犬细小病毒VP2蛋白的原核表达与纯化

为了研究犬细小病毒（canine parovirus，CPV）VP2蛋白的结构和功能，本试验对CPV VP2蛋白进行表达和纯化。采用大肠杆菌表达外源蛋白的方法，将CPV VP2基因插入原核表达载体pET-32a（+）中构建重组原核表达载体pET-VP2，转化至大肠杆菌BL21（DE3）感受态细胞中，在不同IPTG浓度、诱导温度和诱导时间条件下进行原核表达，确定最佳诱导条件。最佳条件下的诱导产物经超声破碎离心后，利用镍柱对重组蛋白进行纯化并用SDS-PAGE和Western blotting进行双重鉴定。结果显示，重组质粒pET-VP2经双酶切鉴定分别获得大小为5 900 bp左右的载体条带和1 755 bp左右的目的基因条带，成功构建了pET-VP2重组质粒；重组VP2蛋白的分子质量约为64 ku，与预期大小一致，且在37℃、1.0 mmol/L IPTG、诱导5 h条件下表达量最高，条带最亮；蛋白超声破碎后经SDS-PAGE发现，只在沉淀中出现了目的条带，而上清中并未出现相应条带，说明重组蛋白均以包涵体的形式存在；纯化后获得的重组蛋白，经SDS-PAGE和Western blotting双重鉴定，均在64 ku处出现条带，说明纯化后的蛋白为重组蛋白pET-VP2。本试验通过对CPV VP2蛋白的原核表达及纯化成功获得了大量纯度较高的CPV VP2蛋白，为今后制备CPV VP2蛋白多克隆抗体及进一步研究该蛋白在对治疗犬细小病毒病中的临床应用价值提供了基础理论依据。

Prokaryotic Expression and Purification of VP2 Protein of Canine Parvovirus

This experiment was aimed to study the structure and function of VP2 protein of canine parvovirus (CPV),and we expressed and purified its VP2 protein.E.coli was used to express foreign proteins,the CPV VP2 gene was inserted into the prokaryotic expression vector pET-32a(+) to obtain the recombinant plasmid pET-VP2 and then transformed into E.coli BL21(DE3),prokaryotic expression was carried out under different concentrations of IPTG,induction temperature and induction time,to find the best induction conditions.The expression product was sonicated and purified by nickel column.The recombinant proteins were identified by SDS-PAGE and Western blotting.The recombinant plasmid pET-VP2 was identified by double enzyme digestion,it appeared about 5 900 bp carrier band and about 1 755 bp target gene band,pET-VP2 recombinant plasmid was successfully constructed;The molecular weight of the recombinant VP2 protein was about 64 ku,the best condition was 37 ℃ induction for 5 h with 1.0 mmol/L IPTG.The result of SDS-PAGE showed that the target band only appeared in the precipitate,and did not appear in the supernatant,which indicated that the recombinant protein existed in the form of inclusion body;The purified recombinant protein was double-stained by SDS-PAGE and Western blotting,and showed a band of 64 ku in size,indicating that the purified protein was the recombinant protein pET-VP2.It provided the basic theoretical basis for the future preparation of polyclonal antibody to VP2 protein of CPV,and further study on the therapeutic effect of VP2 protein in the treatment of CPV.