人源抗狂犬病毒二硫键稳定性Fv抗体片段基因的构建与表达

Construction and expression of human disulfide-stabilized Fv fragment gene to Rabies Virus

目的构建人源抗狂犬病毒二硫键稳定性Fv(dsFv)抗体基因,并在原核系统中实现高效表达,制备有活性的人源抗狂犬病毒dsFv抗体片段。方法采用PCR定点突变的方法,构建抗狂犬病毒dsFv抗体的重、轻链(VH、VL)突变基因,NdeⅠ和NotⅠ双酶切后分别插入pET-22b(+)表达质粒中,转化E.coliBL21(DE3),IPTG诱导表达。将VL、VH包涵体蛋白经盐酸胍变性,按比例混合在复性折叠缓冲液中,使二者折叠形成dsFv抗体片段。并以其母本抗体scFv作对照,对其抗原特异性和稳定性进行初步评价。结果在VH的第44位氨基酸和VL的第100位氨基酸引入了半胱氨酸,成功构建了抗狂犬病毒dsFv抗体基因,并在原核系统中实现了对二者的高效表达,VH、VL蛋白在复性缓冲液中正确折叠形成了dsFv抗体片段。结论对筛选获得的抗狂犬病毒scFv成功进行了稳定性改构,制备了有活性的dsFv抗体片段,改构以后的dsFv保持了对狂犬病毒的特异结合活性,而其在血清中的稳定性有明显改进,而且其热稳定性和抵抗尿素化学变性的能力亦大大改进。

Objective To realize the construction and expression of human dsFv genes to rabies virus, Methods The genes of dsFv VH and VL were amplified by PCR-based point mutagenesis strategy. Then the genes were cloned into plasmid pET-22b （＋）. E. coli BL21（DE3） was transformed with the recombinant expression plasmid and the protein was induced in Luris-Bertani medium by addition of IPTG. The inclusion body proteins of VH and VL were denaturalized by GuHCl, and then re-naturalized in refolding solution to form dsFv fragments. Afterwards, we evaluated the antigen-binding activity of the dsFv and its stability as compared with its originative scFv. Results The fragment genes of dsFv to rabies virus were constructed successfully by PCR-based point mutagenesis strategy. Sequence analysis proved that cysteines were introduced into the position 44 aa of VH and position 100 aa of VL. The dsFv VH and VL genes were expressed in PET22b（＋）/BL21（DE3）. The VH and VL protein folded into the active dsFv antibody fragments which showed specific binding capability to rabies virus. Conclusion We succeeded in achieving the stabilization of the human scFv to rabies virus and obtaining the active human dsFv antibody fragments. This established a solid basis for further study of the biological activaty and clinical application of dsFv to rabies virus.