华支睾吸虫半胱氨酸蛋白酶基因克隆表达及其生物信息学分析

Cloning,expression and bioinformatics analysis of 36.96 ku cysteine protease gene of Clonorchis sinensis

目的:克隆、表达华支睾吸虫的全长分子量为36.96ku半胱氨酸蛋白酶(36.96ku-CsCP)基因,并分析其生物学特性。方法:提取华支睾吸虫成虫总RNA,逆转录合成cDNA,PCR扩增目的基因,构建pET28a(+)-36.96ku-CsCP重组质粒,测序正确后转入E.coliBL21(DE3)中,经IPTG诱导表达,表达产物用SDS-PAGE进行分析;并运用NCBI和ExPASy等有关的生物信息学分析工具,对该基因及其编码蛋白进行预测和分析。结果:测序结果显示,36.96ku-CsCP基因序列与GenBank上已发表的序列有2个氨基酸位点突变(aa32由E→K,aa86由E→G);重组质粒经IPTG诱导后,目的蛋白在大肠杆菌中成功表达;生物信息学分析显示36.96ku-CsCP蛋白由信号肽(aa1-18)和前体蛋白(aa19-327)组成,为胞外分泌性蛋白,属于木瓜蛋白酶样半胱氨酸蛋白酶中C1A超家族,理论等电点为5.11,为亲水性蛋白;在36.96ku-CsCP蛋白的二级结构中,α-螺旋、β-折叠、β-转角和无规则卷曲所占的比例分别为28.75%、18.35%、7.03%和45.87%,含有11个亲水性和7个柔韧性较高的区域(临界值2.0),23个表面可及性较高的区域(临界值1.9),4个保守结构区域,10个翻译后修饰位点,13个潜在B细胞抗原表位与3个T细胞表位。结论:成功克隆、表达了华支睾吸虫36.96ku-CsCP基因,并获得该蛋白的生物信息学数据,可为后续相关研究提供参考。

Objective: To clone and express 36.96 ku cysteine protease (36.96 ku-CsCP) gene of Clonorchis sinensis and analyze the biology characteristics. Methods: A total of RNA was extracted from the Clonorchis sinensis and reversely transcribed into cDNA.The 36.96 ku-CsCP gene was amplified by PCR.The sequence was subcloned into the expression vector pET-28a (+).The accurate recombinant plasmid was transformed into E.coli BL21 (DE3) and the expression protein induced by IPTG.The recombinant protein was analyzed by SDS-PAGE.The 36.96 ku-CsCP gene and its expression protein were predicted and analyzed by bioinformatics analysis tools such as NCBI and ExPASy. Results: The 36.96 ku-CsCP sequence had 2 mutations (aa32 and aa86) compare with the sequence in GenBank.The pET-28a (+)-36.96 ku-CsCP was expressed under induction of IPTG.The bioinformatics analysis tools showed that 36.96 ku-CsCP (pI 5.11) belonged to Papain-like cysteine peptidase C1A superfamily,and was a secretory and hydrophilic protein.It composed of signal peptide (aa1-18) and precursor protein (aa19-327).The secondary structure of 36.96 ku-CsCP consisted of α-helixes (28.75%),β-folds (18.35%),β-corners (7.03%),and random coils (45.87%).36.96 ku-CsCP had 11 hydrophilic regions and 7 flexible regions (critical value:2.0),and 23 accessible regions (critical value:1.9).There are 4 conserved regions,10 post-translational modification sites,13 potential B-cell epitopes and 3 T-cell epitopes in 36.96 ku-CsCP. Conclusion: The 36.96 ku-CsCP gene of clonorchis sinensis was cloned and expressed successfully.The bioinformatics data of the protein were obfained to provide reference for the follow up studies.