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  本文選題：血型糖蛋白B　切入點：真核表達　出處：《揚州大學》2007年碩士論文　論文類型：學位論文

【摘要】： 紅細胞血型是指紅細胞膜上特異性抗原的類型。血型研究在輸血安全,法醫(yī)鑒定,遺傳疾病研究中意義重大。20世紀末,已經(jīng)發(fā)現(xiàn)了29個血型系統(tǒng),600多種血型抗原,使得血型研究成為一門獨立的學科發(fā)展起來。血型糖蛋白B(GPB)是紅細胞膜上重要的抗原之一,分為S和s抗原亞型,屬于MNSsU血型系統(tǒng)。Ss抗原的不同在于:在GPB抗原多肽鏈中,第48位氨基酸為M時,表現(xiàn)為為S型抗原,為T時則表現(xiàn)為s型抗原。它們在人群中約各占48%和52%,正常人紅細胞均表達GPB蛋白�？筍和抗s為溫性IgG抗體,常在輸血免疫后產(chǎn)生,可引起明顯的溶血性輸血反應,因此血型單抗做為臨床上血型定型試劑,是必不可少的。 通過RT-PCR方法從K562細胞中克隆GPB基因,并將其克隆入T載體。將序列正確的GPBs經(jīng)雙酶切后克隆進pcDNA3.1-His-Myc A載體,用重組真核表達載體轉(zhuǎn)染CHO細胞,經(jīng)G418篩選得到了陽性克隆;用RT-PCR及Western blot等方法對穩(wěn)定轉(zhuǎn)染GPBs的CHO細胞進行檢測,獲得了穩(wěn)定表達GPBs的CHO細胞系。同時,將克隆出GPBs的抗原胞外區(qū)基因序列克隆進pGEX 4T-2原核表達載體,將測序結(jié)果正確的原核重組菌經(jīng)IPTG誘導表達,獲得GST-GPBs胞外區(qū)的融合蛋白抗原。 用紅細胞、GPBs轉(zhuǎn)基因細胞和原核表達的純化抗原作為免疫源免疫小鼠,采用B淋巴細胞雜交瘤技術(shù)制備單克隆抗體,通過紅細胞血凝試驗、ELISA、Western blot等方法成功篩選出1株抗GPBs胞外區(qū)的特異性單克隆的雜交瘤株。采用直接、間接血凝試驗檢測雜交瘤上清及部分McAb腹水效價;通過試劑盒亞型鑒定,獲得的抗體為IgG型單抗。
[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.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392

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本文編號：1620785