| Interferon-gama(IFN-?)is a multifunctional protein,which was secreted by the specific immune cells.IFN-?,belonging to Interferons type ?,has weak heat and acid resistance and easily deactivation.IFN-? has the property of inhibiting the growth of tumor cell and regulating the body's nonspecific immune function.IFN-? also has functions of antiviral and parasitic infections.The biologics of IFN-? has used in the treatment of hepatitis b and hepatitis c,which achieved good results.Therefore,IFN-? has good application prospect in the treatment of viral diseases and regulating the body's immunity.Although the research of interferon in fish started relatively late,it has obtained a variety of fish IFN genes through the method of gene engineering.According to IFN-? gene sequence of Takifugu rubripes in Genbank,IFN-? gene from Takifugu rubripes using genetic engineering method to synthesis was cloned into pPICZ?-A and transformed into Pichia pastoris GS115 strains through electrical process.Several Zeocin resistant clones were obtained and integration of IFN-? gene in P.pastoris genome was confirmed by PCR using specific primers.The expression of the strains induced by methanol was proved by SDS-PAGE gel electrophoresis.Cytopathic inhibition method was used to detect the bioactivity of target protein.The result showed that the recombinant protein with a molecular weight of 24,000 Da was detected by SDS-PAGE in culture media.The concentration of recombinant IFN-? protein was 0.282 mg/mL after inducing 96 h.And also recombinant IFN-? protein of Takifugu rubripes could stimulate WISH cell proliferation,which was associated with rIFN-? protein concentration.It was obtained recombinant IFN-? protein from prokaryotic which was made of the existing laboratory.The molecular weight of recombinant IFN-? protein in prokaryotic expression was 37 kDa and the concentration of it was 0.346 mg/mL.The active of recombinant IFN-? protein was 1.7× 105 U/mL detected by Cytopathic inhibition method.The active of recombinant IFN-? protein in the prokaryotic was much higher than it in eukaryotic.The results laied a foundation for subsequent immune experiment.Takifugu rubripes at 6 months were treated with recombinant IFN-? protein produced by prokaryotic in different concentrations(25 ?g/mL rIFNg-? as the experimental group of 1,50 ?g/mL rIFNg-? as experimental group 2).PBS was used as the control.Kibdey tissue was taken at 6,12,24,and 36 hours after intraperitoneal injection.The total RNA was extracted by Trizol method,and RNA was used as template to reverse the first strand of cDNA.Real-time quantitative PCR was used to detect the expression of IFN-?,IFN-?,MHC?,Mx 1 gene in kidney at different time.The results showed that the expression of IFN-? gene increased and reached the highest level at 24 h,then decreased in the experimental group after immunization with rIFN-?.The expression level of IFN-? gene in experimental group 2 was the highest at 6 h.IFN-? and MHC? had the highest expression at 6 h after immunization and the expression in experimental group 1 was higher than that of experimental group 2.The expression of Mx gene was an upward trend,which were highest at 24 h.RNA-Seq was used to analyze the transcriptome of kidney of Takifugu rubripes chanllged by PBS and IFN-?.The Raw data were measured at 29,939,506 and 24,630,588 respectively.The clean reads of the original data are 29,415,565 and 23,943,395,respectively.292 differential genes were screened by differential gene screening,of which 171 were up-regulated in the IFN-? group.GO and KEGG were used to classify and enrich the genes.SNP analysis was performed on the transcriptome.118,876 and 114,091 SNPs were obtained from the two groups,among which the transition of SNP was the largest. |
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