摘要
经生物学软件DNAStar分析,参照已发表的禽流感病毒(avian influenza virus,AIV)(H5N1)基因组序列,设计合成1对特异性引物,RT-PCR扩增了长约750bp的M1基因片段,将目的片段定向克隆至pET30a表达载体,经酶切及测序鉴定正确后转化BL21表达菌,经IPTG诱导获得以包涵体形式表达的重组蛋白。将重组蛋白变性、纯化和复性后,BCA法测定纯化蛋白的浓度为0.656mg/mL,免疫印迹检测结果表明,纯化的重组蛋白具有良好的反应活性。将纯化蛋白免疫BALB/c小鼠,间接ELISA检测结果表明,重组蛋白可产生抗M1蛋白特异性抗体,具有良好的免疫原性。本研究成功表达了流感病毒H5N1的基质蛋白M1,且重组蛋白具有良好的免疫原性,为进一步研制H5N1亚型流感病毒诊断试剂及基因工程疫苗奠定基础。
Based on published AIV（HSN1）genome sequence, a fragment of about 750 bp long was amplified by PCR tech- nique with specific primers using biological software DNAStar to analysis. Then the amplified product was directionally cloning into pET30a expression vector. After identifying with enzyme cut and sequencing, the recombinant plasmid was transformed into E. coli BL21 （DE3）. The recombinant protein M1 was expressed in inclusion body form in E coli after induction with IPTG. After denaturation, purification and renaturation, the concentration of purified protein was 0. 656 mg/mL, Western blotting showed the recombinant protein bad a good immunogenicity. The purified protein immunized BALB/c mice, indirect ELISA showed that the recombinant protein could produce specific antibodies anti-M1 IgG, which had a good immunogenicity. That is a better basic for diagnostic and genetically engineering vaccine research on H5N1 influenza virus.
出处
《中国畜牧兽医》
CAS
北大核心
2012年第6期46-49,共4页
China Animal Husbandry & Veterinary Medicine
基金
内蒙古自治区自然科学基金项目(2009MS1113)
