利用E.coli表达PCV2 Cap蛋白病毒样颗粒的不同策略及免疫原性评价  

Different strategies for preparing PCV2 virus-like particles in Escherichia coli and evaluation of immunogenicity

作　　者：赵强[1,2] 刘莹 赵云环[1] 翟刚 张帅 郭晓旭[1] 范京惠 左玉柱[1,2] ZHAO Qiang;LIU Ying;ZHAO Yunhuan;ZHAI Gang;ZHANG Shuai;GUO Xiaoxu;FAN Jinghui;ZUO Yuzhu(College of Veterinary Medicine,Hebei Agricultural University,Baoding,Hebei 07100l,China;Hebei Veterinary Biotechnology Innovation Center,Baoding,Hebei 071001,China)

机构地区：[1]河北农业大学动物医学院,河北保定071001 [2]河北省兽医生物技术创新中心,河北保定071001

出　　处：《中国兽医学报》2022年第9期1744-1751,共8页Chinese Journal of Veterinary Science

基　　金：河北省重点研发计划资助项目(19226622D);河北省农业产业技术体系生猪创新团队资助项目(HBCT2018110207)。

摘　　要：为得出在大肠杆菌(E.coli)中制备PCV2 VLPs的最佳策略,通过PCR方法,从临床样本中扩增出野生型nCap基因,并对其进行密码子优化合成yCap。以pET-32a为表达载体,构建重组质粒pET-32a-nCap和pET-32a-yCap,以BL21和含有稀有密码子tRNA的Rosetta-gamiB感受态细胞作为表达菌株,以相同体系培养并经IPTG诱导表达,称重比较收获的湿菌体量,通过SDS-PAGE与Western blot对Cap蛋白的表达方式及免疫反应性进行鉴定,使用镍亲和层析法进行纯化并通过SDS-PAGE分析纯化效率,并利用透射电镜观察能否形成VLP,将形成VLP的蛋白以弗氏佐剂为佐剂制备疫苗免疫小鼠,使用ELISA试剂盒测定特异性抗体IgG及细胞因子。结果显示,未经密码子优化的Cap蛋白只能在Rosetta-gamiB中表达,命名为VLP-B,经密码子优化的Cap蛋白在BL21和Rosetta-gamiB中均能表达,分别命名为VLP-A及VLP-C,3种蛋白均主要以可溶性形式表达且VLP-A的产量高于VLP-B、VLP-C;经镍亲和层析法纯化后皆可在透射电镜下观察到大量直径在20 nm左右的VLPs,但VLP-C纯化效率极低,VLP-A效果最佳,VLP-B次之;50μg/只重组VLPs免疫小鼠后(dpi)抗体水平迅速升高,14 d时均已达到阳性临界值,并维持在一个较高的水平直至42 d,同时也诱导产生了细胞因子反应,VLP-C免疫原性最佳,VLP-B次之,VLP-A相对较差。由此可知,将Cap基因密码子优化并提供大肠杆菌所缺少的稀有密码子的tRNA的促进VLPs组装效果要优于提供tRNA,更优于密码子优化,但蛋白表达量低,无法使用镍亲和层析得到理想效果,仍需进一步探索纯化方式。此外,仅将Cap基因密码子优化,产量相对较高,可经镍亲和层析法纯化,但免疫原性相对较弱。结果表明,本研究为PCV2 VLPs疫苗的进一步研究和生产提供了参考依据,对于降低疫苗成本和研制相关生物制品具有重大意义。In order to obtain the best strategy for preparing PCV2 VLPs in E.coli,the wild-type nCap gene was amplified from clinical samples by PCR,and the codon optimized yCap was produced by Sangon Biotech.pET-32 a was used as the expression vector to construct recombinant plasmids pET-32 a-nCap and pET-32 a-yCap, and Rosetta-gamiB containing rare codon tRNA was used as competent cells, and BL21 competent cells was used as expression strains, cells were cultured in the same system and induced by IPTG,the expression mode and immunoreactivity of Cap protein were identified by SDS-PAGE and Western blot, nickel affinity chromatography was used for purification,and purification efficiency was analyzed by SDS-PAGE.And transmission electron microscope was used to observe whether VLP could be formed.VLP-forming protein was prepared and mixed with Freund’s adjuvant to immunize mice,and commercial ELISA kit was used to determine specific antibody IgG and cytokine interleukin 4(IL-4),interferon-γ(IFN-γ)and tumor necrosis factor(TNF-α).The results showed that the Cap protein without codon optimization could only be expressed in Rosetta-gamiB,it was named VLP-B,and the codon optimized Cap protein could be expressed in both BL21and Rosetta-gamiB,it was named VLP-A and VLP-C,respectively.The three proteins were mainly expressed in soluble form and the yield of VLP-A was higher than that of VLP-B,the yield of VLP-B was higher than that of VLP-C.After purification by nickel affinity chromatography,a large number of VLPs with diameters about 20nm could be observed under transmission electron microscope.However,in terms of purification efficiency,VLP-C ranked last,VLP-A ranked first,and VLP-B ranked second;after immunizing mice with 50μg recombinant VLPs,their antibody levels increased rapidly,reaching the positive critical value at 14d,and maintaining at a high level until 42d.At the same time,a cytokine response was induced.The immunogenicity of VLP-C was the best,followed by VLP-B,and VLP-A was relatively poor.Thus,it could be see

关 键 词：CAP蛋白 密码子优化 稀有密码子 病毒样颗粒 免疫原性 

分 类 号：S852.65[农业科学—基础兽医学]