基因Ⅰ型草鱼呼肠孤病毒VP7蛋白合成肽抗体的制备及应用
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摘要
【目的】制备基因I型草鱼呼肠孤病毒(GCRV)VP7蛋白合成肽抗体并验证其特异性,为研究VP7外衣壳蛋白在基因I型GCRV病毒与宿主间的相互作用及研制基因工程疫苗提供参考依据。【方法】以GCRV GZ1208株为模板进行RT-PCR扩增,运用在线生物信息学分析软件对VP7蛋白B细胞表位进行功能预测,选择位于VP7蛋白第26～39位氨基酸的多肽序列进行人工合成,与钥孔血蓝蛋白载体蛋白偶联后免疫新西兰白兔以获得VP7蛋白合成肽抗体,然后采用Western blotting和间接免疫荧光试验验证VP7蛋白合成肽抗体对基因I型GCRV的特异性识别能力。【结果】VP7蛋白氨基酸序列在基因I型GCRV中高度保守,其合成肽抗体效价约1∶256000。Western blotting分析结果显示,融合蛋白VP7约在55 kD处出现单一的特异性条带,而GZ1208株和JX0901株样品约在30 kD处出现对应的特异性条带,与预期结果基本一致;间接免疫荧光试验结果表明,VP7蛋白合成肽抗体可特异性识别感染基因I型GCRV的草鱼脑细胞(GCB),感染细胞中出现特异性绿色荧光信号,而感染其他基因型GCRV及正常未感染病毒的GCB细胞中均无特异性绿色荧光出现。【结论】制备获得的VP7蛋白合成肽抗体能特异性识别基因I型GCRV,而不识别其他基因型毒株,可用于草鱼出血病的临床诊断和基因I型GCRV的病原学研究。
【Objective】Anti-VP7 protein synthetic peptide antibody of grass carp reovirus(GCRV)genotype I was prepare and its specificity was identified to provide reference for the research on the interaction of VP7 capsid protein between GCRV genetype I and the host,and development of genetic engineering vaccine.【Method】GCRV GZ1208 was used as the template to conduct RT-PCR amplification. Online bioinformatics software was used to predict the function of the B cell epitopes of VP7 protein. The polypeptide sequences of amino acid located at 26-39 positions of VP7 protein were selected and synthesized artificially. The New Zealand white rabbits were immunized with the polypeptide of VP7 coupling linked with keyhole limpet hemocyanin carrier protein,and the synthetic peptide antibody against VP7 protein was obtained. Then,Western blotting and indirect immunofluorescence assays were used to test the specific identifiction af anti-VP7 synthetic peptide antibody to GCRV genotype I.【Result】The amino acid sequences of VP7 was highly conserved in genotype I GCRV,and the titer of synthetic peptide antibody was as high as 1∶256000. The results of Western blotting indicated that the fusion protein VP7 showed a single specific band at about 55 kD,while GZ1208 strain and JX0901 strain showed corresponding specific bands at about 30 k D,which was consistent with the expected results.The results of indirect immunofluorescence assays indicated that VP7 protein synthetic peptide antibody could specificallyidentify the grass carp's brain cells(GCB)infected with genetype I GCRV,and the specific green fluorescence appeared in the infected cells. While there was no specific green fluorescence appeared in the cells infected with other genotypes GCRV and in normal cells.【Conclusion】The synthetic peptide antibody against VP7 protein is successfully prepared,which can specifically recognize GCRV genotype I but cannot recognize other genotype strains. The present study can be applied in clinical diagnosis of grass carp hemorrhagic disease as well as pathogenic research of GCRV genotype I.
【Objective】Anti-VP7 protein synthetic peptide antibody of grass carp reovirus(GCRV)genotype I was prepare and its specificity was identified to provide reference for the research on the interaction of VP7 capsid protein between GCRV genetype I and the host,and development of genetic engineering vaccine.【Method】GCRV GZ1208 was used as the template to conduct RT-PCR amplification. Online bioinformatics software was used to predict the function of the B cell epitopes of VP7 protein. The polypeptide sequences of amino acid located at 26-39 positions of VP7 protein were selected and synthesized artificially. The New Zealand white rabbits were immunized with the polypeptide of VP7 coupling linked with keyhole limpet hemocyanin carrier protein,and the synthetic peptide antibody against VP7 protein was obtained. Then,Western blotting and indirect immunofluorescence assays were used to test the specific identifiction af anti-VP7 synthetic peptide antibody to GCRV genotype I.【Result】The amino acid sequences of VP7 was highly conserved in genotype I GCRV,and the titer of synthetic peptide antibody was as high as 1∶256000. The results of Western blotting indicated that the fusion protein VP7 showed a single specific band at about 55 kD,while GZ1208 strain and JX0901 strain showed corresponding specific bands at about 30 k D,which was consistent with the expected results.The results of indirect immunofluorescence assays indicated that VP7 protein synthetic peptide antibody could specificallyidentify the grass carp's brain cells(GCB)infected with genetype I GCRV,and the specific green fluorescence appeared in the infected cells. While there was no specific green fluorescence appeared in the cells infected with other genotypes GCRV and in normal cells.【Conclusion】The synthetic peptide antibody against VP7 protein is successfully prepared,which can specifically recognize GCRV genotype I but cannot recognize other genotype strains. The present study can be applied in clinical diagnosis of grass carp hemorrhagic disease as well as pathogenic research of GCRV genotype I.
引文
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