Prokaryotic Expression, Purification, And Identification Of Plasminogen Activator From Gloydius Brevicaudus

ObjectiveThe subject using gene techniques constructed the prokaryotic expression vector of pET42a(+)-GBV-PA (plasminogen activator of Gloydius brevicaudus venom,GBV-PA),and expressed recombinate GBV-PA(rGBV-PA) fusion protein in Escherichia coli(E. coli). These create the possibility for expression of GBV-PA cloning efficiently in E. coli and mass production of GBV-PA. These also provide the basis for study on the relationship of constitution between function and the pharmacological mechanism of GBV-PA.MethodA couple of specific primers were designed on the basis of known nucleotide sequence of TSV-PA(Plasminogen Activator of Trimeresurus stejnegeri Venom) . The forward primer contained EcoRâ… restriction site,and the reward prime contained Xhoâ… restriction site. GBV-PA gene fragment was amplified from total RNA of Gloydius brevicaudus venom gland with RT-PCR. The PCR products were cloned and inserted into prokaryotic expression plasmid pET42a(+).Recombinant plasmid (pET42a(+)-GBV-PA)were identified by restriction enzymes digestion,then transformed into E. coli strain BL21(DE3). pET42a(+)-GBV-PA was identified by sequencing. Expression of rGBV-PA fusion protein induced by IPTG,and the expressed product was analyzed by SDS- PAGE .The rGBV-PA fusion protein was purified with His-Bind resin protein purification procedure. The antigenicity of expressed product was identified by Western blotting.Results 1.The cDNA in 800 bp was obtained by RT-PCR amplification,which was the same as GBV-PA gene.2.The GBV-PA gene was synthesized sueeessuflly and inserted into the vector PET42a(+).The recombinant expression plasmid pET42a(+)-GBV-PA was constructed. The size of cDNA sequence of GBV-PA gene was 777 bp which encoding 259 amino acids. MW=27921. Homologies of nucleotide sequence and amino acid sequence of GBV-PA between TSV-PA were 90.13% and 81.92%, respectively.3. The rGBV-PA fusion protein was expressed in E. coli BL21(DE3). SDS - PAGE showed that the fusion protein was expressed as inclusion body formation in E. coli BL21(DE3). MW was 61.7kD.4. The rGBV-PA fusion protein was obtained by His-bind Purification system,which has the antigenicity of Gloydius brevicaudus venom.ConclusionsThe prokaryotic expression recombinant plasmid pET42a(+)-GBV-PA were successfully constructed. The GBV-PA gene was expressed in E. coli BL21(DE3). The rGBV-PA fusion protein was obtained by His-bind Purification system,which has the antigenicity of Agkistrodon halys pallas venom. This can provide experimental basis for research and development of GBV-PA as thrombolytic drug.