家蚕类胰凝乳蛋白酶基因的发掘与表达特征分析

Excavating and Expression Pattern Analysis of Chymotrypsin-like Proteinase Gene in Bombyx mori

类胰凝乳蛋白酶(CTLP)是鳞翅目昆虫幼虫中肠中的主要蛋白酶。基于构建的家蚕中肠等组织差异表达基因的SSH文库,发掘和克隆了一条新的家蚕类胰凝乳蛋白酶基因Ctlp(GenBank登录号:JQ081296)。该基因定位于18号染色体nscaf2901,靠近端粒,有5个外显子和4个内含子,全长cDNA序列976 bp,ORF为840 bp,编码279个氨基酸残基,信号肽序列1～18 aa(分值0.985),1～20 aa和50～100 aa区域为2个由内到外的跨膜螺旋,推测蛋白质相对分子质量为29 780.10,pI为8.75,蛋白质功能域(保守区)和分子进化分析显示其为丝氨酸蛋白酶家族的类胰凝乳蛋白酶。家蚕Ctlp基因具有在5龄幼虫中肠特异性高表达和伴随进食量增加而上调表达的特征,同时也具有性别差异性和熟蚕期特异性上调表达的现象,暗示CTLP可能具有促进消化以外的功能。构建pET32a-ctlp重组表达载体,SDS-PAGE分析和Western blot鉴定结果显示,重组蛋白在原核表达系统中的表达量较低。

Chymotrypsin-like proteinase （CTLP） is the predominant proteinase in insect midgut of Lepidoptera. Based on the constructed SSH library of differentially expressed genes in midgut of silkworm, we excavated and cloned a new Ctlp gene from Bombyx mori （GenBank accession No. JQ081296）. This gene is located on nscaf2901 of chromosome 18 and is near telomere. It has 5 exons and 4 introns. The full-length cDNA sequence is 976 bp long with an ORF of 840 bp that encodes 279 amino acids. Amino acid residues of the signal peptide is from 1 to 18 （score =0. 985）, and the regions of 1 -20 aa and 50-100 aa are two transmembrane helices from inside to outside. The protein is predicted to have a relative molecular weight of 29 780. 10 and an isoelectric point of 8.75. Analyses of functional domains （conserved regions） and molecular evolution demonstrated that the protein encoded by this .clene is a chymotrypsin-like in serine prote-ase family. Gene expression profile showed that silkworm Ctlp gene was specifically and highly expressed in midgut of the 5th instar silkworm larvae and was up-regulated with increase of food intake. Simultaneously, its expression differed with sex of the larvae and was specifically up-regulated during maturation stage of the larvae. These results suggest that CTLP also plays a role in other physiological processes except for improving digestion. We constructed the recombinant expression vector pET32actlp for further expression analysis. However, SDS-PAGE and Western blot analyses showed that recombinant protein had a low expression level in prokaryotic expression system.