关键词: 中华蜜蜂, SRP9, 基因克隆, 序列分析, 结构预测, 原核表达

Abstract: 【Aim】 The study aims to clone the SRP9 gene from the Chinese honeybee, Apis cerana cerana, to analyze its sequence, and to gain the purified recombinant protein, so as to lay the foundation for the study of its protein function. 【Methods】 The cDNA sequence of the SRP9 gene was cloned from A. c. cerana by RT-PCR. Then the phylogenetic tree was constructed by MEGA5.1, and the protein structure was predicted and analyzed by software PrediceProtein and SWISS-MODEL. The SRP9 gene of A. c. cerana was expressed in Escherichia coli. The recombinant protein was purified by urea elution. 【Results】 Sequence analysis revealed an open reading frame (ORF) of 237 bp, which was named AccSRP9 and encodes 78 amino acids. The deduced relative molecular weight of the encoded protein is 9.36 kD and the isoelectric point is 9.17. Phylogenetic analysis showed that the SRP9 proteins of A. c. cerana, A. mellifera and A. florea cluster together. Protein secondary structure prediction showed that AccSRP9 contains two α-helix and three β-fold regions. By using homology modeling, the predicted three-dimensional structure of AccSRP9 was obtained. The AccSRP9 ORF was ligated into the expression vector pGEX-6P-1 and then transformed to E. coli BL21(DE3) for prokaryotic expression and the recombinant protein was purified. The results showed that the protein was expressed in inclusion bodies. Using the method of washing inclusion bodies, the N-terminal GST tagged recombinant protein pGEX-6P-1-SRP9 was obtained. 【Conclusion】 This study successfully cloned the SRP9 gene AccSRP9 from A. c. cerana, analyzed its sequences and obtained the purified recombinant protein, providing referencs and materials for further study of the functions of the gene.

Key words:  Apis cerana cerana; SRP9,  gene cloning, sequence analysis;  structure prediction, prokaryotic expression