家蚕溶茧酶基因的克隆、序列分析及原核表达

Cloning and Expression of the Cocoonase Gene from Bombyx mori

【目的】获得家蚕溶茧酶基因的全长序列,实现溶茧酶基因在大肠杆菌中的融合表达。【方法】利用cDNA末端扩增技术(RLM-RACE)克隆了家蚕溶茧酶基因cDNA序列(GenBank登录号:EF428980)。【结果】家蚕溶茧酶基因cDNA序列全长1047bp,其中780bp的蛋白质编码区可编码260个氨基酸,预测蛋白质分子量为27.6kD,等电点(pI)为8.89。家蚕溶茧酶基因包含4个内含子。用Signal P3.0Server程序分析家蚕溶茧酶基因,预测其从第1～22位为信号肽序列。SMART分析结果预测其第34～254位氨基酸序列具有类胰蛋白的丝氨酸蛋白酶活性。重组质粒pET-32a-Cocoonase转化E.coli BL21进行原核表达,SDS-PAGE分析结果表明,家蚕溶茧酶基因以融合蛋白形式表达,相对分子量为48kD。【结论】本研究成功地克隆、表达了家蚕溶茧酶基因,分析和预测了它的结构和功能,为其进一步的生物学功能研究及其应用奠定了基础。

[ Objective] This study was carded out to obtain and analyze the sequence of the Cocoonase gene from Bombyx mori, and to produce the fusion protein of the Cocoonase gene in E. coli. [ Methods ] The cDNA sequence of the Cocoonase gene from Bombyx mori was determined using RLM-RACE method （GenBank accession No. EF428980）. [Results] The full-length eDNA is 1 047 bp with a 780 bp open reading frame （ORF）, which encodes a protein of 260 amino acids. The protein is about 27.6 kD and its pl value is 8.89. From genomic DNA, there are four introns and five exons within the open reading frame of the Cocoonase gene. Amino acid sequence analysed by Signal P 3.0 Server showed that there is a signal peptide of 22 amino acids at the N-terminal. Prediction of protein founctional domain indicated that the trypsin-like serine protease domain of the Cocoonase is from 34 to 254 amino acid. The recombinant pET-32a-Cocoonase was constructed and introduced into E. coli BL21 to express a Trx-His-linked protein by IPTG. The result of SDS-PAGE demonstrated that the expression products migrated at a size of 48 kD. [Conclusion] The Cocoonase gene from Bombyx mori was successfully cloned and expressed. Furthermore, the structure and function of this gene were analyzed and predicted.