摘要
为制备携带猪瘟病毒(CSFV)E2蛋白的猪圆环病毒2型(PCV2)病毒样颗粒(VLP),并在小鼠体内评价其免疫原性,本研究采用PCR扩增PCV2 Cap基因,利用重叠延伸PCR将PCV2 Cap蛋白的诱饵表位(aa169~aa180)替换成编码两个连续的CSFV E2蛋白线性表位(aa829~aa837)的融合基因(PCV2-Cap^(169~180)-E2^(829~837))经测序鉴定正确后克隆至载体pET-32a(+)中,构建重组质粒p-Cap-E2并采用PCR方法鉴定正确后转化大肠杆菌BL21(DE3),经IPTG诱导表达重组蛋白(PCV2-Cap-E2),采用改良的镍亲和层析法纯化重组蛋白,并利用SDS-PAGE与western blot对重组蛋白的表达形式及反应原性鉴定;利用透射电镜观察纯化的重组蛋白能否形成VLP;利用本研究制备的VLP、PCV2及CSFV商品化疫苗分别免疫小鼠,采用ELISA方法检测免疫后不同时间小鼠体内的抗体水平及细胞因子含量。SDS-PAGE结果显示,在49 ku处出现目的条带,且重组蛋白主要以可溶性形式表达,纯化得到单一的目的蛋白;western blot结果显示,重组蛋白能够与猪源PCV2多克隆抗体(PAb)、猪源CSFV PAb及HRP标记的6×His-Tag小鼠单克隆抗体(MAb)发生特异性反应,在49 ku处出现特异性条带;电镜观察可见重组蛋白形成规则的VLP;抗体的ELISA结果显示,与PBS对照相比,VLP能够诱导小鼠产生较高的PCV2及CSFV抗体水平(P<0.01),与各商品化疫苗均无显著差异。细胞因子的ELISA结果显示,与PBS对照组相比,VLP能够诱导小鼠产生较高水平的细胞因子(P<0.01)。体外病毒中和试验结果显示,VLP免疫的小鼠血清中具有中和PCV2的活性。综上所述,本实验首次在大肠杆菌中可溶性表达并获得了纯化的重组蛋白(PCV2-Cap-E2),且其能够在体外自组装成VLP,并可刺激小鼠产生针对PCV2 Cap蛋白及CSFV E2蛋白的特异性抗体,为研制针对PCV2和CSFV的新型二联VLP疫苗提供了物质基础。
The aim of this study is to construct porcine circovirus type 2(PCV2)virus-like particles carrying the CSFV E2 protein(CSFV E2)and to evaluate their immunogenicity in mice.In this study,the PCV2 Cap gene was amplified by PCR,and the decoy epitope(aa169-aa180)of the PCV2 Cap protein was replaced with a fusion gene(PCV2-Cap^(169~180)-E2^(829~837))encoding two consecutive linear epitopes(aa829-aa837)of the CSFV E2 protein by using overlap-extension PCR,and was cloned into the vector pET-32a(+).The resulting recombinant plasmid p-Cap-E2 was constructed and identified by PCR before transforming into Escherichia coli BL21(DE3),and its expression was induced by IPTG.The recombinant proteins from PCV2-Cap-E2 were purified by using the modified nickel affinity chromatography method,the expression and reactivity of the purified proteins were identified with SDS-PAGE and western blot.Transmission electron microscopy was used to observe whether the purified recombinant proteins could form VLP;mice were immunized with homemade VLP,PCV2,CSFV commercial vaccine and PBS,and the antibody levels and cytokine contents of mice were detected by ELISA at different times after immunization;SDS-PAGE showed that the target band appeared at 49ku,and the recombinant proteins were expressed in the form of soluble proteins;the recombinant protein was mainly expressed as soluble protein,and the purification of the recombinant protein resulted in collecting a single target protein.Western blot results showed that the recombinant protein reacted specifically with porcine PCV2 polyclonal antibody(pAb),porcine CSFV PAb,HRP-labeled 6×His Tag mouse monoclonal antibody(MAb),with specific bands appearing at 49ku;electron microscopy showed that the recombinant protein could form regular VLP;antibody ELISA results showed that the homemade VLP induced higher antibody levels in stimulated mice compared to the PBS control.Cytokine ELISA results showed that the homemade VLP induced higher levels of cytokine production in mice compared with the PBS control
作者
李岩岩
张帅
赵云环
任晓祥
李思琪
王文钊
左玉柱
范京惠
LI Yan-yan;ZHANG Shuai;ZHAO Yun-huan;REN Xiao-xiang;LI Si-qi;WANG Wen-zhao;ZUO Yu-zhu;FAN Jing-hui(College of Animal Medicine,Hebei Agricultural University,Baoding 071001,China;Hebei Veterinary Biotechnology Innovation Center,Baoding 071001,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2024年第1期70-77,共8页
Chinese Journal of Preventive Veterinary Medicine
基金
河北省现代农业产业技术体系建设专项资金(HBCT2024220201、HBCT2024220401)。
