【摘要】： Luman是與單純皰疹病毒潛伏感染的發(fā)生以及再次活化密切相關(guān)的細(xì)胞因子,是堿性亮氨酸拉鏈蛋白家族成員之一,是未折疊蛋白反應(yīng)和內(nèi)質(zhì)網(wǎng)應(yīng)激重要調(diào)節(jié)物。其分子結(jié)構(gòu)分兩個(gè)區(qū)域,其中N-端區(qū)域與調(diào)節(jié)轉(zhuǎn)錄的功能有關(guān),Luman-N-端區(qū)域分子量大約為30 ku。Luman所有已知功能區(qū),包括活化區(qū),宿主細(xì)胞因子結(jié)合序列和堿性亮氨酸拉鏈區(qū)域在內(nèi)都位于N-端。Luman-N-端的研究對(duì)于進(jìn)一步揭示Luman調(diào)控機(jī)制將有重大意義。本試驗(yàn)將轉(zhuǎn)化了Luman-N-端融合蛋白基因的BL21在IPTG的誘導(dǎo)下進(jìn)行表達(dá)并進(jìn)行表達(dá)條件的優(yōu)化,利用電洗脫法將重組蛋白純化,以純化的重組蛋白為抗原免疫BALB/c小鼠,建立Luman-N-端融合蛋白抗體間接ELISA檢測(cè)方法,以PEG 4000為融合劑將免疫小鼠脾細(xì)胞與骨髓瘤細(xì)胞SP2/0融合,有限稀釋法篩選陽(yáng)性克隆,制備單克隆抗體并進(jìn)行鑒定。試驗(yàn)結(jié)果如下: 1. Luman-N-端融合蛋白的最佳表達(dá)條件是:37℃,IPTG終濃度為0.1 mmol/L下誘導(dǎo)7 h。經(jīng)電洗脫法純化,SDS-PAGE顯示:得到了分子量大約為56 ku單一條帶。經(jīng)Bradford法檢測(cè),重組蛋白的濃度約為0.65 mg/mL。 2.建立了抗Luman-N-端融合蛋白抗體測(cè)定的間接ELISA方法。方陣滴定法確定包被原最適工作濃度為5.0μg/mL,最佳的包被溫度和時(shí)間為37℃1 h然后在4℃過(guò)夜,最適包被液為pH 9.6,0.05 mol/L的CBS;1.0 %明膠封閉,辣根過(guò)氧化物酶標(biāo)記山羊抗小鼠IgG(酶標(biāo)二抗)最適稀釋倍數(shù)為l∶1 500,作用時(shí)間為30 min。 3.加強(qiáng)免疫后的小鼠脾臟細(xì)胞與骨髓瘤細(xì)胞SP2/0進(jìn)行融合,經(jīng)間接ELISA篩選及 4次亞克隆篩選,獲得了3株可穩(wěn)定分泌抗Luman-N-端融合蛋白單克隆抗體的雜交瘤細(xì)胞(8B2G ,3A5E和1A3F),小鼠體內(nèi)誘生法制腹水。對(duì)雜交瘤細(xì)胞株及其上清單抗與腹水單抗的特性進(jìn)行了一系列鑒定。結(jié)果顯示,雜交瘤細(xì)胞株凍存復(fù)蘇及傳代后仍能穩(wěn)定分泌單抗,細(xì)胞染色體檢查正常,單抗效價(jià)高,親和力不一。Western blot分析證明3株單抗均能與原核表達(dá)的Luman-N-端融合蛋白特異結(jié)合。單抗與Zhangfei融合蛋白,LRF融合蛋白交叉反應(yīng)率均小于5 %。 本試驗(yàn)成功制備了Luman-N-端融合蛋白單克隆抗體,為L(zhǎng)uman的進(jìn)一步研究打下了一定的基礎(chǔ)。
[Abstract]:Luman is a cytokine closely related to the occurrence and reactivation of herpes simplex virus latent infection. It is a member of basic leucine zipper protein family and an important regulator of unfolded protein reaction and endoplasmic reticulum stress. Its molecular structure is divided into two regions, in which the N-terminal region is related to the function of regulating transcription, and the molecular weight of the Luman-N- terminal region is about 30 ku.Luman all known functional regions, including the activation region. Both the host cytokine binding sequence and the basic leucine zipper region are located at the N-terminal. The study of the Luman-N- terminal will be of great significance in further understanding the regulatory mechanism of Luman. In this experiment, BL21, which transformed the Luman-N- terminal fusion protein gene, was expressed under the induction of IPTG and the expression conditions were optimized. The recombinant protein was purified by electroelution. The purified recombinant protein was used as antigen to immunize BALB/c mice. The indirect ELISA detection method of Luman-N- terminal fusion protein antibody was established. The spleen cells of immunized mice were fused with myeloma cells SP2/0 with PEG 4000 as fusion agent. The positive clones were screened by limited dilution method and monoclonal antibodies were prepared and identified. The results are as follows: 1. The optimal conditions for expression of Luman-N- terminal fusion protein were as follows: 37 鈩，

本文編號(hào)：2317085