至今,GenScript的服务及产品已被Cell, Nature, Science, PNAS等1300多家生物医药类杂志引用近万次,处于行业领先水平。NIH、哈佛、耶鲁、斯坦福、普林斯顿、杜克大学等约400家全球著名机构使用GenScript的基因合成、多肽服务、抗体服务和蛋白服务等成功地发表科研成果,再次证明GenScript 有能力帮助业内科学家Make research easy.
Nanostructured TNFα protein targets the zebrafish (Danio rerio) immune system through mucosal surfaces and improves the survival after Mycobacterium marinum
Aquaculture.2019;
Ji Jie, Torrealba Debora, Thwaite Rosemary, Gomez Andrómeda-Celeste, Parra David, Roher Nerea
IBsTNFα were produced as described previously (Torrealba et al., 2016). Briefly, E. colicarrying the TNFα expression plasmid was cultured in LB broth with ampicillin (100 μg/ml) at 37 °C. When OD550 reached 0.5, recombinant protein expression was induced with 1 mM isopropyl-D-thiogalactoside (IPTG, Panreac) for 3 h. To purify IBs, enzymatic and mechanical disruption steps, were carried out as detailed in (Torrealba et al., 2016). Finally, samples were centrifuged at 15,000 ×g for 15 min, and pellets containing purified IBs were stored at −80 °C. IBs were checked for contamination on LB plates without antibiotic. Protein was quantified by western blot using an anti-His-tag antibody (Genscript A00186-100) and a standard curve using a recombinant protein and Quantity One software (Bio-Rad).
Waterborne pathogens are a constant threat to fish. Fish mucosa are the point of entry for the majority of microbes and are key in mounting an effective immune response to block invasion, colonization and spread. In the search for tools to enhance immune responses we have evaluated nanostructured TNFα protein nanoparticles (IBsTNFα) as potential mucosal adjuvants. IBsTNFα are non-toxic, stable, protein-based biologically active nanomaterials which are cheap to produce in microbial cell factories. Here we test the in vivo uptake of IBsTNFα in zebrafish larvae by bath immersion and in adult zebrafish by oral gavage. We used flow cytometry, histology and confocal microscopy, to show IBsTNFα can be effici... More
Waterborne pathogens are a constant threat to fish. Fish mucosa are the point of entry for the majority of microbes and are key in mounting an effective immune response to block invasion, colonization and spread. In the search for tools to enhance immune responses we have evaluated nanostructured TNFα protein nanoparticles (IBsTNFα) as potential mucosal adjuvants. IBsTNFα are non-toxic, stable, protein-based biologically active nanomaterials which are cheap to produce in microbial cell factories. Here we test the in vivo uptake of IBsTNFα in zebrafish larvae by bath immersion and in adult zebrafish by oral gavage. We used flow cytometry, histology and confocal microscopy, to show IBsTNFα can be efficiently taken up by the intestinal mucosa. Fluorescently labelled nanoparticles were ingested by 5 dpf larvae and accumulated in the intestine. In time course studies in adult zebrafish we observed the nanoparticles could penetrate the intestinal epithelia, traverse the lamina propria and enter the muscle layer. Gene expression analysis of intestine and spleen shows that some immune-related genes were significantly modulated. Further, we show that intraperitoneal injected IBsTNFαcould protect fish against a lethal infection of Mycobacterium marinum administered by intraperitoneal injection. IBsTNFα therefore can have positive effects on infection outcome and show potential to be developed as a mucosal adjuvant for aquaculture.