【摘要】 通过生物信息学预测,番茄环纹斑点病毒(Tomato zonate spot virus,TZSV)Gn蛋白为跨膜蛋白,有3个跨膜结构域,抗原表位预测发现非跨膜区包含有较高的抗原指数区域,因此选择Gn蛋白的非跨膜区进行原核表达及多抗血清制备。用特异性引物,通过RT-PCR从感染TZSV的番茄中扩增出部分Gn基因片段,将获得的片段构建到原核表达载体pET-30a中,获得原核表达载体pET-30a-Gn,转化大肠杆菌BL21(DE3),用IPTG诱导表达。SDS-PAGE电泳分析显示,高效诱导表达了40 kD融合蛋白。用回收纯化的融合蛋白免疫家兔,获得多抗血清。间接ELISA测定多抗血清的效价为1/16 384。利用制备好的抗血清对感染TZSV的病样进行Western blotting检测,能检测到特异性条带。结果表明该抗血清特异性良好,可用于TZSV Gn相关的免疫反应实验。更多还原

【Abstract】 Bioinformatics prediction showed that Gn protein in tomato zonate spot virus(TZSV)is a transmembrane protein consistingof 3 transmembrane domains. Antigen epitope prediction showed that there was a high antigen index region,thus non-transmembrane region ofGn was selected to conduct the prokaryotic expression and to prepare polyclonal antiserum. The Gn gene fragment of TZSV was amplified fromTZSV-infected tomato sample by RT-PCR,and then cloned into prokaryotic expression vector p ET-30 a. The recombinant plasmids p ET-30 aGn were transformed into Escherichia coli BL21(DE3),and then induced to express with IPTG. The result of SDS-PAGE analysis showed thespecific fusion protein with molecular weight of 40 k D was highly expressed. Using the purified fusion protein,rabbit was immunized to obtainpolyclonal antiserum against TZSV Gn protein. The titer of antiserum was 1/16384 determined by ID-ELISA. The specific band was detected byWestern blot while applying the prepared antiserum to the TZSV-infected samples. These results reveal that the specificity of the antiserum isfine,and it is applicable for immune-reaction test related to TZSV Gn.更多还原