Engineering, expression, and immuno-characterization of recombinant protein comprising multi-neutralization sites of rabies virus glycoprotein.
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.
Li
Xiangdong
X
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, No. 518, Ziyue Road, Shanghai 200241, PR China.
Luo
Jinyan
J
Wang
Shuiming
S
Shen
Yang
Y
Qiu
Yafeng
Y
Wang
Xiaodu
X
Deng
Xufang
X
Liu
Xuehui
X
Bao
Weidong
W
Liu
Peihong
P
Zhou
Jinping
J
Ding
Chan
C
Ma
Zhiyong
Z
D057134:Antibodies, Neutralizing / Q000235:genetics / Q000276:immunology; D000956:Antigens, Viral / Q000235:genetics* / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics* / Q000276:immunology*; D015202:Protein Engineering; D011820:Rabies virus / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology*
D057134:Antibodies, Neutralizing; D000956:Antigens, Viral; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C064951:glycoprotein G, Rabies virus