發(fā)布時間：2018-03-05 05:08

  本文選題：cyclin　切入點：D1　出處：《吉林大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 細(xì)胞周期蛋白D1(cyclin D1)作為一種重要的周期蛋白,與腫瘤的發(fā)生、發(fā)展及患者的預(yù)后密切相關(guān),并在多種腫瘤中存在著過度表達(dá)。通過反義核酸、導(dǎo)入抗體和降解腫瘤細(xì)胞內(nèi)cyclin D1蛋白均能有效地阻礙腫瘤細(xì)胞的增殖。因此cyclin D1被認(rèn)為是腫瘤基因治療的重要靶點。 單鏈抗體(single chain variable fragment, scFv)作為具有抗原結(jié)合活性的最小的抗體片段,具有分子量小,易于侵入實體瘤,免疫原性較小,能通過基因工程技術(shù)大量生產(chǎn)等特點,具有良好的應(yīng)用前景。 為了有效的滅活腫瘤細(xì)胞內(nèi)過度活化的cyclin D1/CDK4的活性,抑制腫瘤細(xì)胞的增殖,本研究通過PCR方法對從人源噬菌體抗體庫中篩選得到抗cyclin D1單鏈抗體基因wtAD9中的linker區(qū)的終止密碼子實行了定點突變,并在原核細(xì)胞中進(jìn)行了誘導(dǎo)表達(dá)、純化,并通過ELISA、Western blot、競爭性抑制、親和力測定、免疫熒光和免疫共沉淀等實驗方法對AD9進(jìn)行了活性表征。結(jié)果表明AD9能夠特異性結(jié)合重組cyclin D1蛋白和腫瘤細(xì)胞內(nèi)源性的cyclin D1蛋白,并具有較高的親和力。 本實驗得到的結(jié)果對后續(xù)開展抗cyclin D1胞內(nèi)抗體腫瘤基因治療的研究奠定了基礎(chǔ)。
[Abstract]:Cyclin D1), as an important cyclin, is closely related to the occurrence, development and prognosis of tumor, and is overexpressed in many kinds of tumors. Both the introduction of antibodies and the degradation of cyclin D1 protein in tumor cells can effectively inhibit the proliferation of tumor cells, so cyclin D1 is considered as an important target of tumor gene therapy. Single chain variable fragment (scFV), as the smallest antibody fragment with antigen-binding activity, has the characteristics of small molecular weight, easy invading solid tumor, low immunogenicity, and can be produced in large quantities by genetic engineering technology. It has good application prospect. In order to effectively inactivate the activity of cyclin D1 / CDK4 and inhibit the proliferation of tumor cells, In this study, the terminal codon of the linker region of cyclin D1 single chain antibody gene wtAD9 was screened from human phage phage antibody library by PCR, and was induced and purified in prokaryotic cells. The activity of AD9 was characterized by Elisa Western blot, competitive inhibition, affinity assay, immunofluorescence and immunoprecipitation. The results showed that AD9 could specifically bind to recombinant cyclin D1 protein and endogenous cyclin D1 protein in tumor cells. And has high affinity. The results of this study laid a foundation for further research on tumor gene therapy of anti cyclin D1 antibodies.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前2條

1 劉志強(qiáng);抗Cyclin D1人源胞內(nèi)單鏈抗體的原核表達(dá)與鑒定[D];吉林大學(xué);2011年

2 王媚娘;抗CDK4人源單鏈抗體作用機(jī)制的研究[D];吉林大學(xué);2011年

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本文編號：1568849