人破伤风免疫噬菌体单链抗体库的构建及体外亲和筛选

Construction of a human tetanus toxoid immune phage single-chain Fv antibody library and screening of scFv against tetanus toxin Hc fragment

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 a human tetanus toxoid immune phage single-chaln Fv（scFv） antibody library and to screen scFv against tetanus toxin heavy chain C （Hc） fragment （TeNT-He）. Methods The variable heavy （ VH ） and variable light （VL） genes were amplified from peripheral blood lymphocytes from five volunteers with high titer antibodies against tetanus toxoid. Gene encoding scFv fragments were constructed by SOE-PCR and then cloned into the phagemids vector pCANTABSE. The phagemids containing scFv gene were transformed into electrocompetent Escherichia coli TG1 cells to construct the antibody library. ScFv clones were screened by three rounds of panning with TeNT-Hc. ScFv protein was expressed by vector pET32a（ ＋ ） and purified by an affinity chromatography HisTrap FF. The inclusion of 3 scFvs＇protein was via dialysis renaturation. Non-competitive enzyme immunoassay method was used to determine the affinity constant （KD） of 3 scFvs. The rate at which the scFv inhibits TeNT-Hc binding to ganglioside GT1 b showing neutralizing activity in vitro of scFv was calculated. Results A human tetanus toxoid immune phage single-chain Fv antibody library was construc- ted with a size of about 1 x 10s. Three scFvs were selected which bound specifically to TeNT-Hc named 27G ,22D and S-4- 7H-scFv. The expression of 3 scFvs in E. coil BL21 （DE3） as inclusion body accounted for about 40% to 45% of total bac- terial proteins. After the scFv inclusion bodies were washed, purified and renaturated, the seFv proteins maintained specif- ic binding activities to TeNT-Hc and the yield was 4 to 6 mg per litre. The KD of three scFvs was（ 1.25× 10^-7 ）, （2.31 ×10^-7 ） and （ 1.97×10^-7 ） mol/L while the inhibitory rate was 64.5%, 58.6% and 51.5%, respectively. Conclusion Successful construction of a human tentanus immune phage single-chain Fv antibody library and obtaining specific scFv against He which can inhibit the TeNT-He binding to ganglioside GTlbContribute much to the preparation of human engineering antibody against tetanus toxin.