| Interferon is a kind of glycoprotein,which is produced by the body immune cells and has many biological activities such as antivirus,anti-tumor and immunomodulatory.The biological properties and activity of interferon,they were divided into type I and type ? according to the source.Feline interferon ?(FeIFN-?)belongs to type ? interferon,which mainly possesses antiviral and anti-tumor effects.Feline interferon ?(FeIFN-?)belongs to type ? interferon,which plays a key role in host defense against intracellular pathogens.Although the research on interferon has a long history,the soluble expression and their application of FeIFN-? and FeIFN-? are rarely reported.In this study,the cloning,prokaryotic soluble expression and purification of FeIFN-? and FeIFN-? genes were carried out,and their safety was preliminarily evaluated,which will lay a foundation for the clinical application of FeIFN-? and FeIFN-?.The specific primers of FeIFN-? and FeIFN-? genes were designed based on the sequences of FeIFN-? and FeIFN-? in NCBI by using bioinformatics analysis,and the fragments of mature FeIFN-? and FeIFN-? without signal peptide were amplified from the cDNA of cat liver.The recombinant prokaryotic expression vectors for pET28a-SUMO-FeIFN-? and pET28a-SUMO-FeIFN-? were constructed and transformed into E.coli BL21(DE3)and Rosetta(DE3)competent cells,respectively.The expression conditions of recombinant proteins were optimized,and the fusion protein was purified by using Ni column,removing the SUMO-enzyme tag.Finally,the unlabeled and mature FeIFN-? and FeIFN-? were obtained after purification by molecular interception column,and the safety was evaluated by intraperitoneal injection in mice.The results showed that:(1)The length of FeIFN-? and FeIFN-?mature protein genes were 522 bp and 444 bp,respectively,that were amplified using cDNA of cat liver as template,the constructed p MD19-T-FeIFN-? and p MD19-T-FeIFN-? clones were transformed into DH5? cells,the size and sequence of the extracted plasmid were confimred by PCR,double enzyme digestion and sequencing.(2)Prokaryotic expression vectors of pET28a-SUMO-FeIFN-? and pET28a-SUMO-FeIFN-? were transformed into BL21(DE3)and Rosetta(DE3)competent cells,respectively.The size offusion proteins was about 35 ku and 34 ku and the proteisn was mainly expressed in the supernatant of ultrasonically broken cells,which were soluble prokaryotic expressions.The optimal final concentration of IPTG was 0.6 mmol/L and 0.8 mmol/L,the time of induction was 6 h and 9 h,and the temperature was 16?.(3)Purification results showed that single band of the proteins was about 35 ku and 34 ku after purification by Ni column,and the mature FeIFN-?and FeIFN-? proteins of about 18 ku and 17 ku were obtained with concentrations of0.79 mg/m L and 0.86 mg/m L,respectively after the SUMO tag was removed by enzyme.(4)The results of acute toxicity test of mouse,there were no clinical symptoms of discomfort,no changes in tissue necropsy and no significant difference in body weight(P>0.05),and no significant difference in blood biochemical indexes(P>0.05),but the levels of Glu in FeIFN-? 0.2 mg/ group and ALT in FeIFN-? 0.3mg/ group were higher than those in control group(P<0.05).The four tissues of liver,spleen,lung and kidney in each group were normal,and there were no obvious changes of inflammatory cell infiltration and fibrosis.In conclusion,the soluble expression system of FeIFN-? and FeIFN-? was established by using the prokaryotic expression system of Escherichia coli in vitro,and mature FeIFN-? and FeIFN-? proteins were obtained,which laid a foundation for the development and application of FeIFN-? and FeIFN-?. |
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