本文選題：Zhangfei + 融合蛋白�。� 參考：《西北農(nóng)林科技大學(xué)》2008年碩士論文

【摘要】： Zhangfei是堿性亮氨酸拉鏈蛋白家族成員之一,分子量為28.9ku,是參與內(nèi)質(zhì)網(wǎng)應(yīng)激和未折疊蛋白反應(yīng)調(diào)節(jié)的轉(zhuǎn)錄因子之一,一些腫瘤細(xì)胞中也存在未折疊蛋白反應(yīng),其在胚胎發(fā)育、人類疾病等生理和病理過程中具有重要的意義。為此本試驗(yàn)對(duì)Zhangfei融合蛋白進(jìn)行了誘導(dǎo)表達(dá)、純化并免疫動(dòng)物,制備并鑒定了Zhangfei蛋白的單克隆抗體,試驗(yàn)結(jié)果如下: 1篩選了誘導(dǎo)表達(dá)Zhangfei融合蛋白的最佳培養(yǎng)條件,證明在37℃搖床培養(yǎng)條件下,IPTG的終濃度為3 mmol/L,誘導(dǎo)時(shí)間為7 h時(shí),Zhangfei融合蛋白的表達(dá)量相對(duì)最大。 2比較了谷胱甘肽瓊脂糖凝膠和電洗脫兩種純化Zhangfei融合蛋白的方法,得出電洗脫純化方法得到純度較高的目的蛋白。 3用電洗脫純化的Zhangfei融合蛋白免疫Balb/c小鼠和家兔,應(yīng)用間接ELISA常規(guī)方法檢測(cè)血清抗體效價(jià),免疫小鼠的血清抗體效價(jià)均超過1∶5×103,免疫兔的血清抗體效價(jià)高達(dá)1∶3×106,證明Zhangfei融合蛋白有較強(qiáng)的免疫原性。 4用45 %PEG(4000)融合骨髓瘤細(xì)胞和免疫脾細(xì)胞,用間接ELISA法篩選陽性孔,克隆率為98.96 %,陽性克隆率30.18 %。用有限稀釋法篩選得到2株陽性雜交瘤細(xì)胞,分別命名為ZFD1和ZFD2,兩株細(xì)胞株分泌單抗均為針對(duì)Zhangfei蛋白的單克隆抗體。 5應(yīng)用間接ELISA常規(guī)方法檢測(cè)細(xì)胞株培養(yǎng)上清和腹水的陰陽性及其效價(jià),ZFD1雜交瘤細(xì)胞培養(yǎng)上清的效價(jià)為1∶1000,腹水單抗的效價(jià)為1∶2×105以上。ZFD2雜交瘤細(xì)胞培養(yǎng)上清的效價(jià)為1∶1000,腹水單抗的效價(jià)為1∶105。經(jīng)凍存和傳代培養(yǎng)證明均具有穩(wěn)定分泌抗體的能力。2株細(xì)胞株分泌的抗體均屬于IgG1類型。ZFD1和ZFD2分泌單抗的親和常數(shù)分別為2.102×108,2.313×108。 上述結(jié)果為檢測(cè)該蛋白在動(dòng)物組織細(xì)胞中的表達(dá),研究zhangfei蛋白在機(jī)體的生理功能提供奠定了基礎(chǔ)。
[Abstract]:Zhangfei is a member of the alkaline leucine zipper protein family with a molecular weight of 28.9 ku.It is one of the transcription factors involved in the regulation of endoplasmic reticulum stress and unfolded protein reaction. Human diseases and other physiological and pathological processes have important significance. In this experiment, the Zhangfei fusion protein was induced and expressed, purified and immunized, and the monoclonal antibody against Zhangfei protein was prepared and identified. The results were as follows: 1 the optimal culture conditions for the induction and expression of Zhangfei fusion protein were screened. The results showed that the final concentration of IPTG was 3 mmol / L and the induction time was 7 h under the condition of 37 鈩，

本文編號(hào)：2086144