抗IL-4全人源单链抗体的筛选和鉴定

Selection and identification of completely humanized anti-IL-4 single-chain antibodies

目的表达人白细胞介素4(IL-4),从全人源单链抗体(sc Fv)文库中筛选抗IL-4全人源sc Fv并鉴定其特异性。方法采用异丙基-β-D-硫代半乳糖苷(IPTG)诱导p ET102/IL-4/BL21表达IL-4,进行纯化鉴定。利用全人源噬菌体抗体文库表达抗IL-4全人源sc Fv,用表达的IL-4作为抗原,筛选阳性克隆菌株经PCR、酶切及测序鉴定。采用斑点杂交检测抗IL-4sc Fv的特异性。结果表达的IL-4融合蛋白相对分子质量(Mr)为27 000。采用该抗原筛选出了表达抗IL-4的阳性克隆,PCR结果显示,在约1000 bp处有扩增的目的条带。酶切结果显示,有2株克隆的指纹图谱相同,其余均不同。选取指纹图谱不同的且与IL-4结合能力强的4个sc Fv的克隆进行测序,显示有3个的序列正确。斑点杂交显示,这3株克隆表达的sc Fv均具有特异性。结论成功筛选到抗IL-4全人源sc Fv。

Objective To express human interleukin 4（ IL-4）,select completely humanized anti-IL-4 single-chain antibodies（ sc Fvs） from the completely humanized sc Fv library and identify their specificity. Methods With IPTG-induced p ET102/IL-4/BL21,human IL-4 was synthesized,and then purified and identified. Completely humanized sc Fvs against IL-4 were expressed from the completely humanized antibody phage library,and positive clones were selected with human IL-4 as antigen. The positive clones were tested by PCR,enzyme cleavage and sequencing. The specificity of the completely humanized anti-IL-4 sc Fvs was determined by dot blot hybridization. Results The relative molecular mass（ Mr） of IL-4fusion protein was about 27 000. With IL-4 as antigen,we obtained the positive clones expressing anti-IL-4 antibodies. PCR showed amplified target bands of about 1000 bp. Enzyme cleavage analysis revealed that fingerprint maps of the selected clones were diverse except 2 clones. Among 4 clones expressing sc Fvs that had different fingerprint maps and strong binding to IL-4,sequencing showed the sequence of 3 was correct. Then,dot blot hybridization demonstrated that the sc Fvs expressed by the 3 clones were specific. Conclusion Completely humanized sc Fvs against IL-4 has been obtained successfully.