RESUMO
Silkworm sericin has been widely exploited in biomaterials due to its favorable biological activities. However, the extraction processes of sericin from silkworm cocoons can alter the biological and biophysical properties, including a structural diversity of natural sericin. In addition, extracted natural sericin is often contaminated with fibroin that may be harmful to human cells. Induction of tolerogenic dendritic cell (DC) has become a strategy in biomaterial fields because this cell type plays a key role in immune modulation and wound healing. To overcome undesired effects of extracted natural sericin and to improve its biological properties, we biosynthesized sericin 1-like protein that contained only functional motifs and tested its biological activity and immunomodulatory properties in fibroblasts and DCs, respectively. In comparison to natural sericin, biosynthetic sericin 1 promoted collagen production in fibroblasts at a late time point. Furthermore, DCs treated with biosynthetic sericin 1 exhibited a tolerogenic-like phenotype with semimaturation and low production of proinflammatory cytokines, but high production of anti-inflammatory cytokine, IL-10. Biosynthetic sericin 1 might be developed as immunomodulator or immunosuppressant.
Assuntos
Células Dendríticas/metabolismo , Sericinas/biossíntese , Animais , Células Cultivadas , Colágeno/biossíntese , Células Dendríticas/química , Feminino , Fibroblastos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Sericinas/análiseRESUMO
Silk production in Bombyx mori L. is largely determined by the expression of genes encoding fibroin and sericin. Here, we examined the regulatory function of a microRNA (miRNA) on silk gene expression using the sericin-1 gene (BmSer-1). First, we downloaded whole mature miRNAs of silkworm from miRBase and identified bmo-miR-2780a as a candidate miRNA for the regulation of BmSer-1 expression. We used semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) with stem-loop primers to investigate the expression profile of bmo-miR-2780a and its predicted target gene BmSer-1 in seven different tissues from 5th instar day-3 larvae, including head, fat body, anterior silk gland (ASG), middle silk gland (MSG), posterior silk gland (PSG), middle gut, and hemolymph. Our results showed that bmo-miR-2780a was specifically expressed in the MSG and that the expression level of BmSer-1 was significantly higher in the MSG than in other tissues. Recombinant plasmids carrying both pri-mir-2780a and Ser1-3'UTR were constructed and then used to cotransfect BmN cells. We further detected the effect of bmo-miR-2780a on Ser-1 in vivo. These results showed that the target gene was significantly decreased by miR-2780a compared with the control group (p < .05), thus indicating that bmo-miR-2780a might negatively regulate the expression of Ser-1.
Assuntos
Bombyx/genética , Proteínas de Insetos/genética , MicroRNAs/genética , Sericinas/genética , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Proteínas de Insetos/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , MicroRNAs/metabolismo , Sericinas/metabolismoRESUMO
Various genetically modified bioreactor systems have been developed to meet the increasing demands of recombinant proteins. Silk gland of Bombyx mori holds great potential to be a cost-effective bioreactor for commercial-scale production of recombinant proteins. However, the actual yields of proteins obtained from the current silk gland expression systems are too low for the proteins to be dissolved and purified in a large scale. Here, we proposed a strategy that reducing endogenous sericin proteins would increase the expression yield of foreign proteins. Using transgenic RNA interference, we successfully reduced the expression of BmSer1 to 50%. A total 26 transgenic lines expressing Discosoma sp. red fluorescent protein (DsRed) in the middle silk gland (MSG) under the control of BmSer1 promoter were established to analyze the expression of recombinant. qRT-PCR and western blotting showed that in BmSer1 knock-down lines, the expression of DsRed had significantly increased both at mRNA and protein levels. We did an additional analysis of DsRed/BmSer1 distribution in cocoon and effect of DsRed protein accumulation on the silk fiber formation process. This study describes not only a novel method to enhance recombinant protein expression in MSG bioreactor, but also a strategy to optimize other bioreactor systems.
Assuntos
Reatores Biológicos , Bombyx/genética , Bombyx/metabolismo , Interferência de RNA , Proteínas Recombinantes/biossíntese , Sericinas/genética , Seda/biossíntese , Animais , Animais Geneticamente Modificados , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/genética , Seda/genética , Proteína Vermelha FluorescenteRESUMO
Hox genes are well-known master regulators in developmental morphogenesis along the anteroposterior axis of animals. However, the molecular mechanisms by which Hox proteins regulate their target genes and determine cell fates are not fully understood. The silk gland of Bombyx mori is a tubular tissue divided into several subparts along the anteroposterior axis, and the silk genes are expressed with specific patterns. The sericin-1 gene (ser1) is expressed in the middle silk gland (MSG) with sublocal specificity. Here we show that the Hox protein Antp is a component of the middle silk gland-specific complex, MIC (MSG-intermolt-specific complex), binds to the essential promoter element of ser1, and activates its expression. Ectopic expression of Antp in transgenic silkworms induced the expression of ser1 in the posterior silk gland (PSG), but not in the anterior part of MSG (MSG-A). Correspondingly, a MIC-like complex was formed by the addition of recombinant Antp in extracts from PSG with its cofactors Exd and Hth, but not in extracts from MSG-A. Splicing patterns of ser1 mRNA induced by the ectopic expression of Antp in PSG were almost the same as those in MSG at the fifth instar and altered depending on the induction timing of Antp. Other Hox genes were expressed with sublocal specificity in the silk gland. The Bombyx silk gland might provide a useful system for understanding how Hox proteins select and regulate their target genes.
Assuntos
Bombyx/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Sericinas/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Bombyx/metabolismo , Diferenciação Celular , Clonagem Molecular , Feminino , Perfilação da Expressão Gênica , Hibridização In Situ , Larva , Masculino , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Splicing de RNA , Proteínas Recombinantes/metabolismo , Seda/metabolismo , Distribuição Tecidual , TransgenesRESUMO
The domestic silkworm is a type of lepidopteran insect that feeds on mulberry leaves and has high economic value because of its ability to spin cocoons. Sericin 1 is an important component of silkworm cocoons, accounting for approximately 25% of the material. In this study, CRISPR/Cas9-mediated gene editing was successfully used to destroy the sericin 1 gene, and homozygous mutants were obtained after continuous screening. Homozygous mutation resulted in premature termination of the translation of sericin 1 protein at 323 amino acids. Comparative transcriptomic and proteomic analyses of middle silk gland cells from wild-type individuals and mutants were performed on the fourth day of the fifth instar, and the results suggest that sericin 1 plays an important role in the cellular immune system. In addition, the results suggest that sericin 1 has a synergistic effect with some protease inhibitors and that the secretion of these proteins is strictly regulated. These results will provide new insights into the function and expression pattern of sericin 1 and the mechanism of silk secretion.
RESUMO
Sericin is a kind of water-soluble protein expressed specifically in the middle silk gland of Bombyx mori. When the sericin-1 gene promoter was cloned and a transgenic vector was constructed to express a foreign protein, a specific Helitron, Bmhel-8, was identified in the sericin-1 gene promoter sequence in some genotypes of Bombyx mori and Bombyx mandarina. Given that the Bmhel-8 Helitron transposon was present only in some genotypes, it could be the source of allelic variation in the sericin-1 promoter. The length of the sericin-1 promoter sequence is approximately 1063 or 643 bp. The larger size of the sequence or allele is ascribed to the presence of Bmhel-8. Silkworm genotypes can be homozygous for either the shorter or larger promoter sequence or heterozygous, containing both alleles. Bmhel-8 in the sericin-1 promoter exhibits enhancer activity, as demonstrated by a dual-luciferase reporter system in BmE cell lines. Furthermore, Bmhel-8 displays enhancer activity in a sericin-1 promoter-driven gene expression system but does not regulate the tissue-specific expression of sericin-1.