[Construction, expression and functional detection of fusion gene of CD80 extracellular region and anti-human CD33 scFv].
OBJECTIVE
To construct and express the CD80 extracellular region-anti-human CD33 single chain fragment of variable region (ExCD80-CD33scFv) fusion gene, and detect the biological activity of the fusion protein.


METHODS
Extracellular region of CD80 (ExCD80) was amplified and then linked with anti-CD33scFv using 403Aa-427Aa (hydrophilic fragments) in domain III of human serum albumin (HAS) as interlinker. The recombinant fusion gene was subcloned into the prokaryotic expression vector PET22b(+) and expressed in E.coli Rosetta (DE3) after induction by IPTG. The purified fusion protein was obtained after a series of purification steps including cell lysis, inclusion body solubilization, Ni(2+); metal affinity chromatography and protein refolding. The biological activity of the fusion protein was detected with indirect immunofluorescence technique.


RESULTS
The ExCD80 (633bp) was amplified by PCR and 403aa-427aa from domain III of HAS as interlinker was synthesized correctly. SDS-PAGE and Western blotting demonstrated that ExCD80-CD33scFv fusion gene expression vector was successfully constructed and expressed in E.coli Rosetta (DE3). The relative molecular mass (Mr;) of the fusion protein was 55 000, which has human CD33-binding specificity after renaturation as shown by indirect immunofluorescence technique.


CONCLUSIONS
Recombinant ExCD80-CD33scFv fusion gene was successfully constructed and expressed in E.coli Rosetta (DE3), which could provide a foundation for the future target therapy to the myeloid leukemia.
Department of Immunology, School of Basic Medicine, Tianjin Medical University, Tianjin 300070 China.
Xiaojun
X
Chen
Institute of Hematology, Blood Diseases Hospital, Chinese Academy of Medical Sciences, Tianjin 300020, China.
Lu
L
Liang
Institute of Hematology, Blood Diseases Hospital, Chinese Academy of Medical Sciences, Tianjin 300020, China.
Feng
F
Xue
Institute of Hematology, Blood Diseases Hospital, Chinese Academy of Medical Sciences, Tianjin 300020, China.
Tao
T
Sui
Institute of Hematology, Blood Diseases Hospital, Chinese Academy of Medical Sciences, Tianjin 300020, China.
Chunlan
C
Dong
D018122:B7-1 Antigen / Q000235:genetics* / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D018922:HL-60 Cells; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism*; D063268:Sialic Acid Binding Ig-like Lectin 3 / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology
D018122:B7-1 Antigen; D011993:Recombinant Fusion Proteins; D063268:Sialic Acid Binding Ig-like Lectin 3; D057127:Single-Chain Antibodies