目的 检测布鲁杆菌Ⅳ型分泌系统蛋白VirB9的免疫原性,寻找潜在的、新的布鲁杆菌亚单位疫苗靶标.方法 采用双酶切的方法,将布鲁埃希菌VirB9基因全长克隆至原核表达载体pET32a上,将克隆后载有VirB9基因的pET32a质粒转化至大肠杆菌BL21 (DE3)中,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导重组VirB9蛋白在大肠埃希菌中的表达.利用重组VirB9蛋白所携带的His标签,通过Ni-NAT层析,纯化在大肠杆菌中重组表达的VirB9蛋白,再通过十二烷基硫酸钠-聚丙烯酰胺凝胶(SDS-PAGE)电泳和二喹啉甲酸(BCA)蛋白定量试剂盒对纯化的蛋白进行纯度及浓度检测.用布鲁杆菌疫苗株S19株免疫BAL B/c小鼠,并以磷酸盐缓冲液(PBS)为对照.在免疫4周后,鼠尾取血,血清虎红平板凝集试验、试管凝集试验检查小鼠血清抗体；酶联免疫吸附试验(ELISA)检测S19株免疫小鼠体内抗VirB9抗体滴度.在免疫后第35天,取小鼠脾脏,分离脾细胞,利用Elispot技术,检测VirB9蛋白体外再刺激后分泌细胞因子(干扰素-γ,IFN-γ)的脾细胞数,酶联斑点图像仪计数斑点,每个斑点代表1个抗原特异性T淋巴细胞,分析VirB9刺激机体产生细胞免疫反应的情况.结果 通过酶切克隆的方法成功地将VirB9全长基因741 bp克隆至pET32a载体上.SDS-PAGE显示,VirB9蛋白的相对分子质量约43×103,纯度超过97％；BCA法检测,蛋白浓度为1.6 g/L.免疫组小鼠血清虎红平板凝集试验阳性,试管凝集试验小鼠血清抗体滴度＞ 1∶800;对照组小鼠血清虎红平板凝集试验阴性,试管凝集试验小鼠血清抗体滴度阴性.免疫组检测到抗VirB9蛋白抗体,抗体滴度均＞1:3200,对照组小鼠体内未检测到抗VirB9蛋白抗体的存在.酶联斑点图像分析显示,用VirB9蛋白体外刺激,免疫组小鼠5×105个脾细胞中有147个细胞可以分泌IFN-γ,对照组仅有38个细胞.结论 在布鲁杆菌感染过程中,Ⅳ型分泌系统蛋白VirB9具有免疫原性,能够刺激小鼠产生体液免疫反应与细胞免疫反应.%Objective To detect the immunogenicity of VirB9,a protein of type Ⅳ secretion system of Brucella.Methods Full length VirB9 gene was cloned into plasmid pET32a and expressed in Escherichia (E.) coli BL21 (DE3).Expression of recombinant protein was induced by isopropyl beta-D-thiogalactopyranoside (IPTG) and the recombinant fusion protein was purified by affinity chromatography on Ni2+-conjugated chelateing sepharose.The purity of the purified protein was ascertained by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDSPAGE) and the concentration was measured by bicinchoninic acid (BCA) protein assay kit.Animal model was established by immunizing BAL B/c mice with live vaccine strain S19 of Brucella and the mice immunized with phosphate buffered saline (PBS) as control.The blood of immunized mice was acquired after 4 weeks.Antibody against VirB9 in S19 immunized mice was detected by Rose Bengal plate agglutination test and serum tube agglutination test; IgG antibody titers against VirB9 in immunized mice were determined by enzyme linked immunosorbent assay(ELISA).At the 35th day,the immunized mice and control mice were killed and spleens were collected.The splenocytes were harvested and stimulated with each of VirB9,concanvalin A(ConA) or medium in triplicate.Production of gamma interferon (IFN-γ) was determined by enzyme-linked immunospot assay (Elispot).Results The full length of VirB9 gene was cloned into pET32a.The recombinant VirB9 protein was expressed at 43 × 103 in relative molecular mass and the purity of the purified recombinant VirB9 protein was above 97％ in SDS-PAGE and the concentration was 1.6 g/L in BCA protein assay.The antibody of VirB9 was detected in all S19 immunized mice but not PBS immunized mice by Rose Bengal plate agglutination test.The antibody titer in all S19 immunized mice was ＞ 1 ∶ 800 or ＞ 1 ∶ 3200 by tube agglutination test and ELISA,respectively.Meanwhile,the protein stimulated stronger IFN-γresponse in immunized mice than that in the control mice(147 cells Vs 38 cells).Conclusion VirB9 can stimulate humoral and cellular immunity and it might be an appropriate target for developing subunit vaccine against Brucella.
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