College of Food and Biological Engineering, Xiamen Key Laboratory of Marine Functional Food, Fujian Provincial Engineering Technology Research Center of Marine Functional Food, Jimei University, Xiamen, Fujian 361021, China.
Hefei National Laboratory for Physical Sciences at Microscale, CAS Key Laboratory of Innate Immunity and Chronic Disease, Division of Life Sciences and Medicine, University of Science & Technology of China, Hefei, Anhui 230026, China.
J Agric Food Chem. 2021 Jan 13;69(1):428-436. doi: 10.1021/acs.jafc.0c05733. Epub 2020 Dec 30.
In order to reduce the immunoreactivity of sarcoplasmic calcium-binding protein (SCP), site-directed mutations were used to replace key amino acids in the conformational epitopes and calcium-binding sites. The mutant SCPs (mSCPs) were expressed in , and their immunoreactivities were analyzed using iELISA and basophil activation assays. Furthermore, the structural changes of mSCPs were determined from the circular dichroism spectra. The iELISA results showed that mSCPs could effectively inhibit the binding of wild-type SCP (wtSCP) to sensitive serum, with inhibition rates that reached 90%. Moreover, mSCPs could downregulate the expression levels of CD63 and CD203c on the basophil surface. Compared with wtSCP, the peak values were significantly changed, and the calcium binding ability was impaired, which explained the decline in immunoreactivities of the mSCPs. All of the data confirmed that this approach was effective in reducing the immunoreactivity of SCP and could be applied to other shellfish allergens.
为了降低肌浆钙结合蛋白（SCP）的免疫原性，采用定点突变的方法替换构象表位和钙结合位点中的关键氨基酸。突变 SCP（mSCP）在大肠杆菌中表达，并使用 iELISA 和嗜碱性粒细胞激活试验分析其免疫原性。此外，还通过圆二色谱光谱确定了 mSCP 的结构变化。iELISA 结果表明，mSCP 可以有效抑制野生型 SCP（wtSCP）与敏感血清的结合，抑制率达到 90%。此外，mSCP 可以下调嗜碱性粒细胞表面 CD63 和 CD203c 的表达水平。与 wtSCP 相比，峰值明显改变，钙结合能力受损，这解释了 mSCP 免疫原性下降的原因。所有数据均证实，该方法可有效降低 SCP 的免疫原性，可应用于其他贝类过敏原。
