Li Xiangdong, Luo Jinyan, Wang Shuiming, Shen Yang, Qiu Yafeng, Wang Xiaodu, Deng Xufang, Liu Xuehui, Bao Weidong, Liu Peihong, Zhou Jinping, Ding Chan, Ma Zhiyong
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, No. 518, Ziyue Road, Shanghai 200241, PR China.
Protein Expr Purif. 2010 Apr;70(2):179-83. doi: 10.1016/j.pep.2009.09.005. Epub 2009 Sep 13.
The rabies virus (RV) glycoprotein (G protein) induces neutralizing antibodies, which are important in protection against rabies. In the present study, three gene fragments that encode polypeptides (corresponding to amino acid residues 19-60, 181-219, and 300-458) comprising the linear neutralization sites of the G protein were spliced together in tandem by PCR-based site-directed mutagenesis and heterologously expressed in Escherichia coli (DE3). The recombinant protein (designated rRVg) was purified under denaturing conditions and solubilized by stepwise dialysis against an alkaline buffer (0.05 M Na(2)CO(3) pH 9.6). Western blot analysis of the antigenicity of rRVg showed that it was recognized by rabies-immune serum. Competitive neutralization assay revealed that rRVg significantly reduced the RV-neutralizing activity of the rabies-immune serum. These results show potential of rRVg as a diagnostic antigen for detecting RV-neutralizing antibodies in immunized hosts.
狂犬病病毒（RV）糖蛋白（G蛋白）可诱导产生中和抗体，这些抗体对于预防狂犬病至关重要。在本研究中，通过基于PCR的定点诱变将编码包含G蛋白线性中和位点的多肽（对应于氨基酸残基19 - 60、181 - 219和300 - 458）的三个基因片段串联拼接在一起，并在大肠杆菌（DE3）中进行异源表达。重组蛋白（命名为rRVg）在变性条件下纯化，并通过逐步透析至碱性缓冲液（0.05 M Na₂CO₃，pH 9.6）使其溶解。对rRVg抗原性的蛋白质免疫印迹分析表明，它能被狂犬病免疫血清识别。竞争性中和试验显示，rRVg显著降低了狂犬病免疫血清的RV中和活性。这些结果表明rRVg作为检测免疫宿主中RV中和抗体的诊断抗原有潜在应用价值。
