妊娠期特异性beta1糖蛋白9 HLA-A3限制性细胞毒性T淋巴细胞表位的鉴定  

Identification of HLA-A3 restricted cytotoxic T lymphocyte epitopes in pregnancy specific beta1 glycoprotein 9

作　　者：李璐[1] 高艳锋[1] 祁元明[1] 

机构地区：[1]郑州大学生物工程系,郑州450001

出　　处：《郑州大学学报（医学版）》2011年第2期176-179,共4页Journal of Zhengzhou University(Medical Sciences)

基　　金：国家自然科学基金资助项目30872381;30901362

摘　　要：目的:鉴定来自食管癌细胞中特异性高表达的妊娠期特异性beta1糖蛋白9(PSG9)的HLA-A3限制性细胞毒性T淋巴细胞(CTL)表位。方法:首先运用RT-PCR方法检测PSG9mRNA在食管癌细胞EC9706、EC-1及EC-109中的表达情况。然后通过Bimas和Syfpeithi预测,再结合NetCTL1.2选取4条来源于PSG9的HLA-A3限制性的表位。候选表位P336的2位的氨基酸由异亮氨酸替换为亮氨酸,9位的氨基酸由酪氨酸替换为赖氨酸。候选表位P378的9位氨基酸由精氨酸替换为赖氨酸。候选表位通过标准的Fmoc化学法合成,通过结合力实验检测表位与T2A3细胞表面HLA-A3分子的结合力水平,通过细胞毒实验检测对EC-1细胞毒活性。结果:PSG9在肿瘤细胞EC9706、EC-1以及EC-109中都有表达。候选表位P107、P201和P336-2L9K与HLA-A3分子有弱结合力,P336、P378和P378-9K与HLA-A3分子有中等结合力。细胞毒实验结果显示P336-2L9K、P378和P378-9K对EC-1细胞均有一定的杀伤作用。结论:多肽P336-2L9K、P378和P378-9K能诱导体外抗肿瘤免疫反应,有可能成为PSG9阳性肿瘤细胞的共同CTL表位。Aim：To identify the HLA-A3 restricted cytotoxic T lymphocyte（CTL） epitopes from pregnancy specific beta1 glycoprotein 9（PSG9） which specifically over-expressed in esophagus cancer.Methods：The expression of PSG9 mRNA in esophagus cancer cells was determined by RT-PCR.Four novel HLA-A3 restricted epitopes from PSG9 were predicted by Bimas and Syfpeithi,combined with NetCTL 1.2.Candidate peptide P336 was optimized by substituting the isoleucine at position 2 into leucine,and the tyrosine at position 9 into lysine.Candidate peptide P378 was optimized by substituting the arginine at position 9 into lysine.The candidate peptides were synthesized by standard Fmoc chemistry method.Their binding affinities toward HLA-A3 molecule were evaluated by T2A3 cells binding assay.The cytotoxic activities were evaluated by LDH assay.Results：The expression of PSG9 mRNA was observed in EC9706,EC-1 and EC-109.The candidate peptides P107,P201 and P336-2L9K showed weak affinity toward HLA-A3 molecule,while P336,P378 and P378-9K showed moderate affinity.The LDH assay showed that P336-2L9K,P378 and P378-9K could lyse EC-1 cells.Conclusion：The peptides P336-2L9K,P378 and P378-9K could induce anti-tumor immunity in vitro,and might be the common CTL epitopes among PSG9 positive tumors.

关 键 词：妊娠期特异性betal糖蛋白9 细胞毒性T淋巴细胞 HLA-A3 表位 

分 类 号：Q279[生物学—细胞生物学]