本文選題：IL-a + IL-p多克隆抗體�。� 參考：《南昌大學(xué)學(xué)報(bào)(理科版)》2017年01期

【摘要】：利用分子克隆方法構(gòu)建重組載體pGEX-4T-1-IL-12a,轉(zhuǎn)化BL21(DE3),經(jīng)IPTG誘導(dǎo)表達(dá),谷胱甘肽瓊脂糖凝膠4B親和層析柱純化后,獲得重組融合蛋白GST-IL-12p35。以此蛋白為免疫抗原免疫新西蘭大耳白兔制備IL-12p35多克隆抗體。采用ELISA法評(píng)估抗體效價(jià),Western blot檢測(cè)抗體特異性。LPS刺激小鼠脾細(xì)胞,ELISA法檢測(cè)IL-12抗體阻斷后細(xì)胞培養(yǎng)上清中的IFN-γ的分泌量。結(jié)果:成功構(gòu)建重組載體pGEX-4T-1-IL-12a,并獲得重組融合蛋白GST-IL-12p35(約48kDa)。抗原免疫新西蘭大耳白兔制備IL-12p35多克隆抗體,ELISA法測(cè)得IL-12p35多克隆抗體效價(jià)為1鐩256 000,Western blot證實(shí)制備的多克隆抗體能夠識(shí)別結(jié)合重組蛋白。IL-12p35多克隆抗體能夠有效地抑制LPS誘導(dǎo)的脾細(xì)胞IFN-γ的表達(dá)。
[Abstract]:The recombinant plasmid pGEX-4T-1-IL-12awas constructed by molecular cloning and transformed into BL21DE-3. The recombinant fusion protein GST-IL-12p35 was obtained by IPTG induced expression and purified by glutathione agarose gel 4B affinity chromatography. IL-12p35 polyclonal antibodies were prepared by immunizing New Zealand rabbits with this protein. ELISA assay was used to evaluate the titer of antibody. Western blot was used to detect the secretion of IFN- 緯 in the culture supernatant of murine spleen cells stimulated by IL-12 antibody. Results: the recombinant vector pGEX-4T-1-IL-12awas successfully constructed and the recombinant fusion protein GST-IL-12p35 was obtained. Antigen-immunized New Zealand white rabbits with IL-12p35 polyclonal antibodies were obtained by Elisa. The titer of IL-12p35 polyclonal antibodies was 1: 256 000. Western blot confirmed that the prepared polyclonal antibodies could recognize the binding protein. IL-12p35 polyclonal antibodies could be effectively inhibited. LPS induced expression of IFN- 緯 in splenocytes.
【作者單位】： 鄭州大學(xué)生命科學(xué)學(xué)院分子免疫實(shí)驗(yàn)室;鄭州人民醫(yī)院;河南省醫(yī)藥科學(xué)研究院;
【基金】：重大新藥創(chuàng)制科技重大專項(xiàng)(2012ZX09103301-022) 國(guó)家自然科學(xué)基金資助項(xiàng)目(U1204817、81373119、81571526)
【分類號(hào)】：S852.4

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