【摘要】 目的 通过昆虫-杆状病毒表达系统表达猪圆环病毒2型（porcine circovirus 2,PCV2）衣壳（capsid,Cap）蛋白，并评价其作为候选疫苗的免疫原性，探讨其能否诱导BALB/c小鼠的体液免疫和细胞免疫反应。方法 将PCV2 Cap基因进行密码子优化后，克隆至pFastBac Dual载体，并转化至大肠埃希菌DH10 Bac感受态细胞中，提取Bacmid后，使用M13通用引物进行PCR扩增。将鉴定正确的Bacmid转染至Sf9细胞中，表达并定量Cap蛋白。将45只雌性BALB/c小鼠随机分为3组：PBS对照、Cap和商业疫苗YI Cap组，每组15只，间隔14 d经背部皮下免疫1次，共3次，每次PBS 100??L、Cap 10??g、商业疫苗YI Cap 10??g。通过ELISA法测定小鼠血清IgG水平，流式细胞术分析细胞免疫反应类型，H&E染色观察亚单位疫苗的组织相容性。结果 昆虫-杆状病毒表达系统能够有效表达Cap蛋白，带有增强型绿色荧光蛋白（enhanced green fluorescent protein,eGFP）的Cap蛋白能够共定位至细胞质膜中，通过透射电子显微镜观察到Cap蛋白可自组装形成病毒样颗粒（virus-like particles,VLPs），继而构建另一种无eGFP荧光标签的重组载体，经SDS-PAGE和Western blot分析可在目标位置清晰观察到明显条带。Cap候选疫苗组小鼠42 d时小鼠血清中IgG含量达最高，IFNγ与IL-4含量分别为（27.75±0.99）和（17.63±1.13）pg/mL，均显著高于PBS组（F分别为14.89和0.211 9,P分别<0.05和<0.001）;Cap组脾淋巴细胞刺激指数（stimulation index,SI）达6.72，显著高于PBS组（F=1.228,P<0.001）。Cap候选疫苗能够诱导Th1型细胞免疫反应，且具有良好的组织相容性。结论 在昆虫-杆状病毒表达系统中成功表达了Cap蛋白，Cap候选疫苗免疫小鼠后，可诱导小鼠机体产生体液免疫和细胞免疫反应，是进一步开发更有效的PCV相关疾病疫苗的潜在候选疫苗。更多还原

【Abstract】 Objective To express the capsid(Cap) protein of porcine circovirus 2(PCV2) by insect-baculovirus expression system, evaluate its immunogenicity as a vaccine candidate, and explore whether it can induce humoral and cellular immune responses in BALB/c mice.Methods Following codon optimization, the PCV2 Cap gene was cloned into pFastBac Dual vector and transformed into E.coli DH10 Bac cells. After Bacmid extraction, PCR amplification was carried out with M13 universal primers. The appropriately identified Bacmid was then transfected into Sf9 cells to express Cap protein, and the protein was quantified. Forty-five female BALB/c mice were randomly divided into three groups: PBS control, Cap and commercial vaccine YI Cap group, 15 mice in each group, which were immunized subcutaneously on the back once at 14-day intervals, for a total of three times, with PBS 100 ??L, Cap 10 ??g, and commercial vaccine YI Cap 10 ??g respectively each time. The serum IgG level of mice was measured by ELISA, the cellular immune response type was analyzed by flow cytometry, and the histocompatibility of the subunit vaccine was observed by H&E staining.Results The insect-baculovirus expression system effectively expressed the target protein, and the Cap protein with enhanced green fluorescent protein(eGFP) colocalized with the cell’s plasma membrane. Transmission electron microscopy revealed that the Cap protein could self-assemble to produce virus-like particles(VLPs) before constructing another recombinant vector sans the eGFP fluorescent flag. SDS-PAGE and Western blot analysis revealed the conspicuous bands at the desired area. The IgG content of Cap vaccine candidate mice reached the highest level at 42 days. The levels of IFNγ and IL-4 were(27. 75 ± 0. 99) and(17. 63 ± 1. 13) pg/mL, respectively, both of which were significantly higher than those of PBS group(F = 14. 89 and 0. 211 9,P < 0. 05 and < 0. 001, respectively). The stimulation index(SI) of splenic lymphocytes in the Cap group reached 6. 72, which was significantly higher than that in the PBS group(F = 1. 228, P < 0. 001). The Cap vaccine candidate could induce Th1-type cellular immune response and exhibited good histocompatibility.Conclusion The Cap protein was successfully expressed in the insect-baculovirus expression system. The Cap vaccine candidate can induce humoral and cellular immune responses in mice after immunization, which is a potential vaccine candidate for further development of more effective vaccines against PCV-related diseases.更多还原