抗人红细胞单链抗体融合HIV-1gp41抗原肽双功能分子的制备与鉴定

发布时间：2018-04-26 08:20

本文选题：红细胞 + H抗原　；参考：《中国人民解放军军事医学科学院》2007年博士论文

【摘要】： 抗人红细胞抗体是一类针对人红细胞表面各种抗原的单克隆抗体。在抗人红细胞单克隆抗体中，IgG类抗体因其在盐水中能与红细胞结合但不使红细胞凝集，被称为抗人红细胞非凝集型单克隆抗体。近年研究证实，该类单克隆抗体无论是在疾病诊断还是临床治疗方面，都具有很大的潜力。目前，国内外已制备了许多非凝集型抗人红细胞单克隆抗体。实践证明，将抗人红细胞单克隆抗体改造成重组的单链抗体，将进一步拓展该类单抗的应用范围。 H血型抗原是Hh血型系统中唯一的抗原。除稀有的孟买(Bombay)型红细胞外，H抗原分布在所有人红细胞的表面。与红细胞表面其他特有抗原相比，H抗原属于共有抗原，在各型红细胞中分布广泛，应用价值较大。根据自体红细胞凝集试验原理建立的抗原、抗体检测方法是抗红细胞抗体在免疫诊断方面的应用之一。自体红细胞凝集试验的基本原理是：取一滴被检者自身的全血，向其中加入一种双功能分子，这种双功能分子既可以结合全血中的红细胞，又可与待检的抗原或抗体结合，如果该血样中含有待检的抗体或抗原，那么，双功能分子将分别与红细胞和待检物结合，通过该双功能分子的桥联作用，将血中的红细胞连接到一起，从宏观上看，就会呈现肉眼可见的凝集现象，即为自体红细胞凝集试验。本研究在已经制备了一株分泌抗人红细胞H抗原的杂交瘤细胞株2E8的基础上，建立了具有自主知识产权的自体全血凝集试验检测方法，可用于检测血液中是否有抗HIV-1gp41抗体的存在。 1抗体可变区基因的克隆通过RT-PCR的方法，，从分泌抗人红细胞H抗原的杂交瘤细胞株2E8中克隆了抗体重链可变区基因(V_H)和轻链可变区基因(V_L)，基因大小分别为351bp和339bp。其中，VH属于鼠抗体可变区重链基因家族Ⅰ(B)亚群，V_L属于鼠抗体可变区轻链基因家族Ⅰ亚群。 2单链抗体蛋白的表达与鉴定通过重叠引物延伸法(SOE)将克隆的抗体重链和轻链可变区基因拼接成抗人红细胞H抗原单链抗体基因，基因大小为750bp。将序列鉴定正确的单链抗体基因用BamHⅠ和NheⅠ酶从中间载体上酶切下来，与经同样酶切的pET-his载体连接，构建原核表达载体pET-his-scFv。将含单链抗体基因的原核表达载体转化到BL21(DE3)plysS菌中，用IPTG诱导目的基因表达单链抗体蛋白。采用SDS-PAGE、Western blotting法鉴定单链抗体蛋白的表达，证实目的蛋白以包涵体的形式获得了大量表达，其分子量大小约32kDa。经ELISA、竞争ELISA、免疫荧光、凝集抑制等试验证实单链抗体蛋白具有与红细胞表面H抗原结合的活性。 3抗人红细胞单链抗体融合HIV-1gp41抗原肽基因的表达与鉴定在成功亚克隆单链抗体基因的基础上，将2E8 C_H1基因和HIV-1gp41抗原肽基因顺次连接到单链抗体基因的C端，亚克隆抗人红细胞单链抗体融合HIV-1gp41抗原肽基因，基因大小为1150bp。将序列正确的抗人红细胞单链抗体融合HIV-1gp41抗原肽基因从中间载体上酶切下来，与经同样酶切的pET-his载体连接，构建原核表达载体pET-his-scFv-C_H1-gp41。将构建的含抗人红细胞单链抗体融合HIV-1gp41抗原肽基因的原核表达载体转化到BL21(DE3)plysS菌中，用IPTG诱导目的基因表达融合蛋白。采用SDS-PAGE、Western blotting法鉴定融合蛋白的表达，证实目的蛋白以包涵体的形式获得了大量表达，其分子量大小约45kDa。经ELISA、竞争ELISA、免疫荧光、凝集抑制等试验证实融合蛋白具有与红细胞表面H抗原结合的活性。 4基于融合蛋白的全血免疫检测系统模拟检测采用正常O型红细胞、已经确认的含HIV-1gp41抗体血浆、上述表达产物等模拟全血免疫检测系统。结果表明30份经WB确证的阳性HIV血浆均可产生红细胞凝集反应，符合率为100％，其中强凝集样本22份，弱凝集样本8份。用A、B、AB三种血型红细胞分别与6份经WB确证的阳性HIV血浆组成全血与双功能分子融合蛋白作用后，均产生了凝集反应。取30份O型HIV抗体阴性全血与融合蛋白作用，均未产生凝集。 5结论通过以上结果得知，以抗人红细胞H抗原2E8单抗为基础，制备抗人红细胞单链抗体融合HIV-1 gp41抗原肽的双功能分子，能够用于检测血液中抗HIV-1gp41抗体的存在。
[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.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

【参考文献】