人胰腺癌相关免疫原性候选膜抗原的鉴定和验证

摘要

Objective To verify the obtained immunogenic membrane antigens candidate of pancreatic cancer in the performed research. Methods Pancreatic caner cell line SW1990 membrane protein underwent immunoblot with serum IgG purified from clinically collected sera of 66 pancreatic cancer patients. Number 3 and number 8 positive dots of immunoblot were identified by MALDI-TOF mass spectrometry and peptide mass frigerfrint matching. The candidate membrane antigens were further validated in cell lines by RT-PCR, real-time PCR and Western blot, and their different expression level of gene and protein in pancreatic caner cell lines were contrastly studied. Results Number 3 and number 8 positive dots were identified as: voltage-dependent anion channel ( VDAC3 ) and catechol-o-methyltransferase (COMT).RT-PCR,real-time PCR and Western blot showed that gene and protein of VDAC3 and COMT were expressed in the pancreatic cancer cell line SW1990, AsPc and P3 respectively. Conclusion VDAC3 and COMT might be the candidate immunogenic membrane antigens of human pancreatic cancer, and their gene and protein are differently expressed in the pancreatic cancer cell line SW1990,AsPc and P3.%目的 对前期蛋白质组学筛查、鉴定的胰腺癌相关免疫原性候选膜抗原进行验证.方法 应用基质辅助激光解吸离子化飞行时间质谱分析与肽质指纹库对前期从66例胰腺癌患者血清的IgG与人胰腺癌细胞株SW1990的膜蛋白免疫印迹杂交的3号和8号阳性点进行鉴定.再应用RT-PCR、real-time PCR和Western blot方法,在细胞株层面从基因及蛋白水平对筛查出的膜抗原进行有效性验证,并比较不同胰腺癌细胞株中目的膜抗原的基因及蛋白表达水平的差异.结果 3号和8号阳性点经质谱分析鉴定分别为电压依赖性离子通道3(VDAC3)和儿茶酚胺氧位甲基转移酶(COMT).经RT-PCR、real-time PCR和Western blot实验证明,候选膜抗原VDAC3和COMT的基因及蛋白在胰腺癌细胞株SW1990、AsPc、P3中均有表达.结论 人胰腺癌细胞膜蛋白VDAC3、COMT可能是候选的具有免疫原性的胰腺癌相关膜抗原,并且其基因和蛋白在胰腺癌细胞株SW1990、AsPc、P3中均有表达.