犬α干扰素基因的高效表达及其活性测定.pdf

犬 α干扰素基因的高效表达及其活性测定 王 　艳 ,王海震 ,曹瑞兵 ,陈溥言 (南京农业大学农业部动物疫病诊断与免疫重点实验室 ,江苏南京 2 10095) High Level Expression and Activity Assay of Canine Interf eron a Gene WAN G Yan , WAN G Haizhen , CA O Ruibing , C H EN Puyan ( Key laboratory of A nimal D iseases D iag nosis and I mmunology , M inistry of A g ricult ure , N anj ing A g ricultural University , N anj ing 210095 , China ) Abstract : Wit h t he rever se t ran scrip tion chain reaction ( R TPCR) , t he gene of Canine′s int erf e ( α) ronalp ha Ca IFN wa s amp lified and cloned , f ro m t he tot al RN A in t he lymp hocyt e s stimula t ed wit h concanavalin A f ro m t he p erip heral bloo d of do g . The amp lified f ragment wa s in sert ed into t he p ro karyotic exp re ssing vector pBV220 an d t hen sequenced . Sequencin g re sult showed t hat t he ho molo gy wa s 100 % bet ween t he gene we clo ned and t he one p ubli shed in GenBank . SD SPA GE a ssay showed t hat t he t ar get p rot ein , wit h t he molecular weight of 19kDa , coul d be exp re ssed in a high level and up to 27 . 6 % of t he tot al p rot ein in E. col i cell , in t he for m of inclu sio n bo die s. The t ar get p rot ein wa s added to MDC K cell s ,w hich were sub sequent ly inf ect ed by α t he 100 TCID50 V SV . The re sult showed goo d cyto kine activit y . The reco mbinant Ca IFN activ 6 it y wa s up to 5 . 11 ×10 U/ . Key words : Canine int erf ero nalp ha gene ;Pro karyotic exp re ssion ; Reco mbinant int erf eron 摘