DEVELOPMENT OF GENETICALLY ENGINEERED MOUSE/HUMAN CHIMERIC AND SINGLE CHAIN ANTIBODIES AGAINST HUMAN BRAIN GLIOMA:A PRELIMINARY REPORT

In order to improve the clinical usefulness of mAb of mouse origin in targeting diagnosis and therapy for human brain glioma, it is necessary to humanize it and reduce its molecular size. By means of RT-PCR technique, a 348 bp heavy chain variable domain (VH),and a 318 bp light chain variable domain (VL) cDNA fragments were cloned from mouse hybridoma cell line SZ39 secreting mAb against human brain glioma. By recombinant DNA technique, the two cDNA 'fragments were linked to human IgGI heavy chain CHI and light chain K constant regions, respectively, to form a mouse/human chimeric gene which was then inserted into an expression vector pHENI-SZ39 Fab/Hu. In addition,the two cDNA fyagments were linked directly with a universal link6r and inserted into an expression vector pHENI-SZ39ScFv. The two expression vectors were separately introduced into non-suppressor E.coli HB2151to secrete chimeric antibodies and single-chain antibodies,respectively. On ELISA and Western blot assays, the two genetically engineered antibodies were bound specifically to the same 180 kD cell surface membrane antigen on human brain glioma cell line SHG44 as did the parental mAb SZ39.

主要发达国家生物技术关键领域SCI计量对比研究

生物技术是我国战略性高新技术发展领域,也是全球各国在生物经济时代能否获胜的关键技术。通过科学引文索引数据库(SCI)中论文收录文献的文献计量学研究,对单克隆抗体、疫苗、重组蛋白、基因治疗、干细胞研究、组织工程、基因组学、蛋白质组学、芯片技术九类关键技术领域的全球研究分布格局进行了探讨,并结合生物医药产业链进行定标比超研究,以期发现我国在全球生物技术领域所处的位置和产业差距。研究发现美国在以上关键技术领域均处于全球绝对领先地位,而我国在某些技术领域如芯片、组织工程、组学等领域已具有一定比较优势和国际竞争能力,但在较长时期内研发和产品仍将处于跟随地位。

鼠抗人CD14单链抗体ScFv2F9原核表达载体的构建和表达

目的:构建ZCH-7-2F9(简称2F9)单链抗体(ScFv2F9)原核表达载体,获得活性目的蛋白,为2F9免疫毒素及其它类型的基因工程抗体的研究奠定基础。方法:根据VH2F9、VL2F9、(G4S)3基因序列以及pIVEX2.3-MCS空载体多克隆位点内切酶位点NdeI和SmaI的核酸序列设计引物,采用高保真的Taq酶,通过重叠延伸拚接法(splicing by overlap extension,SOE)扩增克隆到ScFv2F9基因,T-A克隆、测序核实后克隆到pIVEX2.3-MCS空载体中,阳性重组质粒转化大肠杆菌菌株E.coli BL21star(DE3)plysS,IPTG诱导目的蛋白表达,镍树脂纯化后体外复性浓缩,流式细胞术观察其识别和结合CD14的能力。结果:成功构建ScFv2F9的原核表达载体pIVEX2.3-MCS/ScFv2F9;对大肠杆菌的包涵体进行纯化复性,其复性率为32.6%;通过流式细胞术观察到复性ScFv2F9对亲本单抗2F9-FITC有部分的阻滞效果,复性ScFv2F9阻滞1次后阳性细胞百分数、平均荧光强度(MFI)和高峰频道(peakCh)分别下降了11.73%、11.96%和31.57%,阻滞2次后分别下降了26.44%、21.75%和42.11%。结论:ScFv2F9在原核细胞中获得成功表达,复性后目的蛋白对人CD14有一定的识别和结合能力,为2F9单抗的进一步改造打下了坚实的基础。

Preparation of single chain variable fragment of MG_7 mAb by phage display technology

AIM: To develop the single chain variable fragment of MG MG(7)murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS: mRNA was isolated from MG MG(7) producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG MG(7) recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATO III of highly expressing MG(7)-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG(7) ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG(7) ScFv. Finally, the relative molecular mass of soluble MG(7) ScFv was measured by SDS-PAGE. RESULTS: The V(H), V(L) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 X 10(6) and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG(7) antibody for binding to the antigen expressed on KATO III cells. Within 2 strong positive phage clones, the soluble MG(7) ScFv from one clone was found to have the binding activity with KATO III cells. SDS-PAGE showed that the relative molecular weight of soluble MG(7) ScFv was 32. CONCLUSION: The MG(7) ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.

针对人DNAH2蛋白的鼠源单克隆抗体的制备

目的：制备特异性的鼠源单克隆抗DNAH2（Homo sapiens dynein，axonemal，heavy chain 2）抗体。方法首先针对人DNAH2蛋白N端1-300 aa序列，构建可表达His标签的免疫原融合表达质粒，在大肠杆菌内以IPTG诱导His-免疫原的表达并纯化；然后免疫BALB/c小鼠，分离出脾淋巴细胞，并与骨髓瘤细胞融合，形成杂交瘤细胞；最后以酶联免疫吸附测定（ELISA）、蛋白免疫印迹法（Western blot）筛选阳性杂交瘤细胞。结果利用IPTG有效诱导了大肠杆菌内DNAH2免疫原的表达；筛选的阳性杂交瘤细胞检测出DNAH2蛋白条带。结论成功制备针对人DNAH2蛋白的鼠源单克隆抗体。