嗜铬粒蛋白N端片段基因在枯草杆菌中的表达及表达产物的活性分析

Expression of N Domain of Chromogranin A in Bacillus subtilisand Its Antifungal Activity

嗜铬粒蛋白 (CGA)是存在于分泌细胞的由 43 9个氨基酸组成的可溶性蛋白。近年的研究发现CGA的N端具有抗血管收缩、抗细菌和抗真菌的功能。为了寻找高效低毒的抗真菌片段 ,利用PCR技术扩增了编码人嗜铬粒蛋白N端 1 76位氨基酸 (CGA1 76)的DNA片段 ,并将之克隆进本实验构建的枯草杆菌诱导型表达载体pSBPTQ ,获得含CGA1 76基因的重组质粒pSVTQ ,转化蛋白酶三缺陷的枯草杆菌DB40 3。经蔗糖诱导后 ,CGA1 76片段在枯草杆菌DB40 3 (pSVTQ)中获得表达 ,产物分泌到细胞外 ,分泌量约为 5mg L ,占总分泌蛋白的 13 3 %。测试了表达产物对几种丝状真菌和酵母的抑制作用 ,发现在 4μmol L的浓度下 ,枯草杆菌表达的重组CGA1 76对镰刀菌。

Chromogranin A(CGA) is a soluble protein existed in most secreted cell s and neurons. It was recently found that the bovine CGA N terminal region has v asoinhibitory, antibacterial and antifungal activities. Since the need for effe ctive antifungal agents increases in parallel with the expanding number of immun ocompromised patients at risk for fungal infections, it becomes imperative to fi nd antifungal compounds with low toxicity toward mammalian cells. To study the a ntifungal activity of CGA N terminal region, the DNA fragment encoding for the N terminal 1-76 amino acid sequence (CGA1-76) of human CGA was amplified by PC R technique. After DNA sequence analysis, the amplified DNA fragment was cloned in to the Bacillus subtilis inducible and expression vector pSBPTQ constructed in this study and the resultant plasmid pSVTQ was then transformed into triple- protease deficient Bacillus subtilis strain DB403 competent cells. The trans formants was screened on LB plates containing 10μg/mL kanamycin. The positive t ransformant DB403(pSVTQ) was grown on kanmycin containing 2×MSR medium and sucr o se was added to 2% final concentration for induction after 2h cultivation. The c ulture supernatant was used to run SDS-PAGE.The result of SDS-PAGE showed tha t the CGA1-76 was expressed by sucrose induction and the expressed product secr eted into the medium with a yield of 5mg/L. The expressed product reacts specifi cally with m ouse anti CGA47-68 monoclonal antibody. The antifungal activity of the expresse d product was examined by adding the culture supernatant to the fungal spore or Candida albican suspensions at appropriate proportion and found that the rec ombinant human CGA1-76 produced in Bacillus subtilis inhibits the growth of Fusarium sp. Alternaria sp. and Candida albican at the concerntrat ion of 4μmol/L. These results demonstrate that human CGA1-76 has expressed in Bacillus subtilis and the expressed product is immunogenic and has the antif ungal activity.