Novel human 3-domain disulfide-stabilized antibody fragment against glycoprotein of rabies virus.
Mutated disulfide bond sites VH (Cys44) and VL (Cys100) were constructed in variable domains (Fvs) of the human anti-glycoprotein antigen of the rabies virus (anti-GPRV), and the light chain variable (VL) and heavy chain variable (VH) fragments were linked using the heavy chain constant region 1 (CH1) of the human immunoglobulin (Ig) to successfully construct a 3-domain disulfide-stabilized fragment of variables (3d-dsFv). 3d-dsFv was mainly expressed as an inclusion body. After refolding by the conventional dilution method, 3d-dsFv was purified using a nickel-nitrilotriacetic acid (Ni-NTA) column. Enyzme-linked immunosorbent assay (ELISA) was used to determine the binding activity of 3d-dsFv to GPRV. Flow cytometry studies and rapid fluorescent focus inhibition test were used to evaluate the function of 3d-dsFv. The results showed that the stability of 3d-dsFv was improved notably in some aspects such as thermal kinetics, ability to withstand urea denaturation, etc. 3d-dsFv could bind specially to infective cells and the GPRV. The titration of 3d-dsFv to RV-CVS is 83.3 IU/mg, and it can easily reach 2.5IU/mL, which is the value suggested by the WHO as effective for neutralization titration of the rabies virus.
Cai
Kun
K
State Key Laboratory of Pathogens and Biosecurity, Institute of Microbiology and Epidemiology, No 20 Dongdajie, Fengtai District, Beijing 100071, PR China.
Wang
Hui
H
Bao
Shizhong
S
Shi
Jing
J
Hou
Xiaojun
X
Gao
Xiang
X
Liu
Hao
H
Yin
Jun
J
D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D000915:Antibody Affinity; D000956:Antigens, Viral / Q000378:metabolism*; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D005822:Genetic Vectors; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000096:biosynthesis; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D010957:Plasmids; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011820:Rabies virus / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017186:Titrimetry; D014759:Viral Envelope Proteins / Q000378:metabolism*
D000914:Antibodies, Viral; D000956:Antigens, Viral; D006023:Glycoproteins; D007127:Immunoglobulin Constant Regions; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C064951:glycoprotein G, Rabies virus