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目的构建人破伤风免疫噬菌体单链抗体库,筛选抗破伤风毒素重链C端(TeNT-Hc)的特异性单链抗体(scFv)。方法利用RT-PCR从5名破伤风抗体滴度较高的健康献浆员外周血淋巴细胞中获得全套人破伤风抗体VH和VL基因,经过重叠PCR将VH和VL基因连接获得scFv片段。将scFv片段克隆至pCANTAB5E载体,电转化大肠TG1感受态菌,获得抗体库。以TeNT-Hc为抗原对抗体库进行3轮筛选,获得特异性人源性抗TeNT-Hc单链抗体。scFv由pET32a(+)表达载体表达,产物包涵体采用HisTrap FF预装柱纯化并透析复性。采用非竞争酶免疫实验法测定scFv亲和常数(affinity constant,KD值)。检测scFv抑制TeNT-Hc与神经节苷脂GT1b结合的体外中和活性,计算抑制率。结果构建库容量为1×108的人破伤风免疫噬菌体单链抗体库,经过筛选获得3株特异性较好的人源性抗TeNT-Hc单链抗体。原核表达结果显示,3株scFv均以包涵体形式存在,表达量占菌体总蛋白的40%～45%。包涵体经洗涤、纯化和复性后,scFv均保持了与TeNT-Hc的特异结合活性,1 L培养物经纯化后可获得4～6 mg scFv蛋白。亲和力测定结果,27G、22D和S-4-7H-scFv的KD值分别为(1.25×10-7),(2.31×10-7)和(1.97×10-7)mol/L。3株抗体对TeNT-Hc与神经节苷脂GT1b结合的抑制率分别为64.5%,58.6%和51.5%。结论人破伤风免疫噬菌体单链抗体库的构建及具有体外中和活性的特异性抗TeNT-Hc单链抗体的获得,为人破伤风基因工程中和抗体的制备奠定了良好基础。
Objective To construct human tetanus immunization phage scFv library and screen for specific single chain antibody (scFv) against tetanus toxin C terminal (TeNT-Hc). Methods A total of human tetanus antibody VH and VL genes were obtained from 5 peripheral blood lymphocytes of healthy donators with high titers of tetanus antibody by RT-PCR. The VH and VL genes were linked by overlapping PCR to obtain scFv fragments. The scFv fragment was cloned into pCANTAB5E vector and transformed into E. coli TG1 competent cells to obtain an antibody library. Three cycles of antibody screening against TeNT-Hc antigen were used to obtain specific human anti-TeNT-Hc single chain antibody. The scFv was expressed by the pET32a (+) expression vector and the inclusion bodies were purified by HisTrap FF pre-packed column and dialyzed. The scFv affinity constant (KD value) was determined by non-competitive enzyme immunoassay. The scFv was tested for in vitro neutralization activity of TeNT-Hc binding to ganglioside GT1b, and the inhibition rate was calculated. Results A tetanus immunization phage single chain antibody library with a capacity of 1 × 108 was constructed. Three specific anti-TeNT-Hc scFv antibodies were obtained by screening. The results of prokaryotic expression showed that all three scFvs were present in inclusion bodies, accounting for 40% -45% of the total bacterial proteins. After washing, purification and renaturation of inclusion bodies, scFv retains the specific binding activity to TeNT-Hc. After purification of 1 L culture, 4-6 scFv proteins are obtained. The KD values of 27G, 22D and S-4-7H-scFv were (1.25 × 10-7), (2.31 × 10-7) and (1.97 × 10-7) mol / L, respectively. The inhibition rates of the three antibodies on the binding of TeNT-Hc to ganglioside GT1b were 64.5%, 58.6% and 51.5%, respectively. Conclusion The construction of human tetanus immunization phage scFv and the obtaining of specific anti-TeNT-Hc scFv with in vitro neutralization activity provide a good foundation for the preparation of neutralizing antibody for human tetanus gene engineering.