Ana C K Pedra, Natasha R de Oliveira, Mara A C Maia, Francisco D S Santos, Tiffany T Bunde, Pedro H F C Souza, Laura de V Maiocchi, Odir A Dellagostin, Thaís L O Bohn
{"title":"生产重组细胞因子和多克隆抗体,用于分析金色叙利亚仓鼠的细胞免疫反应。","authors":"Ana C K Pedra, Natasha R de Oliveira, Mara A C Maia, Francisco D S Santos, Tiffany T Bunde, Pedro H F C Souza, Laura de V Maiocchi, Odir A Dellagostin, Thaís L O Bohn","doi":"10.1007/s11033-024-09940-5","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>The development of therapies and vaccines for various diseases often necessitates the analysis of cellular immunity. However, unlike other rodents, the limited availability of reagents for Syrian hamsters restricts immunological analysis, particularly in the determination of serum effector molecules such as cytokines. In this study, we aim to produce and characterize the cytokines IFN-γ, TGF-β, IL-6, IL-10, and TNF-α from Syrian hamsters in recombinant form and to generate polyclonal antibodies against them in rats.</p><p><strong>Methods and results: </strong>Cytokine transcript sequences were cloned into expression vectors in E. coli. Recombinant proteins were produced, purified through affinity chromatography, and characterized by Western blot using an anti-6xHis monoclonal antibody. Rats were immunized with the recombinant proteins to generate polyclonal antibodies (pAbs). These pAbs were characterized by Western blot and titrated by indirect ELISA. The recombinant cytokines rTNF-α, rIL-10, rIFN-γ, rTGF-β, and rIL-6 were produced and specifically recognized at their expected molecular weights of 22.3 kDa, 19.8 kDa, 18.9 kDa, 11.8 kDa, and 22.9 kDa. pAbs were produced and demonstrated the ability to specifically recognize their target proteins with titers of 409,600 (rIL-10), 204,800 (rTNF-α), 102,400 (rIL-10), 51,200 (rTGF-β), and 25,600 (rIFN-ɣ).</p><p><strong>Conclusions: </strong>The reagents produced represent a starting point for developing immunoassays to detect hamster cytokines, facilitating the analysis of cellular immunity in this biomodel.</p>","PeriodicalId":18755,"journal":{"name":"Molecular Biology Reports","volume":null,"pages":null},"PeriodicalIF":2.6000,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Production of recombinant cytokines and polyclonal antibodies for analysis of cellular immune response in golden Syrian hamster.\",\"authors\":\"Ana C K Pedra, Natasha R de Oliveira, Mara A C Maia, Francisco D S Santos, Tiffany T Bunde, Pedro H F C Souza, Laura de V Maiocchi, Odir A Dellagostin, Thaís L O Bohn\",\"doi\":\"10.1007/s11033-024-09940-5\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>The development of therapies and vaccines for various diseases often necessitates the analysis of cellular immunity. However, unlike other rodents, the limited availability of reagents for Syrian hamsters restricts immunological analysis, particularly in the determination of serum effector molecules such as cytokines. In this study, we aim to produce and characterize the cytokines IFN-γ, TGF-β, IL-6, IL-10, and TNF-α from Syrian hamsters in recombinant form and to generate polyclonal antibodies against them in rats.</p><p><strong>Methods and results: </strong>Cytokine transcript sequences were cloned into expression vectors in E. coli. Recombinant proteins were produced, purified through affinity chromatography, and characterized by Western blot using an anti-6xHis monoclonal antibody. Rats were immunized with the recombinant proteins to generate polyclonal antibodies (pAbs). These pAbs were characterized by Western blot and titrated by indirect ELISA. The recombinant cytokines rTNF-α, rIL-10, rIFN-γ, rTGF-β, and rIL-6 were produced and specifically recognized at their expected molecular weights of 22.3 kDa, 19.8 kDa, 18.9 kDa, 11.8 kDa, and 22.9 kDa. pAbs were produced and demonstrated the ability to specifically recognize their target proteins with titers of 409,600 (rIL-10), 204,800 (rTNF-α), 102,400 (rIL-10), 51,200 (rTGF-β), and 25,600 (rIFN-ɣ).</p><p><strong>Conclusions: </strong>The reagents produced represent a starting point for developing immunoassays to detect hamster cytokines, facilitating the analysis of cellular immunity in this biomodel.</p>\",\"PeriodicalId\":18755,\"journal\":{\"name\":\"Molecular Biology Reports\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.6000,\"publicationDate\":\"2024-09-25\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Molecular Biology Reports\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1007/s11033-024-09940-5\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular Biology Reports","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1007/s11033-024-09940-5","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Production of recombinant cytokines and polyclonal antibodies for analysis of cellular immune response in golden Syrian hamster.
Background: The development of therapies and vaccines for various diseases often necessitates the analysis of cellular immunity. However, unlike other rodents, the limited availability of reagents for Syrian hamsters restricts immunological analysis, particularly in the determination of serum effector molecules such as cytokines. In this study, we aim to produce and characterize the cytokines IFN-γ, TGF-β, IL-6, IL-10, and TNF-α from Syrian hamsters in recombinant form and to generate polyclonal antibodies against them in rats.
Methods and results: Cytokine transcript sequences were cloned into expression vectors in E. coli. Recombinant proteins were produced, purified through affinity chromatography, and characterized by Western blot using an anti-6xHis monoclonal antibody. Rats were immunized with the recombinant proteins to generate polyclonal antibodies (pAbs). These pAbs were characterized by Western blot and titrated by indirect ELISA. The recombinant cytokines rTNF-α, rIL-10, rIFN-γ, rTGF-β, and rIL-6 were produced and specifically recognized at their expected molecular weights of 22.3 kDa, 19.8 kDa, 18.9 kDa, 11.8 kDa, and 22.9 kDa. pAbs were produced and demonstrated the ability to specifically recognize their target proteins with titers of 409,600 (rIL-10), 204,800 (rTNF-α), 102,400 (rIL-10), 51,200 (rTGF-β), and 25,600 (rIFN-ɣ).
Conclusions: The reagents produced represent a starting point for developing immunoassays to detect hamster cytokines, facilitating the analysis of cellular immunity in this biomodel.
期刊介绍:
Molecular Biology Reports publishes original research papers and review articles that demonstrate novel molecular and cellular findings in both eukaryotes (animals, plants, algae, funghi) and prokaryotes (bacteria and archaea).The journal publishes results of both fundamental and translational research as well as new techniques that advance experimental progress in the field and presents original research papers, short communications and (mini-) reviews.