本文選題：白細(xì)胞介素9 + 單克隆抗體�。� 參考：《重慶理工大學(xué)》2012年碩士論文

【摘要】：1988年Uyttenhove等[1]人在建立的Th2細(xì)胞株中發(fā)現(xiàn)白細(xì)胞介素9（Interleukin-9，IL-9），并將其命名為P40（即IL-9）。直到2008年Nature Immunology雜志上報(bào)道了關(guān)于“Th9”細(xì)胞能特異分泌IL-9、IL-10的一類新型輔助性T細(xì)胞，屆時(shí)科學(xué)界對(duì)IL-9的研究又進(jìn)入了一個(gè)新的高潮時(shí)期。 據(jù)文獻(xiàn)報(bào)道，IL-9主要與過(guò)敏性哮喘、寄生蟲(chóng)感染和過(guò)敏性脊髓炎等疾病有關(guān)。哮喘疾病導(dǎo)致IL-9大量表達(dá)，從而激活肥大細(xì)胞、嗜酸性粒細(xì)胞等增殖，進(jìn)而導(dǎo)致氣道炎癥因子增多，分泌大量的粘液并伴隨著高應(yīng)答過(guò)敏反應(yīng)[2]。在寄生蟲(chóng)感染中，IL-9主要參與免疫保護(hù)作用[3]。上述報(bào)道提示我們，通過(guò)制備IL-9單克隆抗體，采取人緣化修飾后在體內(nèi)能有效地阻斷IL-9的表達(dá)，進(jìn)而對(duì)哮喘疾病的治療起到一定的緩解作用，，為下一步單克隆抗體藥物的研發(fā)奠定了基礎(chǔ)。 本實(shí)驗(yàn)采用全基因合成的方式，根據(jù)大腸桿菌密碼子的偏嗜性，對(duì)GeneBank中的IL-9成熟肽進(jìn)行密碼子替換后，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質(zhì)粒中。 以PET-22b-IL-9質(zhì)粒為模板，通過(guò)PCR擴(kuò)增，得到IL-9基因。將得到的目的基因與pGEX-6P-2質(zhì)粒連接，轉(zhuǎn)入XL-1Blue（E.coli）中，通過(guò)試驗(yàn)確定其最佳表達(dá)條件為：30℃，0.2mmol/L IPTG誘導(dǎo)5小時(shí)。破菌沉淀經(jīng)SDS-PAGE電泳分析表明，目的蛋白在上清和包涵體中均有表達(dá)。經(jīng)GST親和層析純化，獲得了純度較高的重組IL-9蛋白。 將上述純化的重組蛋白免疫BALB/C小鼠，獲得效價(jià)為1:16×104的抗IL-9的小鼠血清，取小鼠脾細(xì)胞與SP2/0細(xì)胞在PEG1500的作用下進(jìn)行細(xì)胞融合，融合率為42%。采用間接ELISA方法對(duì)雜交瘤細(xì)胞上清中分泌的單克隆抗體進(jìn)行篩選，陽(yáng)性率為3.6%。經(jīng)過(guò)3次亞克隆篩選后，獲得了1株能穩(wěn)定分泌抗IL-9的雜交瘤細(xì)胞株，命名為2E5。采用秋水仙素鑒定雜交瘤細(xì)胞的染色體數(shù)目，結(jié)果顯示該細(xì)胞株屬于融合細(xì)胞體。通過(guò)分別與重組IL-9、標(biāo)準(zhǔn)IL-9蛋白和GST標(biāo)簽蛋白進(jìn)行交叉反應(yīng)來(lái)鑒定單抗的特異性，結(jié)果表明其有很高的特異性。反復(fù)凍融和傳代的雜交瘤細(xì)胞分泌的上清經(jīng)間接ELISA檢測(cè)，其效價(jià)達(dá)1:320左右，小鼠腹水效價(jià)保持在1:12800。綜上所述，此雜交瘤細(xì)胞的成功獲得將會(huì)在抗體藥物的研發(fā)中發(fā)揮重要作用。
[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.
【學(xué)位授予單位】：重慶理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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