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通过PCR的方法,从番茄黄化曲叶病毒江苏分离物DNA中扩增到V2基因,并克隆到pMD18-T载体,序列测定结果显示其与报道的XH2分离物序列完全一致,核苷酸序列全长351 bp,编码115个氨基酸,推测分子量约13 kD。将该V2基因亚克隆到原核表达载体PET32a中获得重组表达载体PET32a-V2,转化大肠杆菌BL21(DE3),IPTG可诱导1个分子量约为32 kDa的融合蛋白(含His标签)表达,经Ni+NTA亲和柱纯化,获得纯化的重组蛋白,免疫家兔制备TYLCV-V2蛋白的多克隆抗体Anti-V2,间接ELISA测定Anti-V2的效价达1∶655 360,Western blot及DIBA分析结果表明Anti-V2可与V2蛋白发生特异血清反应,可为进一步研究V2蛋白的功能提供参考。
The V2 gene was amplified from the DNA of tomato yellow leaf curl isolate Jiangsu by PCR and cloned into pMD18-T vector. The results of sequencing showed that it was completely consistent with the reported XH2 isolate. The nucleotide sequence The full length of 351 bp, encoding 115 amino acids, suggesting a molecular weight of about 13 kD. The V2 gene was subcloned into the prokaryotic expression vector PET32a to obtain the recombinant expression vector PET32a-V2, which was transformed into E. coli BL21 (DE3). IPTG induced a fusion protein (His-tag) with a molecular weight of about 32 kDa. + NTA affinity column to obtain the purified recombinant protein, immunized rabbits to prepare polyclonal antibody TYLCV-V2 protein Anti-V2, indirect ELISA measured Anti-V2 titer of 1: 655 360, Western blot and DIBA analysis results It indicates that Anti-V2 can react specifically with V2 protein and provide a reference for further study on the function of V2 protein.