為分析藍(lán)舌病I型病毒（BTV-1）VP5蛋白的免疫原性，本研究構(gòu)建了載體PET-28a-VP5并轉(zhuǎn)化BL21（DE3），然后進(jìn)行IPTG誘導(dǎo)表達(dá)，以及SDS-PAGE和Western-blot分析。結(jié)果顯示，重組VP5蛋白相對分子質(zhì)量約為62 kDa，與預(yù)期結(jié)果一致。進(jìn)一步優(yōu)化條件，發(fā)現(xiàn)IPTG為0.2 mmol/L、溫度為25℃、誘導(dǎo)6 h時，重組蛋白在上清中表達(dá)量最高，通過Ni柱純化獲得的重組蛋白純度約為75%。將純化后的重組蛋白VP5分別以40μg/只和20 μg/只免疫Balb/c小鼠，同時設(shè)置PBS為陰性對照，對三免后血清進(jìn)行間接ELISA檢測，發(fā)現(xiàn)高、低劑量免疫小鼠均能產(chǎn)生針對VP5蛋白的特異性抗體。用滅活BTV-1進(jìn)行DOT-ELISA檢測，發(fā)現(xiàn)其能與VP5蛋白免疫的小鼠血清發(fā)生特異性反應(yīng)，說明利用大腸桿菌表達(dá)的重組蛋白VP5具有良好的免疫特性，這為進(jìn)一步開展BTV相關(guān)研究奠定了基礎(chǔ)。
Expression，Purification and Immunogenicity Analysis onVP5 Protein
of Bluetongue Virus Serotype I
In order to analyze the immunogenicity of VP5 protein of bluetonguevirus serotype I（BTV-1），in this paper，the prokaryotic expression vector PET-28a-VP5 was constructed andtransformed into BL21（DE3）.After inducible expression by IPTG，the VP5 protein waspurified and analyzed by means of SDS-PAGE and Western-blot. The results showedthat the relative molecular mass of recombinant VP5 protein was about 62 kDa，which was consistent with the expected result. It was found that，by further optimizing，the expression levelof recombinant protein in supernatant was maximum when IPTG was 0.2 mmol/L，the bacteria solution was inducted for 6 hours at the temperature of25℃，and the purity of recombinant VP5 protein throughNi-sepharose purification was about 75%. Then the purified VP5 protein wasimmunized to Balb/c mice at the doses of 40 and 20 μgper mouse respectively，meantime，PBS was set as the negative control. The serum samples were testedby indirect ELISA after being immunized for 3 times. It was concluded that thespecific antibody against VP5 protein appeared in immunized mice with high orlow dose. In addition，it was found that inactivatedBTV-1 could specifically react with the serum of immunized mice with VP5protein by DOT-ELISA test，indicating that theimmunogenicity of recombinant VP5 protein was good，whichlaid the foundation for further research on BTV.
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