中华蜜蜂王浆多肽Apisimin基因转录、克隆与表达研究

Analysis of Apisimin cDNA from Apis cerana cerana and Its Expression in E.coli

构建中华蜜蜂(Apis cerana cerana)工蜂头部cDNA文库并进行了测序,从8568个表达序列标签(expression sequencetag,EST)中获得96个Apisimin基因克隆,结果表明Apisimin基因是一个高丰度转录的基因。进一步对克隆分析表明该基因cDNA全长为379 nt,包含一个237 nt、可以编码78个氨基酸的ORF。推导的Apisimin蛋白N端含有一个由24个氨基酸残基组成的信号肽,后端为54残基、预计分子量为5.9kDa的成熟肽。该ORF与意大利蜜蜂和印度蜜蜂的Apisimin基因核苷酸同源性分别为100%和95%。将成熟肽编码区进行亚克隆,构建了Apisimin与谷胱甘肽转移酶融合表达的载体pGEX/apisimin,并将重组载体转化入大肠杆菌BL21(DE3)进行融合表达。SDS-PAGE和薄层扫描表明,表达的融合蛋白分子量为31 kDa,表达量占细菌总蛋白的22.1%,约50%是可溶性的。通过亲和柱进一步纯化了表达的融合蛋白。

A cDNA library from worker heads of Apis cerana cerana was constructed and a total number of 8568 expression sequence tags （ESTs） were definite. Ninety-six clones containing Apisirftin gene were identified from the total ESTs, indicating that Apisimin gene is a highly transcriptional gene. The total length of the cDNA was 379 nt, containing an open-reading frame （ORF） of 237 nt. The putative ORF might encode a 78 amino acid residue precursor, which is consisted of a 24 amino acid signal peptide in the N-terminal and a 54 residue mature peptide with a predicted molecular weight of 5.9 kDa in the C-terminal. The Apisimin gene shared 100 % and 95% of homologies with A. mellifera and A.cerana indica at nucleotide level, respectively. The coding region of the matured peptide was sub-cloned into the prokaryotic expression vector pGEX-4T-2 to fuse with GST to form a recombinant vector pGEX/apisimin. The recombinant was then transformed into E. coli BL21 （DE3） for fusion expression. The SDS-PAGE and thin-layer scanning showed that the expressed GST-apasimin fusion protein was about 31 kDa in size and about 22.1% in proportion to the total bacterial proteins as well as about 50% soluble. The fusion protein was further purified through affinity chromatography.