【摘要】 通过重叠区扩增基因拼接法(SOE)合成抗菌肽天蚕素B基因,并在其N端引入Kex2酶切位点。亚克隆天蚕素B并将3个亚克隆串联在一起,每个单体前都加上Kex2酶切位点,将天蚕素B以及串联体克隆至表达载体pPICZαA上,用SacⅠ酶切使之线性化,采用电击法转化毕赤酵母SMD1168,转化子用小瓶发酵。经Tricine-SDS-PAGE检测,在α信号因子的引导下,表达产物可以分泌到培养基中,且具有明显抑菌活性。更多还原

【Abstract】 Cecropin B gene was achieved through the gene splicing by overlap extension(SOE).Especially a Kex2 signal cleavage site was fused in N end of the antibacterial peptide gene.Cecropin B gene was subcloned and ligated tandem.The modified Cecropin B and its tandem genes cloned into the pPICZαA vector to construct the recombinant expression vectors.The recombinant expression vectors were linearized by SacⅠ and transformed into Pichia pastoris SMD1168 strain by electroporation.The positive clones were screened and those clones were fermented in flask.Tricine-SDS-PAGE showed that the Cecropin B protein could be secreted into the culture leading by α-factor from pPICZαA.Antibacterial activities and heat-stability were also found.更多还原