constant formula as 4.12 脳 107M-12.73 脳 108M-1 and 4.71 脳 108M-1, respectively.The mAb2F8 secreted by Mouse Monoclonal Antibody Isotyping Kit was IgG2b, and the other three IgG1.4 strains mAb ascites showed a heavy chain band with a molecular weight of 50kDa and a light chain band with a molecular weight of 26kDa.The changes of chromosome number in 90 ~ (10 ~ 8) range. IIF and indirect ELISA were used to detect 85 clinical specimens. The results showed that self-made mAb could bind to natural antigen.The results are in good agreement with the results of silver-plated staining.Conclusion:1) four hybridoma cell lines secreting stable anti- mAb were screened, and all the mAb secreted by the four hybridoma cells could recognize recombinant Tp0453.(2) the mAb of the recombinant Tp0453 were all IgG class antibodies, and could recognize the natural epitopes of Tp0453 antigen, and had good specificity.(3) the results of self-made mAb and silver staining are in good agreement with each other, and it is expected to be applied to the detection of TP antigen.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 曾令宇,黃強(qiáng),陳利弘,萬(wàn)琳,盧曉風(fēng);表達(dá)于大腸桿菌中的人源性抗CTLA4單鏈抗體復(fù)性方法探索[J];生物醫(yī)學(xué)工程學(xué)雜志;2005年03期

2 黃建生,解詠梅,張潛,沈先榮,任大明,吉冬梅,許諄,賈福星,雷呈祥,蘭和魁,鄭維揚(yáng),張麗蕓,陳立茵,郭明秋;HCV多表位抗原基因重組減毒口服活菌苗的研究[J];微生物學(xué)報(bào);2000年05期

3 趙飛駿,吳移謀,張曉紅,劉雙全,余敏君;梅毒螺旋體外膜蛋白Gpd基因的克隆、表達(dá)及其免疫活性研究[J];微生物學(xué)報(bào);2005年05期

4 曾鐵兵,吳移謀,黃澍杰,吳志周;巢式PCR擴(kuò)增梅毒螺旋體polA及其臨床應(yīng)用的研究[J];中國(guó)皮膚性病學(xué)雜志;2004年02期

5 劉雙全,吳移謀,趙飛駿,顧偉鳴;梅毒螺旋體Tp0453重組蛋白的表達(dá)純化及免疫活性研究[J];中國(guó)皮膚性病學(xué)雜志;2005年11期

6 曾鐵兵,吳移謀,黃澍杰,吳志周;衡陽(yáng)和江門地區(qū)梅毒螺旋體基因分型的初步研究[J];中華皮膚科雜志;2004年12期

7 趙飛駿;吳移謀;張曉紅;劉雙全;楊勝輝;余敏君;;梅毒螺旋體融合雙價(jià)DNA疫苗的構(gòu)建及其免疫活性研究[J];中華皮膚科雜志;2006年05期

8 劉雙全,吳移謀,趙飛駿,曾鐵兵;梅毒螺旋體Tp0453重組蛋白的表達(dá)及在梅毒血清學(xué)診斷中的初步應(yīng)用[J];中華檢驗(yàn)醫(yī)學(xué)雜志;2005年10期

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