呈现新型冠状病毒RBD抗原病毒样颗粒的表达与鉴定*

目的:以乙型肝炎病毒核心抗原HBcAg为载体,构建呈现新冠病毒刺突蛋白受体结合域的病毒样颗粒,并鉴定其免疫原性,为新冠病毒疫苗的开发提供新思路。方法:在乙型肝炎病毒核心蛋白氨基酸编码序列第78和81位插入新冠病毒刺突蛋白受体结合域(RBD),并通过柔性linker(G4S)3进行连接,序列优化后将融合基因克隆到原核表达载体pET-28a(+),转化表达菌Rosetta,在自诱导培养基中诱导表达,菌体破碎后经蔗糖密度梯度离心,透析浓缩的方法纯化病毒样颗粒。SDS-PAGE、Western blot、透射电子显微镜检测和鉴定VLPs。将制备的VLPs与佐剂等比例混合经皮下免疫BALB/c小鼠,ELISA检测小鼠血清中特异性抗体,分析该HBc-RBD VLPs的免疫原性。结果:在自诱导培养基中,大肠埃希菌可表达部分可溶的VLPs,经蔗糖密度梯度离心纯化后在透射电子显微镜下可以观察到病毒样颗粒的存在。动物实验表明HBc-RBD VLPs刺激小鼠产生了特异性抗体。结论:在原核表达系统中成功表达了展示RBD抗原的VLPs,并通过小鼠实验初步验证了免疫原性,为新冠病毒疫苗的研发提供了新方向。

Expression and Identification of Virus-like Particles Presenting Novel Coronavirus RBD Antigen

Objective: Hepatitis B virus core protein HBc was used as vector to construct virus-like particles expressing novel coronavirus spike protein receptor binding domain RBD, and their immunogenicity was identified, which provides a new idea for the development of COVID-19 vaccines. Methods: The amino acid coding sequence 78 and 81 of hepatitis B virus core protein HBc (1-183 aa) were inserted into novel coronavirus spike protein receptor binding domain RBD and ligated by flexible linker (G4S) 3. After sequence optimization, the fusion gene was cloned into prokaryotic expression vector pET-28a (+) and transformed into expression strain Rosetta. After induced expression in self-inducing medium, the virus-like particles (VLPs) were purified by sucrose density gradient centrifugation. VLPs were detected and identified by SDS-PAGE, Western blot and transmission electron microscope. BALB/c mice were immunized subcutaneously with the prepared VLPs in equal proportion with adjuvant. The specific antibodies in the serum of the mice were analyzed by ELISA to verify the immune effect of HBc-RBD VLPs. Results: Escherichia coli can express partially soluble VLPs in self-inducing medium. VLPs could be observed by transmission electron microscope after purification by sucrose density gradient centrifugation. Mice immunized with HBc-RBD VLPs produced specific antibodies against RBD antigen. Conclusion: VLPs displaying RBD antigen were successfully expressed in prokaryotic expression systems, and their immunogenicity was preliminarily verified by mouse experiment, which provides a new direction for the research and development of novel coronavirus vaccines.