土拨鼠肝炎病毒核心蛋白的高效表达和B细胞表位鉴定

Over-expression and B-cell linear epitope identification of Woodchuck hepatitis virus (WHV) core protein

目的建立高效表达土拨鼠肝炎病毒核心蛋白(WHcAg)的方法并对其B细胞表位进行鉴定。方法分别构建不同截短型WHcAg的质粒以了解能大量表达并形成WHV核心颗粒的区段,利用变性WHcAg制备单克隆抗体以寻找能与WHcAg线性B细胞表位结合的单克隆抗体。结果WHcAg前144或149氨基酸残基能够大量表达并能形成颗粒样结构。这2种截短型WHcAg能够在大肠杆菌中表达并组装成直径为34 nm的核心颗粒,最终纯化获得毫克级的核心蛋白。截短型WHcAg保留了全长WHcAg的抗原性,而且变性WHcAg存在另外的B细胞线性表位,利用变性WHcAg进行免疫制备单克隆抗体获得的5株抗体均针对WHcAg的N末端表位,该表位在WHcAg和HBcAg高度保守。结论建立了高效表达WHcAg的方法,制备了针对WHcAg单克隆抗体。并对WHcAg的线性B细胞表位进行了鉴定,发现WHcAg和HBcAg的N末端存在共同的线性B细胞表位。

Objective To establish a method for expression of generous Woodchuck hepatitis virus （WHV） core protein and identification B-cell linear epitope of WHcAg. Methods To construct plasmid expressing WHeAg with earboxyl-temtinal mmcations and to determine the region of WHcAg required for assembly. Monoclonal antibodies against denatured WHcAg were generated and tested for their specificity and linear B-cell epitope of WHcAg. Results The first 144 or 149 amino acid residues of WHcAg were able to efficiently assembly into particular structures. Both mmcated forms of WHcAg were accumulated in E. coli as uniform particles with a diameter of 34 nm in large quantities and could be purified in milligram scale. As what expected, the particles of mmcated WHcAg retained the antigenicity of the full length WHcAg. However, denatured WHcAg remained to be reactive with specific antisera, suggesting that WHcAg may possess additional linear B-cell epitopes. Monoclonal antibodies against denatured WHcAg were generated and tested for their specificity. Five antibodies were found to react with the N-terminal region of WHcAg. Due to the conservation of the amino acid sequence in this region of WHcAg and HBcAg, these antibodies recognized recombinant HBcAg as well. Conclusion We constructed the expression system of WHV core protein with carboxyl-temtinal mmcations, generated monoclonal antibodies against WHcAg B-cell epitope, and defined WHcAg linear B-cell epitope. WHcAg and HBcAg share common linear B cell epitopes which span AA 1-8 respectively.