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目的:构建CD59分子配体肽sp22基因重组真核表达系统,探讨sp22基因对CD59分子的影响。方法:构建含特异性sp22基因的真核重组表达载体sp-pIRES,脂质体法转染人前列腺癌PC-3细胞,G418筛选建立稳定转染细胞系,RT-PCR检测转染细胞中sp22基因mRNA的表达筛选阳性细胞克隆,Western blot、ELISA竞争抑制试验检测转染细胞CD59蛋白的表达;台盼蓝染色实验和LDH实验测sp22基因对CD59分子功能的影响。结果:PCR、酶切及DNA测序鉴定表明成功构建了sp-PIRES重组表达载体;RT-PCR试验证实sp22在spPC-3细胞中成功表达;Westernblot、ELESA竞争抑制试验表明:spPC-3细胞比正常PC-3细胞CD59蛋白表达减少(P<0.05);与正常PC-3细胞相比,补体对spPC-3细胞的溶解杀伤明显增强(P<0.05)。结论:sp22基因可在mR-NA及蛋白水平影响人前列腺癌PC-3细胞表面CD59抗原的表达及功能,降低CD59分子抗补体活性,有望用于肿瘤治疗。
OBJECTIVE: To construct a recombinant eukaryotic expression system of sp59 gene of CD59 and investigate the effect of sp22 gene on CD59. Methods: The eukaryotic recombinant expression vector sp-pIRES containing specific sp22 gene was constructed and transfected into human prostate cancer PC-3 cells by lipofectamine 2000. Stable transfected cell lines were screened by G418. The expression of sp22 Western blotting and ELISA competitive inhibition assay were used to detect the expression of CD59 protein. Trypan blue staining and LDH assay were used to determine the effect of sp22 gene on CD59 molecule function. Results: PCR, restriction enzyme digestion and DNA sequencing showed that sp-PIRES recombinant vector was successfully constructed. The sp-PIRES recombinant vector was successfully constructed. The sp-PIRES recombinant vector was successfully expressed in spPC-3 cells by RT-PCR. Western blot and ELESA competitive inhibition assay showed that spPC- The expression of CD59 in PC-3 cells was decreased (P <0.05). Compared with normal PC-3 cells, the CD59 expression in PC-3 cells was significantly increased (P <0.05). Conclusion: sp22 gene can affect the expression and function of CD59 antigen on human prostate cancer PC-3 cells at mR-NA level and protein level, and reduce the anti-complement activity of CD59 molecule, which is expected to be used in cancer therapy.