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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) and screen the full - length humanized single chain antibody (sc Fv) library for anti - IL - 4 whole - human sc Fv and identify its specificity. Methods The expression of IL-4 in p ET102 / IL-4 / BL21 was induced by isopropyl-β-D-thiogalactoside (IPTG). Anti-IL-4 whole-human sc Fv was expressed by using full-human phage antibody library, and the positive clones screened by using the expressed IL-4 as antigen were identified by PCR, restriction enzyme digestion and sequencing. The specificity of anti-IL-4sc Fv was tested by dot blot hybridization. The relative molecular mass (Mr) of the expressed IL-4 fusion protein was 27,000. The positive clones expressing anti-IL-4 were screened by this antigen. The result of PCR showed that the target band was amplified at about 1000 bp. The results of digestion showed that the fingerprints of the two clones were the same and the rest were different. The four sc Fv clones with different fingerprinting and binding ability to IL-4 were selected and sequenced. Three of them were correctly sequenced. Dot blotting showed that the sc Fv expressed by all three clones was specific. Conclusion The anti-IL-4 whole-human sc Fv was successfully screened.