The Effects Of Gossypol On Lymphocytes Nucleic Acids Of Duolang Sheep And Cloning And Expression Of IFN-γ From Duolang Sheep

Gossypol can affect the function of lymphocytes on duolang sheep, which were cultured in vitro. Observe gossypol in different times at the same concentration under the influence of lymphocytes and nucleic acid. Using Trizol method to extract RNA and DNA from lymphocytes, which was induced by gossypol at different times. nucleic acid was influenced by gossypol.The results showed that the phenomenon of lymphocytes degradation on duolang sheep, which were cultured in vitro appeared after3hours,after4hours appeared ladder stripe,after5hours completely degradation at0.071mol/L. Then RNA were appeared18S degradation after5hours.In this experiment, a pair of primers was designed and synthesized according to the sheep IFN-y gene sequence in Genbank, according to the accession number NM₀01009803from Genbank. Coding sequence of IFN-γ gene was amplified by RT-PCR technology from total RNA of Duolang sheep lymphocytes. The IFN-y target genes was cloned to pMD-18T vector and transfer into competent DH5a bacteria then got the positive recombinant, then recombinant sequence was identified by PCR and double digests method. The recombination strain was cultured overnight. The final recombination strain mixed with30%glycerol,sequenced by BGI LifeTech Co. Ltd. Its length was about582bp by sequencing, and multiple sequence alignment found391nucleotide mutations.Then IFN-γ was ligated into pET-28c vector and was identified by PCR, digestion with endonuelease,which proved to constructed a prokaryotic expression plasmid pET-28c-IFN-γ,then transformed pET-28c-IFN-γ into E.coliBL21and expressed approximately24.4KDa fusion protein induced by IPTG. Optimal expression conditions were determined by SDS-PAGE.The optial induction time was4.0h, the best temperature was37℃, and the optimal concentration of IPTG Was1.5mmol/L.Under the optial expression conditions,the fusion protein account14.2%of the total bacterium protein by Bandsean5.0. The rabbit were immunized by the recombination pET-28c-IFN-γ. After immunization, Western-blotting and the agar diffusion test analysis demonstrated that the expressed pET-28c-IFN-γ protein could be detected by positive serum from rabbit that the expressed pET-28c-IFN-γ protein has good reactivity, immunogenicity.