Luman-N-端融合蛋白单克隆抗体的制备及鉴定
发布时间：2018-11-07 17:38
【摘要】： Luman是与单纯疱疹病毒潜伏感染的发生以及再次活化密切相关的细胞因子,是碱性亮氨酸拉链蛋白家族成员之一,是未折叠蛋白反应和内质网应激重要调节物。其分子结构分两个区域,其中N-端区域与调节转录的功能有关,Luman-N-端区域分子量大约为30 ku。Luman所有已知功能区,包括活化区,宿主细胞因子结合序列和碱性亮氨酸拉链区域在内都位于N-端。Luman-N-端的研究对于进一步揭示Luman调控机制将有重大意义。本试验将转化了Luman-N-端融合蛋白基因的BL21在IPTG的诱导下进行表达并进行表达条件的优化,利用电洗脱法将重组蛋白纯化,以纯化的重组蛋白为抗原免疫BALB/c小鼠,建立Luman-N-端融合蛋白抗体间接ELISA检测方法,以PEG 4000为融合剂将免疫小鼠脾细胞与骨髓瘤细胞SP2/0融合,有限稀释法筛选阳性克隆,制备单克隆抗体并进行鉴定。试验结果如下: 1. Luman-N-端融合蛋白的最佳表达条件是:37℃,IPTG终浓度为0.1 mmol/L下诱导7 h。经电洗脱法纯化,SDS-PAGE显示:得到了分子量大约为56 ku单一条带。经Bradford法检测,重组蛋白的浓度约为0.65 mg/mL。 2.建立了抗Luman-N-端融合蛋白抗体测定的间接ELISA方法。方阵滴定法确定包被原最适工作浓度为5.0μg/mL,最佳的包被温度和时间为37℃1 h然后在4℃过夜,最适包被液为pH 9.6,0.05 mol/L的CBS;1.0 %明胶封闭,辣根过氧化物酶标记山羊抗小鼠IgG(酶标二抗)最适稀释倍数为l∶1 500,作用时间为30 min。 3.加强免疫后的小鼠脾脏细胞与骨髓瘤细胞SP2/0进行融合,经间接ELISA筛选及 4次亚克隆筛选,获得了3株可稳定分泌抗Luman-N-端融合蛋白单克隆抗体的杂交瘤细胞(8B2G ,3A5E和1A3F),小鼠体内诱生法制腹水。对杂交瘤细胞株及其上清单抗与腹水单抗的特性进行了一系列鉴定。结果显示,杂交瘤细胞株冻存复苏及传代后仍能稳定分泌单抗,细胞染色体检查正常,单抗效价高,亲和力不一。Western blot分析证明3株单抗均能与原核表达的Luman-N-端融合蛋白特异结合。单抗与Zhangfei融合蛋白,LRF融合蛋白交叉反应率均小于5 %。 本试验成功制备了Luman-N-端融合蛋白单克隆抗体,为Luman的进一步研究打下了一定的基础。
[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，
本文编号：2317084
[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，
本文编号：2317084
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