深黄被孢霉Δ~6-脂肪酸脱氢酶基因在毕赤氏酵母SMD1168中表达的研究

Study on the Expression of Mortieralla Isabellina Δ~6-fatty Acid Desaturase Gene in the Pichia Pastoris Yeast SMD1168

引物根据△6-脂肪酸脱氢酶（D6D）基因以及酵母蛋白表达载体pGAPZctA的多克隆位点进行引物设计，并在上游插入ECoRI位点、下游插入XhoI位点，从深黄被孢霉中克隆D6D基因，构建pMD18-T-D6D载体与pGAPZtxA—D6D载体。将重组质粒线性化，并将其电转导入到毕赤氏酵母（PichiapastorisSMD1168）中，Zeocin抗生素筛选高拷贝转化子，对重组菌进行诱导表达。经过Tricine—SDS—PAGE分析和蛋白质免疫印迹（Westernbloting）试验，证明△6-脂肪酸脱氢酶融合蛋白具有免疫学活性。结果成功构建了重组酵母pGAPZdA—SMD1168，为利用基因工程菌生产y-亚麻酸（GLA）奠定理论基础。

Primers are designed based on △ 6- fatty acid desaturase （D6D） genes conserved region and pGAPZaA vector cloning sites. For primer design, downstream, respectively, introduced ECoR I and Xho I restriction sites. From Mortieralla isabellina cloned △6-fatty acid desaturase gene （D6D） . D6D gene is cloned from Mortieralla isabellina, vector pMD18-T- D6D with pGAPZaA-D6D. That recombination vector is obtained. After ligation of the plasmids by Avr II , being integrated into the genome of host yeast P. pastoris SMD1168 by electroporation and sereening the converter by Zeocin. To induce expression recombinant bacteria, and the Immunologic competence is detected with Tricine-SDS-PAGE and Western bloting. The Yeast pGAPZa A-SMD1168 plasmid is construeted suceessfully. For the mass production of GLA has laid a solid foundation.