Eukaryotic Expression Of Gene Sheep Wool Follicle Of KAP7 And Preparation Of Polyclonal Antibody In Local Breeds Of Southern Xinjiang

Hetian sheep of wool is excellent raw materials to product of the famous "Hetian carpet",Karakul sheep is a lambskin breed sheep,the wool quality improvement is conducive to support the sustainable development of local economy.The aim of this study was to provide some theoretical support and methods for the effects of KAP7 gene on the yield and quality of semi-wool in three local breeds in Plain-type Hetian sheep,Mountain-type Hetian sheep and Karakur sheep in XinJiang?In this study,extraction the follicle total RNA of Plain-type Hetian sheep,Mountain-type Hetian sheep and Karakur sheep,thus reverse transcription obtain to cDNA;(1)The KAP7 gene of hair follicle keratin was amplified by prokaryotic primers and thus constructed the recombinant plasmid pMD18-T-KAP7 identified by double digestion after,thus construction of pET28a(+)-KAP7 prokaryotic expression recombinant plasmid,it was expressed in BL21(DE3),subjected to SDS-PAGE electrophoresis,the purified protein was obtained by extraction,purification and recovery,Freundadjuvant and passively immunized rabbits obtained polyclonal antibody sera and tested for antibody titers using indirect of ELISA;(2)Real Time fluorescence quantitative PCR was used to detect the difference of expression level of KAP7 gene of hair follicle keratin between the three different types at the mRNA levels.(3)The KAP7 gene of hair follicle keratin was amplified by eukaryotic primers and thus construction of pMD18-T-KAP7 recombinant cloning plasmid and identified by restriction enzyme digestion,thus construction of recombinant plasmid p EGFP-N1-KAP7 was imported into COS-7 cells,the fluorescence was detected and analyzed by SDS-PAGE.The results showed that:(1)The recombinant plasmid pET28a(+)-KAP7 was expressed in prokaryotic expression of KAP7 gene of hair follicle keratin,and the protein was expressed in BL21 in a size of about 10 ku;(2)KAP7 gene of hair follicle keratin was obtained by Real Time fluorescence quantitative PCR in Hetian sheep and Karakur sheep are differences in expression at the mRNA levels.(3)The eukaryotic expression recombinant plasmid pEGFP-N1-KAP7 was successfully constructed,and expressed in COS-7 cells.Conclusion that:(1)KAP7 protein of hair follicle keratin passive immunization of rabbit-derived polyclonal antibody positive sera has a high immunological activity and specificity;(2)Although the expression of KAP7 gene of hair follicle keratin obtained by Real Time fluorescence quantitative PCR at the mRNA levels was similar between Plain-type Hetian sheep and Mountain-type Hetian sheep(P>0.05),was significant difference between the two HeTian sheep and karakur sheep(P<0.05);(3)KAP7 gene of hair follicle keratin can express protein of about 39 ku in COS-7 cells.