厚壳贻贝一种新型贝壳胶原蛋白的重组表达与功能分析

贝壳是一种具有优异力学性能的生物硬组织，贝壳基质蛋白质对贝壳的形成具有重要意义。厚壳贻贝(Mytilus coruscus)贝壳中发现一种类似胶原蛋白质的新型贝壳基质蛋白质，命名为collagen-like protein 2（CLP-2）。然而,该蛋白质的结构与功能以及对贝壳形成的影响机制尚不清楚。为此，本研究对CLP 2开展了序列分析；进一步采取密码子优化结合原核重组表达策略，开展了CLP-2的重组表达；在此基础上分析了重组CLP-2对酸钙结晶的诱导、结晶速率抑制以及碳酸钙结合能力。对CLP-2的序列分析结果表明，该蛋白质序列中含有信号肽及两个Von Willebrand factor A（VWA）结构域。CLP-2在数据库中尚无高同源性蛋白质存在，表明这是一种较为新颖的贝壳基质蛋白。所获得的重组CLP-2对碳酸钙体外结晶表现出明显的诱导作用，扫描电镜以及傅里叶红外光谱结果表明，重组CLP-2可诱导碳酸钙晶体的形貌由立方体形转化为球形，并在高浓度下进一步转化为哑铃形；同时，重组CLP-2可促使碳酸钙晶体的晶型由方解石型向文石型转化；重组CLP-2在体外具有碳酸钙晶体结合作用；此外，重组CLP-2能显著抑制碳酸钙晶体的结晶速度（P<0.01）,并具有浓度依赖性。上述结果表明，厚壳贻贝贝壳CLP-2蛋白质在贝壳，特别是文石型肌棱柱层的生物矿化过程中具有重要作用。上述研究为深入了解贻贝贝壳的形成机制，以及胶原类蛋白质对生物矿化过程的影响奠定了基础。

Recombinant Expression and Functional Analysis of a Novel Type of Shell Collagen from Mytilus coruscus

Mollusk shell is the hard tissue with excellent mechanical properties. A novel shell matrix protein named collagen-like protein 2 (CLP-2) was previously identified from the shell of Mytilus coruscus. However, the structure and the function of CLP-2 are unknown for now. For exploring the possible functions of CLP-2 in the formation of the shell of Mytilus coruscus, the sequence of the CLP-2 protein has been analyzed and recombinant expressed using bioinformatics methods and prokaryotic recombinant systems with codon optimization, respectively. The effects of recombinant CLP-2 in calcium carbonate crystallization were analyzed using scanning electron microscope and Fourier transform infrared spectroscopy, respectively. Sequence analysis showed that the protein sequence of CLP-2 contained signal peptide and two Von Willebrand factor A (VWA) domains. In addition, CLP-2 has no high homologous proteins in the current database. The recombinant CLP-2 could induce the crystallization of calcium carbonate in vitro, including the morphology changing from the cubic shape to dumb bell shape, and the transformation from calcite to aragonite, of the calcium carbonate crystal. Furthermore, the recombinant CLP-2 could bind with calcium carbonate crystals and inhibit the crystallization rate significantly (P<0.01). These results indicated that CLP-2 plays an important role in the biomineralization of the mussel shell, especially in the myostraum layer with aragonite. These studies provided a foundation for further understanding the formation mechanism of mussel shells and the effect of collagen on the process of biomineralization.