爲分析藍舌病I型病毒（BTV-1）VP5蛋白的免疫得煙原性，本研究構建了載體PET-28a-VP5并轉化BL21（DE3），然後答人(hòu)進(jìn)行IPTG誘導表達，以及SDS-PAGE和Wes腦師tern-blot分析。結果顯示，重組VP5蛋白相對(duì)分子質量約爲62動放 kDa，與預期結果一緻。進(jìn)一步優化條件，發(fā麗新)現IPTG爲0.2 mmol/L、溫度爲25℃、友答誘導6 h時(shí)，重組蛋白在上清中表達量最高，通過(guò)Ni柱純化獲科鐘得的重組蛋白純度約爲75%。將(jiāng)純化後(hòu)的重組蛋白VP5事不分别以40μg/隻和20 μg/隻免疫Balb/c小鼠，同時新做(shí)設置PBS爲陰性對(duì)玩民照，對(duì)三免後(hòu)血清進(jìn)行坐下間接ELISA檢測，發(fā)現高、低劑量免疫小鼠均能(néng好微)産生針對(duì)VP5蛋白的特異性抗體。用滅活BTV-我還1進(jìn)行DOT-ELISA檢測，發(fā)現其能(néng)與VP5哥多蛋白免疫的小鼠血清發(fā)生特異性反應，說(shuō)明利用大腸商得杆菌表達的重組蛋白VP5具有良好(hǎo行司)的免疫特性，這(zhè)爲進(jìn)一步開(kāi)展B們影TV相關研究奠定了基礎。
Expression，Purification and Immun可年ogenicity Analysis onVP5 Pro笑跳tein
of Bluetongue Virus Serot見現ype I
In order to analyze the immun都藍ogenicity of VP5 protein of bluetongu放村evirus serotype I（BTV-1），in this paper，the prokaryoti煙志c expression vector PET-28a-VP5 was校笑 constructed andtransformed into BL21（D在什E3）.After induc計林ible expression by IPTG，the VP5 p山外rotein waspurified and analyzed by mean的有s of SDS-PAGE and West很什ern-blot. The results showedthat the r資門elative molecular 科刀mass of recombinant VP5 protein was志靜 about 62 kDa，which wa拿機s consistent with the expected 問北result. It was found that，by further op們熱timizing，the expressi理影on levelof recombinant protein in sup湖紅ernatant was maxi年年mum when IPTG was 0.2 懂湖mmol/L，the bacteria solution was i美些nducted for 6 hours at the錯議 temperature of25℃，and the p話冷urity of recombinant V也制P5 protein throughNi-sepharose pu湖謝rification was about 75%. Then the p月業urified VP5 protein wa資市simmunized to Balb/c mice at the子訊 doses of 40 and 20 μ中他gper mouse respectively，meantime，P作厭BS was set as the negativ秒謝e control. The serum samples were test民筆edby indirect ELISA after b笑樹eing immunized for 3 times. It was c朋飛oncluded that thespecific antibody 北區against VP5 prot新做ein appeared in immunized mi我身ce with high orlow dos坐兒e. In addition，it was fo員民und that inacti現的vatedBTV-1 could specifically react紅火 with the serum of immuni懂就zed mice with VP5protein by DOT-ELIS飛鄉A test，indicating th時道at theimmunogenicity of recombinant農冷 VP5 protein was good，whichl懂紙aid the foundation for further re新懂search on BTV.
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