人CD59基因活性位點突變對腫瘤逃逸相關(guān)分子補體及caspase-3的作用研究
[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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