本研究旨在体外表达质体型淀粉磷酸化酶C端结构域融合蛋白GST-SPI-C，免疫新西兰大白兔制备并纯化SPI多克隆抗体。通过原核表达载体构建、GST融合蛋白诱导表达与纯化制备抗原。以GST-SPI-C端蛋白为抗原免疫新西兰大白兔分离抗血清并进行抗体纯化，成功制备了SPI多克隆抗体。用制备的SPI抗体进行了玉米体内外蛋白Western blot验证。结果表明成功构建的PGEX-6T-1-SPI-C表达载体在大肠杆菌中表达并纯化了GST-SPI-C融合蛋白，通过免疫新西兰大白兔制备并纯化了SPI多克隆抗体。制备的SPI多克隆抗体不但能识别GST-SPI-C融合蛋白，而且能识别玉米体内全长SPI蛋白(112KD)，并具有较好的特异性。玉米籽粒和胚乳的western blot检测表明SPI蛋白主要积累在胚乳中，与mRNA定量结果一致。这些结果表明成功制备了玉米SPI多克隆抗体，为进一步研究SPI蛋白作用机制具有重要意义。
英文摘要:
The aim of this study was to express the plastidial starch phosphorylase C-terminal domain fusion protein GST-SPI-C in vitro, and to immunize New Zealand white rabbits to prepare and purify SPI polyclonal antibody. GST-SPI-C antigen was prepared by prokaryotic expression vector construction, expression and purification of GST-SPI-C protein. With GST-SPI-C as antigen to immunize New Zealand white rabbits, Antiserum was isolated and purified from New Zealand white rabbit to prepare antibody successfully. The prepared SPI antibody was tested by western blot of maize protein in vitro and in vivo. The results showed that the successfully constructed PGEX-6T-1-SPI-C expression vector. GST-SPI-C fusion protein was expressed and purified from E. coli. SPI polyclonal antibody was prepared and purified using GST-SPI-C fusion protein as antigen to immunize New Zealand white rabbits. The prepared SPI polyclonal antibody not only recognizes the GST-SPI-C fusion protein, but also recognizes the full-length SPI protein (112KD) in maize with good specificity. Western blot analysis of maize kernels and endosperm showed that SPI protein mainly accumulated in the endosperm, which was consistent with the quantitative results of mRNA. These results indicate that the successful preparation of maize SPI polyclonal antibody is of great significance for further study of the mechanism of action of SPI protein.
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