嗜铬粒蛋白N区抗真菌活性片段研究

Antifungal Activity Fragments of N Domain of Chromogranin A

为寻找高效低毒的抗真菌药物,利用PCR技术扩增了编码人嗜铬粒蛋白N端18-76、18-66和31-76位氨基酸(CGA18-76、CGA18-66和CGA31-76)的DNA片段,将之克隆进枯草杆菌诱导型表达载体pSBPTQ,获得3种重组质粒pSC18-76、pSC18-66和pSC31-76,转化枯草杆菌DB1342。SDS-PAGE分析结果显示:经蔗糖诱导后,CGA18-76、CGA18-66和CGA31-76片段分别在枯草杆菌工程菌中获得表达,产物分泌到细胞外。表达量分别为5.6 mg/L、5.3 mg/L和5.6 mg/L。利用孔穴琼脂扩散法检测表达产物的抗真菌活性,并与CGA1-76进行比较,发现CGA18-76、CGA18-66和CGA31-76对烟曲霉菌、黄曲霉菌、石膏样小孢子菌和白念珠菌均有抑制作用,并以CGA31-76对白念珠菌的抑制作用为最强,CGA18-66对除白念珠菌之外的另3种测试真菌的抑制作用较强,而CGA18-76对测试真菌的抑制作用最弱。

To find antifungal compounds with low toxicity to mammalian cells, the DNA fraglnents encoding the N terlninal 18-76, 18 -66, and 31 -76 amino acid sequences （CGA18 -76, CGA18 -66, and CGA31 -76） of human CGA were amplified by PCR technique. The amplified DNA fragments were cloned into the Bacillus subtilis inducible expression vector pSBPTQ and the resultant plasmids pSC18 -76, pSC18 -66, and pSC31 -76 were then transformed into B. subtilis strain DB1342 competent cells respectively. SDS - PAGE results showed that CGA18 -76, CGA18 -66 and CGA31 -76 were expressed by sucrose induction and secreted into the medium with a yield of 5.6 mg/L, 5.3 mg/L and 5.6 mg/L respectively. The antifungal activity of the expressed products was examined and compared with CGA1 -76. The results demonstrate that all four fragments CGAI -76, GA18 -76, CGA18 -66 and CGA31 -76 inhibit the growth of A. fumigatus, A. flavus, M. gypseum and C. albican, while CGA31 -76 has the highest inhibition activity specifically on C. albican, and CGA18 -66 shows the strnngest inhibition on these fungi except C. albican, and CGA18 -76 shows the weakest inhibition on these fungi.