| In the present study,the full-length cDNA of erythropoietin(EPO)and granulocyte colony-stimulating factor(G-CSF)were cloned from large yellow croaker by reverse transcription-polymerase chain reaction(RT-PCR)and rapid amplification of cDNA ends-polymerase chain reaction(RACE-PCR).Their molecular characterization and structures were analyzed.The expression profiles in different untreatment tissues and in lipopolysaccharide(LPS),polyinosinic-polycytidylic acid(poly I:C)and vibrio parahemolyticus challenge immune-related tissues were detected by semi-quantitative PCR.In addition,the recombinant EPO protein of large yellow croaker(rLcEPO),rLcG-CSF and rLcIL-6 were expressed by the the Pichia pastoris expression system and the Escherichia coli expression system,respectively.Moreover,the rLcEPO,rLcIL-6 were also overexpressed in Larmichthys crocea kidney(LCK)cell line.The expression of some key immune factors were determined in LCK cells after overexpression or recombinant fusion protein stimulation by semi-quantitative PCR.The detailed results can described as follows:(1)The gene clone and expression analysis of LcEPOThe full-length cDNA of LcEPO was 889 bp,containing a 205 bp-5’ untranslated region(UTR),a 126 bp-3’UTR and a 558 bp open reading frame(ORF)which encoded 185 amino acids(aa).The predicted molecular weight of LcEPO amino acids was 20.47 kilodalton(kDa),with isoeletric point of 6.9.LcEPO amino acids were containing 6 oxygen glycosylation sites and a conserved EPO-TPO domain.LcEPO mRNA was expressed in most examined tissues,with the most predominant expression in liver,followed by spleen and very weak expression in skin.The expression levels of LcEPO after challenged with LPS,poly I:C and Vibrio parahaemolyticus were investigated in spleen,head-kidney and liver.LcEPO transcripts were induced significantly after immune challenge,especially in liver(p<0.05).(2)The recombinant expression,polyclonal antibody product and overexpression of LcEPOWith more oxygen glycosylation sites,rLcEPO were expressed by the Pichia pastoris expression system and confirmed by antibody which was produced by mice stimulating with E.coli-expressed r LcEPO.In addition,the overexpression vector pcDNA3.1-EPO was constructed,which sets a base for its further functional studies.(3)The gene clone and expression analysis of LcG-CSFThe full-length cDNA of LcG-CSF was 1211 bp,containing a 473 bp-5’UTR,a 135 bp-3’UTR and a 603 bp ORF which encoded 200 aa.The predicted molecular weight of LcG-CSF amino acids was 21.89 kDa,with isoeletric point of 9.21.LcG-CSF amino acids were not containing oxygen glycosylation sites but containing a conserved IL-6 domain.LcG-CSF mRNA was expressed in most examined tissues,with the most predominant expression in head-kidney,followed by heart and very weak expression in gill.The expression levels of LcG-CSF after challenged with LPS,poly I:C and Vibrio parahaemolyticus were investigated in spleen,head-kidney and liver.LcG-CSF transcripts were induced significantly after immune challenge(p<0.05),especially stimulated with LPS and poly I:C.(4)The recombinant expression of LcG-CSFRecombinant LcG-CSF fusion protein was expressed by the Escherichia coli expression system and purified by Ni column purification system.The endotoxin was removed.The expression of some key immune factors were determined in LCK cells after recombinant G-CSF fusion protein stimulation by semi-quantitative PCR,tumor necrosis factor-α and mucus virus inhibition of protein were significantly up-regulated(p<0.05).The results indicated that LcG-CSF might play an important role in antibacterial-and antiviral-related fish immune response.(5)The recombinant expression and overexpression of some cytokines in large yellow croakerrLcIL-6 were expressed by the Escherichia coli expression system and the endotoxin was removed.The expression of some key immune factors were determined in LCK cells after recombinant IL-6 fusion protein stimulation by semi-quantitative PCR,tumor necrosis factor-α was significantly up-regulated(p<0.05).After LCK transfected with pcDNA3.1-IL-6 overexpression vector,TNF-α was also significantly up-regulated(p<0.05).These results indicated that LcIL-6 might play an important role in antibacterial-related fish immune response. |
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