摘要
业已明确家蚕30K蛋白不仅作为家蚕生长发育的重要能源物质,还对细胞凋亡具有明显的抑制作用。采用RT-PCR技术从家蚕5龄幼虫脂肪体中克隆了BmLp-C6、BmLip19G1和BmLp-C12p(GenBank登录号分别为X54735、AY568957、NM_001101728)共3种30K蛋白基因的cDNA序列,并利用Bac-to-Bac系统构建含有3种30K蛋白基因的重组杆状病毒表达载体,转染家蚕BmN培养细胞大量表达3种30K蛋白,3种表达产物纯化后的样品经SDS-PAGE检测到明显的30 kD大小的目的蛋白条带,Western blotting分析3种纯化后的蛋白样品与6×His-tag抗体具有良好的特异性反应。纯化后检测3种表达产物对诱导凋亡的抑制作用。经MTT法检测3种纯化30 K蛋白可明显增强过氧化处理的人血管内皮原代培养细胞(EC)的活力,采用ELISA法检测3种纯化30K蛋白能明显减缓EC细胞中的DNA片段化,显示3种30K蛋白对H2O2诱导的EC细胞的凋亡均具有一定的抑制作用。用生物信息学方法预测3种家蚕30K蛋白的氨基酸序列相似性达43%,三级结构极其相似,推测3种蛋白具有相似的生物学功能。
It has been clear that silkworm（Bombyx mori） 30K protein not only acts as an important energy substance during the growth and development of silkworm,but also has significant inhibitory effect on cell apoptosis.By means ofRT-PCR technology,three cDNA sequences（BmLp-C 6,BmLip19G1 and BmLp-C12p,GenBank accession No.was X54735,AY568957,NM_001101728） of 30K protein were cloned from fat body of the 5th instar silkworm larvae.Bac-to-Bac system was used to construct three recombinant baculovirus expression vectors containing the three 30K protein genes which were then used to transfect silkworm BmN cells for expression of 30K proteins in large quantity.SDS-PAGE detection to purified samples of the three expression products showed clear target protein bands of 30 kD.Western blotting analysis of the three purified 30K protein samples displayed good specific reactions with 6 × His-tag antibody.The three purified expression products were tested for their inhibitory effect on induced apoptosis.MTT assay showed that the three purified proteins could obviously enhance the viability of primary cultured human vascular endothelial cells（ECs） that had undergone peroxidation treatment.ELISA assay displayed that the three purified 30K proteins could remarkably slow down DNA fragmentation in ECs,showing that the three 30K proteins had certain inhibitory effect on apoptosis of ECs induced by H2O2.Bioinformatics analysis results showed that the amino acid sequence similarity of these three kinds of 30K proteins reached 43% and their tertiary structures were very similar,suggesting that these three proteins have similar biological functions.
出处
《蚕业科学》
CAS
CSCD
北大核心
2011年第4期630-636,共7页
ACTA SERICOLOGICA SINICA
基金
浙江省自然科学基金项目(No.Y3090339)
国家高技术研究发展计划"863"项目(No.2011AA100603)
