Prokaryotic Expression and Western-blot Analysis of Glucose Oxidase Gene from Aspergillus niger

doi:10.7668/hbnxb.2009.04.017

Abstract

Abstract: :In order to further study expressed level and condition of Glucose oxidase (GOD) from Aspergillus niger 31758 in E. coli,the coding region of GOD from A. niger 3. 758 was inserted into the prokaryotic expression vector pET2 11a,and the recombinant plasmid was transformed into E. coli BL21 (DE3) plysS. The result of SDS2PAGE showed that the GOD gene was expressed by IPTGinduction,and the molecular weight of the fusion protein was about 66. 5 kDa.An2 tiserum was produced in rabbit immunized with commercial GOD,and ELISA analysis proved that the titer of this antibody was 106. The Western2blotting result confirmed that the antibody reacted specifically to the expressed fusion protein. The study established basis for preparation of GOD antibody from Aspergillus niger 3. 758.

Key words: Aspergillus niger 3. 758, Glucose oxidase gene, Fusion expression, Antiserum, ELISA, Western2blot

CLC Number:

TQ920.1

AN Yu-lin, SUN Rui-fen, ZHANG He-ling, GUO Shu-chun, YAN Su-li. Prokaryotic Expression and Western-blot Analysis of Glucose Oxidase Gene from Aspergillus niger[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2009, 24(4): 84-87. doi: 10.7668/hbnxb.2009.04.017.

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References

[1] Katherine R,Frederick,James Tung,et al.Glucose Oxidase from Aspergillus niger[J].The Journal of Biological Chemistry990,265(7):3793-3802.
[2] Pazur J,Kleppe K.The Oxidation of Glucose and Related Compounds by Glucose Oxidase from Aspergillus niger[J].Biochemistry964(3):578-583.
[3] Swoboda B E P,Massey V.Purification and Properties of the Glucose oxidase from Aspergillus niger[J].Biol Chem965(240):2209-2215.
[4] Kusai K,Sekuzu I,Hagihara B,et al.Crystallization of Glucose Oxidase from Penicillium amagasakiense[J].Biochem Biophys Acta960,(40):555-557.
[5] 姚裕琪,巩秀峰,高奇华,等.马铃薯晚疫病抗性鉴定及评价[J].内蒙古农业科技,2001(2):8-9.
[6] Whittington H,Kerry Williams S.Expression of the Aspergillus niger Glucose Oxidase gene in A.nidulans and Sacchanomyces cerevisiae[J].Curr Cenet991(18):531-536.
[7] Witt S,Singh M,Kalisz H M.Strucyural and Kinetic properties of nonglycosylated recombinant Penicillium amagasakiense Glucose oxidase expressed in Escherichia coli[J].Apploed and Environmental M icrobiology998,64(4):1405-1411.
[8] 周亚凤,张先恩,刘虹,等.黑曲霉葡萄糖氧化酶基因的克隆及其在酵母中的高效表达[J].生物工程学报,20017(4):400-405.
[9] 母敬郁,王峤,杨纯中,等.瑞氏木酶表达黑曲霉葡萄糖氧化酶[J].生物工程学报,2006,22(1):82-86.
[10] 押辉远.黑曲霉中国株(3.758)葡萄糖氧化酶(GOD)基因的克隆与结构分析[J].安徽农业科学,2008,36(2):454-456.
[11] 孙瑞芬,安玉麟,张鹤龄,等.葡萄糖氧化酶基因植物表达载体的构建[J].华北农学报,2006,21(6):11-15.
[12] Kieser T.Preparing plasmid DNA:alkaline lysis.plasmid isolation by isolation by alkaline lysis[J].Procedure 2.plasmid984(12):85-92.
[13] 汪家政,范明.蛋白质技术手册[M].北京:科学出版社,2004:77-90.
[14] Frederick M,Ansubel.Short Protocols in Molecular Biology(A Laboratory Manual)(3rd ed)[M].Cold Spring Harbor Laboratory Press998,352.
[15] Sambrook J,Fritsch E F,Maniatis T.Molecular Cloning(A Laboratory Manual)(2nd ed)[M].Cold Spring Harbor Laboratory Press989:891-893.
[16] 戎芳.麻疯树(Jatropha curcas L.)外植体愈伤组织中毒蛋白(curcin)Western-blot鉴定及其烟草转基因研究[D].成都:四川大学,2004.

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