【摘要】： 研究目的:通過細(xì)胞雜交-雜交瘤技術(shù)制備抗幽門螺桿菌細(xì)胞空泡毒素(VacA)和粘附素(HpaA)抗原的雙特異性單克隆抗體,并初步鑒定其生物學(xué)、免疫學(xué)和理化特性。為幽門螺桿菌(H.pylori)感染的防治研究、診斷試劑盒的研制及探討其致病機(jī)制奠定基礎(chǔ)。 方法:自重組幽門螺桿菌細(xì)胞空泡毒素、粘附素(pQE30-V/H-DH5α)基因工程菌提取細(xì)胞空泡毒素(VacA)和粘附素(HpaA)的重組蛋白,12%SDS一PAGE電泳鑒定,以VacA- HpaA重組蛋白為免疫原,免疫BALB/c小鼠。取免疫BALB/c小鼠脾細(xì)胞與骨髓瘤細(xì)胞株SP2/0,用50%聚乙二醇(PEG)融合,HAT培養(yǎng)基選擇培養(yǎng)出雜交瘤細(xì)胞。以純化的rVacA為抗原,間接ELISA法初篩分泌抗VacA抗體的雜交瘤細(xì)胞,有限稀釋法克隆化培養(yǎng)獲得穩(wěn)定分泌抗rVacA單抗的陽性雜交瘤細(xì)胞株。陽性雜交瘤細(xì)胞株再與血清HpaA高滴度的免疫BALB/c小鼠脾細(xì)胞用50%聚乙二醇(PEG)融合,HAT培養(yǎng)基選擇培養(yǎng)出雜交瘤細(xì)胞,分別用純化的rVacA、rHpaA為抗原,間接ELISA法篩選同時分泌抗VacA和HpaA抗體的雜交-雜交瘤細(xì)胞,有限稀釋法克隆化培養(yǎng)獲得穩(wěn)定分泌抗rVacA和rHpaA雙特異性單克隆抗體陽性的雜交-雜交瘤細(xì)胞株。陽性細(xì)胞株體外連續(xù)傳30代、反復(fù)凍存復(fù)蘇6次、液氮中凍存1.5個月后復(fù)蘇,測定細(xì)胞株分泌的雙特異性單克隆抗體的穩(wěn)定性。做雜交-雜交瘤細(xì)胞的染色體計數(shù)并與骨髓瘤細(xì)胞系SP2/0和小鼠骨髓細(xì)胞的染色體計數(shù)進(jìn)行比較。制備腹水型和上清型雙特異性單克隆抗體,以飽和硫酸銨沉淀法和免疫親和層析法進(jìn)行純化,Bradford法測定其蛋白濃度,Western blot檢測其免疫特異性,b.v公司的抗體亞型檢測試劑盒測定雙特異性單克隆抗體的Ig類別,ELISA間接法測定其抗體效價及親和常數(shù)。測定pH值對雙特異性單克隆抗體穩(wěn)定性影響實(shí)驗(yàn),分別以0.05M pH2.2甘氨酸-鹽酸緩沖液、0.05M pH9.6碳酸鹽緩沖液和0.01M pH7.4磷酸鹽緩沖液將腹水型雙特異性單克隆抗體(以免疫前小鼠血清作陰性對照)作1:10稀釋,4℃靜置24h、48h后,用間接ELISA測定其0D450nm與陰性對照0D450nm的比值。 結(jié)果:自基因工程菌pQE30-V/H-DH5α中提取重組VacA- HpaA融合蛋白,12%SDS-PAGE鑒定后,作為免疫原成功免疫小鼠。經(jīng)兩次細(xì)胞融合,HAT選擇培養(yǎng),抗體檢測篩選,獲得37孔分泌同時抗rVacA和rHpaA雙特異性單克隆抗體的融合細(xì)胞孔,經(jīng)克隆化培養(yǎng)得到3株穩(wěn)定分泌雙特異性單克隆抗體的雜交-雜交瘤細(xì)胞株,命名為fB8、fC5和fE7,其分泌的雙特異性單克隆抗體均可與純化的rVacA或rHpaA蛋白特異性結(jié)合。fB8雜交-雜交瘤細(xì)胞株穩(wěn)定性較好,體外連續(xù)傳30代、反復(fù)凍存復(fù)蘇6次、液氮中凍存1.5個月后復(fù)蘇測定細(xì)胞株仍可穩(wěn)定分泌雙特異性單克隆抗體。fB8雜交-雜交瘤細(xì)胞染色體計數(shù)為127±5條,而小鼠脾細(xì)胞為40條,Sp2/0骨髓瘤細(xì)胞為64±3條。三株同時抗rVacA和rHpaA的雙特異性單克隆抗體雜交-雜交瘤細(xì)胞(fB8、fC5和fE7)分泌的抗體均為IgG1。fB8腹水型雙特異性單克隆抗體的效價為5.12×105,蛋白濃度為11.32 mg/ml(Bradford法)。pH值對雙特異性單克隆抗體穩(wěn)定性影響的實(shí)驗(yàn)結(jié)果顯示,酸性和堿性都可使雙特異性單克隆抗體穩(wěn)定性下降。 結(jié)論:初步建立起三株分泌同時抗幽門螺桿菌細(xì)胞空泡毒素和粘附素雙特異性單克隆抗體的雜交-雜交瘤細(xì)胞株.成功制備并純化了抗VacA和HpaA的雙特異性單克隆抗體,其效價較高,親和力較強(qiáng),特異性強(qiáng),將為幽門螺桿菌感染的診斷、預(yù)防和治療發(fā)揮重要作用。
[Abstract]:Objective: To prepare a bispecific monoclonal antibody against Helicobacter pylori (VacA) and adhesin (HpaA) antigen by cell hybridization-hybridoma technique, and to identify its biological, immunological and physical and chemical properties. To study the prevention and treatment of H. pylori infection, the development of the diagnostic kit and the foundation of its pathogenesis. Methods: The recombinant protein of the cell vacuolated toxin (VacA) and the adhesin (HpaA) was extracted from the recombinant H.pylori cell vacuolated toxin and the adhesin (pQE30-V/ H-DH5). The recombinant protein of the cell vacuolated toxin (VacA) and the adhesin (HpaA) was identified by SDS-PAGE. The recombinant protein of vacA-HpaA was used as the immunogen to immunize BALB/ c. C. The spleen cells of the immunized BALB/ c mice were fused with the myeloma cell strain SP2/0, fused with 50% polyethylene glycol (PEG), and the HAT medium selected to culture the hybridization. Tumor cells. The purified rVacuA is an antigen, and the indirect ELISA method is used for the primary screening of a hybridoma cell secreting anti-VacA antibody, and the positive hybridomas stably secrete anti-rVacuA monoclonal antibody are obtained by cloning and culturing by a limited dilution method. The positive hybridoma cell line and the immunized BALB/ c mouse spleen cells with high titer of the serum HpaA were fused with 50% polyethylene glycol (PEG), and the HAT medium was selected to culture the hybridoma cells, and the purified rVacuA and rHpaA were used respectively. Screening of hybrid-hybridoma cells with simultaneous secretion of anti-VacA and HpaA antibodies by indirect ELISA, and the hybrid-hybridomas that stably secrete anti-rVacA and rHpaA bispecific monoclonal antibodies are obtained by cloning and culturing in a limited dilution method. Cell strain. The positive cell line was continuously transmitted for 30 generations in vitro, the recovery was repeated for 6 times, and the recovery was performed in liquid nitrogen for 1.5 months, and the double-specific monoclonal antibody secreted by the cell line was determined. Stability. The chromosome counts of the hybrid-hybridoma cells were counted and the chromosomes of the myeloma cell line SP2/0 and the mouse bone marrow cells were counted Line comparison. Ascites and supernatant type bispecific monoclonal antibodies were prepared, and purified by saturated sulfuric acid precipitation method and immunoaffinity chromatography, and the protein concentration was determined by Bradford method. Western blot was used to detect its immunity. The Ig category of bispecific monoclonal antibody was determined by the antibody subtype detection kit of b. v, and the antibody titer and affinity of the two-specific monoclonal antibody were determined by ELISA. And the stability of the double-specificity monoclonal antibody is measured by measuring the pH value, and the stability of the double-specificity monoclonal antibody is measured with 0.05M pH2. 2glycine-hydrochloric acid, respectively. Ascites-type bispecific monoclonal antibody (negative control with the serum of the pre-immunized mouse) was used as 1:10 for buffer, 0.05M pH9.6 carbonate buffer and 0.01 M pH 7.4 phosphate buffer, and the 0 D450nm and negative control 0D450nm were determined by indirect ELISA after 24 h and 48 h at 4 鈩，

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