烟草花叶病毒运动蛋白的表达及特异性抗体制备

从烟草花叶病毒(Tobacco mosaic virus,TMV)感染的发病烟草叶片中提取总RNA,通过RT-PCR扩增得到其运动蛋白基因,将扩增产物克隆到pMD18-T载体上.DNA序列分析表明,所得运动蛋白基因全长为807 bp,与已报道的TMV-U1株系核苷酸和氨基酸同源性均为100%.将目的基因亚克隆到原核表达载体pET-29a上,并转化大肠杆菌BL21(DE3),IPTG诱导4 h后蛋白表达量达到最大,超声波显示所得融合蛋白以不可溶形式存在.SDS-PAGA检测蛋白表达情况,表达产物与目的蛋白大小一致,割胶免疫注射家兔得到抗体,ELISA测定效价为25600,Western-blot检测证明在烟草叶片被侵染早期MP得到表达,且抗体特异性良好.

Expression and antibody preparation of Tobacco mosaic virus movement protein

The movement protein(MP) gene of Tobacco mosaic virus(TMV) was obtained by RT-PCR from the total RNA of TMV infected leaves and cloned to pMD18-T vector,whose length was 807 bp,compared with the TMV-U1 strain in GenBank,the identities of neucleotides and amino acids were both 100%.The target fragment was subcloned to an expression vector pET-29a,and the fusion protein was expressed in the E.coli BL21(DE3) strain,the highest amount of target protein was gained 4 hours after IPTG inducement,which was insoluble.The antibody against the movement protein was prepared by immunized rabbit with target protein cut from SDS-PAGE,a high titer 25600 was determined by ELISA procedure.Western-blot analysis indicated that movement protein was expressed in infected leaves at an early stage,and the antibody showed a good speciality also.