期刊文献+

黑曲霉外源表达系统对Dactylellina cionopaga基因PrD Ⅰ的表达 被引量:3

Expression of PrD Ⅰ of Dactylellina cionopaga in Aspergillus niger
原文传递
导出
摘要 目的:Dactylellina cionopaga是产生黏性分枝的捕食线虫真菌,进行其基因外源表达是鉴定该菌侵染相关因子的途径之一。方法:该文构建了含有组氨酸标签的黑曲霉表达载体pGT21M-HIS,对D.cionopaga中与侵染机制相关的丝氨酸蛋白酶基因PrD Ⅰ进行了外源表达。结果:RT-PCR鉴定结果显示,PrDⅠ在黑曲霉201中获得了转录。SDS-PAGE和酶活分析结果表明,经亲和层析纯化的重组蛋白分子量约为45kDa,在pH 5.0的条件下具有蛋白水解活性。纯酶液的蛋白浓度为30μg/ml。结论:构建的带有组氨酸标签的表达载体pGT21M-HIS能够用于基因在黑曲霉表达系统中的外源表达,并为目的蛋白的纯化和鉴定提供了极大便利。黑曲霉外源表达系统在表达丝状真菌基因方面相对其他表达系统具有优势。 Objective:Dactylellina cionopaga is an adhesive column-producing nematophagous fungus.To express the genes exogenously is one way of identifying the infection-related factors of the fungus.Method:In this study,a vector pGT21M-HIS with a His-tag was constructed for the heterologous expression in Aspergillus niger.The serine proteinase-encoding gene PrD Ⅰ of D.cionopaga,which was related to the infection of nematodes,was expressed with the vector.Result:The result of RT-PCR presented that the target gene was transcripted in A.niger 201.SDS-PAGE analysis and enzyme activity assay shown that the molecular weight of the recombinant protein purified with affinity chromatography was about 45kDa and possessed the proteinase activity at pH 5.0.The concentration of the purified protein in the enzyme solution was 30μg/ml.Conclusion:This expression indicated that the constructed pGT21M-HIS could be used for the foreign expression of genes in A.niger,which provided the convenience for the purification and identification of target protein.A.niger expression system is advantageous in contrast to other expression systems so far as the expression of filamentous fungi genes concerned.
出处 《生物技术》 CAS CSCD 北大核心 2012年第3期38-43,共6页 Biotechnology
基金 中国博士后科学基金面上项目(20070410155)资助
关键词 食线虫真菌 Dactylellina cionopaga 丝氨酸蛋白酶 外源表达 黑曲霉 表达载体 nematophagous fungi Dactylellina cionopaga serine proteinase exogenous expression Aspergillus niger expression vector
  • 相关文献

参考文献22

  • 1刘丽,刘谨,仇润祥,朱兴国,唐国敏.丝状真菌表达分泌系统中受体菌的构建[J].生物工程学报,2002,18(6):667-670. 被引量:12
  • 2Andreas H. F. J. Roth,Petra Dersch.A novel expression system for intracellular production and purification of recombinant affinity-tagged proteins in Aspergillus niger[J]. Applied Microbiology and Biotechnology . 2010 (2)
  • 3Ines Pisanelli,Magdalena Kujawa,Doris Gschnitzer,Oliver Spadiut,Bernhard Seiboth,Clemens Peterbauer.Heterologous expression of an Agaricus meleagris pyranose dehydrogenase-encoding gene in Aspergillus spp. and characterization of the recombinant enzyme[J]. Applied Microbiology and Biotechnology . 2010 (2)
  • 4Bo-Ra Kwon,Myoung-Ju Kim,Jin-A Park,Hea-Jong Chung,Jung-Mi Kim,Seung-Moon Park,Sung-Hwan Yun,Moon-Sik Yang,Dae-Hyuk Kim.Assessment of the core cryparin promoter from Cryphonectria parasitica for heterologous expression in filamentous fungi[J]. Applied Microbiology and Biotechnology . 2009 (2)
  • 5G. M. Eibes,T. A. Lú-Chau,F. J. Ruiz-Due?as,G. Feijoo,M. J. Martínez,A. T. Martínez,J. M. Lema.Effect of culture temperature on the heterologous expression of Pleurotus eryngii versatile peroxidase in Aspergillus hosts[J]. Bioprocess and Biosystems Engineering . 2009 (1)
  • 6Jaffee,B.A.,Muldoon,A.E.Susceptibility of root-knot and cyst nematodes to the nematode-trapping fungi Monacrosporium ellipsosporum and M. cionopagum. Soil Biology and Biochemisty . 1995
  • 7Huang XW,Zhao NH,Zhang KQ.Extracellular enzymes serving as virulence factors in nematophagous fungi involved in infection of the host. Research in Microbiology . 2004
  • 8Ward M,Wilson L J,Kodama K H,et al.Improved Production of Chymosin in Aspergillus by Expression as a Glucoamylase -Chymosin Fusion. Journal of Biotechnology . 1990
  • 9T Guillemette,NN Peij,T Goosen,K Lanthaler,GD Robson,CA Hondel,H Stam,DB Archer.Genomic analysis of the secretion stress response in the enzyme-producing cell factory Aspergillus niger. BMC Genetics . 2007
  • 10Ahman J,Johansson T,Olsson M,et al.Improving the pathogenicity of a nematode-trapping fungus by genetic engineering of a subtilisin with nematotoxic activity. Applied and Environmental Microbiology . 2002

二级参考文献8

  • 1Wernars K, Goosen T, Swart K et al. Genetic analysis of Asper gillus nidulans amds+ transformant. Mol Gen Genet, 1986, 205:3 12~317
  • 2Kanak L D, Webster D A. Cloning, characterization and expressi on of the bacterial globin gene from Vitreoscilla in Escherichia coli. Gen e, 1988, 70:377~386
  • 3Verwoerd, T C, Dekker, B M M, Hoekema. A. A small-scale procedur e for the rapid isolation of plant RNAs. Nucleic Acids Research, 1989, 17(6):2362
  • 4Berka, R, Ward, M, Wilson, L J et al. Molecular cloning and d eletion of the gene encoding aspergillopesin A from Aspergillus awamori.Gene, 1990, 86:153~162
  • 5Johannes P T W van den Hombergh, Peter J I van de Vondervoort, Laurence F raissinet-Tachet et al. Aspergillusas a host for heterologous protein prod uction: the problem of proteases. TIBTECH, 1997, 15:256~253
  • 6Broekhuijsen M P, Mattern I E, Contreras R et al. Secretion o f heterologous proteins by Aspergillus niger: Production of active human int erleukin-6 in a protease-deficient mutant by KEX2 like processing ofglucoamyla se-hIL6 fusion protein. Journal of Biotechnology, 1993, 31:135 ~145
  • 7Mattern I E, Johannes M van Noort, Paul van den Berg et al. I solation and characterization of mutants of Aspergillus niger deficient in e xtracellular proteases. Mol Gen Genet, 1992, 234:332~336
  • 8ZHANG S Z(张树政). Industrial handbook of enzyme preparation. Bei jing: Science Press (科学出版社), 1984

共引文献11

同被引文献39

  • 1曾铮,吴大洋,李春峰.铜绿假单胞菌弹性蛋白酶基因的克隆及在昆虫细胞中的表达[J].蚕业科学,2006,32(3):345-349. 被引量:2
  • 2杨春晖,王海燕.短小芽孢杆菌碱性蛋白酶基因启动子的克隆、鉴定及其应用[J].遗传,2007,29(7):874-880. 被引量:3
  • 3ALAGARSAMY S, LARROCHE C, PANDEY A. Microbiology and industrial biotechnology of food-grade proteases: a perspective[J]. Food Technology and Biotechnology, 2006, 44(2): 211-220.
  • 4BRAKEBUSCH M, WINTERGERST U, PETROPOULOU T, et al. Bromelain is an accelerator of phagocytosis, respiratory burst and killing of Candida albicans by human granuloeytes and monocytes[J]. European Journal of Medical Research, 2001, 6(5): 193-200.
  • 5FRANTZ C, STEWART K M, WEAVER V M. The extracellular matrix at a glance[J]. Journal of Cell Science, 2010, 123(24): 4195-4200.
  • 6WIEDERANDERS B. Structure-function relationship in class CA1 cysteine peptidase propeptides[J]. Acta Biochemical Polonica, 2003, 50(3): 691-713.
  • 7DAVIES D R. The structure and function of the aspartic proteinases[J]. Annual Review of Biophysics and Biophysical Chemistry, 1990, 19(1): 189-215.
  • 8NAGASE H, VISSE R, MURPHY G. Structure and function of matrix metalloproteinases and TIMPs[J]. Cardiovascular Research, 2006, 69(3): 562-573.
  • 9PICCARD H, van den STEEN P E, OPDENAKKER G. Hemopexin domains as multifunctional liganding modules in matrix metalloproteinases and other proteins[J]. Journal of Leukocyte Biology, 2007, 81(4): 870-892.
  • 10BACH E, DAROIT D J, CORRI~A A P F, et al. Production and properties of keratinolytic proteases from three novel Gram- negative feather-degrading bacteria isolated from Brazilian soils[J]. Biodegradation, 2011, 22(6): 1191-1201.

引证文献3

二级引证文献20

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部