目的:构建一个U251细胞血清饥饿特异抗原ScFv噬菌体抗体库。方法:将U251细胞进行48小时的血清饥饿培养,将其细胞裂解液免疫4周龄的BALB/c小鼠,提取被免疫小鼠脾脏细胞总RNA,反转录成cDNA,利用RT-PCR扩增免疫球蛋白IgG的重链可变区(VH)...目的:构建一个U251细胞血清饥饿特异抗原ScFv噬菌体抗体库。方法:将U251细胞进行48小时的血清饥饿培养,将其细胞裂解液免疫4周龄的BALB/c小鼠,提取被免疫小鼠脾脏细胞总RNA,反转录成cDNA,利用RT-PCR扩增免疫球蛋白IgG的重链可变区(VH)和轻链可变区(VL)基因,用一个柔性片段(Linker)连接VL和VH基因片段,将连接产物重组到pCANTAB-5E载体,转入TG1菌株中,随后加入辅助噬菌体(Help phage)M13K07超感染,构建单链抗体片段(Single chainfragment of variation,ScFv)噬菌体抗体库。结果:经过富集筛选后,库容量达到3×106cfu/L,随机挑取8个克隆进行ELISA检测,获得了1个阳性克隆。结论:成功构建了一个具有一定库容的U251细胞血清饥饿特异抗原的单链抗噬菌体抗体库,为筛选血清应答蛋白抗体、进一步克隆血清应答蛋白的基因奠定基础。展开更多
A murine phage antibody library was constructed.First,the total RNA was extracted from fresh spleens of nonimmunized mice,then the cDNA library was achieved via reverse transcription PCR.Gene fragments encoding V_H an...A murine phage antibody library was constructed.First,the total RNA was extracted from fresh spleens of nonimmunized mice,then the cDNA library was achieved via reverse transcription PCR.Gene fragments encoding V_H and V_L were amplified and assembled into a single gene using a DNA linker encoding a polypeptide of 15 amino acid residues(Gly4Ser)3 through PCR.And finally the recombinant DNA fragments were cloned into the phagemid pCANTAB5E vector and introduced into E.coli TG1.The phagemid particles displaying functional ScFv were rescued by reinfection of helper phage M13K07,thus a murine antibody library was obtained.展开更多
文摘目的:构建一个U251细胞血清饥饿特异抗原ScFv噬菌体抗体库。方法:将U251细胞进行48小时的血清饥饿培养,将其细胞裂解液免疫4周龄的BALB/c小鼠,提取被免疫小鼠脾脏细胞总RNA,反转录成cDNA,利用RT-PCR扩增免疫球蛋白IgG的重链可变区(VH)和轻链可变区(VL)基因,用一个柔性片段(Linker)连接VL和VH基因片段,将连接产物重组到pCANTAB-5E载体,转入TG1菌株中,随后加入辅助噬菌体(Help phage)M13K07超感染,构建单链抗体片段(Single chainfragment of variation,ScFv)噬菌体抗体库。结果:经过富集筛选后,库容量达到3×106cfu/L,随机挑取8个克隆进行ELISA检测,获得了1个阳性克隆。结论:成功构建了一个具有一定库容的U251细胞血清饥饿特异抗原的单链抗噬菌体抗体库,为筛选血清应答蛋白抗体、进一步克隆血清应答蛋白的基因奠定基础。
文摘A murine phage antibody library was constructed.First,the total RNA was extracted from fresh spleens of nonimmunized mice,then the cDNA library was achieved via reverse transcription PCR.Gene fragments encoding V_H and V_L were amplified and assembled into a single gene using a DNA linker encoding a polypeptide of 15 amino acid residues(Gly4Ser)3 through PCR.And finally the recombinant DNA fragments were cloned into the phagemid pCANTAB5E vector and introduced into E.coli TG1.The phagemid particles displaying functional ScFv were rescued by reinfection of helper phage M13K07,thus a murine antibody library was obtained.
