黄曲霉毒素分解酶基因在枯草芽孢杆菌中的表达与应用

Expression and Application of Aflatoxin Detoxification Enzyme Gene in Bacillus subtilis

本试验旨在研究黄曲霉毒素分解酶(ADTZ)基因在枯草芽孢杆菌中的表达与应用。试验通过构建枯草芽孢杆菌ADTZ基因的整合表达载体,将ADTZ基因整合到野生型枯草芽孢杆菌(B.subtilis LN)中,通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)初步检测ADTZ蛋白的表达情况。将重组枯草芽孢杆菌作为发酵菌种接种到发霉玉米中进行发酵试验,检测发霉玉米样品中黄曲霉毒素B1(AFB1)含量的变化,验证ADTZ基因在枯草芽孢杆菌中的表达效果。结果表明,通过构建整合表达载体成功将ADTZ基因整合到枯草芽孢杆菌基因组中,在SDS-PAGE上检测到重组枯草芽孢杆菌能够分泌表达特异性蛋白条带。经过重组菌发酵的发霉玉米AFB1的含量较未接种和接种野生型枯草芽孢杆菌发酵的发霉玉米AFB1的含量差异显著(P<0.05)。由此可见,ADTZ基因可成功整合到野生型枯草芽孢杆菌中,并进行了胞外分泌表达,表达产物具有生物活性,能有效地降解AFB1。

This experiment was conducted to study the expression and application of aflatoxin detoxification enzyme （ ADTZ ） gene in Bacillus subtilis （ B.subtilis ）. The ADTZ gene was transformed into wild-type B.subtilis LN genome by constructing an integrated vector of B.subtilis . The expression of ADTZ protein was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE）. And the recombinant B.subtilis were inoculated in moldy corn as fermentation strains for the fermentation experiment, and the content of aflatoxin B 1 （AFB 1） in moldy corn samples was detected to validate the expression effect of ADTZ gene in B.subtilis . The results showed that the ADTZ gene was successively integrated into wild type B.subtilis genome by constructing integrative expression vector. The recombined B.subtilis could secrete the specific protein and it was detected by SDS-PAGE. The content of AFB 1 in the fermented moldy corn of recombined B.subtilis had significant difference with the fermented moldy corn of uninoculated and wild type B.subtilis LN（ P 〈0.05）. It showed that the ADTZ gene is successfully integrated into wild type B.subtilis LN and expressed in extracellular. The expressed product has biological activity, and can degrade AFB 1 effectively.