Screening of human antibody Fab fragment against HBsAg and the construction of its dsFv form.
The objective of this study was to pursue the techniques involving the screening of the human antibody Fab fragment against hepatitis B virus surface antigen (HBsAg) and the construction of its disulfide-stabilized Fv fragment (dsFv). The phage antibody Fab fragments against HBsAg were screened from the human combinatorial immunoglobulin library. Sequence analysis revealed that its heavy chain gene was complete, but the light chain gene was lost. To improve the affinity of the antibody by chain shuffling, a human antibody light chain gene repertoire was generated by reverse transcriptase-polymerase chain reaction (RT-PCR) from the human peripheral blood lymphocytes. A phage antibody sub-library was then constructed by inserting the light chain gene repertoire into the phagmid that contained the Fd gene. Five clones with appreciably higher absorbance than that of the original clones were obtained, which indicated that the affinity of the light chain-shuffled phage antibodies was improved. Then, the mutated genes of dsFv against HBsAg were constructed by using PCR-based point mutagenesis method. Purified V(H) and V(L) proteins were folded into a 25-kDa protein, designated as anti-HBsAg dsFv. ELISA and competition ELISA revealed that the dsFv maintained the specificity of the Fab by binding to HBsAg, even through with a lower binding activity. These results have facilitated the undertaking of further functional analyses of the constructed dsFv, and may therefore provide an improved technique for the production and application of dsFvs against HBsAg.
Jia
Leili
L
Center for Disease Control, Prevention, Academy of Military Medical Sciences, Beijing, China.
Yu
Jiyun
J
Song
Hongbin
H
Liu
Xuelin
X
Ma
Weina
W
Xu
Yuanyong
Y
Zhang
Chuanfu
C
Dong
Shicun
S
Li
Qiao
Q
D005786:Gene Expression Regulation; D015723:Gene Library; D006514:Hepatitis B Surface Antigens / Q000378:metabolism*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism*; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction
D006514:Hepatitis B Surface Antigens; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; C055966:immunoglobulin Fv