【摘要】 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。更多还原

【Abstract】 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 gene is a chymotrypsin-like in serine protease 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 pET32a-ctlp for further expression analysis.However,SDS-PAGE and Western blot analyses showed that recombinant protein had a low expression level in prokaryotic expression system.更多还原