本文選題：紅細胞 + H抗原　； 參考：《中國人民解放軍軍事醫(yī)學科學院》2007年博士論文

【摘要】： 抗人紅細胞抗體是一類針對人紅細胞表面各種抗原的單克隆抗體。在抗人紅細胞單克隆抗體中，IgG類抗體因其在鹽水中能與紅細胞結(jié)合但不使紅細胞凝集，被稱為抗人紅細胞非凝集型單克隆抗體。近年研究證實，該類單克隆抗體無論是在疾病診斷還是臨床治療方面，都具有很大的潛力。目前，國內(nèi)外已制備了許多非凝集型抗人紅細胞單克隆抗體。實踐證明，將抗人紅細胞單克隆抗體改造成重組的單鏈抗體，將進一步拓展該類單抗的應(yīng)用范圍。 H血型抗原是Hh血型系統(tǒng)中唯一的抗原。除稀有的孟買(Bombay)型紅細胞外，H抗原分布在所有人紅細胞的表面。與紅細胞表面其他特有抗原相比，H抗原屬于共有抗原，在各型紅細胞中分布廣泛，應(yīng)用價值較大。根據(jù)自體紅細胞凝集試驗原理建立的抗原、抗體檢測方法是抗紅細胞抗體在免疫診斷方面的應(yīng)用之一。 自體紅細胞凝集試驗的基本原理是：取一滴被檢者自身的全血，向其中加入一種雙功能分子，這種雙功能分子既可以結(jié)合全血中的紅細胞，又可與待檢的抗原或抗體結(jié)合，如果該血樣中含有待檢的抗體或抗原，那么，雙功能分子將分別與紅細胞和待檢物結(jié)合，通過該雙功能分子的橋聯(lián)作用，將血中的紅細胞連接到一起，從宏觀上看，就會呈現(xiàn)肉眼可見的凝集現(xiàn)象，即為自體紅細胞凝集試驗。 本研究在已經(jīng)制備了一株分泌抗人紅細胞H抗原的雜交瘤細胞株2E8的基礎(chǔ)上，建立了具有自主知識產(chǎn)權(quán)的自體全血凝集試驗檢測方法，可用于檢測血液中是否有抗HIV-1gp41抗體的存在。 1抗體可變區(qū)基因的克隆 通過RT-PCR的方法，，從分泌抗人紅細胞H抗原的雜交瘤細胞株2E8中克隆了抗體重鏈可變區(qū)基因(V_H)和輕鏈可變區(qū)基因(V_L)，基因大小分別為351bp和339bp。其中，VH屬于鼠抗體可變區(qū)重鏈基因家族Ⅰ(B)亞群，V_L屬于鼠抗體可變區(qū)輕鏈基因家族Ⅰ亞群。 2單鏈抗體蛋白的表達與鑒定 通過重疊引物延伸法(SOE)將克隆的抗體重鏈和輕鏈可變區(qū)基因拼接成抗人紅細胞H抗原單鏈抗體基因，基因大小為750bp。將序列鑒定正確的單鏈抗體基因用BamHⅠ和NheⅠ酶從中間載體上酶切下來，與經(jīng)同樣酶切的pET-his載體連接，構(gòu)建原核表達載體pET-his-scFv。 將含單鏈抗體基因的原核表達載體轉(zhuǎn)化到BL21(DE3)plysS菌中，用IPTG誘導(dǎo)目的基因表達單鏈抗體蛋白。采用SDS-PAGE、Western blotting法鑒定單鏈抗體蛋白的表達，證實目的蛋白以包涵體的形式獲得了大量表達，其分子量大小約32kDa。經(jīng)ELISA、競爭ELISA、免疫熒光、凝集抑制等試驗證實單鏈抗體蛋白具有與紅細胞表面H抗原結(jié)合的活性。 3抗人紅細胞單鏈抗體融合HIV-1gp41抗原肽基因的表達與鑒定 在成功亞克隆單鏈抗體基因的基礎(chǔ)上，將2E8 C_H1基因和HIV-1gp41抗原肽基因順次連接到單鏈抗體基因的C端，亞克隆抗人紅細胞單鏈抗體融合HIV-1gp41抗原肽基因，基因大小為1150bp。將序列正確的抗人紅細胞單鏈抗體融合HIV-1gp41抗原肽基因從中間載體上酶切下來，與經(jīng)同樣酶切的pET-his載體連接，構(gòu)建原核表達載體pET-his-scFv-C_H1-gp41。 將構(gòu)建的含抗人紅細胞單鏈抗體融合HIV-1gp41抗原肽基因的原核表達載體轉(zhuǎn)化到BL21(DE3)plysS菌中，用IPTG誘導(dǎo)目的基因表達融合蛋白。采用SDS-PAGE、Western blotting法鑒定融合蛋白的表達，證實目的蛋白以包涵體的形式獲得了大量表達，其分子量大小約45kDa。經(jīng)ELISA、競爭ELISA、免疫熒光、凝集抑制等試驗證實融合蛋白具有與紅細胞表面H抗原結(jié)合的活性。 4基于融合蛋白的全血免疫檢測系統(tǒng)模擬檢測 采用正常O型紅細胞、已經(jīng)確認的含HIV-1gp41抗體血漿、上述表達產(chǎn)物等模擬全血免疫檢測系統(tǒng)。結(jié)果表明30份經(jīng)WB確證的陽性HIV血漿均可產(chǎn)生紅細胞凝集反應(yīng)，符合率為100％，其中強凝集樣本22份，弱凝集樣本8份。用A、B、AB三種血型紅細胞分別與6份經(jīng)WB確證的陽性HIV血漿組成全血與雙功能分子融合蛋白作用后，均產(chǎn)生了凝集反應(yīng)。取30份O型HIV抗體陰性全血與融合蛋白作用，均未產(chǎn)生凝集。 5結(jié)論 通過以上結(jié)果得知，以抗人紅細胞H抗原2E8單抗為基礎(chǔ)，制備抗人紅細胞單鏈抗體融合HIV-1 gp41抗原肽的雙功能分子，能夠用于檢測血液中抗HIV-1gp41抗體的存在。
[Abstract]:Anti human erythrocyte antibody is a class of monoclonal antibodies against various antigens on the surface of human erythrocytes. In the anti human erythrocyte monoclonal antibody, IgG antibody is called anti human erythrocyte non agglutinative monoclonal antibody because it can bind to red blood cell but not agglutinate red cell in salt water. At present, many non agglutinative anti human erythrocyte monoclonal antibodies have been prepared at home and abroad. Practice has proved that the transformation of anti human erythrocyte monoclonal antibody into recombinant single chain antibody will further expand the scope of the application of this kind of monoclonal antibody.
H blood group antigen is the only antigen in the Hh blood group system. In addition to the rare Mumbai (Bombay) type red cells, the H antigen is distributed on the surface of all human erythrocytes. Compared with the other specific antigens on the surface of the red cell, H antigen is a common antigen. It is widely distributed in all types of red cells and should be of great value. Based on the principle of autologous red cell agglutination test Antigen detection and antibody detection are one of the applications of anti erythrocyte antibodies in immunodiagnosis.
The basic principle of autologous red cell agglutination test is to take one drop of the whole blood of the examiner to add a double functional molecule, which can combine the red blood cells in the whole blood, and combine with the antigen or antibody to be tested. If the blood sample contains the antibody or antigen to be tested, then the bifunctional molecules will be separated. Combined with red blood cells and samples, the red blood cells in the blood are linked together through the bridging action of the bifunctional molecules. From the macroscopic view, the agglutination of the naked eye can be seen, that is, the autologous red cell agglutination test.
On the basis of a hybridoma cell strain 2E8 secreting anti human erythrocyte H antigen, an autologous whole blood agglutination test method with independent intellectual property right has been established in this study, which can be used to detect the presence of anti HIV-1gp41 antibodies in the blood.
Cloning of the 1 antibody variable region gene
By means of RT-PCR, the antibody heavy chain variable region gene (V_H) and light chain variable region gene (V_L) were cloned from the hybridoma cell line 2E8 secreting anti human erythrocyte H antigen. The size of the gene was 351bp and 339bp., respectively. VH belonged to the mouse antibody variable region heavy chain gene family I (B) subgroup and V_L belonged to the mouse antibody variable region light chain gene family I. Subgroup.
Expression and identification of 2 single chain antibody protein
The cloned antibody heavy chain and light chain variable region gene were spliced into anti human erythrocyte H antigen single chain antibody gene by overlapping primer extension method (SOE). The size of the gene was 750bp. and the sequence identified the correct single chain antibody gene was cut from the intermediate vector with the BamH I and Nhe I enzyme and connected with the same enzyme as the pET-his carrier to construct the prokaryotic cell. Expression vector pET-his-scFv.
The prokaryotic expression vector containing single chain antibody gene was transformed into BL21 (DE3) plysS bacteria, and the target gene was induced by IPTG to express single chain antibody protein. The expression of single chain antibody protein was identified by SDS-PAGE and Western blotting. It was proved that the target protein was expressed in a large amount in the form of inclusion body. The molecular weight of the protein was 32kDa. through ELISA, and was competing for ELI. SA, immunofluorescence and agglutination inhibition test confirmed that single chain antibody protein had binding activity with H antigen on red cell surface.
Expression and identification of 3 anti human red cell scFv fusion HIV-1gp41 antigen peptide genes
On the basis of the successful subclone single chain antibody gene, the 2E8 C_H1 gene and the HIV-1gp41 antigen peptide gene are connected to the C terminal of the single chain antibody gene, and the subclone anti human red cell single chain antibody fusion HIV-1gp41 antigen peptide gene, and the gene size is 1150bp. to fuse the correct anti human red cell single chain antibody to the HIV-1gp41 antigen peptide gene. The enzyme was cut from the intermediate vector and connected with the pET-his vector with the same enzyme cut, and the prokaryotic expression vector pET-his-scFv-C_H1-gp41. was constructed.
The prokaryotic expression vector containing the recombinant human erythrocyte single chain antibody fusion HIV-1gp41 antigen gene was transformed into the BL21 (DE3) plysS bacteria, and the target gene was induced by IPTG to express the fusion protein. The expression of the fusion protein was identified by SDS-PAGE and Western blotting. It was proved that the target egg white was expressed in the form of inclusion body, and its molecules were expressed. The size is about 45kDa.. After ELISA, competitive ELISA, immunofluorescence and agglutination inhibition test, the fusion protein has the activity of binding to the H antigen on the red cell surface.
4 simulation test of whole blood immune detection system based on fusion protein
Using the normal O type erythrocytes, the confirmed HIV-1gp41 antibody plasma and the above expression products were used to simulate the whole blood immune detection system. The results showed that 30 positive HIV plasma confirmed by WB could produce red cell agglutination reaction with the coincidence rate of 22, 8 weak agglutination samples and three blood group red blood cells with A, B, AB. The agglutination reaction was produced when the whole blood and the bifunctional molecular fusion protein composed of 6 positive HIV plasma confirmed by WB proved to be agglutinative reaction. 30 O type HIV antibody negative whole blood and fusion protein were taken, and no agglutination was produced.
5 Conclusion
Based on the anti human erythrocyte H antigen 2E8 monoclonal antibody, the preparation of bifunctional molecules against human erythrocyte single chain antibody fusion HIV-1 gp41 antigen peptide can be used to detect the presence of anti HIV-1gp41 antibody in the blood.

【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R392

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