蜱抗凝血肽与RGDS肽重组基因的酵母表达载体构建

目的构建蜱抗凝血肽与RGDS肽重组基因,将其克隆到甲醇酵母,建立分泌表达载体pPIC9K。方法设计、合成蜱抗凝血肽与RGDS肽的双功能分子的重组基因,对人工合成的目的基因片段进行酶切、测序鉴定。运用PCR技术,扩增目的基因,PCR产物经胶回收后克隆至毕赤酵母表达载体pPIC9K,用双酶切和DNA测序鉴定重组质粒。结果PCR、酶切鉴定和DNA测序证明双功能基因重组质粒构建成功。结论该方法能方便、高效地获得所需的多拷贝基因,为下一步在毕赤酵母中表达双功能分子蛋白建立了基础。

Construction of recombinant plasmid pPIC9K inserted with genes of tick anticoagulant peptide and RGDS peptide

To construct a recombinant plasmid pPIC9K inserted with the genes of tick anticoagulant peptide and RGDS peptide(TAP-RGDS),the TAP-RGDS gene was designed and synthesized artificially,then confirmed by restriction enzyme analysis and DNA sequencing.The TAP-RGDS gene was amplified by polymerase chain reaction(PCR).and the PCR product was purified after agarose gel electrophoresis,and was cloned into an expression vector pPIC9K for yeast Pichia pastoris.The recombinant vector was confirmed by restriction enzyme analysis and DNA sequencing.It was confirmed that TAP-RGDS gene was inserted correctly into the vector pPIC9K by the above techniques.The desired multi-copy gene can be obtained conveniently and efficiently by these methods,which provides a basis to express the recombinant protein in yeast P.pastoris.