Expression and bioactivity identification of soluble MG7 scFv.
OBJECTIVE
To examine the molecular mass and identify the bioactivity of MG7 scFv for its application as a targeting mediator in gene therapy of gastric cancer.


METHODS
Two strongly positive recombinant phage clones screened from MG7 recombinant phage antibody library were separately transfected into E.coli TG1. Plasmid was isolated from the transfected E.coli TG1 and digested by EcoR I and Hind III to examine the length of exogenous scFv gene. Then, the positive recombinant phage clones were individually transfected into E.coli HB2151. The transfectant was cultured and induced by IPTG. Perplasmic extracts was prepared from the induced transfectant by osmotic shock. ELISA was used to examine the antigen-binding affinity of the soluble MG7 scFv. Immunodotting assay was adopted to evaluate the yield of soluble MG7 scFv produced by transfected E.coli HB2151. Western blot was used to examine the molecular mass of MG7 scFv. Finally, the nucleotide sequence of MG7 scFv was examined by DNA sequencing.


RESULTS
Two positive recombinant phage clones were found to contain the exogenous scFv gene. ELISA showed that MG7 scFv had strong antigen-binding affinity. Immuodotting assay showed that transfected E.coli HB2151 could successfully produce the soluble MG7 scFv with high yield via induction by IPTG. The molecular mass of MG7 scFv was 30 kDa by western blot. DNA sequencing demonstrated that the VH and VL genes of MG7 scFv were 363 bp and 321 bp,respectively.


CONCLUSIONS
We have successfully developed the soluble MG7 scFv which possessed strong antigen-binding affinity.
Yu
Zhao-Cai
ZC
Department of Gastroenterology, Xijing Hospital, Fourth Military Medical University, Xi'an 710033, Shaanxi Province, China.
Ding
Jie
J
Pan
Bo-Rong
BR
Fan
Dai-Ming
DM
Zhang
Xue-Yong
XY
D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D015316:Genetic Therapy; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015183:Restriction Mapping; D012995:Solubility; D013274:Stomach Neoplasms / Q000276:immunology / Q000628:therapy*
D007128:Immunoglobulin Fragments; C055966:immunoglobulin Fv