發(fā)布時(shí)間：2018-08-04 19:20

【摘要】：人B淋巴細(xì)胞刺激因子(hBLyS)是1999年發(fā)現(xiàn)的腫瘤壞死因子超家族新成員,它與某些免疫疾病的制病機(jī)制密切相關(guān),其低量或過(guò)量表達(dá)均能引起機(jī)體的免疫失衡。本研究以大腸桿菌BL21(λDE3)表達(dá)重組hBLyS的可溶性蛋白為抗原,分別免疫日本大耳白兔和BALB/c小鼠,制備出抗hBLyS的多克隆和單克隆抗體,并對(duì)這兩種抗體的一些特性進(jìn)行鑒定。此外,應(yīng)用這兩種抗體建立hBLyS的雙抗夾心ELISA檢測(cè)方法。 1 抗hBLyS多克隆抗體的制備與鑒定 選取2～3kg的雄性日本大耳白兔,淋巴結(jié)注射弗氏佐劑乳化的可溶性hBLyS蛋白,每間隔2周注射一次;第3次免疫后11天,耳動(dòng)脈采血,間接ELISA檢測(cè)抗血清的效價(jià)為50萬(wàn)倍;頸總動(dòng)脈放血,制備抗血清,并應(yīng)用分級(jí)飽和硫酸銨鹽析法進(jìn)行純化,westen-blot分析抗體的特異性。 2 抗hBLyS單克隆抗體的制備與特性鑒定 用hBLyS蛋白免疫BALB/c小鼠,取免疫脾細(xì)胞與骨髓瘤SP2/0細(xì)胞進(jìn)行融合,通過(guò)5次亞克隆獲得1株分泌抗hBLyS單克隆抗體的雜交瘤細(xì)胞,,染色體數(shù)目在87～101之間:該抗體屬于IgG1類蛋白,其腹水效價(jià)可達(dá)2×10~6通過(guò)western-blot鑒定其特異性良好。采用小鼠腹腔注射法大量制備抗體,A蛋白親和層析法純化抗體。 3 建立檢測(cè)hBLyS蛋白含量的ELISA方法 選用2種ELISA方案對(duì)hBLyS蛋白含量的測(cè)定方法進(jìn)行研究,方案一用鏈霉親和素做間接包被,方案二用生物素-親和素系統(tǒng)做終反應(yīng)放大;優(yōu)化選擇2種檢測(cè)方法的反應(yīng)條件;方法一的敏感度較低:方法二在hBLyS蛋白濃度為3.2～400 ng/mL范圍內(nèi),標(biāo)準(zhǔn)曲線的線性關(guān)系良好;從而建立了一種可檢測(cè)hBLyS蛋白濃度為ng級(jí)水平的ELISA方法。
[Abstract]:Human B lymphocyte stimulating factor (hBLyS) is a new member of the tumor necrosis factor superfamily discovered in 1999. It is closely related to the pathogenesis of some immune diseases, and its low or excessive expression can cause immune imbalance. In this study, the soluble protein of Escherichia coli BL21 (位 DE3) expressing recombinant hBLyS was used as antigen, Japanese white rabbits and BALB/c mice were immunized, polyclonal and monoclonal antibodies against hBLyS were prepared, and some characteristics of these two antibodies were identified. In addition, Using these two antibodies to establish a double antibody sandwich ELISA detection method for hBLyS. 1 preparation and identification of anti hBLyS polyclonal antibody Male Japanese white rabbits with 2~3kg, Lymph nodes were injected with soluble hBLyS protein emulsified by Freund's adjuvant and injected once every 2 weeks. 11 days after the third immunization, blood was collected from ear artery, the titer of antiserum detected by indirect ELISA was 500000 times. The specificity of the antibody was analyzed by Western blot with the method of fractionated saturated ammonium sulfate saltout. 2 preparation and characterization of monoclonal antibody against hBLyS BALB/c mice were immunized with hBLyS protein. Immune spleen cells were fused with myeloma SP2/0 cells and a hybridoma cell line secreting monoclonal antibody against hBLyS was obtained by five subclones. The chromosome number of the hybridoma cells was between 87101.The antibody belongs to IgG1 protein. Its ascites titer can reach 2 脳 10 ~ (-6). Its specificity is good by western-blot. The antibody was purified by affinity chromatography of antibody A protein by intraperitoneal injection of mice. (3) A ELISA method for the detection of hBLyS protein was established. Two kinds of ELISA schemes were used to determine the content of hBLyS protein. In scheme one, streptavidin was used as indirect coating, and biotin-avidin system was used to amplify the final reaction, and the reaction conditions of two detection methods were optimized. The first method has low sensitivity: the second method has a good linear relationship in the range of 3.2O400 ng/mL of hBLyS protein concentration, thus a ELISA method for detecting hBLyS protein concentration at ng level was established.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 張益鵠,徐艷娟,王央共,崔武任,沈關(guān)心;兔抗嗎啡多克隆抗體制備[J];中國(guó)法醫(yī)學(xué)雜志;2000年02期

2 董國(guó)偉,王沫,劉賢進(jìn),余向陽(yáng);兔抗甲胺磷多克隆抗體的制備[J];華中農(nóng)業(yè)大學(xué)學(xué)報(bào);2001年04期

3 童貽剛,王海濤;重組完整抗體研究進(jìn)展[J];生物技術(shù)通訊;2001年03期

4 李楚芳,劉萱,李平,馬清鈞,曹誠(chéng)軍;hPLIC-2可溶性表達(dá)及多克隆抗體的制備[J];生物技術(shù)通訊;2004年05期

5 李愛(ài)鳳;單克隆抗體與抗癌研究進(jìn)展[J];山西職工醫(yī)學(xué)院學(xué)報(bào);2003年01期

6 張明娟,呂卓人,袁育康,賀浪沖,王顥;抗哇巴因多克隆抗體F(ab)_2片段的研制及鑒定[J];西安醫(yī)科大學(xué)學(xué)報(bào)(中文版);2000年06期

7 萬(wàn)亞坤,張俊杰,陳南春,陳蘇民;兔抗人ERA多克隆抗體的純化[J];細(xì)胞與分子免疫學(xué)雜志;2001年02期

8 鄭夢(mèng)杰,楊純正;基因工程抗體的設(shè)計(jì)和應(yīng)用[J];中國(guó)藥學(xué)雜志;2002年04期

，

本文編號(hào)：2164887