日本血吸虫钙磷蛋白基因的克隆、表达及其进化分析

目的 克隆和表达日本血吸虫钙磷蛋白基因，纯化表达产物。 方法 从日本血吸虫cDNA文库中挑出钙磷蛋白基因进行体外扩增，将目的基因亚克隆至原核表达载体pET-28a中进行表达，组氨酸标签亲和柱层析法纯化表达产物，并用蛋白质印迹（Western blotting）分析其免疫原性。然后用生物信息学方法对其结构域和功能作用位点进行分析和预测，并绘制该蛋白的进化树。 结果 构建了重组原核表达载体，并在大肠埃希菌中获得了表达。纯化的重组蛋白可以被日本血吸虫感染的兔血清识别。经生物信息学分析发现该基因的编码蛋白含有4个EF手型（exchange factor hand，EF-hand）功能结构域，另外具有3种潜在的功能作用位点，即2个蛋白激酶C磷酸化位点、8个酪蛋白激酶II磷酸化位点和1个N-肉豆蔻酰化位点，属于II型（type-II）钙磷蛋白。 结论 日本血吸虫钙磷蛋白基因编码蛋白为钙结合蛋白，有可能成为潜在的日本血吸虫病疫苗候选抗原和药物及诊断靶点。

Cloning, Expression and Phylogenetic analysis of Schistosoma japonicum Calcyphosine Gene

OBJECTIVE: To clone and express Schistosoma japonicum (Sj) calcyphosine gene, and purify the expressed protein. METHOD: The encoding sequence selected from Sj cDNA library was amplified by PCR. After subcloned into prokaryotic expression vector pET-28a, the expressed protein was purified with His -Tag affinity chromatography. Western blotting was used to detect the immunogenicity. The structure and functions of the protein were analyzed by bioinformatics method, and the phylogenetic tree of the protein was drawn. RESULT: The recombinant protein was specifically recognized by the Sj infected rabbit serum. The bioinformatics analysis showed 4 EF-hand domains. Besides, it was predicted that Sj calcyphosine contains two phosphorylation sites for protein kinase C, eight phosphorylation sites for casein kinase II and one N-myristoylation site. The Sj calcyphosine belonged to type-II calcyphosine. CONCLUSION: The calcyphosine gene is a calcium-binding protein and might be a potential candidate for diagnosis, vaccine or drug target.