Construction, expression and in vitro biological behaviors of Ig scFv fragment in patients with chronic B cell leukemia.
The expression vector of SmIg scFv fragment was constructed in patient with B cell chronic lymphocyte leukemia (B-CLL) and expressed in E. coli to obtain scFv fragment, and the effect of the protein on the proliferation of stimulated peripheral blood mononuclear cells (PBMC) was investigated in vitro. Two pairs of primers were designed, and variable region genes of light chain and heavy chain were amplified by PCR respectively from the pGEM-T vectors previously constructed in our laboratory which containing light chain gene or Fd fragment of heavy chain gene. The PCR product was digested, purified and inserted into pHEN2 vector to construct the soluble expression vector pHEN2-scFv. After the induction by IPTG, the scFv protein was identified by SDS-PAGE electrophoresis and purified by Ni-NTA-Chromatography. MTT was used to determine the effect of purified protein on the proliferation of stimulated PBMC in vitro. Plasmid PCR and restriction enzyme digestion of pHEN2-scFv revealed the pHEN2-scFv vector was constructed successfully. Id-scFv protein was expressed in positive clone after induced by IPTG. SDS-PAGE analysis showed that the relative molecular weight of fusion protein was about 30 kD (1 kD= 0.9921 ku), which was consistent with the theoretically predicted value. Proliferation of PBMC could be induced by purified Id-scFv. It was suggested that the expression vector of SmIg scFv fragment was constructed successfully, and scFv protein was expressed and secreted from E. coli, which could induce proliferation of PBMC. This may lay an experimental foundation for further research of Id-HSP complex vaccine for B-CLL.
Zhu
Lijuan
L
Department of Immunology, School of Basic Medical Sciences, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Liao
Wenjun
W
Zhu
Huifen
H
Lei
Ping
P
Wang
Zhihua
Z
Shao
Jingfang
J
Zhang
Yue
Y
Shen
Guanxin
G
D049109:Cell Proliferation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D050438:Genes, Immunoglobulin Heavy Chain / Q000235:genetics; D050439:Genes, Immunoglobulin Light Chain / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics*; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000276:immunology* / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D057127:Single-Chain Antibodies
D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C114805:scFv fragment SW1