辣根过氧化物酶同工酶C基因在巴斯德毕赤酵母中的克隆与鉴定

目的：克隆辣根过氧化物酶同工酶C基因，为此基因的表达作准备。方法：用PCR方法从辣根的总DNA中扩增得到一种辣根过氧化物酶同工酶C基因HRPC2，通过PCR的方法去除内含子后连接到pMD18-T载体上，测序证明正确后，用限制性内切酶切下目的基因，插入到巴斯德毕赤酵母表达载体pPIC9K中，构建成重组质粒pC2EX9K。再将辣根过氧化物酶同工酶C基因在毕赤酵母中进行克隆、鉴定。结果：重组质粒pC2EX9K转化毕赤酵母后，经PCR鉴定，证明形成了目的基因的克隆。结论：应用毕赤酵母作为受体菌，pPIC9K为载体，成功克隆了HRPC2。

Cloning and Identification of Horseradish Peroxidase Isozyme C Gene in Pichia pastoris

Objective:To clone the Horseradish peroxidase isozyme C(HRPC2) of horseradishfor the expression of the gene.Methods:The HRPC2 gene was amplified with PCR from the total DNA of Horseradish.After introns of HRPC2 were cutted,it was linked into pMD-18Tvector.When it was confirmed by sequencing,the target gene was cut down by restriction endonuclease.Then it was inserted into expression vector pPIC9K of Pichia pastoris ,and the recombinant plasmid pC2EX9K was obtained.The HRPC2 gene was cloned in Pichia pastoris and was identified.Results:The recombinant plasmid pC2EX9K was transformed into Pichia pastoris .Then,the cloned target gene was confirmed by PCR analysis.Conclusion:The HRPC2 gene was cloned successfully using pPIC9K as the vector and using Pichia pastoris as host bateria.