中国生物工程杂志

ABSTRACT AIM:Cloning two genes of heparin-binding domain of fibronectin into vector of insect expression and expressing two polypeptide in insect cell Sf9.Purifing polypeptide with Ni-NTA and Testing biological activity of polypeptide binding with heparin and antygen of FN with Western-blot.METHODS:The gene of Two heparin-binding domain of fibronectin were amplified from FNcDNA by high fidelity PCR. The aim DNA sequences were cloned into Bac-to-Bac HT Vector(AorB) to get recombinant plasmid. The recombinant plasmid was transformed into E.coli DH5α And the positive plasmid was selected throuth ampicillin resistance to get positive recombinant donor.After being sequenceed and determined right read frame,positive donor was transformed into MAX Efficiency DH10BacTM Competent E.coli to get positive bacmid (white colony )by gentamicin,kanamycin and tetracycline resistance with IPTG and X-gal.recombinant bacmid transformed into Sf9 cell and in it recombinant baclovirus was formed.Isolating baclovirus from supernatant of culture Sf9 cell and performing a viral plaque assay,then recombinant baclovirus were transformed into Sf9 cell again , polypeptide was expressed in it. Polypeptide in supernatant of insect cell lysate were purified with Ni-NTA colummn .Binding heparin and FN antygen activity of polypeptides were detected by western blot assay. Tt was determined in mice that rhFNHC-36 polypeptide antagonizes endotoxemia with thrombocytopenia. RESULTS: Two heparin-binding polypeptide of fibronectin were expressed in insect cell sf9.Expressed polypeptides can bind with heparin in vivo and have losted antygen of FN . The rhFNHC-36 polypeptide has function of antagonizing endotoxemia with thrombocytopenia in mice. CONCLUSION: Two heparin-binding polypeptide of fibronectin can be expressed in insect expression system.Expressed polypeptide can bind with heparin in vivo ,not with antygen of FN and have function of antagonizing endotoxemia with thrombocytopenia in mice. Exprssion ratio is low,more method must be found to elevate it.