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
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摘要
目的:克隆、表达华支睾吸虫的全长分子量为36.96 ku半胱氨酸蛋白酶(36.96 ku-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(+)-36.96 ku-CsCP重组质粒,测序正确后转入E.coli BL21(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,36.96 ku-CsCP基因序列与GenBank上已发表的序列有2个氨基酸位点突变(aa_(32)由E→K,aa_(86)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36.96 ku-CsCP蛋白由信号肽(aa _(1-18))和前体蛋白(aa _(19-327))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36.96 ku-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36.96 ku-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-28 a(+).The accurate recombinant plasmid was transformed into E.coliBL21(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-28 a(+)-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 C1 A 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.
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-28 a(+).The accurate recombinant plasmid was transformed into E.coliBL21(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-28 a(+)-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 C1 A 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.
引文
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[18] LI Y,HU X,LIU X,et al.Molecular cloning and analysis of stage and tissue-specific expression of Cathepsin L-like protease from Clonorchis sinensis[J].Parasitol Res,2009,105(2):447-452.
[19] LI Y,HU X,LIU X,et al.Serological diagnosis of clonorchiasis:using a recombinant propeptide of cathepsin L proteinase from Clonorchis sinensis as a candidate antigen[J].Parasitol Res,2012,110(6):2197-2203.
[20] 梁前进,王鹏程,白燕荣.蛋白质磷酸化修饰研究进展[J].科技导报,2012,30(31):73-79.
[21] 牛书俐,庞骁,周莉,等.RGD三肽的临床应用进展[J].基础医学与临床,2018,38(1):107-111.
[22] 刘宜升.华支睾吸虫的生物学和华支睾吸虫病防治[M].北京:科学出版社,2012:420.
[23] CHOI M H,PARK I C,LI S,et al.Excretory-secretory antigen is better than crude antigen for the serodiagnosis of clonorchiasis by ELISA[J].Korean J Parasitol,2003,41(1):35-39.
[2] FURST T,KEISER J,UTZINGER J.Global burden of human food-borne trematodiasis:a systematic review and meta-analysis[J].Lancet Infect Dis,2012,12(3):210-221.
[3] 杨平,石武祥,康敏,等.广西少数民族农村地区人体重点寄生虫感染状况调查[J].安徽医科大学学报,2017,52(3):372-375.
[4] DVORAK J,MASHIYAMA S T,SAJID M,et al.SmCL3,a gastrodermal cysteine protease of the human blood fluke Schistosoma mansoni[J].PLoS Negl Trop Dis,2009,3(6):e449.
[5] GUILIANO D B,HONG X,MCKERROW J H,et al.A gene family of cathepsin L-like proteases of filarial nematodes are associated with larval molting and cuticle and eggshell remodeling[J].Mol Biochem Parasitol,2004,136(2):227-242.
[6] LARSON E T,PARUSSINI F,HUYNH M H,et al.Toxoplasma gondii cathepsin L is the primary target of the invasion-inhibitory compound morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl[J].J Biol Chem,2009,284(39):26839-26850.
[7] TSUBOKAWA D,HATTA T,MAEDA H,et al.A cysteine protease from Spirometra erinaceieuropaei plerocercoid is a critical factor for host tissue invasion and migration[J].Acta Trop,2017(167):99-107.
[8] HERNANDEZ H M,MARCET R,SARRACENT J.Biological roles of cysteine proteinases in the pathogenesis of Trichomonas vaginalis[J].Parasite,2014(21):54.
[9] BERASAIN P,CARMONA C,FRANGIONE B,et al.Specific cleavage sites on human IgG subclasses by cruzipain,the major cysteine proteinase from Trypanosoma cruzi[J].Mol Biochem Parasitol,2003,130(1):23-29.
[10] BERASAIN P,CARMONA C,FRANGIONE B,et al.Fasciola hepatica:parasite-secreted proteinases degrade all human IgG subclasses:determination of the specific cleavage sites and identification of the immunoglobulin fragments produced[J].Exp Parasitol,2000,94(2):99-110.
[11] ZAWISTOWSKA-DENIZIAK A,WASYL K,NORBURY L J,et al.Characterization and differential expression of cathepsin L3 alleles from Fasciola hepatica[J].Mol Biochem Parasitol,2013,190(1):27-37.
[12] WONGKHAM C,TANTRAWATPAN C,INTAPAN P M,et al.Evaluation of immunoglobulin G subclass antibodies against recombinant Fasciola gigantica cathepsin L1 in an enzyme-linked immunosorbent assay for serodiagnosis of human fasciolosis[J].Clin Diagn Lab Immunol,2005,12(10):1152-1156.
[13] WONGKHAM C,TANTRAWATPAN C,INTAPAN P M,et al.Evaluation of immunoglobulin G subclass antibodies against recombinant Fasciola gigantica cathepsin L1 in an enzyme-linked immunosorbent assay for serodiagnosis of human fasciolosis[J].Clin Diagn Lab Immunol,2005,12(10):1152-1156.
[14] WESOLOWSKA A,BASALAJ K,NORBURY L J,et al.Vaccination against Fasciola hepatica using cathepsin L3 and B3 proteases delivered alone or in combination[J].Vet Parasitol,2018(250):15-21.
[15] LARSON E T,PARUSSINI F,HUYNH M H,et al.Toxoplasma gondii cathepsin L is the primary target of the invasion-inhibitory compound morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl[J].J Biol Chem,2009,284(39):26839-26850.
[16] LV X,CHEN W,WANG X,et al.Molecular characterization and expression of a cysteine protease from Clonorchis sinensis and its application for serodiagnosis of clonorchiasis[J].Parasitol Res,2012,110(6):2211-2219.
[17] LI S,CHUNG Y B,CHUNG B S,et al.The involvement of the cysteine proteases of Clonorchis sinensis metacercariae in excystment[J].Parasitol Res,2004,93(1):36-40.
[18] LI Y,HU X,LIU X,et al.Molecular cloning and analysis of stage and tissue-specific expression of Cathepsin L-like protease from Clonorchis sinensis[J].Parasitol Res,2009,105(2):447-452.
[19] LI Y,HU X,LIU X,et al.Serological diagnosis of clonorchiasis:using a recombinant propeptide of cathepsin L proteinase from Clonorchis sinensis as a candidate antigen[J].Parasitol Res,2012,110(6):2197-2203.
[20] 梁前进,王鹏程,白燕荣.蛋白质磷酸化修饰研究进展[J].科技导报,2012,30(31):73-79.
[21] 牛书俐,庞骁,周莉,等.RGD三肽的临床应用进展[J].基础医学与临床,2018,38(1):107-111.
[22] 刘宜升.华支睾吸虫的生物学和华支睾吸虫病防治[M].北京:科学出版社,2012:420.
[23] CHOI M H,PARK I C,LI S,et al.Excretory-secretory antigen is better than crude antigen for the serodiagnosis of clonorchiasis by ELISA[J].Korean J Parasitol,2003,41(1):35-39.