大蜡螟（Galleria mellonella L.）血淋巴中雄性特异性蛋白（MSP）的克隆与表达

Cloning and expression of male-specific protein (MSP) from the hemolymph of greater wax moth, Galleria mellonella L.

作者信息

Han Jikhyon, Lee Chang Seok, Yun Chi Young, Lee Bong Hee, Ko Young Gyu, Kang Chang Soo, Lee Sang Dae, Hwang Jae Sam, Kang Seok Woo, Kim Hak R

机构信息

Laboratory of Animal Macromolecules Physiology and Biochemistry, School of Life Sciences and Biotechnology, Korea University, Seoul, Korea.

出版信息

Arch Insect Biochem Physiol. 2003 Nov;54(3):110-20. doi: 10.1002/arch.10106.

DOI:10.1002/arch.10106

PMID:14571505

Abstract

Male-specific protein (MSP) is a soluble protein that accumulates in high amounts in the hemolymph and other organs of adult male wax moth. The MSP was purified from adult male wax moth by gel filtration and reversed phase column chromatography, and its amino acid sequence was determined. Because of blocked N-terminus, several internal amino acid sequences of MSP were obtained by the in-gel digestion method using trypsin. RT-PCR was conducted using degenerate primers designed from the internal amino acid sequences. 5'-RACE PCR was used to obtain the complete coding region and 5'-UTR sequence. The full length MSP cDNA sequence encodes a 239 amino acid polypeptide with an 18 amino acid signal peptide. The putative mature MSP has a molecular mass of 24,317 Da and an isoelectric point (pI) of 6.00, but shows a molecular mass of 27 kDa on SDS-PAGE. Sequence alignment showed a significant similarity between MSP and juvenile hormone binding proteins (JHBPs) of several lepidopteran species, including G. mellonella.

摘要

雄性特异性蛋白（MSP）是一种可溶性蛋白，在成年雄性蜡螟的血淋巴和其他器官中大量积累。通过凝胶过滤和反相柱色谱法从成年雄性蜡螟中纯化出MSP，并测定了其氨基酸序列。由于N端封闭，使用胰蛋白酶通过胶内消化法获得了MSP的几个内部氨基酸序列。使用从内部氨基酸序列设计的简并引物进行RT-PCR。5'-RACE PCR用于获得完整的编码区和5'-UTR序列。全长MSP cDNA序列编码一个含有18个氨基酸信号肽的239个氨基酸的多肽。推测的成熟MSP分子量为24,317 Da，等电点（pI）为6.00，但在SDS-PAGE上显示分子量为27 kDa。序列比对显示MSP与几种鳞翅目物种（包括大蜡螟）的保幼激素结合蛋白（JHBPs）之间存在显著相似性。