Fh8明显增强PCV2 Cap蛋白的可溶性表达及抗原性

摘要

猪圆环病毒2型(Porcine circovirus type 2,PCV2)的主要功能蛋白Cap蛋白,很难在体外获得可溶性表达.本研究旨在以Fh8蛋白为融合标签实现PCV2 ORF2基因在大肠杆菌中的可溶性表达,以小鼠评价可溶性Cap蛋白的抗原性.以病毒基因组为模板,扩增ORF2靶基因,插入pCold原核表达质粒,成功构建的pCold-ORF2阳性质粒转入大肠杆菌BL21中.利用SDS-PAGE分析Cap蛋白的可溶性表达情况,利用Western blotting鉴定Cap蛋白的反应原性.重组蛋白与佐剂乳化制备免疫原,免疫Balb/c小鼠,ELISA方法检测血清中Cap抗体,利用PCR检测组织脏器中病毒载量,以评估可溶性Cap蛋白的免疫原性.PCR结果表明,Fh8和PCV2 ORF2基因被成功扩增.SDS-PAGE结果表明Fh8标签显著提高了ORF2在大肠杆菌中的可溶性表达,分子量35000.Western blotting结果显示,猪PCV2阳性血清能够特异性的识别可溶性Cap蛋白.小鼠一次接种Cap蛋白28 d后,抗体全部转阳,免于PCV2的攻击.表明通过新型小标签实现了PCV2 ORF2基因的可溶性表达,且具有良好的免疫原性.%Cap protein is the main functional protein of porcine circovirus type 2(PCV2),but it is difficult to obtain soluble expression in vitro.Our object was to realize soluble expression of PCV2 ORF2 gene by Fh8-tagged in Escherichia coli ,and to evaluate the antigenicity of the recombinant Cap protein in mice. PCV2 DNA was used as template to amplify ORF2 gene and was cloned into prokaryotic expression vector pCold. The resultant pCold-ORF2 plasmid was transformed into E.coli BL21. SDS-PAGE and Western blotting assay were used to analyze the solubility of the recombinant protein and to identify its reactivity,respectively.The recombinant protein and adjuvant were emulsified to prepare vaccine to immune Balb/c mice.The specific antibody of Cap recombinant protein in sera was detected by ELISA method,and virus loading in tissues was detected by PCR. So,the immunogenicity of Cap protein could be assessed. The PCR results showed that Fh8 and PCV2 ORF2 genes were successfully amplified. SDS-PAGE results showed that soluble expression of ORF2 in E. coli was significantly increased by Fh8 tag,and the molecular weight was 35 000. Western blotting results showed that the soluble Cap protein was recognized specifically by porcine PCV2 positive sera. Mice inoculated with the Cap protein vaccine produced high antibody titers at 28 d after initial immunization and could be well protected from PCV2 attack. The soluble expression of PCV2 ORF2 gene was achieved by a novel small label,and the Cap vaccine had good immunogenicity.