摘要
为制备猫传染性腹膜炎病毒N蛋白特异性多克隆抗体,研究根据HLJ/HRB/2016/11毒株N基因序列设计引物,通过PCR进行基因扩增,并将目的基因在Bam HⅠ、HindⅢ酶切位点克隆至pET32a(+)原核表达载体上,将其转化到BL21(DE3)感受态细胞,经终浓度为1 mmol·L^(-1)的IPTG在16℃诱导16 h后,SDS-PAGE检测表达产物,结果显示,在约69 kda处出现目的条带,且该蛋白主要以可溶形式表达。将重组N蛋白经Ni柱纯化后,按每只BALB/c小鼠免疫50μg,三免后7 d采血分离血清,经间接ELSA检测结果显示,获得的N蛋白可被特异性识别且效价可达到1∶12800。研究制备的猫传染性腹膜炎病毒N蛋白及其多克隆抗体为后续猫传染性腹膜炎的疫苗提供基础。
In order to prepare polyclonal antibodies specific for N protein of feline infectious peritonitis virus,primers were designed based on the N gene sequence of HLJ/HRB/2016/11 strain,and the gene was amplified by PCR.The target gene was cloned into p ET32a(+)prokaryotic expression vector at the Bam HⅠand HindⅢrestriction sites.The protein was transformed into BL21(DE3)competent cells and induced by IPTG with a final concentration of 1 mmol·L^(-1)for 16 h at 16℃.SDS-PAGE detected the expression product.The results showed that the target band appeared at about 69 kda,and the protein was mainly expressed in soluble form.The recombinant N protein was purified by Ni column and immunized BALB/C mice at the rate of 50μg/mouse.Blood samples were collected for serum separation at 7 d after the third immunization.Indirect ELSA detection showed that the obtained N protein could be specifically recognized with a titer up to 12800.The feline infectious peritonitis virus N protein and its polyclonal antibody prepared in this study would provide the basis for the subsequent feline infectious peritonitis vaccine.
作者
赵飞宇
杨丹
赵鹏宇
李梓健
李璐
孙东波
Zhao Feiyu;Yang Dan;Zhao Pengyu;Li Zijian;Li Lu;Sun Dongbo(College of Animal Science and Technology,Heilongjiang Bayi Agricultural University,Daqing163319)
出处
《黑龙江八一农垦大学学报》
2023年第1期46-52,共7页
journal of heilongjiang bayi agricultural university
基金
黑龙江八一农垦大学三横三纵科研团队支持计划(TDJH201804)。
关键词
猫传染性腹膜炎病毒
衣壳蛋白
原核表达
多克隆抗体
feline infectious peritonitis virus
capsid protein
prokaryotic expression
polyclonal antibody
