鼠TNF家族的B细胞活化因子ORF基因的克隆及原核表达

目的克隆鼠TNF家族的B细胞活化因子(Bcell activating factor of TNF family,BAFF)ORF基因,并进行原核表达及纯化。方法提取鼠新鲜脾脏组织总RNA,经RT-PCR扩增BAFF ORF基因,测序后,克隆入载体pMAL-c2x,构建重组表达质粒pMAL-c2x/BAFF ORF,转化大肠杆菌Rosseta(DE3),IPTG诱导表达,表达产物经亲和层析纯化后,进行Western blot分析。结果 RT-PCR扩增得到约930bp的cDNA,其序列与GenBank中登录的鼠BAFF ORF cDNA序列的同源性达99.9%;重组表达质粒pMAL-c2x/BAFF ORF经酶切鉴定构建正确;表达的重组蛋白相对分子质量约为76500,IPTG终浓度为0.9mmol/L,诱导时间为4h,目的蛋白的表达量最高,破菌上清和沉淀中均可见目的蛋白条带,可溶性蛋白的表达量占全菌总蛋白的49%;纯化的重组蛋白纯度可达90%,含量为0.9mg/ml,且具有良好的反应原性。结论已成功克隆了鼠BAFF ORF基因,并进行了原核表达及纯化,为进一步研究其生物功能奠定了基础。

Cloning and Prokaryotic Expression of ORF of Murine B Cell Activating Factor of TNF Family

Objective To clonethe ORF of murine B cell activating factor of TNF family(BAFF),express in prokaryotic cells and purify the expressed product.Methods Total RNA was extracted from fresh spleen of mice,from which the ORF of BAFF was amplified by RT-PCR and cloned into vector pMAL-c2x.The constructed recombinant plasmid was transformed to E.coli Rosseta(DE3)for expression under induction of IPTG.The expressed product was purified by affinity chromatography and analyzed by Western blot.Results A cDNA fragment at length of about 930 bp was amplified by RT-PCR,of which the homology was 99.9% to the ORF cDNA sequence reported in GenBank.Restriction analysis proved that recombinant plasmid pMAL-c2x /BAFF ORF was constructed correctly.The relative molecular mass of expressed recombinant protein was about 76 500.The expression level reached a peak value after induction with IPTG at a final concentration of 0.9 mmol/L for 4 h.Target protein bands were observed in both supernatant and precipitate of ultrasonicated E.coli.The expressed product in soluble form contained 49% of total somatic protein.The purified recombinant protein reached a purity of 90% and a content of 0.9 mg/ml,and showed good reactogenicity.Conclusion Murine BAFF ORF was successfully cloned,expressed in prokaryotic cells and purified,which laid a foundation of further study on its biological function.