[Eukaryotic expression and determination of ZCH-7-2F9 single chain antibody against human CD14].
OBJECTIVE
Acute monocytic leukemia (AML)-M5 is the common type of acute myeloid leukemias in children. Studies have shown that there are abundant lipopolysaccharide (LPS) receptor (designated as CD14) molecules on the cell membrane of M5 cells and they play an important role in the diagnosis of M5, since they can be recognized and bound by mouse-anti-human CD14 monoclonal antibody (McAb). However, mouse-originated antibodies are largely not suitable for clinical application due to the severe side effects, thus "humanized antibody" is desired. As the first step for developing humanized antibody, the construction and expression of single chain antibody (scFv) with functional protein are necessary. The present study aimed to express ZCH-7-2F9 ScFv (scFv(2F9)) in eukaryotic cells and obtain the scFv(2F9) protein with a high biological activity.


METHODS
Four primers were synthesized to construct the eukaryotic expressional vector, which included SfiI and EcoRI enzyme cleaving site, 6 x His and stop code TGA sequences. scFv(2F9) gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Positive recombinants (pSectag2A/scFv(2F9)) were identified through enzyme cleaving and sequenced before expression and were transformed into Chinese hamster ovary (CHO) cells for expression. Western-Blot and flow cytometry (FCM) were carried out to determine the relative molecular mass (Mr) and binding activity of scFv(2F9).


RESULTS
The cloned scFv(2F9) gene was identified to be functional by sequencing and expressing. The interested protein could be detected in the culture supernatant of transformed CHO cells with an Mr of 31 000. The blocking test showed that the positive cell percentages, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 90.02%, 63.30% and 63.38%, respectively after blocking with scFv(2F9), while those were 4.55%, 10.09% and 5.02% after blockage using the supernatant from the CHO cells transfected with blanked vector pSectag2A.


CONCLUSIONS
The scFv(2F9) against human CD14 antigen was successfully expressed in eukaryotic cells and showed a high biological activity, which may be useful for the further studies on its humanized antibodies.
Ning
Bo-tao
BT
Department of Hematology and Oncology, the Children's Hospital of Zhejiang University School of Medicine, Hangzhou, China.
Tang
Yong-min
YM
Cao
Jiang
J
Shen
Hong-qiang
HQ
Qian
Bai-qin
BQ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D016466:CHO Cells; D002648:Child; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000276:immunology*; D008969:Molecular Sequence Data; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D018950:Lipopolysaccharide Receptors; D057127:Single-Chain Antibodies