猪瘟病毒E2-GM-CSF融合蛋白在HEK293T细胞中的表达和免疫原性分析

Expression and Immunogenicity Analysis of Classical Swine Fever VirusE2-GM-CSF Fusion Protein in HEK293T Cells

【目的】设计构建分泌表达E2-GM-CSF融合蛋白的HEK293T重组细胞,并评估表达的E2-GM-CSF融合蛋白的免疫原性,为猪瘟病毒(Classical swine fever virus,CSFV)的防控提供新的候选亚单位疫苗。【方法】利用PCR方法扩增E2-GM-CSF基因并克隆入慢病毒表达载体pCDH,将得到的重组慢病毒质粒pCDH-E2-GM-CSF包装成E2-GM-CSF慢病毒颗粒,转导HEK293T细胞,经嘌呤霉素加压筛选获得分泌表达E2-GM-CSF融合蛋白的HEK293T重组细胞,纯化表达的E2-GM-CSF融合蛋白经小鼠免疫试验验证其免疫原性。【结果】pCDH-E2-GM-CSF质粒经酶切鉴定,得到大小分别为8172 bp的载体片段和1521 bp的E2-GM-CSF基因片段,表明E2-GM-CSF基因成功克隆入pCDH载体;细胞基因组DNA PCR扩增结果为一条大小为1686 bp的条带,表明E2-GM-CSF基因成功整合至HEK293T细胞基因组;Western blotting分析获得约70 ku的条带,证明E2-GM-CSF融合蛋白在HEK293T细胞中成功分泌表达;SDS-PAGE验证显示,纯化后的E2-GM-CSF融合蛋白为单一条带,纯度较高,大小约70 ku;小鼠免疫血清ELISA检测结果表明,表达的E2-GM-CSF融合蛋白具有良好的免疫原性,免疫后第14天在小鼠血清中检测到效价为1∶300的E2特异性抗体,免疫后第21天E2特异性抗体效价达1∶900,免疫后第28天小鼠血清中的E2特异性抗体效价最高达到1∶8100,远高于E2蛋白的1∶1800。【结论】利用慢病毒载体构建的表达E2-GM-CSF融合蛋白的HEK293T重组细胞可正确分泌表达具有免疫原性的E2-GM-CSF融合蛋白,为CSFV的防控提供了新的候选亚单位疫苗,同时其构建、开发与评估过程也可为其他亚单位疫苗在哺乳动物细胞中的快速表达提供借鉴。

【Objective】A HEK293T cell pool expressing E2-GM-CSF fusion protein was established to evaluate the immunogenicity of E2-GM-CSF fusion protein,in order to provide a new option for the development of Classical swine fever virus(CSFV)subunit vaccine.【Method】Firstly,E2-GM-CSF fusion gene was amplified by PCR and cloned into the pCDH lentivector.Then,the recombinant plasmid pCDH-E2-GM-CSF was constructed and packaged into lentiviral particles.Secondly,recombinant HEK293T cell pools expressing E2-GM-CSF fusion protein were obtained by the transduction of the lentiviral particles and the selection of puromycin.The immunogenicity of the purified E2-GM-CSF fusion protein was evaluated in mice.【Result】The pCDH-E2-GM-CSF plasmid was identified by double-enzyme digestion,and the vector fragments and E2-GM-CSF gene fragments with sizes of 8172 and 1521 bp were obtained respectively,indicating that E2-GM-CSF gene was successfully cloned into pCDH vector.A 1686 bp band was obtained by PCR amplification of cells genomic DNA,which showed that E2-GM-CSF gene was integrated into the genomic DNA of HEK293T cells.Western blotting analysis obtained a band of about 70 ku,proving the successful expression of E2-GM-CSF fusion protein.The SDS-PAGE verification showed that the purified E2-GM-CSF fusion protein was a single band about 70 ku with high purity.The immunization trail showed the good immunogenicity of E2-GM-CSF fusion protein produced by HEK293T cells.The E2 specific antibody of 1∶300 was detected in mouse serum after 14 days of immunization,and the titer of antibody reached 1∶900 after 21 days of immunization.At the 28th day post immunization,the E2 specific antibody titer reached a maximum of 1∶8100,which was much higher than 1∶1800 of E2 protein.【Conclusion】In this study,a HEK293T cell pool expressing E2-GM-CSF fusion protein was established,and the immunogenicity of E2-GM-CSF fusion protein was preliminarily confirmed,providing a novel candidate subunit vaccine against CSFV.Therefore,the development of this vaccine could also provide a reference for the rapid expression of other subunit vaccines in mammalian cells.