The Effect Of Androgens On Sheep KAP6.1 Gene Polyclonal Antibody Preparation And KAP4.2 Gene Eukaryotic Expression

[Objective]To preliminarily screen the effects of different androgen concentrations on the expression of sheep keratin and on the preparation of sheep hair follicle keratin polyclonals.[Method]Hetian sheep?Mountain type,Plain type?and Karakul sheep were used as experimental materials.Extract three kinds of sheep skin total RNA and reverse transcribed into template c DNA.And directional insert p MD19-T cloning vector,construct p MD19-T-KAP6.1 cloning recombinant plasmid,then double enzyme digestion,and construct p ET28a?+?-KAP6.1 prokaryotic expression recombinant plasmid,and import it into BL21?DE3?E.coli Expression was detected by SDS-PAGE and Western Blot.Different concentrations of androgen were added in the culture of protein-expressing bacteria,and protein expression was detected with protein quantitative kit to detect the protein expression to obtain the optimal androgen concentration,then set the experimental group with the optimal concentration,and immunize female young rabbits with adjuvant to obtain polyclones Antibody serum,use indirect ELISA to detect polyclonal antibody titer.Design specific primers for hair follicle keratin KAP4.2 gene,PCR amplify the target fragment,construct p MD19-T-KAP4.2 cloned recombinant plasmid,after successful sequencing,construct p EGFP-N1-KAP4.2eukaryotic expression recombinant plasmid,then transfer Stain COS-7 cells,culture the cells with cell culture medium containing different concentrations of androgens,detect fluorescence,quantitative protein analysis,and do Western Blot detection.[Results]?1?The prokaryotic expression of hair follicle keratin KAP6.1 gene was detected by Western Blot and the expressed protein was about 11KDa.?2?The prepared polyclonal antibody to hair follicle keratin KAP6.1 has a higher titer by indirect ELISA.?3?The hair follicle keratin KAP4.2 gene was transfected into COS-7 cells,and the green fluorescence was observed by fluorescence microscope.[Conclusion]When the concentration of androgen is 10^-8mol/L,it can promote the expression of KAP6.1 gene keratin in three kinds of sheep hair follicles,and also promote the preparation of polyclonal hair follicle keratin KAP6.1,and it can also promote the KAP4.2 gene of hair follicles.Keratin eukaryotic protein expression also has a corresponding enhancement effect.