猪圆环病毒2型山西流行株Cap蛋白可溶性表达与免疫原性分析

Soluble Expression and Immunogenicity Analysis of Cap Protein of Porcine Circovirus Type 2 Epidemic Strain from Shanxi Province

为建立猪圆环病毒2型(PCV2)山西流行毒株Cap蛋白的原核可溶性表达体系,并分析比较重组蛋白与2种商品化基因工程Cap亚单位疫苗在小鼠体内不同阶段的免疫原性,本试验以PCV2e SXJX株(GenBank登录号:MH922991.1)的ORF 2基因为基础,根据大肠埃希菌密码子偏性优化序列,将其插入原核表达载体pET-28a-c(+)中,鉴定为阳性后转入宿主菌BL21(DE3)中,经优化表达条件后,对超声破碎后的菌体上清进行纯化,分析纯化后产物的纯度和抗原性,并利用负染透射电镜观察重组Cap蛋白体外自组装成病毒样颗粒(VLPs)。分别将同等免疫剂量的PCV2e重组Cap蛋白和2种商品化PCV2基因工程亚单位疫苗免疫健康BALB/c雌鼠,应用ELISA方法检测不同阶段小鼠体内特异性抗体的水平。结果显示:重组Cap蛋白可在大肠埃希菌中表达,以完全可溶性形式存在于菌体超声波破碎后的上清中,表达产物纯化效果良好,并能被PCV2单克隆抗体识别;透射电镜观察结果发现,表达的重组Cap蛋白可以自组装成约20 mm大小的VLPs;动物免疫试验结果显示,在首免后第21天PCV2e Cap试验组抗体水平显著高于勃林格疫苗组(P<0.05),但极显著低于普莱柯疫苗组(P<0.01),首免后第42天3个免疫组的抗体水平差异不显著(P>0.05),表明重组蛋白在小鼠体内免疫原性良好。本试验成功构建了PCV2e Cap蛋白原核可溶性表达体系,其重组蛋白具有较好的免疫原性,为PCV2的诊断检测和亚单位基因工程疫苗的研制提供了候选抗原,并为该蛋白功能的深入研究提供了科学依据。

In order to establish a prokaryotic soluble expression system for Cap protein of Shanxi porcine circovirus type 2(PCV2),and to analyze and compare the immunogenicity of recombinant Cap protein and two commercial genetically engineered Cap subunit vaccines at different stages in mice,this study inserted the ORF 2 gene of PCV2e SXJX strain(GenBank accession number:MH922991.1)into prokaryotic expression vector pET-28a-c(+),and subsequently transferred into Escherichia coli strain BL21(DE3).After optimization of expression conditions,cell supernatant was subjected to ultrasonic fragmentation and purified,and the purity and antigenicity of the recombinant Cap protein were analyzed.The virus capsid-like particles(VLPs)assembly of the recombinant Cap protein was observed by negative staining transmission electron microscope.Healthy female BALB/c mice were immunized with the recombinant PCV2e Cap protein and two commercial PCV2 genetically engineered subunit vaccines,and the levels of specific antibodies in mice at different stages were assessed by ELISA.The results showed that the recombinant Cap protein was highly expressed in E.coli and existed in the supernatant after ultrasonic fragmentation in complete soluble form,and the expressed product was readily purified and recognized by PCV2 monoclonal antibody.Transmission electron microscope observation showed that the expressed Cap protein could self-assemble into VLPs with a size of about 20 mm.Animal immunization test showed that the antibody levels in the PCV2e Cap test group on day 21 after the first immunization were significantly higher than those in the Boehringer vaccine group(P<0.05),but significantly lower than those in the Pulike vaccine group(P<0.01),and the antibody levels of the three test groups on day 42 after the first immunization were not significant(P>0.05),indicating that the recombinant protein was immunocompetent in mice.Thus,the present study describes the successful construction of a prokaryotic soluble expression system for PCV2e Cap protein,and the recombinant Cap protein displays strong immunogenicity.This provides options for diagnosis of PCV2 and development of a subunit genetically engineered vaccine against PCV2,as well as further characterization of the function of the PCV2e Cap protein.