与烯醇酶基因家族同源的日本血吸虫cDNA的克隆及功能表达

Cloning and functional expression of a Schistosoma japonicum cDNA homologous to the enolase gene family.

作者信息

Waine G J, Becker M, Kalinna B, Yang W, McManus D P

机构信息

Molecular Parasitology Unit, Queensland Institute of Medical Research, Bancroft Centre, Brisbane, Australia.

出版信息

Biochem Biophys Res Commun. 1993 Sep 30;195(3):1211-7. doi: 10.1006/bbrc.1993.2173.

DOI:10.1006/bbrc.1993.2173

PMID:8216251

Abstract

A cDNA encoding the complete open reading frame of Schistosoma japonicum enolase has been cloned. The 1494bp cDNA (C30) was isolated from a S. japonicum cDNA expression library immunoscreened with hyperimmune rabbit sera raised against soluble adult S. japonicum proteins. The ORF encodes a protein of 434 amino acids exhibiting 72% identity to human, murine, and rat enolases, and 62% identity to Saccharomyces cerevisiae enolase. The inferred molecular mass of the protein is 47,251 Daltons, similar to that reported for the enolases of other species. In vitro translation of C30 also generated a protein of 47kDa. After subcloning and expression, the recombinant protein was purified by affinity chromatography under non-denaturing conditions and shown to exhibit functional enolase enzymatic activity.

摘要

编码日本血吸虫烯醇化酶完整开放阅读框的cDNA已被克隆。从用针对可溶性日本血吸虫成虫蛋白产生的超免疫兔血清进行免疫筛选的日本血吸虫cDNA表达文库中分离出1494bp的cDNA（C30）。该开放阅读框编码一种由434个氨基酸组成的蛋白质，与人类、小鼠和大鼠的烯醇化酶具有72%的同一性，与酿酒酵母烯醇化酶具有62%的同一性。该蛋白质的推断分子量为47251道尔顿，与其他物种烯醇化酶的报道分子量相似。C30的体外翻译也产生了一种47kDa的蛋白质。亚克隆和表达后，重组蛋白在非变性条件下通过亲和层析纯化，并显示具有功能性烯醇化酶的酶活性。