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生物工程学报 2014, Vol. 30 (9) :1372 -1380 <<-上一篇 下一篇 ->>
引用: 于慧,姜艳平,崔文,武啸,何佳,乔薪瑗,李一经,唐丽杰.猪乳铁蛋白在4种重组乳杆菌中的表达及抑菌活性比较[J].生物工程学报,2014,30(9):1372~1380
猪乳铁蛋白在4种重组乳杆菌中的表达及抑菌活性比较
于慧, 姜艳平, 崔文, 武啸, 何佳, 乔薪瑗, 李一经, 唐丽杰
于慧  1 东北农业大学生命科学学院,黑龙江 哈尔滨 150030
姜艳平  2 东北农业大学动物医学学院,黑龙江 哈尔滨 150030
崔文  2 东北农业大学动物医学学院,黑龙江 哈尔滨 150030
武啸  2 东北农业大学动物医学学院,黑龙江 哈尔滨 150030
何佳  2 东北农业大学动物医学学院,黑龙江 哈尔滨 150030
乔薪瑗  2 东北农业大学动物医学学院,黑龙江 哈尔滨 150030
李一经  2 东北农业大学动物医学学院,黑龙江 哈尔滨 150030
唐丽杰  1 东北农业大学生命科学学院,黑龙江 哈尔滨 150030;2 东北农业大学动物医学学院,黑龙江 哈尔滨 150030
Comparison of expression and antibacterial activities of recombinant porcine lactoferrin expressed in four Lactobacillus species
全文:     原文
摘要: 为了获得优化的猪乳铁蛋白乳杆菌表达系统,并比较重组猪乳铁蛋白的抑菌活性,根据乳杆菌使用密码子的偏嗜性优化合成猪乳铁蛋白成熟肽编码序列,将其克隆到乳杆菌表达载体pPG612.1的Xho Ⅰ/ BamH Ⅰ位点,获得了plf乳杆菌表达载体质粒pPG612.1-plf。将获得的重组质粒分别电转化入干酪乳杆菌ATCC393、戊糖乳杆菌KLDS1.0413、植物乳杆菌KLDS1.0344和副干酪乳杆菌KLDS1.0652细胞内,获得4种表达猪乳铁蛋白的重组乳杆菌。经木糖诱导,通过Western blotting和激光共聚焦检测重组猪乳铁蛋白的表达,用ELISA方法检测和比较4种重组菌上清中表达猪乳铁蛋白的量,并用琼脂孔穴扩散抑菌法检测4种重组乳杆菌表达乳铁蛋白的抑菌活性。结果表明,乳铁蛋白在4种重组乳杆菌中均得到正确表达,其产物分子量约73 kDa,重组干酪乳杆菌、重组戊糖乳杆菌、重组植物乳杆菌和重组副干酪乳杆菌的重组猪乳铁蛋白表达量分别为9.6 μg/mL、10.8 μg/mL、12.5 μg/mL、9.9 μg/mL。重组猪乳铁蛋白对大肠杆菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、巴氏杆菌和李氏杆菌均有一定的抑菌作用,对金黄色葡萄球菌的抑菌作用最强,且4种重组乳杆菌中重组植物乳杆菌表达产物的抑菌效果优于其他重组菌的表达产物。结果表明在4种乳杆菌中重组猪乳铁蛋白的最佳表达系统为植物乳杆菌,该结果为猪乳铁蛋白的乳杆菌表达系统进一步开发与应用奠定了基础。
关键词猪乳铁蛋白,干酪乳杆菌,戊糖乳杆菌,植物乳杆菌,副干酪乳杆菌,表达,抑菌活性
Abstract: The coding sequence for the mature peptide of porcine lactoferrin (Plf) was synthesized according to the codon usage of lactobacillus, to establish optimized porcine lactoferrin Lactobacillus expression system. The gene was ligated into the XhoⅠ/BamHⅠsite of Lactobacillus expression vector pPG612.1 and the recombinant plasmid pPG612.1-plf was transformed individually into Lactobacillus casei ATCC393, Lactobacillus pentosus KLDS1.0413, Lactobacillus plantarum KLDS1.0344 or Lactobacillus paracasei KLDS1.0652 by electroporation. After induction with xylose, expression of the recombinant proteins was detected by Western blotting and confocal laser scanning microscopy. Secretion of recombinant Plf proteins from four recombinant species was determined quantitatively by ELISA. The antibacterial activities of recombinant proteins were measured by agar diffusion method. The result shows that Plf was correctly expressed in four species of recombinant lactobacillus, with molecular weight of about 73 kDa. The expression levels in recombinant Lactobacillus casei, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus paracasei were 9.6 μg/mL, 10.8 μg/mL, 12.5 μg/mL and 9.9 μg/mL, respectively. Antimicrobial activity experiment shows that the recombinant proteins were active against E. coli, Staphylococcus aureus, Salmonella typhimurium, Listeria, Pasteurella. The recombinant Plf expressed by recombinant Lactobacillus plantarum showed the best antibacterial activity among all recombinant lactobacillus species. These data represent a basis for the development and application of porcine lactoferrin from recombinant lactobacillus.
Keywords: porcine lactoferrin, Lactobacillus casei, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus paracasei, expression, antibacterial activity
基金支持:国家自然科学基金 (Nos. 31072057, 30871809), 黑龙江省大学科技创新项目基金 (No. 2011TD001) 资助。
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