抗细粒棘球蚴人工重组B抗原人源单链可变区抗体的筛选与在原核系统中的表达

SCREENING AND EXPRESSION IN PRONUCLEUS SYSTEM OF HUMAN PHAGE ANTIBODY AGAINST RECOMBINANT ANTIGEN B OF ECHINOCOCCUS GRANULOSUS

目的 筛选并表达抗细粒棘球蚴人工重组 B抗原 (r- Ag B)的人源单链可变区抗体 (Sc Fv) ,为细粒棘球蚴 B抗原蛋白质功能的研究及包虫病的基因治疗开辟新途径。 方法 采用噬菌体表面展示技术 ,以重组纯化的细粒棘球蚴 B抗原为固相抗原 ,经过 5轮“吸附 -洗脱 -扩增”的筛选过程 ,从半合成的噬菌体抗体库中获得了抗原结合活性和特异性较强的含抗 r- Ag B的 Sc Fv基因片段的阳性克隆 ,提取质粒 ,经 Sfi /Not 酶切鉴定后 ,亚克隆到 p CANTAB5 E载体上 ,转化大肠杆菌 XL1 - Blue,经 IPTG诱导 ,表达可溶性 Sc Fv。 结果 筛选得到的 Sc Fv片段基因为 76 8bp,经 SDS- PAGE鉴定 ,在大肠杆菌 XL1 - Blue中表达的 Sc Fv分子质量约 2 8ku,经过 EL ISA和 Western- Blot检测 ,该单链抗体具有较强的抗原结合特性和特异性。 结论 细粒棘球蚴重组 B抗原人源单链可变区抗体筛选和表达成功 。

Objective To study the antigen B and the gene therapy of hydatid disease, human single chain fragment of variable antibody (ScFv) against recombinant antigen B (r AgB) of Echinococcus granulosus was screened from a phage antibody library using phage display technology, then it was characterized and expressed in pronucleus system. Methods The phage library was panned by r AgB of E. granulosus which was coated in a microtiter plate. After 5 rounds of biopanning, 30 clones were demonstrated specific to rAgB of E. granulosus . The specificity of ScFv has been demonstrated by ELISA. The plasmid digested by SfiⅠ/NotⅠ was sub cloned into pCANTAB5E. Soluble ScFv was expressed when IPTG was added under 30 ℃ for 30 h. Results The selected phage antibody had both the standard construction of light and heavy chains of variable fragment and the specific combination capacity with r AgB. The DNA sequence data showed that the ScFv gene was composed of 768 bp and the expression product had 255 amino acids. Molecular weight of ScFv was 28 ku. Conclusion ScFv against r AgB of E. granulosus has been produced and identified by the phage display technology.