Robust Systemic and Mucosal Immune Responses to Coxsackievirus B3 Elicited by Spider Silk Protein 下载文件详细资料

Coxsackievirus B3 (CVB3) is a member of the enterovirus genus and linked to several diseases, including myocarditis, which can progress to dilated cardiomyopathy. Despite ongoing preclinical efforts, no clinically approved vaccines against CVB3 are currently available, highlighting the urgent need for effective prophylactic solutions. In this study, a nanovaccine platform based on spider minor ampullate silk protein (MiSp) is introduced. This platform utilizes protein nanoparticles engineered from chimeric proteins that incorporate CVB3 antigenic peptides into customized MiSp, subsequently loaded with all-trans retinoic acid (RA). These functional nanovaccines are capable of eliciting both mucosal and systemic immune responses following subcutaneous administration and demonstrate significant protective effects against CVB3 infection in mice. This study signifies an approach in peptide-based parenteral vaccine strategies, utilizing engineered MiSp nanoparticles combined with RA. This methodology represents a promising pathway for preventing enterovirus infections by leveraging the unique immunomodulatory properties of spidroins and RA to combat these pathogens effectively.

柯萨奇病毒B3（CVB3）是肠病毒属的成员，与多种疾病有关，包括心肌炎，心肌炎可进展为扩张型心肌病。尽管正在进行临床前研究，但没有临床批准
目前已有针对CVB3的疫苗，这突显了对有效预防方案的迫切需求。本研究介绍了一种基于蜘蛛小安瓿丝蛋白（MiSp）的纳米疫苗平台。该平台利用嵌合蛋白工程化的蛋白质纳米颗粒，将CVB3抗原肽掺入定制的MiSp中，随后负载全反式视黄酸（RA）。这些功能性纳米疫苗能够在皮下给药后引发粘膜和全身免疫反应，并对小鼠CVB3感染显示出显著的保护作用。这项研究标志着一种基于肽的肠外疫苗策略的方法，利用工程化的MiSp纳米颗粒与RA相结合
这项研究标志着基于肽的肠外注射的一种疫苗策略，利用工程化的MiSp纳米颗粒与RA相结合。该方法通过利用蜘蛛丝蛋白和RA的独特免疫调节特性有效对抗这些病原体，为预防肠道病毒感染提供了一条有前景的途径。

Protein Preparation: The chimeric protein sequences encoding NM-VP1T and NM-VP1B, respectively, were synthesized and cloned into the pET-32a plasmid using NdeI and XhoI restriction sites. These plas-mids were subsequently transformed into E. coli BL21 (DE3) competent cells, which were cultured at 37 °C in LB medium supplemented with100 μg mL. ampicillin. Growth continued until OD600 reached ≈0.8, after which the temperature was lowered to 25 °C and 1 mm Isopropyl 𝛽-D-Thiogalactoside (IPTG) was added to induce protein expression. The cells were incubated for an additional 12 h. For protein purification, the cells were harvested via centrifugation and lysed using a high-pressure homogenizer (PhD Technology LLC, USA). The resulting insoluble pellets were thoroughly washed three times with 30 mL of washing buffer (20 mm Tris, 300 mm NaCl, 1 mm EDTA, 1% Triton X-100, 1 m urea, pH 8.0). To remove any residual detergent, the purified inclusion bodies were finally rinsed with 20 mm Tris (pH 8.0) and solubilized using a freeze-thaw method.

蛋白质制备：编码NM的嵌合蛋白序列-VP1T和NM-VP1B分别被合成并克隆到pET-32a质粒使用NdeI和XhoI限制性位点。随后将这些质粒转化到大肠杆菌BL21（DE3）感受态细胞中，在37°C下在添加了100μg mL氨苄青霉素的LB培养基中培养。生长持续到OD600达到≈0.8，之后温度降至25°C，加入1mm异丙基-D硫代半乳糖苷（IPTG）以诱导蛋白质表达。将细胞再孵育12小时。为了纯化蛋白质，通过离心收集细胞，并使用高压均质机（美国PhD Technology LLC）破碎细胞。用30mL洗涤缓冲液（20mm Tris、300mm NaCl、1mm EDTA、1%Triton X-100、1m尿素，pH 8.0）彻底洗涤所得不溶性颗粒3次。为了去除任何残留的洗涤剂，最终用20mm Tris（pH 8.0）冲洗纯化的包涵体，并使用冻融法溶解。

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文件名称：	Robust Systemic and Mucosal Immune Responses to Coxsackievirus B3 Elicited by Spider Silk Protein
公司名称：	PhD Technology LLC
下载次数：	85
文件详细说明：
Coxsackievirus B3 (CVB3) is a member of the enterovirus genus and linked to several diseases, including myocarditis, which can progress to dilated cardiomyopathy. Despite ongoing preclinical efforts, no clinically approved vaccines against CVB3 are currently available, highlighting the urgent need for effective prophylactic solutions. In this study, a nanovaccine platform based on spider minor ampullate silk protein (MiSp) is introduced. This platform utilizes protein nanoparticles engineered from chimeric proteins that incorporate CVB3 antigenic peptides into customized MiSp, subsequently loaded with all-trans retinoic acid (RA). These functional nanovaccines are capable of eliciting both mucosal and systemic immune responses following subcutaneous administration and demonstrate significant protective effects against CVB3 infection in mice. This study signifies an approach in peptide-based parenteral vaccine strategies, utilizing engineered MiSp nanoparticles combined with RA. This methodology represents a promising pathway for preventing enterovirus infections by leveraging the unique immunomodulatory properties of spidroins and RA to combat these pathogens effectively. 柯萨奇病毒B3（CVB3）是肠病毒属的成员，与多种疾病有关，包括心肌炎，心肌炎可进展为扩张型心肌病。尽管正在进行临床前研究，但没有临床批准目前已有针对CVB3的疫苗，这突显了对有效预防方案的迫切需求。本研究介绍了一种基于蜘蛛小安瓿丝蛋白（MiSp）的纳米疫苗平台。该平台利用嵌合蛋白工程化的蛋白质纳米颗粒，将CVB3抗原肽掺入定制的MiSp中，随后负载全反式视黄酸（RA）。这些功能性纳米疫苗能够在皮下给药后引发粘膜和全身免疫反应，并对小鼠CVB3感染显示出显著的保护作用。这项研究标志着一种基于肽的肠外疫苗策略的方法，利用工程化的MiSp纳米颗粒与RA相结合这项研究标志着基于肽的肠外注射的一种疫苗策略，利用工程化的MiSp纳米颗粒与RA相结合。该方法通过利用蜘蛛丝蛋白和RA的独特免疫调节特性有效对抗这些病原体，为预防肠道病毒感染提供了一条有前景的途径。 Protein Preparation: The chimeric protein sequences encoding NM-VP1T and NM-VP1B, respectively, were synthesized and cloned into the pET-32a plasmid using NdeI and XhoI restriction sites. These plas-mids were subsequently transformed into E. coli BL21 (DE3) competent cells, which were cultured at 37 °C in LB medium supplemented with100 μg mL. ampicillin. Growth continued until OD600 reached ≈0.8, after which the temperature was lowered to 25 °C and 1 mm Isopropyl 𝛽-D-Thiogalactoside (IPTG) was added to induce protein expression. The cells were incubated for an additional 12 h. For protein purification, the cells were harvested via centrifugation and lysed using a high-pressure homogenizer (PhD Technology LLC, USA). The resulting insoluble pellets were thoroughly washed three times with 30 mL of washing buffer (20 mm Tris, 300 mm NaCl, 1 mm EDTA, 1% Triton X-100, 1 m urea, pH 8.0). To remove any residual detergent, the purified inclusion bodies were finally rinsed with 20 mm Tris (pH 8.0) and solubilized using a freeze-thaw method. 蛋白质制备：编码NM的嵌合蛋白序列-VP1T和NM-VP1B分别被合成并克隆到pET-32a质粒使用NdeI和XhoI限制性位点。随后将这些质粒转化到大肠杆菌BL21（DE3）感受态细胞中，在37°C下在添加了100μg mL氨苄青霉素的LB培养基中培养。生长持续到OD600达到≈0.8，之后温度降至25°C，加入1mm异丙基-D硫代半乳糖苷（IPTG）以诱导蛋白质表达。将细胞再孵育12小时。为了纯化蛋白质，通过离心收集细胞，并使用高压均质机（美国PhD Technology LLC）破碎细胞。用30mL洗涤缓冲液（20mm Tris、300mm NaCl、1mm EDTA、1%Triton X-100、1m尿素，pH 8.0）彻底洗涤所得不溶性颗粒3次。为了去除任何残留的洗涤剂，最终用20mm Tris（pH 8.0）冲洗纯化的包涵体，并使用冻融法溶解。了解更多，欢迎下载全文，或请联系 15221020121交流。
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