免疫沉淀法与质谱联用检测RHD蛋白序列方法学的建立*

doi:10.3969/j.issn.1671-2587.2021.05.016

临床输血与检验 ›› 2021, Vol. 23 ›› Issue (5): 623-627.DOI: 10.3969/j.issn.1671-2587.2021.05.016

免疫沉淀法与质谱联用检测RHD蛋白序列方法学的建立^*

钟福铃, 梁延连, 苏宇清, 吴凡, 梁爽, 张海璇, 洪文旭, 徐筠娉

518035 深圳市血液中心（钟福铃,梁延连,苏宇清,吴凡,梁爽,洪文旭,徐筠娉）; 北京百泰派克生物科技有限公司（张海璇）

收稿日期:2021-03-19 发布日期:2021-10-20
通讯作者: 苏宇清,女,主任技师,主要从事免疫血液学与输血医学研究,（E-mail） 1435133589@qq.com。共同通信作者：徐筠娉,女,博士,副主任技师,主要从事免疫血液学与输血医学研究,（E-mail）yunpingxu1982@qq.com。
作者简介:钟福铃（1976-）,女,广东梅州人,主管护师,学士,主要从事输血医学相关研究,（E-mail）1225694439@qq.com。并列第一作者：梁延连（1973-）,女,广西平南人,主任技师,学士,主要从事红细胞血型与免疫血液学研究,（E-mail）lianviky@126.com。
基金资助:
*本课题受广东省医学基金（No.B2020179）,深圳市卫生防控策略研究项目（No.SZGW2018008）,深圳市医药三名工程（No.SZSM201811092）,深圳市医学重点学科（No.SZXK070）资助

RHD Protein Sequences by Immunoprecipitation and Mass Spectrometry

ZHONG Fu-ling, LIANG Yan-lian, SU Yu-qing, et al

Shenzhen Blood Center,Guangdong 518035

Received:2021-03-19 Published:2021-10-20

摘要/Abstract

摘要： 目的建立人类红细胞RHD蛋白序列分析的方法,为检测RhD血型基因在红细胞膜上抗原量的表达奠定基础。方法随机选取10例无偿献血者的抗凝血5 mL,均为已知RhD阳性,对标本中的Rh血型抗原蛋白进行免疫沉淀。采用蛋白酶解法从红细胞膜中提取RH蛋白并定量。经蛋白酶切割后,利用LC-MS质谱平台对Rh蛋白序列进行鉴定。结果建立了检测RHD蛋白序列的方法,制定出适合检测RHD蛋白的标准曲线,得到10例样本的RHD蛋白浓度,鉴定到的RHD目标蛋白序列与Blast平台检索到的RHD蛋白片段序列吻合度达100%,获得红细胞膜RHD蛋白肽段二级图谱。结论免疫沉淀法与质谱联用可以鉴定红细胞RHD蛋白的氨基酸序列,为检测RhD抗原在红细胞上的表达量奠定基础,为精确鉴定RhD血型提供可行的方法,具有广泛的应用价值。

关键词: RHD蛋白序列, 红细胞膜蛋白, 免疫沉淀法, 质谱技术

Abstract: Objective We established a method of sequence analysis for human erythrocyte Rhesus D （RhD） blood group protein to lay a foundation for identifying the expression of RhD blood group gene in erythrocyte membrane. Methods Five mL of anticoagulant blood from 10 blood donors with RhD positive were randomly selected. The Rh blood group antigen protein was immunoprecipitated. Rh（Rhesus）protein was extracted from erythrocyte membrane by protease hydrolysis and quantified. After being cleaved by protease,the Rh protein sequences were identified by mass spectrometry （LC-MS）. Results The method for detecting RhD protein sequence was established and the standard curve of RhD protein was developed. The concentrations of RhD proteins in 10 samples were obtained. The RhD target protein and RhD protein fragment from Basic Local Alignment Search Tool（BLAST）platform exhibited 100% sequence identity,and a secondary mapping of RhD protein peptide in the erythrocytic membrane was obtained. Conclusions Immunoprecipitation coupled to mass spectrometry can define amino acid sequence of RhD protein in red blood cells to lay a solid foundation for verifying RhD antigens expressed on red blood cells. It can accurately determine RhD blood group and has a very wide application.

Key words: RhD blood group protein sequence, Erythrocyte protein, Immunoprecipitation method, Mass spectrometry

中图分类号:

R446.6

钟福铃, 梁延连, 苏宇清, 吴凡, 梁爽, 张海璇, 洪文旭, 徐筠娉. 免疫沉淀法与质谱联用检测RHD蛋白序列方法学的建立^*[J]. 临床输血与检验, 2021, 23(5): 623-627.

ZHONG Fu-ling, LIANG Yan-lian, SU Yu-qing, et al. RHD Protein Sequences by Immunoprecipitation and Mass Spectrometry[J]. JOURNAL OF CLINICAL TRANSFUSION AND LABORATORY MEDICINE, 2021, 23(5): 623-627.

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免疫沉淀法与质谱联用检测RHD蛋白序列方法学的建立^*

RHD Protein Sequences by Immunoprecipitation and Mass Spectrometry

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