Molecular Cloning and Expression of Cu/Zn-Containing Superoxide Dismutase from Fasciola hepatica

Author:

Kim Tong-Soo1,Jung Younghun2,Na Byoung-Kuk3,Kim Ki-Sun2,Chung Pyung-Rim2

Affiliation:

1. Department of Parasitology,1 and

2. Department of Parasitology, Inha University College of Medicine, Inchon 400-103,2 Korea

3. Division of Respiratory Viruses,3 National Institute of Health, Seoul 122-701, and

Abstract

ABSTRACT The cytosolic superoxide dismutase (SOD) of Fasciola hepatica , a causative agent of fascioliasis, was purified and characterized. The enzyme consists of two identical subunits, each with an apparent molecular mass of 17.5 kDa. An analysis of the enzyme's primary structure and inhibition studies revealed that the enzyme is a copper/zinc-containing SOD (Cu/Zn-SOD). The enzyme activity was relatively stable in a broad pH range, from pH 7.0 to 10.0, and the enzyme showed maximum activity at pH 7.5. This enzyme also displayed strong antigenicity against sera of bovine and human subjects with fascioliasis. The SOD gene fragment was amplified by PCR with degenerate oligonucleotide primers derived from amino acid sequences conserved in the Cu/Zn-SODs of other organisms. An F. hepatica cDNA library was screened with the SOD gene fragment as a probe. As a result, a complete gene encoding the Cu/Zn-SOD was identified, and its nucleotide sequence was determined. The gene had an open reading frame of 438 bp and 146 deduced amino acids. Comparison of the deduced amino acid sequence of the enzyme with previously reported Cu/Zn-SOD amino acid sequences revealed considerably high homologies. The coding region of the F. hepatica Cu/Zn-SOD was cloned and expressed in Escherichia coli . Staining of native polyacrylamide gel for SOD activity of the expressed protein revealed SOD activity that was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide. This means that the presence of the recombinant fusion protein is indicative of Cu/Zn-SOD. The expressed protein also reacted with sera of bovine and human subjects with fascioliasis, but it did not react with sera of uninfected bovine and human subjects.

Publisher

American Society for Microbiology

Subject

Infectious Diseases,Immunology,Microbiology,Parasitology

Reference61 articles.

1. Asada K. Kanematsu S. Okaka S. Hayakawa T. Phylogenetic distribution of three types of superoxide dismutase in organisms and in cell organelles Chemical and biochemical aspects of superoxide and superoxide dismutases. Bannister J. V. Hill H. 1980 136 153 Elsevier/North-Holland New York N.Y

2. Human fascioliasis in Shropshire;Ashton W. L. G.;Br. Med. J.,1970

3. Active oxygen species and the functions of phagocytic leukocytes;Badwey J. A.;Annu. Rev. Biochem.,1980

4. Aspects of the structure, function and application of superoxide dismutases;Bannister J. V.;Crit. Rev. Biochem.,1987

5. Monoclonal antibodies demonstrate that superoxide dismutase contributes to protection of Nocardia asteroides within the intact host

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3