人红细胞血型糖蛋白B单克隆抗体的制备

发布时间：2018-03-16 16:47

本文选题：血型糖蛋白B　切入点：真核表达　出处：《扬州大学》2007年硕士论文　论文类型：学位论文

【摘要】： 红细胞血型是指红细胞膜上特异性抗原的类型。血型研究在输血安全,法医鉴定,遗传疾病研究中意义重大。20世纪末,已经发现了29个血型系统,600多种血型抗原,使得血型研究成为一门独立的学科发展起来。血型糖蛋白B(GPB)是红细胞膜上重要的抗原之一,分为S和s抗原亚型,属于MNSsU血型系统。Ss抗原的不同在于:在GPB抗原多肽链中,第48位氨基酸为M时,表现为为S型抗原,为T时则表现为s型抗原。它们在人群中约各占48%和52%,正常人红细胞均表达GPB蛋白。抗S和抗s为温性IgG抗体,常在输血免疫后产生,可引起明显的溶血性输血反应,因此血型单抗做为临床上血型定型试剂,是必不可少的。通过RT-PCR方法从K562细胞中克隆GPB基因,并将其克隆入T载体。将序列正确的GPBs经双酶切后克隆进pcDNA3.1-His-Myc A载体,用重组真核表达载体转染CHO细胞,经G418筛选得到了阳性克隆;用RT-PCR及Western blot等方法对稳定转染GPBs的CHO细胞进行检测,获得了稳定表达GPBs的CHO细胞系。同时,将克隆出GPBs的抗原胞外区基因序列克隆进pGEX 4T-2原核表达载体,将测序结果正确的原核重组菌经IPTG诱导表达,获得GST-GPBs胞外区的融合蛋白抗原。用红细胞、GPBs转基因细胞和原核表达的纯化抗原作为免疫源免疫小鼠,采用B淋巴细胞杂交瘤技术制备单克隆抗体,通过红细胞血凝试验、ELISA、Western blot等方法成功筛选出1株抗GPBs胞外区的特异性单克隆的杂交瘤株。采用直接、间接血凝试验检测杂交瘤上清及部分McAb腹水效价;通过试剂盒亚型鉴定,获得的抗体为IgG型单抗。
[Abstract]:RBC blood group is the type of specific antigen on erythrocyte membrane. Blood group research is of great significance in blood transfusion safety, forensic identification and genetic disease research. At the end of the 20th century, more than 600 kinds of blood group antigens have been found in 29 blood group systems. The study of blood group has become an independent subject. Blood group glycoprotein (BGP) is one of the important antigens on erythrocyte membrane, which can be divided into S and s antigen subtypes. The difference of Ss antigen belongs to the MNSsU blood group system: in the polypeptide chain of GPB antigen, When the 48th amino acid is M, it is S antigen, and T is s antigen. They account for about 48% and 52 parts respectively in the population. The normal erythrocytes all express GPB protein, and anti-S and anti-s are warm IgG antibodies. Blood group monoclonal antibody (BBA) is necessary as a clinical blood typing reagent because it can cause obvious hemolytic transfusion reaction after transfusion and immunization. GPB gene was cloned from K562 cells by RT-PCR method and cloned into T vector. The GPBs with correct sequence was cloned into pcDNA3.1-His-Myc A vector after double enzyme digestion. CHO cells were transfected with recombinant eukaryotic expression vector, and the positive clones were obtained by G418 screening. RT-PCR and Western blot were used to detect CHO cells stably transfected with GPBs, and CHO cell lines expressing GPBs stably were obtained. Meanwhile, the gene sequence of the extracellular domain of GPBs was cloned into pGEX 4T-2 prokaryotic expression vector. The recombinant prokaryotic cells which were sequenced correctly were induced by IPTG to obtain the fusion protein antigen of the extracellular domain of GST-GPBs. Mice were immunized with GPBs transgenic cells and purified antigen expressed in prokaryotic cells. Monoclonal antibodies were prepared by B lymphocyte hybridoma technique. A monoclonal hybridoma strain was successfully screened by erythrocyte hemagglutination assay (ELISAL) and Western blot. Direct and indirect hemagglutination tests were used to detect the ascites titers of supernatant and partial McAb of hybridoma. The obtained antibody is a IgG type monoclonal antibody.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

【参考文献】