RESUMEN
A fragment containing amino acid residues 561～578 of HSV-2 glycoprotein G(gG2) was obtained by PCR assembling technique,and doubly cloned into vector pET-KDO.The recombinant plasmid was transformed to BL21(DE3)plysS.Fusion protein,of molecular weight about 39kDa was highly expressed by induction of IPTG.Western blot result showed the fusion protein had good antigenicity.After putification and digestion,the purity reached 95%.The digested purified protein was analysed by ELISA and showed good sensitivity and specificity.The recombinant protein should be useful for type-specific serodiagnosis of HSV-2.
RESUMEN
PBD-1 is an antibacterial peptide that plays an important role in defence system of porcine. To produce PBD-1 with bioactivity in Pichia pastoris, according to published amino acid sequence of porcine beta-defensin 1(PBD-1) and the partiality codon of yeast, the PBD-1 gene was synthesized by PCR and cloned into pPIC9K to construct the recombinant expression vector pPIC9K-PBD-1, the obtained recombinant plasmid was linearized by Sal I, and then transformed into SMD1168 by electroporation. Under the control of the promoter AOX1, an approximately 4.5 kD PBD-1 peptide was expressed. Antibacterial activity assay shows that the PBD-1 has the antibacterial activity on Staphylococcus aureus. This is the first secreted expression of porcine beta-defensin 1 gene in Pichia pastoris.
Asunto(s)
Animales , Antibacterianos , Farmacología , Clonación Molecular , Expresión Génica , Vectores Genéticos , Genética , Pichia , Genética , Ingeniería de Proteínas , Staphylococcus aureus , Porcinos , Genética , beta-Defensinas , Genética , FarmacologíaRESUMEN
In present study,bovine Lactoferricin was first secretly expressed in Pichia pastors yeast expression system.The synthesized LfcinB gene fragment was cloned into expression vector pPIC9K,and then obtained recombinant plasmid,designated as pPIC9K-LfcinB,was linearized and transformed into Pichia pastors strains SMD1168 by electroporation.The transformants were screened with Geneticin and multiply-copy colonies were harvested,in which LfcinB gene was verified to inserted into yeast chromosome stably.The positive recombinant Pichia strains were induced with methanol to express LfcinB in culture supernatant.It's expressive products has high activity of killing bacteria.We concluded that LfcinB gene was cloned and integrated into yeast chromosomes,and obtained expression peptide was tested to have high antibacterial activity.