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目的构建糖类标记载体筛选克隆。方法以pPIC9K-βGl2为模板,通过PCR扩增得到全长的Bgl2基因片段,克隆至pET-28a载体,转化E.coli BL21(DE3),Cel-M9培养基筛选阳性克隆,经DNA测序分析,成功构建pET28a-Bgl2表达载体。SDS-PAGE显示,重组菌经IPTG诱导后表达了目的蛋白,其相对分子质量与预期结果相符。结果 PCR扩增得到Bgl2基因片段,SDS-PAGE显示蛋白以包涵体形式表达,克隆可用Cel-M9培养基筛选。结论用Cel-M9培养基筛选出阳性克隆为构建食品级载体奠定基础。
Objective To construct a carbohydrate marker vector for screening clones. Methods The full-length Bgl2 gene fragment was amplified by PCR using pPIC9K-βGl2 as template. The full length Bgl2 gene fragment was cloned into pET-28a vector and transformed into E.coli BL21 (DE3). The positive clones were screened by Cel-M9 medium. The pET28a-Bgl2 expression vector was successfully constructed. SDS-PAGE showed that the target protein was expressed after induced by IPTG, and the relative molecular mass was consistent with the expected result. Results The fragment of Bgl2 gene was amplified by PCR. SDS-PAGE showed that the protein was expressed in inclusion bodies. The clones could be screened by Cel-M9 medium. Conclusion The positive clones screened with Cel-M9 medium laid the foundation for the construction of food-grade vector.