α2b干扰素基因与Ag85B基因在毕赤氏酵母中的融合表达

Fusion Expression of Genes IFNα2b and Ag85B in Pichia pastoris

Ag85是结核分枝杆菌的一种分枝菌酸转移酶复合体,在结核分枝杆菌的细胞壁合成过程中起着重要作用.Ag85B是该复合体的一个组分,具有较好的免疫原性.通过连接多肽(G4S)4的编码序列,将α2b干扰素基因与Ag85B基因连成融合基因,然后克隆到毕赤氏酵母表达载体pPIC9上,转化PichiapastorisSMD1168后获得重组酵母工程菌SMD1168/pPIC9-α2bAg85B,经甲醇诱导培养,发酵上清液经抗病毒活性测定,Westernblot鉴定以及初步纯化,结果表明,分泌表达的融合蛋白具有一定的抗病毒活性.

Ag85 complex is the mycolic acid transferase of Mycobacteriurn tuberculosis, and it is important in the process of forming the cell wall. Ag85B has good immunity and it is one part of Ag85 complex. Genes human IFNα2b and Ag85B were amplified by PCR,and joined by the DNA linker which encodes polypeptide （G4S）4. The recombinant fusion gene was inserted into Pichia pastoris expression vector pPIC9. The recombinant plasmid was transformed into Pichia pastoris SMD1168 and the transformants SMD1168/pPIC9-α2bAg85B were obtained. The transformants were fermented in flasks and induced by methanol. The fusion protein was identified by Western blot and was further purified. The secreted fusion protein was also identified by activity of IFNα and proved to have high activity of antivirus.