【摘要】： 目的構(gòu)建兩種人CD59基因突變的重組體pALTER-MAX-CD59,轉(zhuǎn)染入HeLa細(xì)胞,篩選高表達(dá)人突變CD59的陽性細(xì)胞克隆,檢測HeLa細(xì)胞中突變CD59對腫瘤逃逸相關(guān)分子(caspase-3)的抑制效應(yīng)及突變?nèi)薈D59的補體抑制活性,研究CD59基因突變與腫瘤逃逸的相關(guān)性,進(jìn)一步證實人CD59與腫瘤逃逸相關(guān)的活性位點,為腫瘤逃逸機制的進(jìn)一步闡明提供新的理論依據(jù)。 方法用重組聚合酶鏈反應(yīng)定點誘變技術(shù)構(gòu)建第40位色氨酸缺失的人的CD59突變體(突變1)和第39-41位氨基酸突變?yōu)樯彼岬娜说腃D59突變體(突變2);與pALTER-MAX質(zhì)粒連接后,用陽離子脂質(zhì)體法轉(zhuǎn)染入HeLa細(xì)胞,以新霉素類似物G418篩選陽性細(xì)胞克隆,以免疫熒光、免疫酶標(biāo)、ELISA、Western-blot、流式細(xì)胞術(shù)進(jìn)一步篩選高表達(dá)突變?nèi)薈D59的細(xì)胞株,用免疫組化法檢測轉(zhuǎn)染人突變CD59的細(xì)胞中腫瘤逃逸相關(guān)分子caspase-3的表達(dá)變化,用乳酸脫氫酶(LDH)測試法檢測突變?nèi)薈D59的抗補體活性。 結(jié)果酶切鑒定及序列測定均證實成功構(gòu)建了兩種人CD59基因突變的重組質(zhì)粒;免疫熒光、免疫酶標(biāo)、ELISA、Western-blot、流式細(xì)胞術(shù)篩選出高表達(dá)突變?nèi)薈D59的細(xì)胞株;免疫組化檢測發(fā)現(xiàn)轉(zhuǎn)染突變1后的HeLa細(xì)胞中caspase-3的含量較轉(zhuǎn)染野生型人CD59的HeLa細(xì)胞中的caspase-3的含量明顯增加,轉(zhuǎn)染突變2后的HeLa細(xì)胞中caspase-3的含量較轉(zhuǎn)染野生型人CD59的HeLa細(xì)胞中的caspase-3的含量明顯減少。LDH法檢測轉(zhuǎn)染突變CD59后HeLa細(xì)胞具有抗補體活性,但其活性減弱。 結(jié)論成功構(gòu)建了兩種CD59突變的重組質(zhì)粒,并獲得高表達(dá)兩種突變CD59的HeLa細(xì)胞株,初步研究了CD59基因突變后抗補體活性改變及caspase-3的變化,證實了CD59引起腫瘤逃逸的途徑除了通過調(diào)節(jié)其抗補體活性來影響MAC的形成外還能通過影響caspase-3的表達(dá)來實現(xiàn)。
[Abstract]:Objective to construct two recombinant human CD59 gene mutants pALTER-MAX-CD59, and transfect them into HeLa cells to screen the positive cell clones expressing human mutant CD59. The inhibitory effect of mutated CD59 on tumor escape associated molecule (caspase-3) and complement inhibitory activity of mutant human CD59 in HeLa cells were detected, and the correlation between CD59 gene mutation and tumor escape was studied. The active sites associated with tumor escape were further confirmed by human CD59, which provided a new theoretical basis for further elucidation of the mechanism of tumor escape. Methods Human CD59 mutants (mutant 1) and CD59 mutants (mutagen2) were constructed by site-directed mutagenesis of recombinant polymerase chain reaction (RPCR) for the 40 th position tryptophan deficient human and the 39-41 amino acid mutagenesis for tryptophan. After ligation with pALTER-MAX plasmid, HeLa cells were transfected into HeLa cells by cationic liposome method. Positive cell clones were screened by neomycin analogue G418. Immunofluorescence, immunoenzyme labeling and ELISA,Western-blot, were used to screen the positive cells. Flow cytometry was used to screen the cell lines with high expression of human CD59, and the expression of tumor escape related molecule caspase-3 was detected by immunohistochemical method in the cells transfected with human mutant CD59. The anti complement activity of mutant human CD59 was detected by lactate dehydrogenase (LDH) assay. Results two recombinant plasmids of human CD59 gene mutation were successfully constructed by restriction endonuclease digestion and sequence analysis, immunofluorescence, immunoenzyme labeling and ELISA,Western-blot, flow cytometry were used to screen the cells with high expression of human CD59. Immunohistochemical analysis showed that the content of caspase-3 in HeLa cells transfected with mutant 1 was significantly higher than that in HeLa cells transfected with wild type CD59. The content of caspase-3 in HeLa cells after transfection of mutant 2 was significantly lower than that in HeLa cells transfected with wild type CD59. LDH assay showed that HeLa cells had anti-complement activity after transfection of mutant CD59, but its activity decreased. Conclusion two recombinant plasmids with CD59 mutation were successfully constructed and HeLa cell lines with high expression of CD59 were obtained. The changes of complement resistance and caspase-3 after CD59 gene mutation were preliminarily studied. It is confirmed that the pathway of escape induced by CD59 can not only affect the formation of MAC by regulating its anticomplement activity, but also affect the expression of caspase-3.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392.12;R730.3

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