Identification and location of sericin in silkworm with anti-sericin antibodies.

Sericin, as the main component of silkworm cocoon silk, surrounds and protects the silk fibroin. Sericin is a natural macromolecular protein complex encoded by the genes Ser1, Ser2, and Ser3. At present, there are no available antibodies against sericin that may be used to identify and locate it at the protein level, hindering the study of its secretion mechanism and materials application. Therefore, the development of effective antibodies against sericin is an urgent necessity. To address this problem, we prepared polyclonal antibodies against the Ser1, Ser2 and Ser3 proteins using synthesized peptides for the first time. The specificity of the antibodies was confirmed using dot blot, immunoblotting and mass spectrometry on the hybrid bands of the middle silk gland. The immunoblotting results of anti-sericin antibodies showed that sericin has different molecular weights in different regions of the middle silk gland and strains in the 5th instar. Through immunohistochemistry, anti-sericin antibodies revealed that sericin presented different distributions in the anterior part of the middle silk gland of 872 strain at the 7th day of 5th instar. In addition, the prepared antibodies not only detected intact sericin molecules, but also detected degraded sericin that was dissolved in five different solvents. In summary, this work prepared effective sericin antibodies for silk protein synthesis and secretion research and provides a possible molecular detection method for biological products containing silkworm sericin.

In Vivo Characterizations of the Immune Properties of Sericin: An Ancient Material with Emerging Value in Biomedical Applications.

The biosafety of sericin remains controversial. The misunderstanding regarding sericin causing adverse biological responses have been clarified by extensively reviewing relevant literatures and experimentally demonstrating that sericin exhibits mild inflammatory responses, negligible allergenicity, and low immunogenicity in vivo. This study supports that sericin is biosafe as a natural biomaterial.

Tolerogenic responses of CD206+, CD83+, FOXP3+, and CTLA-4 to sericin/polyvinyl alcohol/glycerin scaffolds relevant to IL-33 and HSP60 activity.

Silk sericin-releasing (sericin/polyvinyl alcohol (PVA)/glycerin) scaffolds have been designed for wound dressing applications using different fabrication techniques that influence scaffold antigenicity. The immunological tolerance of scaffolds depends on the balance of immunogenic and tolerogenic responses modulated by dendritic cells (DCs). An in vivo skin implantation model was used to compare the tolerogenic effect of sericin/PVA/glycerin scaffolds prepared by freeze-drying versus salt-leaching techniques, using an Allevyn® scaffold as a control. Immunohistochemical and histopathological studies were performed to evaluate tolerogenic DCs (CD206+), immunogenic DCs (CD83+), regulatory T-cells (FOXP3+ and CTLA-4), a proinflammatory cytokine (interleukin 33: IL-33), a stress marker (heat shock protein 60; HSP60), histopathological changes and related inflammatory cells. It was found that both sericin/PVA/glycerin scaffolds were tolerogenic and induced early activated Treg functions, while the Allevyn® scaffold was immunogenic. However, the tolerance of the freeze-dried sericin/PVA/glycerin scaffolds was not as consistent as the salt-leached sericin/PVA/glycerin scaffolds, indicated by the low level of CTLA-4 expression. This was probably due to molecular cross-linking and the morphological and mechanical properties of the freeze-drying technique, which would enhance the immune response. Severe inflammatory responses (including mast cell degranulation and foreign body giant cell accumulation) and histopathological changes (including fat infiltration and fibrosis formation) were mainly found with the Allevyn® scaffold, presumably from its architecture and chemical composition, especially polyurethane. The up-regulation of IL-33 and HSP60 with the Allevyn® scaffold was correlated with the inflammatory and pathological levels. Our findings suggested that salt-leached sericin/PVA/glycerin scaffolds were tolerogenic, induced a low inflammatory response and were appropriate for wound dressing applications.

Silk sericin-insulin bioconjugates: synthesis, characterization and biological activity.

When silk fiber derived from Bombyx mori was subjected to degumming treatments twice in water and subsequent degraded processing in slightly alkaline aqueous solution under high-temperature and high-pressure, the water-soluble silk sericin peptides (SS) with different molecular mass from 10 to 70 kDa were obtained. The sericin peptides could be conjugated covalently with insulin alone with cross-linking reagent glutaraldehyde. The physicochemical properties of the silk sericin-insulin (SS-Ins) conjugates were determined by Enzyme-Linked Immunosorbent Assay (ELISA). The biological activities of SS-Ins bioconjugates were investigated in vitro and in vivo. The results in human serum in vitro indicated that the half-life of the synthesized SS-Ins derivatives was 2.3 and 2.7 times more than that of bovine serum albumin-insulin (BSA-Ins) conjugates and intact insulin, respectively. The pharmacological activity of SS-Ins bioconjugates lengthened to 21 h in mice in vivo, which was over 4 times longer than that of the native insulin. The immunogenicity of silk sericin and the antigenicity of SS-Ins derivatives were not observed in both rabbits and mice. The bioconjugation of insulin with silk sericin protein evidently improved both physicochemical and biological stability of the polypeptide.