茶树α-tubulin基因的原核表达及其多克隆抗体的制备

Prokaryotic Expression of α-tubulin Gene of Camellia sinensis and Preparation of α-tubulin Polyclonal Antibody

根据茶树α-微管蛋白(α-tubulin)的mRNA全长序列(GenBank登录号DQ340766)设计引物,以RT-PCR方法扩增茶树α-tubulin基因,将扩增产物克隆到原核表达载体pET-32a(+)上,并转化至大肠杆菌BL21trxB(DE3)中,经异丙基-β-D硫代半乳糖苷(IPTG)诱导后以包涵体形式表达。以纯化后的α-tubulin融合蛋白制备兔多克隆抗体,用Westernblot方法检测抗体特异性。结果显示α-tubulin蛋白以包涵体形式大量表达,以融合蛋白制备的抗体对茶树体内的α-tubulin具有良好的特异性。

According to the relevant complete mRNA sequence of α-tubulin gene of Camellia sinensis （accession NO. DQ340766）, the coding region of α-tubulin gene was amplified using RT-PCR method, and its products were expressed in Escherichia coli using the pET-32a（＋） vector. The fusion protein was expressed in the form of inclusion body when induced with isopropyl-D-thiogalatopyranoside （IPTG）, then was purified to immunize the rabbit to produce the polyclonal antibody against α-tubulin. The specificity of the antibody was confirmed by Western blot analysis. The results of Western blot revealed well specificity of the antibody against α-tubulin in Camellia sinensis plant.