摘要
目的:实现基于中和性单抗4C13单链抗体的真核表达,并对其生物学活性进行评价.方法:用TRIzol提取抗ricin中和性单抗杂交瘤细胞4C13总RNA,扩增其轻、重链可变区基因,登陆GenBank;用Linker(GGGGS)3将VH和VL连接,用重叠延伸PCR克隆入真核表达载体pCDNA3.1,用脂质体转染293T细胞,对瞬时表达产物进行生物学活性检测.结果:中和性单抗杂交瘤细胞4C13轻、重链可变区基因在GenBank中的登录号为DQ389248和DQ389245,ScFv与ricin可特异性结合,培养上清原液和2倍稀释液的抑制率分别为33.7%和29%;ScFv在ricin浓度为0.01～0.005ng/mL时可中和ricin对SP2/0细胞的细胞毒,随着ricin浓度逐渐增大,细胞存活率逐渐减低.结论:研究结果为研制基于中和性单抗的其他ricin拮抗剂奠定了重要的理论和实践基础.
AIM ： To realize an anti-ricin neutralizing monoclonal antibody-based ScFv proteins with high biological activity in the pCDNA3. 1 vector expression system and to evaluate the neutralizing activities of ScFv. METHODS： The total RNA was obtained by TRIzol from an anti-ficin neutralizing monoclonal anti- body hybridoma cell named 4C13 and heavy-chain and light-chain variable fragments （VH, VL） were amplified by overlap PCR and accepted by Genbank of NCBI. V. and VL were linked by （GGGGS）3 with a direction of VH-linker-VL and cloned to pCDNA3.1 vector expression system. The biological activities of ScFv obtained from 293T cell culture fluids infected by liposome were evaluated. RESULTS： The accession number of VH and VL genes of anti-ricin neutralizing monoclonal antibody was DQ389248 and DQ389245. ScFv could bind with ricin specifically. Inhibition percentage of culture liquid of 293T cell without dilution and with 2 times dilution was 33.7% and 29%, respectively. ScFv could neutralize the cytotoxic effects of ricin to SP2/0 cells at the concentration of 0.01 -0. 005 ng/mL in a nice dose-effect relation. CONCLUSION： The nice biological activi- ties of ScFv have established an important basement for further study of other anti-ricin neutralizing monoclonal antibody-based antidotes.
出处
《第四军医大学学报》
北大核心
2007年第14期1275-1278,共4页
Journal of the Fourth Military Medical University
基金
国家高技术研究发展计划(863)(2002AA232021和2004AA002020)
中国博士后基金(2004035289)
