本文選題：顆粒溶素　切入點(diǎn)：純化　出處：《蘭州大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的：純化原核表達(dá)的重組人顆粒溶素(GNLY),并制備其單克隆抗體(mAb),為GNLY相關(guān)疾病的診斷提供一種新指標(biāo)。 方法：Ni離子親和層析法純化重組人GNLY,用其免疫BALB/c小鼠,采用常規(guī)雜交瘤技術(shù)制備相應(yīng)mAb。間接ELISA法測雜交瘤細(xì)胞培養(yǎng)上清和腹水mAb的效價(jià),ELISA相加試驗(yàn)測定抗原表位,硫氰酸鹽洗脫法測定相對(duì)親和力指數(shù),并行Ig亞類、特異性及雜交瘤細(xì)胞分泌mAb的穩(wěn)定性檢測。 結(jié)果：純化的重組GNLY融合蛋白,其純度和含量分別為95%和0.8g/L。共篩選出能穩(wěn)定分泌抗人GNLY mAb的雜交瘤細(xì)胞4株,分別命名為5E5、5G7、6C8和9C6。4株雜交瘤細(xì)胞培養(yǎng)上清及腹水中mAb的效價(jià)分別為1：100-1：3200和(0.2-8)×10-4。5E5雜交瘤細(xì)胞株分泌mAb的Ig亞類為IgM,其余3株mAb為IgG1。4株雜交瘤細(xì)胞體外連續(xù)培養(yǎng)2個(gè)月及液氮凍存6個(gè)月后復(fù)蘇,分泌mAb的水平仍能達(dá)到之前的水平。4株mAb的相對(duì)親和力指數(shù)為1.5-3.0mol/L；5G7、6C8和9C63株雜交瘤細(xì)胞株分泌mAb的相加指數(shù)(AI)＜50%,而5E5分泌mAb與上述3株細(xì)胞分泌mAb的AI＞50%。4株純化后的mAb與GNLY抗原有較高的吸光度值,而與其他包被的抗原不反應(yīng)。 結(jié)論：利用Ni離子親和層析法獲得可溶性重組GNLY;通過雜交瘤技術(shù),獲得4株能分泌特異性強(qiáng)、效價(jià)高的GNLY mAb雜交瘤細(xì)胞株。
[Abstract]:Objective: to purify the recombinant human granulysin (GNLY) expressed in prokaryotic expression and prepare its monoclonal antibody (McAb) to provide a new index for the diagnosis of GNLY related diseases. Methods the recombinant human GNLYY mice were immunized with the purified recombinant human GNLYY by affinity chromatography. The corresponding mAbs were prepared by conventional hybridoma technique. Indirect ELISA assay was used to detect the antigenic epitopes in the supernatant of hybridoma cell culture and ascites mAb. The relative affinity index, Ig subclass, specificity and stability of mAb secreted by hybridoma cells were determined by thiocyanate elution method. Results: the purity and content of purified recombinant GNLY fusion protein were 95% and 0.8 g / L, respectively. Four hybridoma cells secreting anti-human GNLY mAb stably were screened. The titers of mAb in the supernatant and ascites of the hybridoma cell lines named 5E5, 5G7C8 and 9C6.4 were 1: 100-1: 3200 and 0.2-8 respectively) 脳 10-4.5E5 hybridoma cell lines secreting mAb. The Ig subclasses of mAb secreted by the hybridoma cells of the other three strains of mAb were IgG1.4 hybridoma cells in vitro for 2 months, respectively. Liquid nitrogen was frozen for six months and then recovered. The relative affinity index of mAb was 1.5-3.0mol / L 5G7C8 and 9C63 hybridoma cell lines secreted mAb (AI < 50), while 5E5 secreted mAb and the AI of mAb secreted by the above three strains were higher than 50.4%. GNLY antigen has high absorbance value. It does not react with other coated antigens. Conclusion: the soluble recombinant GNLY was obtained by Ni ion affinity chromatography, and four GNLY mAb hybridoma cell lines with strong specificity and high titer were obtained by hybridoma technique.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392.11

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