信号肽对猪瘟病毒E2蛋白在杆状病毒表达系统中分泌表达的影响

旨在通过引入信号肽序列实现猪瘟病毒E2蛋白在杆状病毒表达系统中的高效表达。首先将内源信号肽（简称endo）、蜂素信号肽（简称mel）及gp67信号肽（简称gp67）分别引入pFastBac1载体，随后插入E2基因，构建重组质粒。随后将重组质粒转化至DH10Bac感受态细胞，经蓝白斑筛选得到重组黏粒，将重组黏粒转染sf21细胞以得到杆状病毒。经扩增后大量感染细胞，检测E2蛋白表达情况，利用镍填料纯化得到E2蛋白，并进行蛋白糖基化水平鉴定及蛋白免疫原性分析。PCR鉴定结果显示成功构建了重组质粒。挑选白斑进行PCR鉴定，证实得到重组黏粒。Western blot检测结果表明，E2蛋白实现了分泌表达，进一步比较细胞培养上清中E2蛋白的表达水平，确定gp67信号肽介导的E2蛋白分泌表达量最高。经镍填料纯化得到E2蛋白，SDS-PAGE结果显示其纯度较高，产率为25 mg·L^-1。通过ELISA分析其抗原性，结果表明纯化所得E2蛋白能特异性识别猪瘟阳性血清中的抗体，其抗原性良好。将纯化后的E2蛋白添加ISA201佐剂乳化后免疫BALB/c小鼠，阻断ELISA结果显示，免疫后14 d即产生较高水平的猪瘟特异性抗体，表明E2蛋白免疫原性良好。本研究为E2亚单位疫苗的研制奠定了基础。

Effect of Signal Peptide on the Secretory Expression of CSFV E2 Protein in Baculovirus Expression System

This study aimed to achieve the effective secretory expression of E2 of classical swine fever virus (CSFV) using baculovirus by introducing the signal peptide. Firstly, the pFastBac1 vector was modified by adding the endogenous E2 signal peptide, the melittin signal peptide or the gp67 signal peptide, respectively. Then the E2 gene fragment was cloned into the modified vectors. The resulting plasmids were then transformed into the DH10Bac competent cells to get recombinant bacmids through blue-white screening. The bacmids were subsequently transfected into the sf21 cells to generate recombinant baculoviruses. Secondly, the sf21 cells were infected for large-scale protein production. The E2 protein was purified by nickel-affinity chromatography and the purity was identified using SDS-PAGE. Finally, the glycosylation pattern and the immunogenicity of the purified E2 were analyzed. Results were as follows:Consequently, the recombinant plasmids were generated. The recombinant bacmids were acquired through blue-white screening. The Western blot analysis showed that the secretory expression of E2 was achieved by introducing signal peptides. Among the three signal peptides, the gp67 mediated the highest secretion efficiency. It was shown that the E2 protein was pure and the yield was about 25 mg·L^-1. Indirect ELISA results demonstrated that the purified E2 protein could be specifically recognized by the CSFV positive serum, indicating that the purified E2 protein was immunogenic. The purified E2 protein was used to immunize BALB/c mice after emulsification with ISA201 adjuvant. The blocking ELISA result showed that the antibody could be detected after 14 days of immunization, indicating that the E2 protein had good immunogenicity. Taken together, the study is useful for developing a subunit vaccine against CSFV using the E2 expressed in the sf21 cells.