抗猪流行性腹泻病毒纳米单链抗体CNC-CBD-scFv的制备及鉴定

Production and identification of nano-single chain antibody CNC-CBD-scFv against porcine epidemic diarrhea virus

为防治猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV),本文制备纳米化抗PEDV单链抗体(singlechain fragment,scFv)并验证其生物学特性。通过PCR方法扩增含有纤维素结合结构域(cellulose binding domain,CBD)的单链抗体基因CBD-scFv,构建抗PEDV的原核重组表达载体pET19b-CBD-scFv,鉴定正确后进行蛋白表达与鉴定。经硫酸水解法制备纳米纤维素(cellulose nanocrystal,CNC)后,将纯化复性后的CBD-scFv蛋白与CNC孵育结合得到纳米抗体CNC-CBD-scFv,通过SDS-PAGE和透射电镜鉴定复合物的结合效果。SDS-PAGE与Westernblot结果表明抗体蛋白CBD-scFv正确表达;制备的纳米纤维素粒度均达到纳米材料级别;CNC与CBD-scFv结合后SDS-PAGE与透射电镜结果说明CNC与CBD-scFv在室温条件下可高效结合。本试验在高效表达合成复合纳米抗体材料的同时为PEDV的防治及应用奠定了一定的试验基础。

In order to prevent porcine epidemic diarrhea virus(PEDV),anti-PEDV nanometer single chain antibody(scFv)was prepared and its biological characteristics was verified in the present study.The CBD-scFv gene was amplified by PCR,the prokaryotic recombinant expression vector pET19b-CBD-scFv was constructed,and then the recombinant protein CBD-scFv was expressed and identified.Cellulose nanocrystal(CNC)was prepared by hydrolysis of sulfuric acid,and purified CBD-scFv was binded with CNC to obtain the nanometer antibody CNC-CBD-scFv.The results of SDS-PAGE and Western blot showed that the antibody protein CBD-scFv was correctly expressed and the nanocellulose particles reached the nanomaterial grade.The results of SDS-PAGE and transmission electron microscopy showed that the antibody CBD-scFv and CNC could bind efficiently at room temperature.This study successfully synthesized the composite nano-antibody materials, and meanwhile laid a certain experimental basis for the prevention and control of PEDV.