蛋白激酶R的表达纯化及多克隆抗体和单克隆抗体的制备

发布时间：2018-04-29 05:21

本文选题：PKR + 原核表达　；参考：《甘肃农业大学》2015年硕士论文

【摘要】：蛋白激酶R(PKR)可被病毒感染后诱导产生的干扰素和双链RNA激活,是宿主天然免疫系统的重要抗病毒效应因子。但是,许多病毒可以通过自身编码的蛋白拮抗PKR的抗病毒作用,如流感病毒的NS1蛋白。为了给研究PKR与病毒蛋白的相互作用及其机制提供条件,本研究表达纯化了PKR蛋白,并通过免疫动物制备了PKR蛋白的多克隆抗体和单克隆抗体。试验1.蛋白激酶R的原核表达纯化及多克隆抗体的制备利用RT-PCR方法扩增人的PKR基因,定向克隆到原核表达载体pGEX-6P-1中。经序列测定分析后,将阳性重组质粒转化大肠杆菌BL21(DE3)感受态细胞,经终浓度0.5mmol/L IPTG诱导表达GST-PKR融合蛋白,表达产物经SDS-PAGE分析后确定融合蛋白获得稳定表达。随后通过Glutathione-sepharose 4B亲和层析纯化系统对融合蛋白进行初步纯化,用离子交换层析及分子筛在AKTA Explorer上进一步纯化,得到高纯度无标签的PKR蛋白。将获得的PKR蛋白与等量的弗氏佐剂乳化后免疫新西兰大白兔收集血清,制备多克隆抗体。通过斑点杂交方法检测其效价达到1:50000以上,并经Western blot和间接免疫荧光(IFA)试验分析显示,多克隆抗体对PKR蛋白具有高度特异性。试验2.蛋白激酶R单克隆抗体的制备利用前面试验表达纯化的PKR蛋白为免疫原,免疫8周龄左右的SPF雌性BALB/C小鼠,进行4次免疫,第4次免疫后3天无菌条件下取小鼠脾细胞与处于对数生长期的SP2/0小鼠骨髓瘤细胞进行融合。通过间接ELIAE方法鉴定阳性杂交瘤细胞,之后利用有限稀释法对效价高的杂交瘤细胞进行4次亚克隆,最终获得持续分泌抗PKR的单克隆杂交瘤细胞3株,分别命名为2E8、4A7、5A4,检测培养基上清中抗体滴度分别为1:3200、1:3200、1:6400,取抗体滴度最高的5A4杂交瘤细胞腹腔注射10周龄左右SPF的BALB/C小鼠,7天后收集腹水,测定腹水效价为1:512000。3株杂交瘤细胞抗体亚型鉴定均为IgG1型。间接免疫荧光(IFA)试验分析显示,单克隆抗体对PKR蛋白具有高度特异性。经间接ELISA检测,获得的3株杂交瘤细胞体外传代培养多代和冻存复苏后细胞培养基上清中抗体的滴度和OD值基本一致,抗体能持续的分泌,表明PKR单克隆抗体制备成功。
[Abstract]:Protein kinase (PKR), which can be activated by interferon and double-stranded RNA induced by virus infection, is an important antiviral effector of host innate immune system. However, many viruses can antagonize the antiviral effects of PKR through their own encoded proteins, such as the NS1 protein of influenza viruses. In order to study the interaction between PKR and virus protein and its mechanism, the PKR protein was expressed and purified, and the polyclonal antibody and monoclonal antibody of PKR protein were prepared by immunizing animals. Experiment 1. Prokaryotic expression and purification of protein kinase R and preparation of polyclonal antibody human PKR gene was amplified by RT-PCR and cloned into prokaryotic expression vector pGEX-6P-1. After sequence analysis, the positive recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, and the GST-PKR fusion protein was induced by the final concentration of 0.5mmol/L IPTG. The expression product was confirmed by SDS-PAGE analysis to obtain stable expression of the fusion protein. The fusion protein was purified by Glutathione-sepharose 4B affinity chromatography system, and then purified on AKTA Explorer by ion exchange chromatography and molecular sieve. The obtained PKR protein was emulsified with Freund's adjuvant to immunize New Zealand white rabbits to prepare polyclonal antibodies. The titer of the polyclonal antibody was over 1: 50000 by dot blot. The results of Western blot and indirect immunofluorescence assay showed that the polyclonal antibody was highly specific to PKR protein. Experiment 2. Preparation of Monoclonal Antibody against protein Kinase R Immunization of SPF female BALB/C mice at about 8 weeks of age by immunizing the purified PKR protein expressed in the previous test for 4 times. Three days after the fourth immunization, murine spleen cells were harvested and fused with myeloma cells of SP2/0 mice in logarithmic phase. The positive hybridoma cells were identified by indirect ELIAE method, and then subcloned the hybridoma cells with high titer by limited dilution method for 4 times. Finally, 3 monoclonal hybridoma cell lines secreting anti-PKR were obtained. The antibody titers in the supernatant of the culture medium were 1: 3 200 and 1: 6400, respectively. The 5A4 hybridoma cells with the highest antibody titers were injected intraperitoneally into the BALB/C mice of about 10 weeks old to collect ascites after 7 days. The titer of ascites was 1: 512000.3 strain hybridoma cell antibody subtypes were identified as IgG1 type. Indirect immunofluorescence assay showed that monoclonal antibody was highly specific to PKR protein. The antibody titers and OD values in the supernatant of the three hybridoma cells cultured in vitro and resuscitated by cryopreservation were basically the same, and the antibody could be secreted continuously, which indicated that the monoclonal antibody against PKR was successfully prepared.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.4

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