The Study On Gene Cloning And Prokaryotic Expression Of Urate Oxidase In Silkworm

Urate oxidase (UO), an enzyme that catalyzes the oxidation of uric acid to allantoin, plays an important role in the metabolism of purines. Most prokaryote and eukaryote have intact urate oxidase gene (wo) that produces urate oxidase activity and excretes allantoin as the end product of purine metabolism. Because of a single nonsense mutation, human and certain non-human primates lack urate oxidase and excrete uric acid as the end product. Utilizing gene engineering methods to construct epression vector of high efficiency, and expression of uric acid in proper expression system which can elevate yield of uricase. There are several uricases been cloned and expression of biology activity protein in the E. coli overseas. But there is no research interiorly. In this paper we studied on the uricase of silkworm, which utilized homolog DNA sequence of Drosophila melanogaster and PCR to get DNA sequence of uricase in silkworm, and then expression in the Prokaryotic Expression System.Cloning and identification of the uricase gene in B. mori, herein we present a molecular characterization of B. mori which includes:1. Identification the sequence of the uricase gene in B. mori: The amino acid sequence of Drosophila uricase was used to search against the silkworm genome database with BLAST program, and we predicted the uricase gene of silkworm, Bombyx mori.2. Cloning, analysis the expression pattern of uricase gene by RT-PCR and uricase gene of B. mori expression in E. coli: The gene was cloned and sequenced, termed it as Bmuo, and the fragment was cloned into the eukaryote expression vector pET-28a. The recombinant uricase under the induction of IPTG After the SDS-PAGE we found the uricase expressed. But in our experiment, we can't detect the activity of the uricase, maybe in the Prokaryotic Expression, the protein of Eukaryotes expressed in the E. coli could not correct posttranslation modify. RT-PCR analysis the expression pattern indicated that Bmuo was expressed only in the fat body and Malpighian tubules of the fifth-instar larva.3. Sequence analysis of uricase in the B. mori: The gene comprised an open reading frame of 1014 bp, which was split into five parts by four introns. The encoded uricase was 337 amino acids with 45.6% sequence identity with that of Drosophila melanogaster. The molecular weight deduced was 38.18 kD, and the predicted isoelectric point was 6.90. The putative protein has two uricase domains, an uricase signature and a high conserved region. The latter maybe have relation with activity of urate oxidase.