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  本文選題：配體　切入點：CD59　出處：《青島大學》2010年碩士論文　論文類型：學位論文

【摘要】： 目的構建CD59分子配體肽(sp22)基因真核表達重組體系,探討sp22基因?qū)毎鸆D59分子表達及功能的影響,為進一步研究sp22分子與CD59分子的作用機制及利用短肽封閉CD59分子表達,為腫瘤基因靶向治療開辟新途徑。方法構建含特異性sp22基因的真核重組表達載體sp-pIRES,脂質(zhì)體法轉(zhuǎn)染人前列腺癌PC-3細胞,G418篩選建立穩(wěn)定轉(zhuǎn)染細胞系,RT-PCR檢測轉(zhuǎn)染細胞中sp22基因mRNA的表達并篩選陽性細胞克隆,RT-PCR檢測轉(zhuǎn)染細胞表面CD59mRNA表達量的變化,Western Blot、ELISA競爭抑制試驗檢測轉(zhuǎn)染細胞CD59蛋白的表達；制備抗CD59多克隆抗體,臺盼藍染色試驗和LDH試驗檢測sp22分子對CD59分子功能的影響,MTT法測定PC-3細胞增殖抑制率。 結果PCR、雙酶切及DNA測序鑒定表明成功構建了sp-PIRES真核重組表達載體；RT-PCR試驗證實sp22在spPC-3細胞中成功表達；Western Blot、ELESA競爭抑制試驗表明：spPC-3細胞比正常PC-3細胞CD59蛋白表達量減少,二者比較其差別有統(tǒng)計學意義(P0.05)；與正常PC-3細胞相比,補體對spPC-3細胞的溶解殺傷能力明顯增強,二者比較其差別有統(tǒng)計學意義(P0.05); MTT結果測定spPC-3細胞與正常PC-3細胞相比較細胞增殖抑制率增高,差別有統(tǒng)計學意義(P0.05)。 結論sp22基因可在mRNA及蛋白水平抑制人前列腺癌PC-3細胞表面CD59蛋白分子的表達及功能,降低CD59分子抗補體活性,為進一步研究利用SP22阻斷CD59分子在腫瘤細胞上的表達創(chuàng)造了有利條件,為腫瘤的免疫治療研究奠定基礎。
[Abstract]:Objective to construct a recombinant eukaryotic expression system of CD59 ligand peptide sp 22) and to investigate the effect of sp22 gene on the expression and function of CD59 molecule. In order to further study the interaction mechanism between sp22 molecule and CD59 molecule and block the expression of CD59 molecule by short peptide. Methods Eukaryotic recombinant expression vector sp-pIRESwith specific sp22 gene was constructed and transfected into human prostate cancer PC-3 cell line G418 by liposome method to establish a stable transfection cell line to detect the expression of SP-pIRESin the transfected cells by reverse transcription-polymerase chain reaction (RT-PCR). The expression of sp22 gene mRNA and the expression of CD59mRNA on transfected cells were detected by RT-PCR. The expression of CD59 protein in transfected cells was detected by Western blottELISA competitive inhibition assay. Anti CD59 polyclonal antibody was prepared, trypan blue staining and LDH assay were used to detect the effect of sp22 on the function of CD59. The inhibitory rate of PC-3 cell proliferation was determined by MTT assay. Results PCR, double enzyme digestion and DNA sequencing showed that the sp-PIRES eukaryotic expression vector was successfully constructed. RT-PCR assay confirmed that sp22 was successfully expressed in spPC-3 cells. The results of competitive inhibition test showed that the expression of CD59 protein in PC-3 cells was lower than that in normal PC-3 cells. Compared with normal PC-3 cells, the cytotoxicity of complement to spPC-3 cells was significantly increased. Compared with normal PC-3 cells, the proliferation inhibition rate of spPC-3 cells was higher than that of normal PC-3 cells, and the difference was statistically significant (P 0.05). Conclusion sp22 gene can inhibit the expression and function of CD59 protein molecules on the surface of human prostate cancer PC-3 cells at the level of mRNA and protein, and decrease the anti-complement activity of CD59 molecule. It provides a favorable condition for the further study of SP22 blocking the expression of CD59 molecules on tumor cells and lays a foundation for the study of tumor immunotherapy.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R737.25;R392

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本文編號：1561139