牛外周血淋巴细胞PD-1胞外区基因的原核表达及多抗制备

Prokaryotic expression of PD-1 extracellular domain gene of bovine peripheral blood lymphocytes and preparation of polyclonal antibody

目的原核表达牛外周血淋巴细胞(peripheral blood lymphocytes,PBL)表面程序性细胞死亡蛋白-1(programmed cell death protein 1,PD-1),并制备其多抗血清。方法以PBL的cDNA为模板扩增牛PD-1胞外区片段,与载体pET-28a(+)连接,构建重组质粒pET-28a-PD-1,并进行生物信息学分析;重组质粒转化大肠埃希菌BL21(DE3),经不同浓度的IPTG诱导表达牛PD-1胞外区重组蛋白(简称牛PD-1蛋白),产物进行SDS-PAGE及Western-blot分析;纯化的目的蛋白免疫小鼠制备血清多抗,与CP、NCP型牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)体外孵育牛PBL 72 h,检测BVDV病毒拷贝数。结果经双酶切及测序鉴定,重组质粒pET-28a-PD-1构建正确;牛PD-1胞外区基因读码框编码147个氨基酸,相对分子质量约为16040,其二级结构主要由α螺旋、延伸链、无规则卷曲构成,与人及鼠序列的同源性分别为79%和71%;在37℃,0.8 mmol/L IPTG诱导条件下,牛PD-1蛋白相对分子质量约19000,以包涵体的形式表达;制备的多抗血清效价最高为1∶102400,能与目的蛋白发生特异性反应,显著降低CP、NCP型BVDV病毒拷贝数(P<0.05)。结论成功构建了重组表达质粒pET-28a-PD-1,获得了牛PD-1蛋白及其多抗血清,且免疫原性较好,PD-1阻断抑制BVDV在PBL细胞中的复制,为开发具有调节及治疗慢性病毒感染的生物制品奠定基础。

Objective To express programmed cell death protein 1(PD-1)on the surface of bovine peripheral blood lymphocytes(PBL)in prokaryotic cells and prepare its polyclonal antibody.Methods The extracellular region of bovine PD-1 was amplified by PCR using the cDNA of PBL as a template and inserted into prokaryotic expression vector pET-28 a(+).The constructed recombinant plasmid pET-28 a-PD-1 was analyzed for bioinformatics and transformed to E.coli BL21 for expression under induction of IPTG at various concentrations.The expressed protein was analyzed by SDS-PAGE and Western blot.Polyclonal antibody was prepared by immunizing mice with the purified target protein,and used for incubation of bovine PBL together with bovine viral diarrhea virus(BVDV)types CP and NCP in vitro,and the copy number of BVDV was determined.Results Restriction analysis and sequencing proved that recombinant plasmid pET-28 a-PD-1 was constructed correctly.The ORF of Bovine PD-1 extracellular domain gene encoded 147 amino acids.The relative molecular mass of the domain was about 16040,while the secondary structure mainly consisted of alpha helix,extended chain and random coil.The homologies of the domain were 79%and 71%to those of humans and mice respectively.After induction with 0.8 mmol/L IPTG at 37℃,the expressed protein,with a relative molecular mass of about 19000,existed in a form of inclusion body.The prepared polyclonal antibody,with a titer of 1∶102400 at most,showed specific reaction with the target protein,which decreased the copy number of BVDV of type CP and NCP significantly(P<0.05).Conclusion Recombinant plasmid pET-28 a-PD-1 was successfully constructed,and recombinant protein and polyclonal antibody with good immunogenicity were obtained.PD-1 blockade inhibited the replication of BVDV in PBL,which laid a foundation of development of biological products with regulation and treatment of chronic viral infection.