人破伤风免疫噬菌体单链抗体库的构建及体外亲和筛选

发布时间：2018-01-19 22:23

本文关键词： 破伤风破伤风毒素单链抗体逆转录聚合酶链反应细菌噬菌体　出处：《军事医学》2013年02期 　论文类型：期刊论文

【摘要】：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。
[Abstract]:Objective to construct human tetanus immunized phage scFv library. Screening of specific scFv antibody against tetanus toxin heavy chain TeNT-Hc). Methods A complete set of human tetanus antibody VH and VL genes were obtained from peripheral blood lymphocytes of 5 healthy donors with high tetanus antibody titer by RT-PCR. The VH and VL genes were ligated by overlapping PCR to obtain the scFv fragment. The scFv fragment was cloned into pCANTAB5E vector and electrotransformed into Escherichia coli TG1 receptive bacteria. The antibody library was obtained. Using TeNT-Hc as antigen, the antibody library was screened for 3 rounds, and the specific human anti TeNT-Hc single chain antibody. ScFv was expressed by pET32a () expression vector. The inclusion bodies were purified by HisTrap FF preloading column and renatured by dialysis. The affinity constant of scFv was determined by non-competitive enzyme immunoassay. The neutralization activity of scFv on the binding of TeNT-Hc to ganglioside GT1b in vitro was detected. Results 1 脳 108 human tetanus immunized phage scFv library was constructed. After screening, three human anti TeNT-Hc scFv with good specificity were obtained. The prokaryotic expression results showed that all of the scFv strains were in the form of inclusion bodies. The specific binding activity of inclusion bodies to TeNT-Hc was maintained after washing, purification and renaturation. 1L culture was purified and 4mg scFv protein was obtained. The result of affinity test was 27G. The KD values of 22D and S-4-7H-scFv were 1.25 脳 10 ~ (-7). The inhibition rate of TeNT-Hc binding to ganglioside GT1b was 64.5%, respectively. Conclusion the construction of human tetanus immunized phage scFv library and the acquisition of specific anti TeNT-Hc scFv with neutralizing activity in vitro. The preparation of neutralizing antibody against human tetanus gene has laid a good foundation.
【作者单位】：军事医学科学院生物工程研究所疫苗与抗体工程研究室全军生物武器损伤防治药物重点实验室;解放军第302医院临床检验医学中心全军感染病临床实验诊断中心;解放军第302医院感染性疾病研究与诊疗中心;
【基金】：国家杰出青年科学基金资助项目(81025018) 国家自然科学基金青年基金资助项目(81102360,81172980)
【分类号】：R392
【正文快照】： 破伤风是由破伤风梭菌产生的破伤风外毒素引起的一种急性危重疾病。该致病菌在自然界中广泛存在,其产生的破伤风痉挛毒素毒性非常强烈,仅次于肉毒毒素[1]。世界卫生组织报道,全世界每年约有100万人感染发病,在一些国家人感染破伤风后的死亡率仍可高达80%。联合国儿童基金会公

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