Secretory expression of porcine interferon-gamma in baculovirus using HBM signal peptide and its inhibition activity on the replication of porcine reproductive and respiratory syndrome virus.
The gene sequence encoding mature porcine interferon-gamma (PoIFN-gamma) fused with a C-terminal 6x histidine tag was cloned into the baculovirus pFastBac Dual vector of the Bac-to-Bac Baculovirus expression system under the control of PH promoter. The authentic signal sequence of porcine interferon-gamma was substituted with the honeybee melittin (HBM) signal sequence, and expressed in insect cells. The recombinant proteins were detected by SDS-PAGE and immunofluorescence assay. The nickel affinity column purified recombinant porcine interferon-gamma with HBM signal peptide (rPoIFN-gammaH) was shown to be a 19kDa protein as confirmed by Western blot analysis. The recombinant PoIFN-gammaH was shown to have cytokine activity, inhibiting the cytopathic effect of vesicular stomatitis virus (VSV) in PK-15 cells at about 1.07x10(6)U/mL. The 2(-7) dilution of the rPoIFN-gammaH in culture supernatant protected the MARC-145 cells from the cytopathic effect caused by 100TCID(50) of porcine reproductive and respiratory syndrome virus.
Wang
Yan-Bin
YB
Key Laboratory of Animal-derived Food Safety of Henan Province, College of Husbandry and Veterinary, Henan Agricultural University, Zhengzhou, Henan 450002, China.
Wang
Zhen-Ya
ZY
Chen
Hong-Ying
HY
Cui
Bao-An
BA
Wang
Ya-Bin
YB
Zhang
Hong-Ying
HY
Wang
Rui
R
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D001516:Bees / Q000235:genetics; D002460:Cell Line; D003588:Cytopathogenic Effect, Viral / Q000187:drug effects; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000235:genetics* / Q000494:pharmacology; D008555:Melitten / Q000235:genetics; D019318:Porcine Reproductive and Respiratory Syndrome / Q000188:drug therapy; D019316:Porcine respiratory and reproductive syndrome virus / Q000472:pathogenicity* / Q000502:physiology*; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology; D011994:Recombinant Proteins; D018411:Spodoptera; D013552:Swine / Q000235:genetics* / Q000276:immunology* / Q000821:virology; D018116:Vesiculovirus / Q000187:drug effects / Q000472:pathogenicity; D014779:Virus Replication / Q000187:drug effects
D000998:Antiviral Agents; D017931:DNA Primers; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D008555:Melitten; D007371:Interferon-gamma