[Construction of a non-viral vector H1s-EGFc and a preliminary study on its function].
OBJECTIVE
To construct a non-viral vector for targeting cancer gene therapy.


METHODS
The coding sequence of H1s-EGFc was inserted into the expression vectors of Pichia pastoris, and the fusion protein was expressed in secretary way. H1s-EGFc was purified by anion exchange chromatography and size exclusion chromatography. H1s-EGFc fusion protein and "killing gene" expression recombinant pKG plasmid DNA were dissolved in serum-free RPMI-1640 culture to produce H1s-EGFc/pKG complex. HeLa cells, an epidermal growth factor receptor (EGFR) highly expressing cell line, and Jurkat cells, an EGFR non-expressing cell line, were cultured and transfected with H1s-EGFc/pKG complex of different concentrations. Trypan blue staining was used to calculate the number of live cells and the killing rate of H1s-EGFc/pKG.


RESULTS
H1s-EGFc fusion protein was constructed and expressed with a purity of over 90%. When the concentrations of H1s-EGFc/pKG complex were 3 microg/ml, 6 microg/ml, and 9 microg/ml respectively the killing rates were 30.6%, 36.2%, and 58.1% respectively.


CONCLUSIONS
The fusion protein H1s-EGFc binds functional gene efficiently and targets it into specific cells. It can be used as non-viral vector in target cancer gene therapy.
Guo
Lei
L
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China.
Yang
Kui
K
Li
Yong
Y
Situ
Hui-fen
HF
Chen
Wei-jing
WJ
Hong
Mei
M
Lu
Jin-zhi
JZ
Lu
Sheng-dong
SD
D066246:ErbB Receptors / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006367:HeLa Cells; D006657:Histones / Q000235:genetics*; D006801:Humans; D010843:Pichia / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014162:Transfection
D006657:Histones; D011993:Recombinant Fusion Proteins; D066246:ErbB Receptors