摘要
目的在HEK293细胞中高效表达抗CEA scFv和抗CA19-9 scFv两种单链抗体的Fc融合蛋白(anti-CEA scFv-Fc和anti-CA19-9 scFv-Fc),并测定两种单链抗体亲和力。方法从GenBank中获取两种scFv基因序列;经优化后合成,并与pET32a(+)构成原核表达载体anti-CEA scFv/pET32a(+)和anti-CA19-9 scFv/pET32a(+),在E.coli BL21(DE3)中进行IPTG诱导表达;两种scFv经PCR扩增技术删除末端终止序列,利用重叠区基因扩增法在anti-CEA scFv上游加入人IL-2信号肽,分别与含人IgG Fc段基因的pTT5载体(Fc/pTT5)构成真核表达载体(anti-CEA scFv(-t)-Fc/pTT5、IL2-antiCEA scFv(-t)-Fc/pTT5和anti-CA19-9 scFv(-t)-Fc/pTT5);使用脂质体转染法,将3个重组质粒转入HEK293细胞中进行表达;产物经rProtein-A FF亲和层析纯化,竞争性细胞ELISA和Western blot检测。结果免疫印迹显示,3个scFv-Fc融合蛋白均在HEK293细胞中表达;竞争结合实验表明,antiCEA scFv-Fc和anti-CA19-9 scFv-Fc的抗体浓度分别为8.2 nmol/L和6.0 nmol/L。结论 anti-CEA scFv和anti-CA19-9scFv能在HEK293细胞内表达;虽然表达量较低,但纯化后表达产物能够识别肿瘤细胞表面抗原CEA和CA19-9。
Objective To express two scFv-Fc fusion proteins of anti-CEA scFv-Fc and anti-CA19-9 scFv-Fc in HEK293 cells efficiently,and to determinate the affinity of two single chain antibodies.Methods Two scFv gene sequences obtained from GenBank were optimized and synthesized.They and pET32a(+)formed double prokaryotic expression vectors,anti-CEA scFv/pET32a(+)and anti-CA19-9 scFv/pET32a(+),respectively,which were translated into E.coli BL21(DE3)and induced expression with IPTG.Two scFv genes(deleted terminators)were amplified by PCR;in addition,through overlapping region amplification method,we added IL-2 signal peptide into the upstream of anti-CEA scFv gene,getting three scFv genes,and connected these genes respectively with pTT5 containing human IgG1 Fc gene(Fc/pTT5)to constitute three eukaryotic expression vectors(anti-CEA scFv(-t)-Fc/pTT5,IL2-anti-CEA scFv(-t)-Fc/pTT5 and anti-CA19-9 scFv(-t)-Fc/pTT5).Three recombinant plasmids of scFv were transferred into HEK293 cells by Lipofectamine.We purified and identified expressing products by rProtein-A FF affinity chromatography,competitive cellular Elisa and Western blot.Results Western blot analysis showed that three scFv-Fc fusion proteins were expressed in HEK293 cells.Competitive binding test showed that antibody concentrations of anti-CEA scFv-Fc and anti-CA19-9 scFv-Fc were 8.2 nmol/L and 6.0 nmol/L,respectively.Conclusion anti-CEA scFv and anti-CA19-9 scFv could express in HEK293 cells,and their purified products could recognize tumor cell surface antigens CEA and CA19-9,although their expression efficiency is relatively low.
作者
陆文斌
宫雪超
金建华
胡文蔚
王月
张华
LU Wenbin;GONG Xuechao;JIN Jianhua;HU Wenwei;WANG Yue;ZHANG Hua(Department of Medical Oncology,Wujin People’s Hospital Affiliated to Jiangsu University,Changzhou 213017,China;Department of Medical Oncology,The First People’s Hospital of Changzhou,The Third Affiliated Hospital of Soochow University,Changzhou 213003,China)
出处
《肿瘤防治研究》
CAS
CSCD
2019年第1期7-13,共7页
Cancer Research on Prevention and Treatment
基金
常州市武进区科技局社会发展类科技支撑项目(WS201606)
常州市卫生计生委重大科技项目(ZD201608)
常州市高层次卫生人才培养工程(2016CZBJ054)
常州市科技支撑-社会发展项目(CE20165051)
