畜牧与兽医2023，Vol.55Issue(12)：68-74,7.
猪δ冠状病毒结构蛋白的表达及其多克隆抗体的制备
Construction of structural protein of porcine deltacoronavirus and preparation of its polyclonal antibody
摘要
Abstract
In order to obtain the structural protein of the porcine deltacoronavirus(PDCoV)and to evaluate its immunogenicity,the com-plete S,E,M and N genes of the PDCoV CZ2020 strain were amplified by RT-PCR,and cloned to pCAGGS eukaryotic expression vectors;and then eucaryotic expression plasmids pCAGGS-PDCoV-S,pCAGGS-PDCoV-E,pCAGGS-PDCoV-M and pCAGGS-PDCoV-N were constructed.The plasmids were identified by sequencing and were transfected to HEK293T cells,and the expressions of PDCOV S,E,M and N proteins were detected by indirect immunofluorescence test(IFA)and Western blot(WB).BALB/c mice were prepared for subcuta-neous injection of the proteins,and recombinant proteins polyclonal antibodies were produced in immunized mice.Next,the titers of the anti-bodies were measured by WB and IFA,and their immunogenicity was evaluated.The results showed that the recombinant plasmids pCAGGS-PDCoV-S,pCAGGS-PDCoV-E,pCAGGS-PDCoV-M,pCAGGS-PDCoV-N were successfully constructed and expressed in HEK293T cells.The recombinant proteins were specifically bounded to anti-pig PDCoV serum.The polyclonal potency of pCAGGS-PDCoV-S,pCAGGS-PDCoV-E,pCAGGS-PDCoV-M,pCAGGS-PDCoV-N was as high as 1∶10 000.These results indicated that vaccines prepared by recombinant proteins would be able to induce mice to produce higher levels of antibodies.This study suggested that pCAGGS-PDCoV-S,pCAGGS-PDCoV-E,pCAGGS-PDCoV-M and pCAGGS-PDCoV-N eucaryotic expression vectors were successfully constructed.The poly-clonal antibodies prepared were highly immunogenic and could serve as a basis for development of diagnostic kits and new vaccines.关键词
PDCoV/S、E、M、N基因/真核表达/抗原性Key words
porcine deltacoronavirus/S,E,M,N genes/eukaryotic expression/antigenicity analysis分类
农业科技引用本文复制引用
张宝太,李基棕,李彬,主性,肖丽,钟纯燕,周金柱,黄金,袁雪松,蔡旭航,李思远,郭荣利..猪δ冠状病毒结构蛋白的表达及其多克隆抗体的制备[J].畜牧与兽医,2023,55(12):68-74,7.基金项目
国家重点研发计划(2022YFD1800803) （2022YFD1800803）
江苏省自然科学基金(BK20221432) （BK20221432）
