目的:研究Ⅲ价轮状病毒基因重配疫苗对SD大鼠的免疫原性及对造血系统的长期毒性。方法:疫苗组大鼠(n=30)免疫3次,每次每只动物2 mL ig,第1次免疫后3周、第3次免疫后1和4周各处死大鼠10只,检测血清特异性抗体,血液学和骨髓。结果:在各时...目的:研究Ⅲ价轮状病毒基因重配疫苗对SD大鼠的免疫原性及对造血系统的长期毒性。方法:疫苗组大鼠(n=30)免疫3次,每次每只动物2 mL ig,第1次免疫后3周、第3次免疫后1和4周各处死大鼠10只,检测血清特异性抗体,血液学和骨髓。结果:在各时间点疫苗组血清抗体滴度均明显高于对照组。第1次免疫后3周时可测出针对G1,G2,G3,G4和G10型抗原的抗体,逐渐升高。针对G2,G3和G4型的抗体滴度明显高于G1和G10型(P<0.01或<0.05)。疫苗组大鼠LY计数和MCH出现了可逆性变化(P<0.05),但无明显毒性,大鼠骨髓象无明显改变。结论:Ⅲ价轮状病毒基因重配疫苗对大鼠有明显免疫原性,对造血系统无明显毒性。展开更多
Objective. Using a recombinant human adenovirus to express modified VP4 gene of rotavirus SA11 strain. Methods. A whole VP4 gene was obtained with PCR and induced the signal peptide at the gene N terminal. The chimera...Objective. Using a recombinant human adenovirus to express modified VP4 gene of rotavirus SA11 strain. Methods. A whole VP4 gene was obtained with PCR and induced the signal peptide at the gene N terminal. The chimera gene was cloned into pCMV plasmid that consists of human cytomegalovirus promoter, and then the gene was cloned to the transfer vector of human adenovirus type 5. Homologous recombination was performed by co- transfection to 293 cell lines with recombinant plasmid and viral genome using CaPO4 precipitation. Results. No mutation was found in the whole VP4 gene sequence of 2362 base pair. The expressed product in recombinant adenovirus was confirmed to be specific and more antigenicity by indirect immunofluorescence assay. Both the Western blot and immunoprecipitation assay showed that the molecular mass of the expressed protein was higher than the wild type VP4 protein, and that the modified product was corresponding to a glycosylation of VP4 protein. Conclusion. To modify the target gene might be an effective method to enhance the stability, antigenicity and immunogenicity of expressed protein.展开更多
The human pathogen, group A rotavirus, is the most prevalent cause of acute infantile and pediatric gastroenteritis worldwide, especially in developing countries. There is an urgent demand for safer, more effective an...The human pathogen, group A rotavirus, is the most prevalent cause of acute infantile and pediatric gastroenteritis worldwide, especially in developing countries. There is an urgent demand for safer, more effective and cheaper vaccines against rotavirus infection. Plant-derived antigens may provide an exclusive way to produce economical subunit vaccines. Virus-like particles, constituting viral capsid proteins without viral nucleic acids, are considered a far safer candidate compared with live attenuated viral vaccines. In this study, the rotavirus capsid proteins VP2, VP6 and VP7 were co-expressed in transgenic tobacco plants, and their expression levels, formation of rotavirus-like particles (RV VLPs) and immunogenicity were extensively studied. Quantitative real-time RT-PCR and Western blot analysis revealed that the expression level of vp6 was the highest while vp7 was expressed at the lowest levels. The RV VLPs were purified from transgenic tobacco plants and analyzed by electron microscopy and Western blot. Results indicated that the plant-derived VP2, VP6 and VP7 proteins self-assembled into 2/6 or 2/6/7 RV VLPs with a diameter of 60-80 nm. When orally delivered into mice with cholera toxin as an adjuvant, the total soluble protein extracted from transgenic tobacco plants induced rotavirus-specific antibodies comparable with those of attenuated rotavirus vaccines, while VP 2/6/7 induced higher serum IgG and fecal IgA titers compared with VP 2/6.展开更多
文摘目的:研究Ⅲ价轮状病毒基因重配疫苗对SD大鼠的免疫原性及对造血系统的长期毒性。方法:疫苗组大鼠(n=30)免疫3次,每次每只动物2 mL ig,第1次免疫后3周、第3次免疫后1和4周各处死大鼠10只,检测血清特异性抗体,血液学和骨髓。结果:在各时间点疫苗组血清抗体滴度均明显高于对照组。第1次免疫后3周时可测出针对G1,G2,G3,G4和G10型抗原的抗体,逐渐升高。针对G2,G3和G4型的抗体滴度明显高于G1和G10型(P<0.01或<0.05)。疫苗组大鼠LY计数和MCH出现了可逆性变化(P<0.05),但无明显毒性,大鼠骨髓象无明显改变。结论:Ⅲ价轮状病毒基因重配疫苗对大鼠有明显免疫原性,对造血系统无明显毒性。
文摘Objective. Using a recombinant human adenovirus to express modified VP4 gene of rotavirus SA11 strain. Methods. A whole VP4 gene was obtained with PCR and induced the signal peptide at the gene N terminal. The chimera gene was cloned into pCMV plasmid that consists of human cytomegalovirus promoter, and then the gene was cloned to the transfer vector of human adenovirus type 5. Homologous recombination was performed by co- transfection to 293 cell lines with recombinant plasmid and viral genome using CaPO4 precipitation. Results. No mutation was found in the whole VP4 gene sequence of 2362 base pair. The expressed product in recombinant adenovirus was confirmed to be specific and more antigenicity by indirect immunofluorescence assay. Both the Western blot and immunoprecipitation assay showed that the molecular mass of the expressed protein was higher than the wild type VP4 protein, and that the modified product was corresponding to a glycosylation of VP4 protein. Conclusion. To modify the target gene might be an effective method to enhance the stability, antigenicity and immunogenicity of expressed protein.
基金supported by the National High Technology Research and Development Program of China (Grant No. 2007AA100505)
文摘The human pathogen, group A rotavirus, is the most prevalent cause of acute infantile and pediatric gastroenteritis worldwide, especially in developing countries. There is an urgent demand for safer, more effective and cheaper vaccines against rotavirus infection. Plant-derived antigens may provide an exclusive way to produce economical subunit vaccines. Virus-like particles, constituting viral capsid proteins without viral nucleic acids, are considered a far safer candidate compared with live attenuated viral vaccines. In this study, the rotavirus capsid proteins VP2, VP6 and VP7 were co-expressed in transgenic tobacco plants, and their expression levels, formation of rotavirus-like particles (RV VLPs) and immunogenicity were extensively studied. Quantitative real-time RT-PCR and Western blot analysis revealed that the expression level of vp6 was the highest while vp7 was expressed at the lowest levels. The RV VLPs were purified from transgenic tobacco plants and analyzed by electron microscopy and Western blot. Results indicated that the plant-derived VP2, VP6 and VP7 proteins self-assembled into 2/6 or 2/6/7 RV VLPs with a diameter of 60-80 nm. When orally delivered into mice with cholera toxin as an adjuvant, the total soluble protein extracted from transgenic tobacco plants induced rotavirus-specific antibodies comparable with those of attenuated rotavirus vaccines, while VP 2/6/7 induced higher serum IgG and fecal IgA titers compared with VP 2/6.
