IL-9重组表达及其单克隆抗体的制备

发布时间：2018-06-04 00:06

本文选题：白细胞介素9 + 单克隆抗体　；参考：《重庆理工大学》2012年硕士论文

【摘要】：1988年Uyttenhove等[1]人在建立的Th2细胞株中发现白细胞介素9（Interleukin-9，IL-9），并将其命名为P40（即IL-9）。直到2008年Nature Immunology杂志上报道了关于“Th9”细胞能特异分泌IL-9、IL-10的一类新型辅助性T细胞，届时科学界对IL-9的研究又进入了一个新的高潮时期。据文献报道，IL-9主要与过敏性哮喘、寄生虫感染和过敏性脊髓炎等疾病有关。哮喘疾病导致IL-9大量表达，从而激活肥大细胞、嗜酸性粒细胞等增殖，进而导致气道炎症因子增多，分泌大量的粘液并伴随着高应答过敏反应[2]。在寄生虫感染中，IL-9主要参与免疫保护作用[3]。上述报道提示我们，通过制备IL-9单克隆抗体，采取人缘化修饰后在体内能有效地阻断IL-9的表达，进而对哮喘疾病的治疗起到一定的缓解作用，，为下一步单克隆抗体药物的研发奠定了基础。本实验采用全基因合成的方式，根据大肠杆菌密码子的偏嗜性，对GeneBank中的IL-9成熟肽进行密码子替换后，送入上海捷瑞生物有限公司合成IL-9基因全序列，并克隆到PET-22b质粒中。以PET-22b-IL-9质粒为模板，通过PCR扩增，得到IL-9基因。将得到的目的基因与pGEX-6P-2质粒连接，转入XL-1Blue（E.coli）中，通过试验确定其最佳表达条件为：30℃，0.2mmol/L IPTG诱导5小时。破菌沉淀经SDS-PAGE电泳分析表明，目的蛋白在上清和包涵体中均有表达。经GST亲和层析纯化，获得了纯度较高的重组IL-9蛋白。将上述纯化的重组蛋白免疫BALB/C小鼠，获得效价为1:16×104的抗IL-9的小鼠血清，取小鼠脾细胞与SP2/0细胞在PEG1500的作用下进行细胞融合，融合率为42%。采用间接ELISA方法对杂交瘤细胞上清中分泌的单克隆抗体进行筛选，阳性率为3.6%。经过3次亚克隆筛选后，获得了1株能稳定分泌抗IL-9的杂交瘤细胞株，命名为2E5。采用秋水仙素鉴定杂交瘤细胞的染色体数目，结果显示该细胞株属于融合细胞体。通过分别与重组IL-9、标准IL-9蛋白和GST标签蛋白进行交叉反应来鉴定单抗的特异性，结果表明其有很高的特异性。反复冻融和传代的杂交瘤细胞分泌的上清经间接ELISA检测，其效价达1:320左右，小鼠腹水效价保持在1:12800。综上所述，此杂交瘤细胞的成功获得将会在抗体药物的研发中发挥重要作用。
[Abstract]:In 1988, Uyttenhove and other [1] people found interleukin 9 (Interleukin-9, IL-9) in the Th2 cell line established, and named them P40 (IL-9) until 2008 Nature Immunology magazine reported a new type of auxiliary cells that secreted IL-9, IL-10. A new period of climax.
According to the literature, IL-9 is mainly related to diseases such as allergic asthma, parasitic infection and allergic myelitis. Asthma causes a large number of expressions of IL-9, which activates the proliferation of mast cells, eosinophils and so on, which leads to the increase of airway inflammatory factors, the secretion of large amounts of mucus and the high response anaphylaxis [2]. in the parasitic infection. IL-9 mainly participates in the protective effect of [3]., the above-mentioned reports suggest that we can effectively block the expression of IL-9 through the preparation of IL-9 monoclonal antibodies in vivo, and thus relieve the treatment of asthma, and lay a foundation for the development of the next monoclonal antibody drug.
In this experiment, the whole gene synthesis method was used. According to the bias of the codon of Escherichia coli, the codon of IL-9 mature peptide in GeneBank was replaced and sent to Shanghai Jilli bio Co., Ltd. to synthesize the whole sequence of IL-9 gene and clone it into the PET-22b plasmid.
Using the PET-22b-IL-9 plasmid as the template, the IL-9 gene was amplified by PCR. The target gene was connected with the pGEX-6P-2 plasmid and transferred into the XL-1Blue (E.coli). The optimum expression condition was determined by the experiment: 30 C and 5 hours induced by 0.2mmol/L IPTG. The protein breaking precipitation was analyzed by SDS-PAGE electrophoresis and the target protein was both in the supernatant and inclusion body. Purified by GST affinity chromatography, the recombinant IL-9 protein with high purity was obtained.
The recombinant protein of the purified recombinant protein was immunized with BALB/C mice, the mice serum was titer at 1:16 x 104, and the mouse spleen cells and SP2/0 cells were fused under the action of PEG1500. The fusion rate was 42%. using indirect ELISA method to screen the monoclonal antibodies secreted in the hybridoma cell supernatant. The positive rate was 3 3.6%.. After the sub clone screening, 1 hybridoma cell lines, which could stabilize the secretion of anti IL-9, were obtained. It was named 2E5. to identify the number of chromosomes of hybridoma cells using colchicine. The results showed that the cell line belonged to the fusion cell body. The specificity of the monoclonal antibody was identified by the cross reaction with the recombinant IL-9, the standard IL-9 protein and the GST labeled protein. The results showed that the hybridoma cells secreted by repeated freezing and passages were detected by indirect ELISA. The titer of the hybridoma cells was about 1:320, and the mouse ascites titer remained in the 1:12800.. The successful acquisition of the hybridoma cells would play an important role in the development of antibody drugs.
【学位授予单位】：重庆理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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