Study on Secretory Expression and Immunogenicity of PPRV F Gene in Insect Cells

This study was aimed to explore the use of baculovirus expression system to secrete peste des petits ruminants virus (PPRV) F gene protein and use it as subunit vaccine. The gene fragment encoding F protein of PPRV was cloned into the baculovirus pFastBac Ⅰ transfer vector with a honeybee melittin signal peptide.The constructed F-pFastBac was transformed into Escherichia coli DH10Bac,resulting the recombinant baculovirus DNA (F-Bacmid) which was confirmed by blue-white plaque assay and antibiotic resistance selection.The F-Bacmid was then transfected into Sf9 insect cells by the cellfectin transfection reagent.The recombinant F protein was expressed in High Five cells in the serum-free medium.The SDS-PAGE and Western blotting analysis of recombinant protein showed that the protein could be expressed in insect cells and secreted into the culture medium. For the immunogenicity study,the recombinant protein was then inoculated into BALB/c mice, the results showed that the recombinant protein was able to stimulate B cells to produce special antibodies. In conclusions,the recombinant baculovirus expressing F protein of PPRV were successfully constructed.This study applied a basis for the development of PPRV subunit vaccine.     

鸭坦布苏病毒E基因在昆虫细胞中的分泌表达及免疫原性与保护性研究

鸭坦布苏病毒(DTMUV)为新出现的病毒,主要引起鸭产蛋量急剧下降。本研究通过杆状病毒表达系统,利用蜂素信号肽(honeybee melittin signal peptide,Mels)构建分泌表达DTMUV JSXZ株E蛋白的重组杆状病毒,将其感染Sf9昆虫细胞,从而分泌表达DTMUV E蛋白,并对该表达E蛋白的免疫原性和保护效果进行了研究。经Western blotting、IFA试验证明E蛋白可以在Sf9昆虫细胞培养上清中分泌表达;通过免疫雏鸭试验、间接ELISA结果证明E蛋白可以诱导产生高水平的IgG血清抗体;MTT试验结果显示E蛋白能刺激T 淋巴细胞增殖;攻毒保护试验结果显示0.3 mL组免疫保护率为80%,0.6 mL组和0.8 mL组保护率均为100%,而PBS对照组雏鸭均表现为典型的DTMUV感染症状,其中3只死亡。上述研究结果为DTMUV亚单位疫苗的研究奠定基础。     

小反刍兽疫病毒(PPRV)F基因在昆虫细胞中的分泌表达及免疫原性研究

试验旨在探索利用杆状病毒表达系统分泌表达小反刍兽疫病毒（peste des petits ruminants virus,PPRV） F基因蛋白及将其作为亚单位疫苗的应用价值。将PPRV F基因克隆到已插入蜂毒信号肽（melittin）的转移载体pFastBacⅠ中,将鉴定正确的重组质粒转化大肠杆菌DH10Bac感受态细胞,经抗性及蓝白斑筛选得到含F基因的重组杆状病毒DNA （F-Bacmid）,转染提取的F-Bacmid DNA至Sf9昆虫细胞,获得重组杆状病毒（F-rBac）,应用无血清培养的High Five细胞进行重组蛋白的表达及条件优化。SDS-PAGE结果表明,重组蛋白在重组杆状病毒感染的High Five昆虫细胞中获得分泌表达,并可产生较高浓度的重组蛋白;Western blotting结果显示,重组蛋白可被PPRV的特异性抗体所识别,表明重组蛋白具有反应原性;应用重组蛋白免疫BALB/c小鼠试验结果显示,该蛋白可刺激小鼠产生较高水平的特异性抗体。本研究成功分泌表达了PPRV F蛋白,该蛋白能够刺激机体产生免疫应答,为小反刍兽疫亚单位疫苗的研制奠定基础。     

Secretory Expression of DTMUV E Gene in Insect Cells and Study on its Immunogenicity and Protective Efficacy

Duck Tembusu virus (DTMUV) was newly emerged which caused severe egg-drop syndrome in duck.Here,we constructed the recombinant baculovirus by honeybee melittin signal peptide (Mels) which expressed E protein of DTMUV JSXZ strain,and its immunogenicity and protective efficacy were investigated in duck infection model.Western blotting and indirect immunofluorescence assay (IFA) showed E protein was effectively secretory expressed in supernatant of Sf9 insect cells.Indirect ELISA result showed E protein could induce high levels of anti-E IgG in the sera.MTT result showed that E protein could stimulate the proliferation of T lymphocytes.Moreover,the protection rates were 80% in 0.3 mL group and 100% in both 0.6 mL and 0.8 mL groups,respectively,whereas,all ducks in PBS control group showed the typical clinical signs with 3 died post challenge.These results laid a foundation for the study of DTMUV subunit vaccine.