摘要
目的用大肠埃希菌表达布鲁氏菌S2株L7/L12蛋白,制备其多克隆抗体。方法设计引物,以S2弱毒株DNA基因组为模板扩增目的基因片段L7/L12,构建pET28a-L7/L12质粒并转化入E.coli BL21(DE3),IPTG诱导目的蛋白表达,SDS-PAGE检测表达蛋白的可溶性及其分子质量;金属亲和层析法纯化目的蛋白,BCA法对纯化蛋白定量。将rL7/L12蛋白与弗氏完全佐剂混合(终浓度为0.5mg/ml),每次1ml分两次间隔21d免疫新西兰大白兔,制备多克隆抗体。免疫结束后12d,颈静脉采血,分离血清,采用间接ELISA法检测血清抗体效价。以免疫血清1∶200倍稀释作为一抗,采用Western blot分析其与r-L7/L12、布鲁氏菌S2株全菌体蛋白及大肠埃希菌DH5α全菌体蛋白结合的特异性。结果经测序与酶切鉴定,成功构建表达载体pET28a-L7/L12,SDS-PAGA分析表达产物部分为可溶性,分子质量单位约为14ku;SDS-PAGE分析亲和纯化蛋白为单一条带,蛋白质含量为0.8mg/ml。间接ELISA检测免疫血清抗体效价为1∶12 800,该抗体能够与r-L7/L12、布鲁氏菌S2株全菌体蛋白结合,而不与大肠埃希菌DH5α菌体蛋白结合。结论成功克隆L7/L12基因并表达出目的蛋白,制备的抗布鲁氏菌S2株r-L7/L12多克隆抗体能特异识别该表达蛋白。
Objective To express the L7/L12 protein of Brucella suis in E.coli BL21 and prepare polyclonal antibodies. Methods Primers were designed in accordance with the gene encoding B.suis strain S2,and genomic DNA of B.suis strain S2 was extracted for amplification of L7/L12 gene fragments using PCR.The fragments were ligated into a pET-28 aplasmid.The constructed vector pET28a-L7/L12 was transformed into E.coli BL21 for expression via induction with IPTG.Soluble protein expression and the molecular weight of that protein were analyzed with SDS-PAGE.The protein was purified with immobilized metal ion affinity chromatography and its purity was determined with a bicinchoninic acid assay（BCA）and SDS-PAGE.An anti-L7/L12 polyclonal antibody was generated in New Zealand rabbits after two rounds of immunization with purified rL7/L12 and IFA（final concentration：250μg/ml）21days apart.Twelve d after immunization,serum was collected and the antibody titer was determined via indirect-ELISA.Rabbit serum was diluted1：200-fold to serve as the primary antibody,and Western blot was used to detect r-L7/L12 and total protein fromB.suis and E.coli DH5α. Results The expression vector pET28a-L7/L12 was successfully constructed as indicated by sequencing and restriction enzyme digestion.SDS-PAGE results indicated that rL7/L12 appeared as a band with a molecular mass of about 14 ku.Purified protein appeared as a single band on gels in SDS-PAGE,and protein content was 0.8mg/ml.Indirect-ELISA indicated the titer of the anti-L7/L12 serum was 1：128 000.Western blot indicated that the polyclonal antibody was highly specific to rL7/L12 and total protein fromB.suis but did not react with total protein fromE.coli DH5α. Conclusion The L7/L12 gene was successfully cloned and expressed in prokaryotic cells,and polyclonal antibodies against L7/L12 that recognized the L7/L12 protein were successfully prepared.
出处
《中国病原生物学杂志》
CSCD
北大核心
2014年第12期1067-1070,共4页
Journal of Pathogen Biology
基金
内蒙古自治区自然科学基金项目(No.2011MS1107)
