两个家蚕FKBP12基因的研究
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摘要
FKBPs是免疫抑制剂FK506的胞内受体蛋白,这些蛋白参与多种生理学作用,包括蛋白折叠,T细胞激活的抑制调节,以及细胞周期抑制等。12 kD的FKBPs蛋白是其代表性成员,在各物种细胞内广泛表达且高度保守,具有PPIase活性。目前只是发现在脊椎动物中有多个FKBP12亚型,而我们通过NCBI家蚕基因库中找到两条完整编码家蚕FKBP12蛋白的nRNA序列,命名为Bm-FKBP12A和Bm-FKBP12B,基因登录号分别为DQ443197和DQ443423。多物种序列比对发现,FKBP12同源基因以及同源异性基因在氨基酸水平上具有高度的相似性,Bm-FKBP12A蛋白与人的FKBP12蛋白同源性为77%Bm-FKBP12B蛋白与人的FKBP12蛋白同源性为73%,Bm-FKBP12A和Bm-FKBP12B之间的相似性为83%。Bm-FKBP12A蛋白推测的的分子量、等电点分别为11.82kD和7.86,Bm-FKBP12B蛋白的分子量、等电点分别为11.63kD和8.79,这些特征与已知的FKBP12蛋白相似。
将两种FKBP12基因的cDNA序列与家蚕全基因组序列进行比对,两个基因都由3个外显子和2个内含子组成,但Bm-FKBP12A基因的内含子序列要比Bm-FKBP12B长。通过在线网站分析了Bm-FKBP12A和Bm-FKBP12B的启动子序列并对两个基因进行了染色体定位预测,发现两个基因都定位在20号染色体上,距离相差1500kbp。Bm-FKBP12A在5′端上游(-361)—(-311)bp的区域有一个启动子序列,Bm-FKBP12B在5′端上游(-161)—(-121)bp的区域有一个启动子序列。组织特异性顺式作用元件没有在Bm-FKBP12A和Bm-FKBP12B基因中发现,说明家蚕FKBP12蛋白在各组织中广泛表达。
Bm-FKBP12B基因的ORF被克隆到pGEX-4T-3载体中,在大肠杆菌BL21中表达融合蛋白GST-Bm-FKBP12B,通过GST亲和层析,得到较纯的融合蛋白GST-Bm-FKBP12B,凝血酶将融合蛋白中的GST切除后,再通过分子筛SephadexG-75进一步纯化。将经过纯化的Bm-FKBP12B作为抗原免疫新西兰大白兔制备多克隆抗体,ELISA检测该抗血清的效价达到1：12800以上。对纯化的Bm-FKBP12B蛋白进行了PPIase活性测定,结果显示具有较高活性。通过相似EST搜索、实时荧光相对定量PCR和Western blotting分析了两种FKBP12蛋白在家蚕各个时期的表达以及各个组织的分布。发现Bm-FKBP12存在于家蚕各个组织及各个时期,Bm-FKBP12A的转录本在各个组织均比Bm-FKBP12B高,而Bm-FKBP12B的转录本在蛾期和胚胎期最高。Western blotting显示FKBP12在丝腺和肠的表达最高,暗示在吐丝前期FKBP12蛋白可能对丝腺的基因表调控以及丝蛋白的形成具有重要作用。GST-Pull down显示家蚕FKBP12与Hmo1蛋白不能发生体外互作。以上这些结果为深入研究家蚕FKBP12基因奠定了基础。
FKBPs are cytosolic receptors for the immunosuppressive drug FK506, which participate in a variety of pathways, including protein folding, down-regulation of T-cell activation and inhibition of cell-cycle progression. The 12 kD FKBPs (also known as FKBP12) are the most comprehensively studied proteins of FKBPs,which are highly conserved, abundant in various cells and possess peptidyl-prolyl cis-trans isomerase(PPIase) activity. It is also of note that among the animal taxa, only the vertebrates were found to possess multiple FKBP12 genes. We searched two complete mRNA sequences encoding the 12 kD FKBP gene orthologue (FKBP12) in Bombyx mori by NCBI GenBank database,named Bm-FKBP12A and Bm-FKBP12B, of which the accession number was DQ443197 and DQ443423. A multiple sequence alignment among species from various taxonomic groups shows high similarity among FKBP 12 paralogues and orthologues at the amino acid level. Homogeneous analysis showed that Bm-FKBP12A shared 77% of identity with that in homo-sapiens, and Bm-FKBP12B shared 73% of identify to that in homo-sapiens respectively. Each other's similarity of 83%. According to the sequencing data, the molecular weights, and isoelectric points were estimated as 11.82kD and 7.86 for Bm-FKBP12A, and 11.63kD and 8.79 for Bm-FKBP12B. These characterizations of two FKBP 12 proteins were similar with other FKBP 12 proteins identified.
The cDNA sequences of the two genes was blasted with Whole-genome sequence of silkworm database on NCBI, two genes both composed of by three exons and two introns,and Bm-FKBP12A have long-introns than Bm-FKBP12B. We analysis the promoter sequences,and precipite chromosomal localizationof them by online web. They both was located on chromosome 20,have the 1500 kbp away. Reporter gene analyses revealed that the regions of (-361)—(-311)bp of Bm-FKBP12A and (-161)—(-121) bp of Bm-FKBPI2B are important for promoter activities in the 5'flanking region. Tissue specific cis-elements were found in the 5'flanking region of neither the Bm-FKBP12A nor Bm-FKBP12B. These results are consistent with the fact that Bm-FKBP12 mRNAs are expressed ubiquitously in a variety of tissues and cells.
The gene of Bm-FKBP12B was cloned into pGEX-4T-3 vector and expressed in Escherichia coli BL21 (E. coli). After the recombinant protein purified by the GST-affinity chromatography, digested by the thrombin and further purified by the Sephadex G-75 chromatography, we attained highly purified Bm-FKBP12B. The purified protein was injected into the New Zealand white rabbit to produce the polyclonal antibody, the titer of the antibodies was determined to be above 1:128000 by ELISA.The purified Bm-FKBP12B show a high activity of peptidyl-prolyl cis-trans isomerase (PPIase). Using similar EST searching, real-time quantitative PCR and Western blotting, the expression of two silkworm FKBP12 proteins in every stage and every tissue was investigated. Bm-FKBP12 localized in almost every organ no matter which development stage the silkworm in was. We found that transcripts of the Bm-FKBP12A gene were more abundant in every development stage than Bm-FKBP12B;And transcripts of Bm-FKBP12B were more abundant in the stage of moth and embryo.According to Western blotting, the Bm-FKBP12 expression levels are higher in silk gland and gut,suggest that Bm-FKBP12 before spinning stage might play important roles in development of silkworm particular the gene expression regulation of silkgland and the formation of silk fiber.There are no interation between FKBP12 and Hmol by GST-Pull down essay. These results laid a good foundation of further studies of Bm-FKBP12.
将两种FKBP12基因的cDNA序列与家蚕全基因组序列进行比对,两个基因都由3个外显子和2个内含子组成,但Bm-FKBP12A基因的内含子序列要比Bm-FKBP12B长。通过在线网站分析了Bm-FKBP12A和Bm-FKBP12B的启动子序列并对两个基因进行了染色体定位预测,发现两个基因都定位在20号染色体上,距离相差1500kbp。Bm-FKBP12A在5′端上游(-361)—(-311)bp的区域有一个启动子序列,Bm-FKBP12B在5′端上游(-161)—(-121)bp的区域有一个启动子序列。组织特异性顺式作用元件没有在Bm-FKBP12A和Bm-FKBP12B基因中发现,说明家蚕FKBP12蛋白在各组织中广泛表达。
Bm-FKBP12B基因的ORF被克隆到pGEX-4T-3载体中,在大肠杆菌BL21中表达融合蛋白GST-Bm-FKBP12B,通过GST亲和层析,得到较纯的融合蛋白GST-Bm-FKBP12B,凝血酶将融合蛋白中的GST切除后,再通过分子筛SephadexG-75进一步纯化。将经过纯化的Bm-FKBP12B作为抗原免疫新西兰大白兔制备多克隆抗体,ELISA检测该抗血清的效价达到1：12800以上。对纯化的Bm-FKBP12B蛋白进行了PPIase活性测定,结果显示具有较高活性。通过相似EST搜索、实时荧光相对定量PCR和Western blotting分析了两种FKBP12蛋白在家蚕各个时期的表达以及各个组织的分布。发现Bm-FKBP12存在于家蚕各个组织及各个时期,Bm-FKBP12A的转录本在各个组织均比Bm-FKBP12B高,而Bm-FKBP12B的转录本在蛾期和胚胎期最高。Western blotting显示FKBP12在丝腺和肠的表达最高,暗示在吐丝前期FKBP12蛋白可能对丝腺的基因表调控以及丝蛋白的形成具有重要作用。GST-Pull down显示家蚕FKBP12与Hmo1蛋白不能发生体外互作。以上这些结果为深入研究家蚕FKBP12基因奠定了基础。
FKBPs are cytosolic receptors for the immunosuppressive drug FK506, which participate in a variety of pathways, including protein folding, down-regulation of T-cell activation and inhibition of cell-cycle progression. The 12 kD FKBPs (also known as FKBP12) are the most comprehensively studied proteins of FKBPs,which are highly conserved, abundant in various cells and possess peptidyl-prolyl cis-trans isomerase(PPIase) activity. It is also of note that among the animal taxa, only the vertebrates were found to possess multiple FKBP12 genes. We searched two complete mRNA sequences encoding the 12 kD FKBP gene orthologue (FKBP12) in Bombyx mori by NCBI GenBank database,named Bm-FKBP12A and Bm-FKBP12B, of which the accession number was DQ443197 and DQ443423. A multiple sequence alignment among species from various taxonomic groups shows high similarity among FKBP 12 paralogues and orthologues at the amino acid level. Homogeneous analysis showed that Bm-FKBP12A shared 77% of identity with that in homo-sapiens, and Bm-FKBP12B shared 73% of identify to that in homo-sapiens respectively. Each other's similarity of 83%. According to the sequencing data, the molecular weights, and isoelectric points were estimated as 11.82kD and 7.86 for Bm-FKBP12A, and 11.63kD and 8.79 for Bm-FKBP12B. These characterizations of two FKBP 12 proteins were similar with other FKBP 12 proteins identified.
The cDNA sequences of the two genes was blasted with Whole-genome sequence of silkworm database on NCBI, two genes both composed of by three exons and two introns,and Bm-FKBP12A have long-introns than Bm-FKBP12B. We analysis the promoter sequences,and precipite chromosomal localizationof them by online web. They both was located on chromosome 20,have the 1500 kbp away. Reporter gene analyses revealed that the regions of (-361)—(-311)bp of Bm-FKBP12A and (-161)—(-121) bp of Bm-FKBPI2B are important for promoter activities in the 5'flanking region. Tissue specific cis-elements were found in the 5'flanking region of neither the Bm-FKBP12A nor Bm-FKBP12B. These results are consistent with the fact that Bm-FKBP12 mRNAs are expressed ubiquitously in a variety of tissues and cells.
The gene of Bm-FKBP12B was cloned into pGEX-4T-3 vector and expressed in Escherichia coli BL21 (E. coli). After the recombinant protein purified by the GST-affinity chromatography, digested by the thrombin and further purified by the Sephadex G-75 chromatography, we attained highly purified Bm-FKBP12B. The purified protein was injected into the New Zealand white rabbit to produce the polyclonal antibody, the titer of the antibodies was determined to be above 1:128000 by ELISA.The purified Bm-FKBP12B show a high activity of peptidyl-prolyl cis-trans isomerase (PPIase). Using similar EST searching, real-time quantitative PCR and Western blotting, the expression of two silkworm FKBP12 proteins in every stage and every tissue was investigated. Bm-FKBP12 localized in almost every organ no matter which development stage the silkworm in was. We found that transcripts of the Bm-FKBP12A gene were more abundant in every development stage than Bm-FKBP12B;And transcripts of Bm-FKBP12B were more abundant in the stage of moth and embryo.According to Western blotting, the Bm-FKBP12 expression levels are higher in silk gland and gut,suggest that Bm-FKBP12 before spinning stage might play important roles in development of silkworm particular the gene expression regulation of silkgland and the formation of silk fiber.There are no interation between FKBP12 and Hmol by GST-Pull down essay. These results laid a good foundation of further studies of Bm-FKBP12.
引文
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