Cloning and expression of canine interferon-gamma gene in mouse SP2/0 cell line.
- Author:
Qi YANG
1
;
Chun XIA
;
De-Ming ZHAO
;
Ming WANG
Author Information
1. College of Animal Medicine, China Agricultural University, Beijing 100094, China.
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Animals;
Base Sequence;
Bone Marrow Neoplasms;
metabolism;
Cattle;
Cell Line;
Cloning, Molecular;
DNA, Complementary;
analysis;
chemistry;
Dogs;
Interferon-gamma;
biosynthesis;
genetics;
pharmacology;
Mice;
Molecular Sequence Data;
Recombinant Proteins
- From:
Chinese Journal of Biotechnology
2002;18(3):365-368
- CountryChina
- Language:Chinese
-
Abstract:
Canine Interferon-gamma (CaIFN-gamma) cDNA was cloned from spleen T cells of dog by reverse transcription-polymerase chain reaction (RT-PCR). CaIFN-gamma cDNA were digested with Hind III and Not I, and inserted into pRc/CMV2 expression vector. The pRc/CMV2/CaIFN-gamma vector was sequenced, and predicted to produce a signal peptide of 23 amino acids and a mature protein of 143 amino acids with a molecular weight of 19 kD. Two potential N-glycosylation sites are located at positions 16 and 83 of the mature protein. Comparison of the CaIFN-gamma protein sequence with that of CaIFN-gamma reported from DDBJ/GenBank revealed a homology of 99%. To establish a long time expression system, pRc/CMV2/CaIFN-gamma vector was transfected into mouse SP2/0 cell line. The SP2/0 cells culture supernatants was harvested and the antiviral activity was measured following cytopathic-effect inhibition assay using Madin-Darby Canine Kidney (MDCK)-vesicular stomatitis virus(VSV) system. Initial transformants with G418 phenotype produced recombinant CaIFN-gamma titers ranging from 2,500 to 5,000 u/mL of culture medium.
