一种新型广谱的原核型分泌表达载体的建立及人表皮生长因子的表达

Construction of a novel secretory expression vector and expession of human epidermal growth factor in E. coli

通过Cyanobase藻类学数据库查询,从集胞藻6803转译后加工过程中的某些蛋白的分泌信号序列得到了可以用于分泌型表达的信号序列,进一步通过SignalP3.0Server分析软件预验证,从集胞藻6803得到4种不同蛋白的信号序列A、B、C、D,将这些信号序列插入pET-His-EGF表达载体hegf基因上游,得到了带有分泌型信号的pS-X系列分泌型表达载体,人表皮生长因子（hEGF）在其中的pS-A载体中实现了分泌型表达,hEGF分泌到周质空间的相对表达量约为1%,在其它3个载体中未见表达。获得的pS-A分泌型表达载体是一种新型分泌型表达载体,它利用蓝藻信号序列作为分泌表达的信号肽,实现了hEGF多肽在大肠杆菌中分泌表达,通过融合白亚细胞定位法处理得到的hEGF多肽在胞质、周质空间和培养基中表达的比例为85∶33∶25（density intensity/mm^2）,利用渗透休克处理方法得到的比例为50∶37∶25。由于A信号序列本身是集胞藻6803膜蛋白（ID：s110172/NCBI-GI：1001433）的分泌信号肽,说明pS-A载体是一种广谱的原核型分泌表达载体。这一结果为实现hEGF在螺旋藻中的分泌型表达奠定了基础。

To construct a novel secretory expression vector which can be used in cyanobacterium and bacterium, signal sequences for secretory expression were searched from Cyanobase and comfirmed by SignalP3.0 Server. Four types of signal sequences in Synechocytis sp. PCC6803 were obtained and named A, B, C and D respectively. These signal sequences were inserted into 5＇ end of hegf gene in pET-His-EGF vector, and the secretory expression vectors with secretory signals, named pS-A, pS-B, pS-C and pS-D respectively, were obtained. The human epidermal growth factor （hEGF） was expressed and collected from different fractions including culture medium, periplasm, cell plasm and pellet and analyzed by SDS-PAGE and Western blotting. The results showed that pS-A vector was the only one that expressed hEGF, and hEGF yeild was about 1% in periplasmic proteins and less than that in the culture medium. This A sequence came from a kind of periplasm protein signal peptide, which is a membrane protein of Synechocytis sp. PCC6803（ID： sll0172/ NCBI-GI： 1001433）. By the sub-cellular fractionation method, the ratio of hEGF expression in cell plasm, periplasmic, culture medium was 85： 33：25 （density intensity / mm^2） ; by cold osmotic shock method, the ratio was 50： 37： 25. Thus, a novel prokaryotic secretory expression vector with a functional signal sequence from cyanobacterium was constructed and it was confirmed by its expression in E. coli. This novel vector pS-A is a pan-vector of prokaryotic expression vector.