携带BCoV抗原表位的乙型肝炎病毒核心抗原病毒样颗粒的制备及免疫效果评价

Preparation of hepatitis B virus core antigen VLPs carrying BCoV epitopes and immunization effect evaluation

为了制备可用于牛冠状病毒(BCoV)亚单位疫苗的含BCoV S蛋白抗原表位的病毒样颗粒(VLP),本研究将BCoV S1和S2蛋白中含B细胞表位的aa351~aa403(159 bp)和aa771~aa784(42 bp)对应的密码子按大肠杆菌偏好性原则优化后插入乙型肝炎病毒核心抗原(HBcAg)主要免疫显性区域(MIR),合成该重组基因后克隆至pET-32a(+),经酶切鉴定显示正确构建了重组质粒pET-32a(+)-HBcAg-BCoV S1+S2。将该重组质粒分别转化E.coli BL21(DE3)和伴侣感受态细胞BL21(DE3)(含pG-KJE8分子伴侣质粒),经诱导表达后采用镍柱亲和层析法纯化两种不同表达形式的重组蛋白,SDS-PAGE检测重组蛋白的表达及纯化效果,结果显示两种表达系统均正确表达了重组蛋白,分别命名为VLP-A、VLP-B,前者为包涵体表达,后者为可溶性表达。纯化后浓度分别为0.9 mg/mL、1.4 mg/mL。利用透射电镜观察纯化后的两种重组蛋白是否形成VLP,结果显示二者均形成VLP,且可溶性表达的重组蛋白形成的VLP产量略高。将两种VLP乳化后以50μg/只分别免疫小鼠,于免疫后7 d、14 d、21 d、28 d、35 d和42 d采血制备血清,采用ELISA方法检测小鼠血清中的BCoV IgG特异性抗体水平及细胞因子IFN-γ、IL-4,结果显示,将两种VLP乳化后免疫小鼠产生的BCoV IgG抗体(P<0.01)和两种细胞因子水平(P<0.05)均显著高于对照组,且可溶性表达的VLP诱导产生的抗体水平和细胞因子水平更高。本研究首次制备了含BCoV S蛋白抗原表位的VLP,两种不同表达形式的重组蛋白均形成VLP,且在小鼠体内均能够产生免疫应答,为BCoV亚单位疫苗及相关生物制品的制备奠定了基础。

In order to prepare BCoV virus-like particles(VLA)that carrying BCoV S surface antigen,optimized sequence containing B cell epitopes of BCoV S1(aa351-aa403)and S2 proteins(aa771-aa784)was linked into the MIR of HBcAg.Recombinant plasmid pET-32a(+)-HBcAg-BCoV S1+S2 was constructed,and then transformed into E.coli BL21(DE3)and chaperone sensing cell BL21(DE3)(containing pG-KJE8 chaperone plasmid).The two recombinant proteins were purified by affinity chromatography on nickel column.SDS-PAGE was used to detect the expression and purification effect of the recombinant proteins.Transmission electron microscopy was used to observe whether the purified recombinant protein formed VLPs.After emulsifying,two kinds of VLPs were inoculated subcutaneously into the mouse(50μg/mouse).Blood samples were collected at 7 days,14 days,21 days,28 days,35 days and 42 days after immunization.The levels of IgG specific antibodies and cytokines IFN-γand IL-4 were detected by ELISA.The results showed that mice immunized with two kinds of VLPs produced significantly higher levels of BCoV IgG antibody than that of the control group.The soluble VLPs collected from the supernatant induced the highest levels of antibodies and cytokines.These results showed that VLPs containing epitopes of BCoV S protein were prepared for the first time and the two recombinant proteins expressed in different forms could both produce immune responses in mice.Our study laid the foundation for the preparation of BCoV subunit vaccine and related biological products,which has a great influence on the prevention and control of BCoV.