抗人结肠癌单抗MC5的功能性ScFv在大肠杆菌中的可溶性表达(英文)

Soluble expression in Escherichia coli of functional ScFv of monoclonalantibody MC5 against human colorectal carcinoma

目的 制备抗人结肠癌单抗MC5的具功能活性的可溶性单链可变区片段 (ScFv)。方法 从分泌MC5的杂交瘤细胞分离mRNA ,RT PCR分别扩增抗体质量、轻链可变区基因 (VH和VLDNA) ,经linkerDNA连接形成ScFvDNA。将ScFvDNA与噬粒载体pCANTAB5E的连接产物转化于大肠杆菌TG1,转化菌经辅助噬菌体M13KO7感染后 ,获得重组噬菌体。以高表达MC5结合抗原的人结肠癌细胞系SW4 80对重组噬菌体进行 2轮筛选后 ,经ELISA筛选呈现抗体ScFv片段的噬菌体单克隆。取 2个强阳性克隆的噬菌体感染大肠杆菌HB2 15 1,用于表达可溶性ScFv。经点印迹和Western印迹对可溶性ScFv进行鉴定 ,经ELISA检测可溶性ScFv的抗原结合活性。结果 所获得的VH、VL和ScFvDNA分别约为 340、32 0和 75 0bp。对重组噬菌体经 2轮亲和筛选后 ,经ELISA从 2 5个克隆中筛检出 10个抗原阳性克隆。源于 2个强阳性克隆的可溶性ScFv的分子质量为32 0 0 0u ,且浓集于细菌周质腔中 ,可溶性ScFv具有抗原结合活性 ,其结合位点与亲本单抗MC5相同。结论 针对MC5的具功能活性的可溶性ScFv的成功制备 ,为人结肠癌的体外 (乃至体内 )

ObjectiveTo prepare functional soluble single chain va ri able fragment(ScFv) of monoclonal antibody(McAb) MC5 against human colorectal ca rcinoma.Methods mRNA was isolated from the MC5 producing hybr idoma cell line and the DNAs encoding variable domains of heavy and light chains (VH and VL)of the antibody were amplified separately by RT PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vect or pCANTAB5E and the ligated sample was transformed into E.coli TG1. The tra nsfo rmed cells were infected with M13KO7 helper phage to yield recombinant phages. A fter two rounds of panning with human colorectal carci noma cell line SW480 highl y expressing MC5 binding antigen, the phage clones displaying ScFvs of MC5 were selected by ELISA. Two phage clones showing strong signal in ELISA were used to infect E.coli HB2151 to express soluble ScFvs. The soluble ScFvs were ident ified by Dot blot and Western blot, and their antigen binding activities were a ssayed by ELISA.Results VH,VL and ScFv DNAs were about 340, 320 and 750 bp respectively. 10 antigen positive phage clones were selected from 25 preselected phage clones by ELISA. Both the soluble ScFvs derived from the 2 out of the 10 antigen positive phage clones were about 32 000 u and concentra ted in periplasmatic space, and could bind the antigen, and shared the same bind ing site with parental McAb MC5. Conclusion The functional solu ble ScFv of MC5 is successfully produced, which may provide a possible novel tar geting vehicle for in vitro and potential in vivo studies on human color ectal carcinoma.