Li Wei, Li Xiujin, Zhong Fei, Jin Huijun, Xie Min, Liu Yuzhi, Liu Longfei, Su Qingjie
Department of Basic Veterinary Medicine, College of Animal Science and Technology, Agricultural University of Hebei, Baoding 071001, China.
Sheng Wu Gong Cheng Xue Bao. 2009 Oct;25(10):1470-6.
To prepare recombinant fox growth hormone (fGH), we amplified its cDNA from silver fox pituitary tissue by RT-PCR and cloned into yeast shuttle vector pPIC9K down stream of a-factor signal peptide sequence by SnaB I and Not I restriction sites. The recombinant secretion vector pPIC9K/fGH, linearized by Sal I, was transformed into histidine-deficient Pichia pastoris strain GS115 by electroporation. We selected His+ -transformed methylotropic (His+, Mut+) yeast using histidine-absent medium containing dextrose (MD) or methanol (MM) as the only carbon source, and then screened the recombinant GS115 with multi-copy fGH genes by G418. The secretive expression of fGH was performed under the induction of methanol in shaking flask culture. The results showed that the fGH cDNA sequence amplified in this paper was basically in consistence with the published in GenBank. We achieved the secretive expression of recombinant fGH identified by SDS-PAGE and Western blotting. The fGH expression level was 119 mg/L, accounted for 34% of total proteins in fermentation medium.
为制备重组狐生长激素（fGH），我们通过逆转录聚合酶链反应（RT-PCR）从银狐垂体组织中扩增出其cDNA，并利用SnaB I和Not I限制性酶切位点将其克隆到酵母穿梭载体pPIC9K的α-因子信号肽序列下游。经Sal I线性化的重组分泌载体pPIC9K/fGH通过电穿孔法转化入组氨酸缺陷型毕赤酵母菌株GS115。我们使用以葡萄糖（MD）或甲醇（MM）作为唯一碳源的无组氨酸培养基筛选出His⁺转化的甲基营养型（His⁺，Mut⁺）酵母，然后用G4筛选出具有多拷贝fGH基因的重组GS115。在摇瓶培养中，在甲醇诱导下进行fGH的分泌表达。结果表明，本文扩增的fGH cDNA序列与GenBank中公布的序列基本一致。我们通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹法（Western blotting）实现了重组fGH的分泌表达。fGH的表达水平为119 mg/L，占发酵培养基中总蛋白的34%。
