Cloning and sequencing of a cDNA for the hemolymph juvenile hormone binding protein of larval Manduca sexta.
Cloning and sequencing of a cDNA for the hemolymph juvenile hormone binding protein of larval Manduca sexta.
复制标题
DOI：
10.1016/s0021-9258(17)45443-3
复制
发表时间：
1990-11
期刊：
影响因子：
--
通讯作者：
K. Lerro;G. Prestwich
中科院分区：
文献类型：
--
作者：
K. Lerro;G. Prestwich
本文克隆并测序了六纹夜蛾幼虫血淋巴保幼激素结合蛋白(JHBP)的基因。用凝胶过滤层析、离子交换层析和制备型十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法从5龄幼虫血淋巴中分离纯化JHBP。针对该蛋白产生的多克隆兔抗体用于从噬菌体Lambda ZAPII的脂肪体表达文库中鉴定和分离JHBP cDNAs。共分离到11个JHBP基因克隆，并将其亚克隆到蓝本载体中，插入片段大小约为750个碱基对。从这些质粒中表达了一个36 kDa的免疫活性蛋白；这种β-半乳糖苷酶融合蛋白与真正的32 kDa JHBP一样，是用[~H]环氧高氟烷基重氮乙酸酯(EHDA)标记的特异性光亲和性蛋白。一个克隆的单链DNA用Sanger双脱氧核苷酸方法进行测序，使用缺失和定制引物技术。确定了一个成熟的翻译产物，含有226个氨基酸残基，分子质量为25,111道尔顿，预测等电点为5.40。该基因正确地预测了一株正宗的六倍子血淋巴JHBP的N-端氨基酸序列和氨基酸组成。计算机对蛋白质和核酸数据库的搜索没有发现任何相关的序列。因此，六纹夜蛾血淋巴JHBP似乎是昆虫激素结合蛋白超家族的第一个成员。
A cDNA for the hemolymph juvenile hormone binding protein (JHBP) of larval Manduca sexta has been cloned and sequenced. The JHBP was purified to homogeneity from fifth instar larval hemolymph using gel filtration chromatography, ion exchange chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyclonal rabbit antibodies, generated in response to this protein, were used to identify and isolate JHBP cDNAs from a fat body expression library in bacteriophage lambda ZAPII. Eleven putative JHBP cDNA clones were isolated and subcloned into Bluescript plasmid; cDNA inserts were approximately 750 base pairs in length. A 36-kDa immunoreactive protein was expressed from these plasmids; this beta-galactosidase fusion protein, like the authentic 32-kDa JHBP, was specifically photoaffinity labeled with [3H] epoxyhomofarnesyl diazoacetate (EHDA). Single-stranded DNA from one clone was sequenced by the Sanger dideoxynucleotide method, using deletion and custom primer techniques. A mature translation product was identified which had 226 amino acid residues, a molecular mass of 25,111 daltons, and a predicted isoelectric point (pI) of 5.40. The cDNA correctly predicts the N-terminal amino acid sequence and the amino acid composition of an authentic M. sexta hemolymph JHBP. A computer search of protein and nucleic acid data bases failed to reveal any related sequences. Thus, M. sexta hemolymph JHBP appears to be the first member of a new superfamily of insect hormone binding proteins.