在毕赤酵母中表达的重组人血管内皮生长因子(rhVEGF_(121))为氨基端4个氨基酸缺失的异构体

Recombinant Human VEGF_(121) Expressed in Pichia pastoris Is an Isomer Lacking Its 4 N-terminal Amino Acids

为获得重组人血管内皮生长因子 ,采用重叠PCR扩增出含Kozak顺序、α因子前导肽和hVEGF12 1的融合基因 ,经DNA测序无误后 ,插入Pichiapastoris酵母载体 ,并体外构建 4拷贝表达单元质粒pAO81 5 4KαVEGF12 1。表达载体转化酵母宿主菌GS1 1 5 ,筛选出高效表达hVEGF的转化子。摇瓶培养并 1 %甲醇诱导 80h的VEGF分泌性表达量可达 30mg L。表达产物经S Sepharose离子交换层析纯化后测活表明表达产物二聚体具有良好生物活性。SDS PAGE分析、Western印迹表明表达产物具有适宜的分子量和抗原性。VEGF蛋白经氨基端氨基酸测序证实纯化后的不同糖化程度产物为同一蛋白质 ,但均为氨基端缺失了 4个氨基酸的全长为 1 1 7个氨基酸的VEGF。

The human VEGF 121 cDNA\, Kozak and α factor sequences were amplified by PCR.After confirming the fused gene by DNA sequence analysis,hVEGF 121 with Kozak and α factor was inserted into the Pichia Pastoris expression system vector pAO815. A recombinant expression plasmid pAO815\|4KαVEGF 121 was formed by multimerization in vitro , and then this recombinant plasmid was transformed into GS115.The recombinant transformants with high expressed hVEGF 121 were cultured in flasks and induced by the addition of 1% methanol. After 80 hours of methanol induction, the expressed hVEGF 121 came up to about 30mg/L culture as shown by SDS\|PAGE. Western blot assay proved that the expressed hVEGF 121 had good antigenicity. The recombinant protein was further purified by S\|Sepharose anion exchange column, and was proved that it is a single protein by N\|terminal sequencing, but it is an isomer lacking N\|terminal 4 amino acids.