[Construction and expression of a fusion protein of scFv against human acetylcholine receptor-human serum albumin in E. coli].
OBJECTIVE
To prepare the fusion protein of a single chain variable fragment 637 (scFv637) against human acetylcholine receptor (AChR) and human serum albumin (HSA) to increase the stability of scFv637.


METHODS
HSA gene was amplified by PCR and cloned into vector pHEN2 containing a scFv637 gene to construct recombinant vector pHEN2-scFv637-HSA and then transformed into E. coli HB2151 for expression. The expression of fusion protein in periplasm of E. coli was detected by dot hybridization. Relative molecular mass of fusion protein was checked by SDS-PAGE and Western blot.


RESULTS
Agarose gel electrophoresis analysis showed that the size of HSA gene and fusion gene was about 1,770 bp and 7,054 bp, respectively. DNA sequencing proved that the nucleotide sequence of constructed scFv637-HSA gene was correct and it was cloned into the open reading frame (ORF) of pHEN2. scFv637-HSA fusion protein only existed in periplasm of E. coli HB2151 transformed with pHEN2-scFv637-HSA. SDS-PAGE and Western blot analysis indicated that relative molecular mass of fusion protein was about 95,900.


CONCLUSIONS
The scFv637-HSA fusion protein can be successfully expressed in E. coli HB2151, which provides a sound basis for further research into its function and clinical application.
Fan
Hua-ying
HY
School of Pharmacy, Yantai University, Yantai 264005, China. huayingf@ytu.edu.cn
Meng
Fan-ping
FP
Qi
Dong
D
Wei
Jing
J
Li
Qiang
Q
Li
Ying-xin
YX
Li
Hong-hua
HH
D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D051379:Mice; D011950:Receptors, Cholinergic / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D012709:Serum Albumin / Q000276:immunology*
D007135:Immunoglobulin Variable Region; D011950:Receptors, Cholinergic; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin