人源抗狂犬病毒单链二硫键稳定抗体的重组设计与表达

Recombinant design and expression of human anti-rabies virus ScdsFv

以人源抗狂犬病毒糖蛋白母本单链抗体ScFv为模板,利用PCR点突变分别在重链FR可变区VH(44)和轻链FR可变区VL(100)分别引入一个半胱氨酸,成功构建了重组单链二硫键稳定抗体基因。连接pET22b(+)载体,转化入E.coli BL21(DE3)得到工程菌,IPTG诱导表达。体外复性并经Ni-NTA亲和层析对目的蛋白ScdsFv进行纯化;利用荧光抗体实验和ELISA检测抗体活性及稳定性。结果表明重组ScdsFv蛋白实现了原核高效表达,通过体外复性和Ni-NTA柱纯化获得纯度大于90%的ScdsFv蛋白。荧光抗体实验和ELISA结果表明ScdsFv具有特异的抗原结合活性,与母本ScFv比较,稳定性有明显提高。这种具有特异抗原结合活性的稳定ScdsFv蛋白的获得为其进一步的功能研究提供了材料。

To constructed the recombinant human anti-rabies virus ScdsFv, cys sites were introduced into framework region （FR） of VH and VL genes which were amplified from human anti-rabies virus ScFv respectively using genetic point mutation technology. Cloned the ScdsFv gene into expression vector pET22b（ ＋ ） and transformed into E. coli BL21 （DE3）. The target protein was expressed by inducing with IPTG. Followed by renaturation in vitro and purified by NiNTA. The binding activity of ScdsFv was identified by Fluorescent antibody test （FAT） and ELISA. Results showed that recombinant ScdsFv were expressed at high level. Purity of the protein 〉 90% after purified by Ni-NTA and renaturaton in vitro. FAT and ELISA results demonstrated that ScdsFv could binding antigen specificity and was more stable than ScFv. Recombinant ScdsFv provided experiment materials for further functional study.