全人源抗乳腺癌噬菌体单链抗体库的构建

目的：利用噬菌体展示技术构建全人源性抗乳腺癌单链抗体库。方法：从临床获取未化疗乳腺癌病人外周血样30份,分离出单个核细胞（PBMC）,提取总RNA,用RT-PCR技术逆转录获得cDNA,并扩增出全套人抗体重链（VH）和轻链（VL）基因,经重叠延伸PCR（SOE-PCR）,在体外将两者连接成单链抗体（scFv）基因片段,将该片段用Sfi I和Not I酶切后克隆至pCantab5E噬菌体载体,电转化TG1感受态菌,收集培养后平板上的菌落,即构建初级噬菌体单链抗体库。结果：得到长度约为360bp和340bp的VH和VL,拼接后得到的scFv长度约为750bp;经PCR初步鉴定插入率约为80%,BstN 1多样性酶切检验,酶切图谱呈多样性。经测序验证,最终获得库容约为2.4×106pfu/ml初级单链抗体库。结论：本研究获得了全人源抗乳腺癌噬菌体单链抗体库,为下一步筛选抗人乳腺癌细胞特异性单链抗体奠定了基础。

Construction of human phage display scFv library against mastocarcinoma

Objective：To construct a human anti-mastocarcinoma cells phage single-chain Fv antibody library by phage display technology.Methods：About 30 peripheral blood samples of breast cancer patients without chemotherapy were got from hospital.Then RNA was isolated from the peripheral blood mononuclear cells（PBMC）and reverse transcribed to cDNA by RT-PCR.VH and VL were amplified and linked together with a linker segment by overlapping PCR（SOE-PCR）to form a human scFv gene.Cut the scFv gene and phagmid vector pCantab5E by SfiⅠand Not,ligated them and electrically transformed to TG1.Consequently,a primary scFv phage display library was constructed.Results：The fragment of VH and VL were about 360 and 340 bp.They were linked in vitro to form scFv of about 750 bp.The insertion efficiency was about 80% tested by PCR method.Randomized clones from unselected library digested with BstNⅠshowed different patterns.Sequencing indicated that VH and VL genes were homologous with the variable region of human antibody.A recombinant phage display library with total of 2.4×106pfu/ml was established.Conclusion：We constructed a primary human anti-mastocarcinoma cells phage scFv antibody library by phage display technology.It can be used for the next selection of specific anti-mastocarcinoma cells phage antibody.