Cloning, Prokaryotic Expression Of The CDNA Of Coturnix's IFN-α And Heat Shock Protein 70

In resent years, coming with the development of the poultry industry, there are moreand more poultry diseases that threat the health of poultry, for example, NDV, Duck Plague,poultry Influenza, et al. The interferon system is the firstline of defense against viralinfection in organism. Type interferon-αis famous as the potent antiviral activity andinterferon gamma manifested stronger immune modifcation activity besides antiviralactivity. That HSP70 may play a role as a carrier molecule in eliciting immune responses tocertain peptideantigens. But control of these diseases have been hampared by frequentreinfection and high cost of chemotherapy. To further investigate the immune responsesand its cytoprotective functions, so we cloned and expressed the gene coturnix'sinterferon-αand HSP70 family in E.coli and have developed a method for its purification.Total RNA was abstracted from Coturnix's spleen lymphocytes after being activated byNDV. The coturnix's interferon-αcDNA was amplified by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and ligated into pET-28α(+).Recombinant plasmidpET-CoINF-αwas indentified by PCR and DNA sequencing. The recombinant CoINF-αwas ananlyzed by SDS-PAGE. Total RNA was abstracted from Coturnix's spleenlymphocytes after being activated by heat. The coturnix's HSP70 was amplified byReverse Transcription-nested Polymerase Chain Reaction (RT-PCR).COHSP70 wasindentified by PCR using the outer primers. It was ligated into pET-28α(+) after indentifiedby PCR.Recombinant plasmid pET-COHSP70 was indentified by PCR and DNAsequencing. The recombinant COHSP70 was ananlyzed by SDS-PAGE.The result showed that coturnix's CoIFN-αcDNA and HSP70 cDNA was successfullyexpressed in E.coli BL21. The nucleotide sequence of coturnix's CoIFN-αcDNA is641bp, it has 192 amino, has 100％identities with Coturnix japonica inGenBank(AB154298.1).But the coturnix's HSP70's nucleotide sequence is 2341bp, ithas 643 amino, has 99％identities with Coturnix japonica in GenBank (AB262971.1AB259847.1).As a fusion protein with a molecular weight of CoIFN-αis about 30KDa.But the HSP70's molecular weight is about 70KDa.