[Secretory expression of chimeric Fab antibody HAb18 against human hepatocellular carcinoma in Pichia pastoris].
OBJECTIVE
To express secretively chimeric Fab antibody HAb18 (cFab) against human hepatocellular carcinoma in Pichia pastoris.


METHODS
Genes encoding CL chain and Fd fragment of cFab antibody HAb18 were subcloned into vectors pPIC9K and pPICZalphaA, respectively. After confirmed by DNA sequence analysis, the recombinant plasmids pPIC9K/CL and pPICZalphaA/Fd were transformed into the genome of Pichia pastoris GS115. Mut(+) multiple insert transformants were screened by G418 and Zeocin and then induced with 5 mL/L methanol to express cFab.


RESULTS
4 days after methanol induction, 26 mg/L of the cFab fragment was detected in the culture supernatant. Western blot proved that the expressed protein could specifically bind with HAb18GEF antigen.


CONCLUSIONS
The successful expression of cFab/HAb18 in Pichia pastoris lays the foundation for large-scale production and further application of the antibody.
Dong
Hong-lin
HL
Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Xing
Jin-liang
JL
An
Jia-ze
JZ
Yang
Xiang-min
XM
Yao
Xi-ying
XY
Dou
Ke-feng
KF
Chen
Zhi-nan
ZN
D000912:Antibodies, Neoplasm / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000276:immunology* / Q000473:pathology; D045744:Cell Line, Tumor; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D008113:Liver Neoplasms / Q000276:immunology* / Q000473:pathology; D010843:Pichia / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology
D000912:Antibodies, Neoplasm; D007140:Immunoglobulin Fab Fragments; D011993:Recombinant Fusion Proteins