摘要
目的分析MNS血型相关基因GYPA、GYPB mRNA剪接体多态性,探讨各种剪接体编码的GPA和GPB蛋白异构体的亚细胞定位与MNS血型抗原表达的相关机理。方法随机选取无偿献血者10份血液,提取外周血总mRNA,反转录为cDNA后,以巢式PCR方法扩增GYPA、GYPB基因的开放阅读框并进行Sanger测序,碱基序列与GYPA(NCBI:NM_002099)、GYPB(NCBI:NM_002100.5)比对。得到的GYPA、GYPB开放阅读框的野生型及各种剪接异构体编码序列后,通过融合PCR技术分别与绿色荧光蛋白(GFP)编码基因融合并克隆后转染到HEK293细胞进行过表达,通过聚焦激光扫描显微镜监测GPA-GFP和GPB-GFP融合荧光蛋白的亚细胞定位。结果2例标本的GYPA mRNA中缺失外显子1与外显子2,预测的GPA蛋白异构体缺失2~26位氨基酸,GYPB mRNA全长序列完整。6例标本的GYPA mRNA完整,GYPB mRNA中缺失外显子2,预测的GPB蛋白异构体缺失13~45位氨基酸,其他外显子序列完整。1例标本的GYPA mRNA完整,GYPB mRNA的外显子5中364~385位碱基被AG替换,显示氨基酸信号肽截短。其他标本的GYP mRNA全长序列完整。GP-GFP融合蛋白的荧光信号的观察结果表明,根据各RNA剪接体克隆表达的GPA、GPB糖蛋白异构体均可表现出细胞膜表面定位分布,其中的一些可变剪接导致蛋白异构体发生不同程度的细胞内弥散,影响蛋白在细胞表面分布的速度和比例,可能构成MNS抗原强弱的原因之一。结论GYP mRNA剪接体有明显多态性特征,但GYP mRNA的部分片段缺失不影响其编码的蛋白异构体在细胞表面的定位分布,能够确保其展示MNS抗原特性。
Objective To analyze the polymorphisms of GYPA and GYPB mRNA spliceosomes associated with MNS blood group,and to explore the mechanism of subcellular localization of GPA and GPB protein isomerism encoded by various spliceosomes as well as the expression of MNS blood group antigen.Methods Ten blood samples of voluntary blood donors were randomly selected.The total mRNA of peripheral blood was extracted and reversed into cDNA.Nested PCR was used to amplify reading open frame of GYPA and GYPB gene,and sequencing was performed by Sanger.The base sequence obtained was compared with GYPA(NCBI:NM_002099)and GYPB(NCBI:Nm_002100.5).After the wild type and various splicing isomer of the open reading frame of GYPA and GYPB had been obtained,they were fused with the encoding gene of green fluorescent protein(GFP)by fusion PCR technology,then cloned and transfected into HEK293 cells for over expression.The subcellular localization of GPA-GFP and GPB-GFP fused fluorescent proteins was monitored by focusing laser scanning microscope.Results Exon-1 and Exon-2 were missing in GYPA mRNA of the 2 samples,and 2~26 amino acids were missing in the predicted GPA isomer,and the full length sequence of GYPB mRNA was complete.GYPA mRNA was intact in 6 samples,exon-2 was missing in GYPB mRNA,13~45 amino acids were missing in the predicted GPB protein isomer,and other exon sequences were intact.One sample had intact GYPA mRNA,and 364~385 bases in exon-5 of GYPB mRNA were replaced by AG,indicating truncation of amino acid signal peptide.The GYP mRNA sequences of other samples were complete.The fluorescence signal of GP-GFP fusion protein showed that all GPA and GPB glycoprotein isomers,cloned according to various RNA splicing,could demonstrate the orientation distribution on the cell membrane surface,while some alternative splicing leaded to different degrees of protein dispersion in the cell,and affected the distribution speed and proportion of protein on the cell surface,which might be one of the reasons for the strength variation of MNS antigen.Conclusion The GYP mRNA spliceosome is obviously polymorphic,but the partial deletion of GYP mRNA fragment does not affect the localization and distribution of the protein isomers encoded by GYP mRNA on the cell surface,which can ensure the expression of MNS antigen characteristics.
作者
梁延连
梁燕文
林建梭
王红星
陈帅
徐筠娉
LIANG Yanlian;LIANG Yanwen;LIN Jiansuo;WANG Hongxing;Chen Shuai;XU Yunping(Shenzhen Blood Center,Shenzhen 518035,China;Shenzhen Reke Biotechnology Co.,LTD.;Guangdong Medical University)
出处
《中国输血杂志》
CAS
2022年第9期887-891,共5页
Chinese Journal of Blood Transfusion
基金
广东省医学基金(B2020179)
深圳市医学三名工程(SZSM201811092)
深圳市医学重点学科(SZXK070)。
