【摘要】 采用RT-PCR方法自甘蔗花叶病毒北京玉米分离物(SCMV-BJ)的基因组中分离出其HC-Pro基因,通过T4DNA聚合酶连接到原核表达载体pET-22b(+)上,获得的重组子pETHC转化大肠杆菌BL21(DE3),用IPTG在37℃进行诱导表达。SDS-PAGE和Westernblot分析表明,HC-Pro基因在大肠杆菌中获得了高效表达,产生分子量约为52kDa的融合蛋白。将融合蛋白纯化后免疫兔子,获得了特异性的抗血清并提取了抗体IgG。ACP-ELISA测定抗血清的效价为1/8192。结果表明,该血清可用于SCMV-BJ的检测,并为进一步分析HC-Pro的功能奠定基础。更多还原

【Abstract】 The HC-Pro gene of the Beijing maize isolate of Sugarcane mosaic virus(SCMV-BJ) was amplified by RT-PCR, and ligated to the expression vector pET-22b(+). The recombinant plasmid pETHC was transformed into E.coli BL21(DE3) and then induced to express by IPTG. The results of SDS-PAGE and Western blot analysis showed that the specific fusion protein with molecular weight of 52kDa was highly expressed by IPTG induction under the condition of 37℃ and was immunoreactive. Rabbit was immunized with purified fusion protein to obtain the antiserum with high specificity. The titer of the antiserum was 1∶8192 determined by antigen coating plate-ELISA (ACP-ELISA). The antiserum is useful for identification of SCMV-BJ in plant materials and further study on HC-Pro function.更多还原