The Cloning And Expression Of TGF-βl And IL-6in Dairy Goat And Their Immune Activity

Th17cells, as a novel subset, played a pivotal role in regulating autoimmune diseasesand defensing the extracellular bacterias infection. The induction and differentiation conditionof Th17was verificated in the human and mouse. The th17cells were induced with the IL-6and TGF-β1on the activated T cells. However, the differentiation and influential factors ofth17in others spcies was not reported, including goats. Therefore, the research on thedifferentiation of goat’s th17could provide guidance for interpreting the immune mechanismof goats’ th17differentiation.In the present research, the genes of IL-6and TGF-β1were cloned from the goat’ssplenocytes which were stimulated by ConA. The total RNA was extracted and targetsequence was amplified, then the mature domains were expressed by prokaryotic vector. ThePBMCs were stimulated by recombinant proteins and the proliferation was detected by MTT,meanwhile, the expression level of the IL-17mRNA was monitored. These results weredemonstrated,1. The length of TGF-β1and IL-6were1173bp and627bp respectively. The sequencesanalyses indicated that the molecular weight of IL-6was21ku, coding208amino acids andincluding N-terminal signal peptide of25AA. High degree of conservation was found byalignment of the amino acid sequences of goat IL-6with that of O. aries, B. taurus, H.sapiens and M. musculus, with amino acid identity98.75%,93.75%,63.75%, and56.25%,respectively. The sequences analyses showed that the TGF-β1was1173bp, coding390aminoacids and including N-terminal signal peptide of29AA. And the mature peptide was codedby112AA and the molecular weight was12.5ku. High degree of conservation was found byalignment of the amino acid sequences of goat TGF-β1with that of O. aries, B. taurus, H.sapiens and M. musculus, with amino acid identity99.23%,99.23%,95.13%, and89.49%,respectively.2. The goat IL-6with length of552bp was constructed the expression vector with pET32a,while the signal peptide sequence was cut. The recombinant plasmid was expressed with theIPTG of0.3mmol/L for4h at37℃in the E.coli and the product was in the supernatantpremodinantly. The mature peptide of goat TGF-β1was expressed with the IPTG of0.5mmol/L for14h at16℃in the E.coli and the product was in the supernatant partially. 3. The proliferation of PBMCs could be stimulated by goat recombinant mature TGF-β1,with statically significant at dose of1ng/mL. The proliferation was inhibited with theaccumulation of the recombinant protein. However, the proliferation of PBMCs stimulatedrecombinant protein together with ConA was inhibited and more severe than recombinantprotein alone.The proliferation of PBMCs could be stimulated by goat recombinant IL-6, with staticallysignificant at dose of10ng/mL and25ng/mL. The proliferation was enhanced with theaccumulation of the recombinant protein. And the proliferation of PBMCs stimulatedrecombinant protein together with ConA was more notable and the proliferation was weakenwith the accumulation of the recombinant protein.4. The PBMCs were stimulated by recombinant TGF-β1, IL-6and ConA, and theconcentrations were10ng/mL,1ng/mL and20μg/mL, meanwhile, the PBMCs were5×106/mL, then the relative expression of IL-17mRNA was detected, which promoted toaccomplish expectant target. The results showed that relative expression of IL-17mRNA wassignificantly increased at24h,48h,72h and120h in the PBMCs. The relative expression ofIL-17mRNA was increased with the time of stimulation.Conclusion: In the present research, the genes of goat TGF-β1and IL-6were clonedsuccessfully, and the recombinant proteins were harvested by prokaryotic expression in E.coli.The proliferation of goat’s PBMCs was stimulated with TGF-β1or IL-6. And relativeexpression of IL-17mRNA was significantly increased in the activated PBMCs with thestimulation by TGF-β1together with IL-6.