旋毛虫钙网蛋白表达纯化及其与补体C1q互作位点的初探

EXPRESSION AND PURIFICATION OF TRICHINELLA SPIRALIS CALRETICULIN AND PRELIMINARY EXPLORATION OF ITS INTERACTION SITES WITH COMPONENT C1Q

旋毛虫钙网蛋白(Trichinella spiralis calreticulin,Ts-CRT)可以通过与宿主补体C1q互作后,帮助虫体逃避宿主补体系统介导的免疫杀伤。为研究Ts-CRT与C1q相互作用的分子结构基础,本研究首先通过分子克隆和亲和层析等方法获得了高纯度的重组蛋白Ts-CRT^(380);其次通过微量热涌动(microscale thermophoresis,MST)实验测定Ts-CRT^(380)与C1q相互作用能力;然后利用Discovery Studio软件模拟Ts-CRT与C1q的分子对接,分析Ts-CRT上参与互作的关键位点,并与4种蠕虫CRT相应位点进行序列比对,分析其保守性。结果显示,Ts-CRT^(380)与C1q解离常数K_(d)值为149μmol/L,证明二者存在中等强度的相互作用,Ts-CRT通过K145、W323、F77和K47等17个氨基酸残基与人源C1q互作,序列比对证实这17个氨基酸位点在5种蠕虫CRT中高度保守。本研究结合MST实验与生物信息学分析,获得了Ts-CRT与人源补体组分C1q的互作位点,有助于理解旋毛虫逃避宿主补体攻击的分子结构和机制;通过序列比对发现旋毛虫CRT与C1q互作位点在其他蠕虫CRT中高度保守,可为以CRT为靶点研制抗蠕虫新药提供依据。

Trichinella spiralis calreticulin(Ts-CRT)could help the parasite evade host complement system-mediated immune attacking by interacting with complement C1q.To explore the structural basis of the interaction between Ts-CRT and C1q,series of experiments were conducted as follows.Firstly,the recombinant protein Ts-CRT^(380)in high purity was obtained through molecular cloning,affinity chromatography and other purification methods.Secondly,the affinity for the interaction between Ts-CRT^(380)and C1q was quantified by Microscale thermophoresis(MST)assay.Thirdly,molecular docking was conducted using Discovery Studio software to identify the key amino acids involved in the interaction between Ts-CRT and C1q.And finally align these key amino acids with the corresponding sites of other four helminths′calreticulin to analyze the conservation.The calculated dissociation constant K_(d)of Ts-CRT^(380)interacting with C1q being 149μmol/L indicated a moderate interaction.17 amino acid residues of Ts-CRT including K145,W323,F77 and K47 participated in the interaction with human C1q.Sequence alignment confirmed that these 17 residues were highly conserved among five helminths′calreticulin.In summary,the interaction sites between Ts-CRT and human complement component C1q were obtained through combining the results of MST experiment and bioinformatics analysis,contributing to understand the structural basis and molecular mechanism of Trichinella spiralis evading host complement attack.Amino acids of Ts-CRT participating in the interaction with C1q were found to be highly conserved among calreticulin of other four helminths according to sequence alignment results,which provided a basis for the development of new anti-helminthic drugs targeting CRT.