枯草芽孢杆菌表达人ADH1B和ALDH2及其活性与稳定性研究  

The enzyme activity and stability of recombinant human ADH1B and ALDH2 expressed in Bacillus subtilis

作　　者：王念 黄利思[1,2,3,4] 张转玲 张森[1,2,3] 刘潇逸 徐龙舟 朱一丹[5] 余新炳 黄艳 WANG Nian;HUANG Lisi;ZHANG Zhuanling;ZHANG Sen;LIU Xiaoyi;XU Longzhou;ZHU Yidan;YU Xinbing;HUANG Yan(Zhongshan School of Medicine,Sun Yat-sen University,Guangzhou,Guangdong 510080,China;Key Laboratory for Tropical Disease Control,Sun Yat-sen University,Guangzhou,Guangdong 510080,China;Provincial Engineering Technology Research Center for Biological Vector Control,Guangzhou,Guangdong 510080,China;Sun Yat-sen Memorial Hospital,Sun Yat-sen University,Guangzhou,Guangdong 510120,China;School of Public Health,Sun Yat-sen University,Guangzhou,Guangdong 510080,China)

机构地区：[1]中山大学中山医学院,广东广州510080 [2]中山大学教育部热带病防治重点实验室,广东广州510080 [3]广东省媒介生物防治研究中心,广东广州510080 [4]中山大学孙逸仙纪念医院,广东广州510120 [5]中山大学公共卫生学院,广东广州510080

出　　处：《热带医学杂志》2024年第5期617-622,共6页Journal of Tropical Medicine

基　　金：国家重点研发计划(2021YFC2300804);国家自然科学基金(81772212)。

摘　　要：目的构建可表达具有活性的人源乙醇脱氢酶(ADH1B)和乙醛脱氢酶(ALDH2)的重组枯草芽孢杆菌(B.subtilis)并验证其稳定性。方法将ADH1B和ALDH2的编码基因adh1B和aldh2分别克隆至表达载体pBE2R中,构建重组质粒pBE2R-adh1B和pBE2R-aldh2,使用化学转化法将重组质粒分别转化B.subtilis WB600;在37℃,200r/min条件下进行表达;Western blotting鉴定表达结果,检测目的蛋白酶活性,体外模拟人工胃液环境评价重组工程菌对酸性环境的耐受程度。结果构建的重组工程菌在37℃,200 r/min条件下培养24 h后,培养基上清及细菌沉淀中均可表达ADH1B和ALDH2;上清中的ADH1B和ALDH2酶活性分别为3.67 U/mL、1.61 U/mL;重组工程菌在pH为2、3、4的人工胃液中消化0~2 h后活菌量与处理前相当。结论B.subtilis WB600可表达可溶性且具有酶活性的人ADH1B和ALDH2,重组工程菌可良好耐受强酸环境。Objective To obtain active and stable expression of recombinant human alcohol dehydrogenase 1B(ADH1B) and aldehyde dehydrogenase 2(ALDH2) from Bacillus subtilis(B. subtilis). Methods Encoding sequencing of human ADH1B and ALDH2 were obtained and cloned into expression vector pBE2R. The recombinant plasmids pBE2R-adh1B and pBE2R-aldh2 were constructed and transformed into B. subtilis WB600 using the chemical transformation method.Recombinant B. subtilis WB600 was cultured at 37 ℃, 200 r/min for 24 h;the expression of recombinant human ADH1B and ALDH2 were confirmed by Western blotting with the specific antibodies. The enzyme activities were confirmed by classic method using specific kits. The stability of the recombinant B. subtilis WB600 strains was analyzed by treatment with artificial gastric fluid in vitro. Results After 24 hours of incubation at 37 ℃ and 200 r/min, recombinant human ADH1B and ALDH2 were successfully identified in the supernatant of the culture medium and the bacterial sediment. The enzyme activities of recombinant human ADH1B and ALDH2 in the supernatant were respectively 3.67 U/mL and 1.61 U/mL. The amounts of recombinant strains after treatment with artificial gastric fluid under pH 2, 3, or 4 for different time were close to those before treatment. Conclusion Recombinant human ADH1B and ALDH2 were successfully expressed in B. subtilis WB600 with enzyme activities and the recombinant B. subtilis WB600 strains could tolerate the strong acid environment.

关 键 词：乙醇脱氢酶 乙醛脱氢酶 枯草芽孢杆菌 酶活性 

分 类 号：Q78[生物学—分子生物学]