家蝇抗菌肽Attacin在毕赤酵母中的异源表达

Heterologous Expression of Attacin Gene from Musca domestica in Pichia pastoris

目的构建家蝇抗菌肽(攻击素,attacin)成熟肽的重组表达质粒并转化巴斯德毕赤酵母(Pichia pastoris)GS115,诱导attacin表达。方法 PCR扩增家蝇抗菌肽attacin基因成熟肽编码区,克隆至酵母表达载体pPIC9K中,将重组质粒pPIC9K-attacin用SacⅠ酶切线性化后,采用电转化法将其转入毕赤酵母GS115中,通过抗性、表型和PCR筛选多拷贝整合株。甲醇诱导毕赤酵母GS115/pPIC9K-attacin的表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Western blotting)分析鉴定表达产物,采用琼脂糖孔穴扩散法检测其对大肠埃希菌K12D31的抑制作用。结果重组表达质粒pPIC9K-attacin构建成功,电转化获得多拷贝整合转化子毕赤酵母GS115/pPIC9 K-attacin,表达产物经SDS-PAGE和Western-blotting鉴定,在约Mr22000处有蛋白条带,表达产物可抑制E.coli K12D31的生长。结论家蝇抗菌肽attacin在巴斯德毕赤酵母中成功进行了表达。

Objective To construct recombinant expression plasmid with an antibacterial peptide （attacin） mature peptide gene from Musca domestica and express an antibacterial peptide attacin in Pichia pastoris. Methods The coding region of attacin mature peptide was cloned into the expression vector pPIC9K. The recombinant plasmid was lin- earized by digestion with SacⅠand transformed into P. pastoris GS115 by electroporation. The insert clones containing multiple copies were selected with geneticin, pha, enotype and PCR. The expression of P. pastoris GS115/pPIC9K-attacin were induced with methanol. The supernatant of the expressed protein was analyzed by SDS-PAGE and Western blotting. The anti-bacterial activity of expression product was analyzed by agarose diffusion assay. Results The expression plasmid was constructed and transformed into P. pastoris GS115. SDS-PAGE and Western blotting analysis indicated that the recombinant containing recombinant plasmid pPIC9K-attacin expressed a Mr 22 000 protein. The expression product inhibited the growth of E. coli K12D31. Conclusion The attacin gene from Musca domestica has been expressed in P. pastoris.