炭疽芽胞杆菌治疗性抗体的初步研究

发布时间：2018-03-31 14:43

本文选题：炭疽　切入点：嵌合抗体　出处：《中国人民解放军军事医学科学院》2007年硕士论文

【摘要】： 炭疽是严重威胁人类健康的传染性疾病，接种炭疽疫苗是预防炭疽的有效手段，但是疫苗产生保护性免疫需要一定时间，在疾病流行和某些突发事件发生时无法提供有效的紧急免疫防护。而中和性抗体作为被动免疫制剂可以弥补炭疽疫苗的不足，直接提供有效的保护。因此，本研究选择在中国仓鼠卵巢(Chinese hamster ovary，CHO)细胞中表达具有中和活性的抗炭疽芽胞杆菌重组保护性抗原(recombinant protective antigen，rPA)的嵌合抗体，为炭疽的治疗提供被动免疫制剂。首先克隆3株具有中和活性的抗炭疽芽胞杆菌重组保护性抗原(rPA)的鼠源单克隆抗体(monoclonal antibody，nAb)的V_L和V_H基因。对获得的可变区基因序列进行生物信息学的分析，初步判断为鼠的免疫球蛋白(Immunoglobin，Ig)基因。为了快速验证获得的可变区基因是否具有生物学活性，将5E1和4B2的V_L、V_H基因分别通过(Gly4Ser)_3的连接肽融合构建了scFv基因，在大肠杆菌中表达并纯化了单链抗体，实验表明scFv能够与rPA特异结合，且在细胞模型上具有中和活性，为表达具有中和活性的嵌合抗体奠定了实验基础。为表达全分子嵌合抗体，通过重叠延伸PCR技术将5E1的V_H、V_L基因分别与人IgG1亚类的C_H和C_K基因融合，构建了完整人-鼠嵌合抗体基因。将人-鼠嵌合抗体基因克隆到表达载体的多克隆位点(mutiple cloning site，MCS)，构建了嵌合抗体的表达质粒。将构建的表达质粒转染CHO／dhfr~-(DG44)细胞，经氨甲喋呤(Methotrexate，MTX)初步加压(MTX浓度为5×10~(-8)mol／L)筛选，获得最高表达量为18mg／L的细胞株。对此细胞株扩大培养并纯化嵌合抗体。纯化的嵌合抗体在非还原SDS-PAGE中表现为一条约150kD的带，在还原SDS-PAGE中表现为一条约50kD和一条约25kD的带；Western印迹结果显示，全分子蛋白和重链(heavy chain，H链)分子均可与羊抗人IgG Fc发生特异性免疫反应；全分子蛋白和还原后的轻链(light chain，L链)重链(high chain，H链)分子均可与羊抗人IgG发生特异性免疫反应。ELISA检测结果显示，纯化的嵌合抗体与rPA发生反应，，说明表达的嵌合抗体保留了亲本鼠源mAb的抗原结合活性。在毒素作用的J774A．1细胞模型和Fisher 344大鼠模型上，纯化的嵌合抗体表现出与鼠源mAb 5E1大致相同的中和活性。以上实验结果表明，制备的嵌合抗体能作为被动保护制剂，在炭疽的治疗上具有潜在应用价值。
[Abstract]:Anthrax is a serious threat to human health infectious disease, anthrax vaccine is an effective means to prevent anthrax, but the vaccine production of protective immunity takes some time, The neutralizing antibody as a passive immune agent can make up for the deficiency of anthrax vaccine and provide effective protection directly. Therefore, in order to provide passive immunoassay for anthrax treatment, we selected chimeric antibodies against recombinant protective antigens of Bacillus anthracis expressed in Chinese hamster ovaryary Cho cells of Chinese hamster ovary. The VL and VSP genes of three murine monoclonal antibodies (McAb) against Bacillus anthracis, recombinant protective antigen rPA), were first cloned. Bioinformatics analysis of the obtained variable region gene sequences was carried out. It was preliminarily identified as immunoglobulin (immunoglobin) IgA gene in mice. In order to quickly verify the biological activity of the obtained variable region gene, scFv gene was constructed by fusion of 5E1 and 4B2's VL / VH gene via Gly4Serap3 ligand peptide respectively, and the single chain antibody was expressed and purified in E. coli. The results showed that scFv could specifically bind to rPA and had neutralization activity in cell model, which laid the experimental foundation for the expression of chimeric antibody with neutralizing activity. In order to express the whole molecular chimeric antibody, the 5E1's V _ S / V _ S _ L gene was fused with the human IgG1 subclass C _ (th) and C _ (K) genes by overlapping extension PCR technique, respectively. A complete human-mouse chimeric antibody gene was constructed. The human mouse chimeric antibody gene was cloned into the polyclonal site of the expression vector, and the expression plasmid of chimeric antibody was constructed. The constructed expression plasmid was transfected into Cho / dhfr-mG44 cells. A cell line with the highest expression of 18mg/L was obtained by screening with methotrexate methotrexate MTX at a concentration of 5 脳 10 ~ (-8) mol 路L ~ (-1). The chimeric antibody was cultured and purified in this cell line. The purified chimeric antibody was expressed as a treaty 150kD band in non-reduced SDS-PAGE. The results of Western blotting with a treaty 50kD and a treaty 25kD in the reductive SDS-PAGE showed that the whole protein and heavy chain heavy chain chain H chain could react specifically with sheep anti-human IgG FC. The whole molecular protein and the reduced light chain light chain L chain) heavy chain and high chain chain H) could react with sheep anti-human IgG. The results of Elisa showed that the purified chimeric antibody reacted with rPA. The results showed that the expressed chimeric antibody retained the antigen-binding activity of the parental murine mAb, and the purified chimeric antibody exhibited approximately the same neutralization activity as the murine mAb 5E1 in the J774A.1 cell model and the Fisher 344 rat model. The results show that the chimeric antibody can be used as a passive protective agent and has potential application value in the treatment of anthrax.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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