title: Expression, purification, and characterization of humanized anti-HBs Fab fragment.
abstract: Anti-HBs Fab fragment has considerable potential for use in the prevention and treatment of liver diseases by HBV. Here we established a high-level expression system to directly produce anti-HBs Fab fragment in Pichia pastoris. This was achieved by co-integration of the genes encoding the heavy and light chains both under the genome of the yeast cells. The Fab fragment was efficiently secreted into medium at a concentration of 50 mg/liter. The authenticity of the Fab fragment was confirmed by immunoblot analysis, which yielded one band of approximately 50 kDa under nonreducing conditions and two bands of approximately 28 kDa under reducing conditions. The anti-HBs Fab fragment was prepared with a purity of 95% by affinity chromatography. The affinity activity of the recombinant Fab was detected by ELISA, which indicated that 1 mg of recombinant Fab was equivalent to 40 IU HBIG (20 IU/mg). The results demonstrated that the recombinant Fab fragment could sufficiently neutralize the HBsAg.
authors.affiliation: Life Science and Technology College in Jinan University, Guangzhou, 510632, China. dengning123@sohu.com
authors.forename: Deng
authors.initials: D
authors.lastname: Ning
authors.forename: Xiang
authors.initials: X
authors.lastname: Junjian
authors.forename: Wang
authors.initials: W
authors.lastname: Xunzhang
authors.forename: Chen
authors.initials: C
authors.lastname: Wenyin
authors.forename: Zhang
authors.initials: Z
authors.lastname: Qing
authors.forename: Su
authors.initials: S
authors.lastname: Kuanyuan
authors.forename: Rao
authors.initials: R
authors.lastname: Guirong
authors.forename: Ren
authors.initials: R
authors.lastname: Xiangrong
authors.forename: Long
authors.initials: L
authors.lastname: Qingxin
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhouyao
mesh_terms: D001666:Binding Sites, Antibody; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005822:Genetic Vectors; D006031:Glycosylation; D006510:Hepatitis B Antibodies / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006510:Hepatitis B Antibodies; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins