Spider silk proteins (spidroins) are particularly attractive due to their excellent
biocompatibility. Spider can produce up to seven different types of spidroins,
each with unique properties and functions. Spider minor ampullate silk protein
(MiSp) might be particularly interesting for biomedical applications, as the
constituent silk is mechanically strong and does not super-contract in water,
attributed to its amino acid composition. In this study, we evaluate the potential
of recombinant nanoparticles derived from Araneus ventricosus MiSp as a
protein delivery carrier. The MiSp-based nanoparticles were able to serve as an
effective delivery system, achieving nearly 100% efficiency in loading the model
protein lysozyme, and displayed a sustained release profile at physiological
pH. These nanoparticles could significantly improve the delivery efficacy of
the model proteins through different administration routes. Furthermore,
nanoparticles loaded with model protein antigen lysozyme after subcutaneous
or intramuscular administration could enhance antigen-specific immune
responses in mouse models, through a mechanism involving antigen-depot
effects at the injection site, long-term antigen persistence, and efficient uptake
by dendritic cells as well as internalization by lymph nodes. These findings
highlight the transnational potential of MiSp-based nanoparticle system for
protein drug and vaccine delivery.
蜘蛛小壺腹腺絲（spidroins）因其出色的生物相容性，可以產(chǎn)生多達(dá)七種不同類型的蜘蛛紅素，
每個都有獨特的屬性和功能。蜘蛛小壺腹腺絲（MiSp）可能對生物醫(yī)學(xué)應(yīng)用特別有趣，因為
成分絲具有機械強度，在水中不會過度收縮，歸因于其氨基酸組成。在這項研究中，我們評估了其潛力
來源于Araneus ventricosus MiSp的重組納米粒子作為蛋白質(zhì)遞送載體。
基于MiSp的納米粒子能夠作為有效的交付系統(tǒng)，在裝載模型時實現(xiàn)了近100%的效率
蛋白質(zhì)溶菌酶，并在生理學(xué)上顯示出緩釋特性pH值。這些納米顆粒可以顯著提高
模型蛋白通過不同的給藥途徑。此外，皮下注射后負(fù)載模型蛋白抗原溶菌酶的納米粒子
或肌肉注射可以增強抗原特異性免疫通過涉及抗原庫的機制在小鼠模型中的反應(yīng)
注射部位的影響、長期抗原持久性和有效攝取通過樹突細(xì)胞以及**結(jié)內(nèi)化。
Construction of recombinant plasmid and protein expression
The correct plasmid (pET-NM) was transformed into E. coli BL21 (DE3) competent cells. For protein expression, the E. coli cells were grown at 37 °C in LB medium containing 100 μg/mL ampicillin until OD600 is around 0.8–1.0. Then 1 mM Isopropyl β-D-Thiogalactoside (IPTG , final concentration) was added to the culture for protein expression at 25°C for 12 h. For protein purification, the cells were harvested by centrifugation and lysed using High Pressure Homogenizer (PhD Technology LLC, USA). In order to obtain pure NM IBs, the insoluble pellets were resuspended in 30 ml of washing buffer (20 mM Tris, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 M urea, pH 8.0) and extensively washed for three times. Finally, the inclusion bodies were washed with 20 mM Tris pH 8.0 to remove contaminating detergent and the purified inclusion bodies (NM-IBs) were used for subsequent solubilization study.
重組質(zhì)粒的構(gòu)建及蛋白表達(dá)
將正確的質(zhì)粒（pET-NM）轉(zhuǎn)化到大腸桿菌BL21（DE3）感受態(tài)細(xì)胞中。對于蛋白質(zhì)表達(dá)，大腸桿菌細(xì)胞在37°C下在含有100μg/mL氨芐青霉素的LB培養(yǎng)基中生長，直到OD600約為0.8-1.0。然后將1 mM異丙基β-D-硫代半乳糖苷（IPTG，終濃度）加入培養(yǎng)物中，在25°C下表達(dá)蛋白質(zhì)12小時。為了純化蛋白質(zhì)，通過離心收集細(xì)胞，并使用高壓均質(zhì)機（美國PhD Technology LLC）裂解細(xì)胞。為了獲得純NM-IBs，將不溶性沉淀物重新懸浮在30ml洗滌緩沖液（20mM Tris、300mM NaCl、1mM EDTA、1%Triton X-100、1M尿素，pH 8.0）中，并廣泛洗滌三次。最后，用20mM Tris pH 8.0洗滌包涵體以去除污染的洗滌劑，純化的包涵體（NM IB）用于隨后的增溶研究。