广西巴马小型猪CTSK基因CDS区克隆与生物信息学分析
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摘要
目的对广西巴马小型猪组织蛋白酶K(cathepsin K,CTSK)基因CDS区进行克隆及生物信息学分析。方法从广西巴马小猪的皮下脂肪中提取总RNA,通过RT-PCR方法扩增获得CTSK基因CDS区序列,并采用相关生物信息学软件对其序列特征、系统进化、编码蛋白结构和理化性质进行分析。结果广西巴马小型猪CTSK基因CDS全长993 bp,编码330个氨基酸,与NCBI上公布的普通猪(NM_214302.1)CTSK基因序列完全一致。同源性比对结果发现,CTSK基因在进化过程中具有较高的保守性。广西巴马小型猪CTSK蛋白具有明显的亲水性区域,在第17-330氨基酸位置区域存在一条信号肽序列,不存在跨膜结构域,二级结构元件由α螺旋、β折叠、无规卷曲和延伸链组成。修饰结构预测发现CTSK蛋白存在两处N糖基化位点,亚细胞定位发现其可能主要分布于内质网和细胞核。结论本研究成功克隆了广西巴马小型猪CTSK基因CDS序列并进行分析,为今后进一步深入研究该基因功能和建立相关疾病动物模型提供理论基础。
Objective This study aimed to clone the CDS region of cathepsin K(CTSK) from Guangxi Bama mini-pig and analyze its genetic structure with bioinformatics software. Methods Total RNA was extracted from subcutaneous fat tissue of a Guangxi Bama mini-pig. The CTSK gene CDS region sequence was obtained through RT-PCR. Its sequence characteristics, evolution, encoded protein structure and physical and chemical properties were analyzed using relevant bioinformatics softwares. Results The CDS of the CTSK gene of Guangxi Bama mini-pig was 993 bp and encoded 330 amino acids, which was completely consistent with the published CTSK gene sequence of pig(NM_214302.1) on NCBI. Homology comparison showed that the CTSK gene has been highly conserved during evolution. The CTSK protein of Guangxi Bama mini-pig had an obvious hydrophilic region and a signal peptide sequence in the region of amino acids 17-330, but had no transmembrane structural domain. Structural prediction indicated that there were two n-glycosylation sites in the CTSK protein, and subcellular localization analysis indicated that it was mainly distributed in the endoplasmic reticulum and nucleus. Conclusions In this study, the CDS region sequence of the CTSK gene in Guangxi Bama mini-pig is successfully cloned and analyzed, which provides a theoretical basis for further study on the function of this gene and the establishment of animal models of related diseases.
Objective This study aimed to clone the CDS region of cathepsin K(CTSK) from Guangxi Bama mini-pig and analyze its genetic structure with bioinformatics software. Methods Total RNA was extracted from subcutaneous fat tissue of a Guangxi Bama mini-pig. The CTSK gene CDS region sequence was obtained through RT-PCR. Its sequence characteristics, evolution, encoded protein structure and physical and chemical properties were analyzed using relevant bioinformatics softwares. Results The CDS of the CTSK gene of Guangxi Bama mini-pig was 993 bp and encoded 330 amino acids, which was completely consistent with the published CTSK gene sequence of pig(NM_214302.1) on NCBI. Homology comparison showed that the CTSK gene has been highly conserved during evolution. The CTSK protein of Guangxi Bama mini-pig had an obvious hydrophilic region and a signal peptide sequence in the region of amino acids 17-330, but had no transmembrane structural domain. Structural prediction indicated that there were two n-glycosylation sites in the CTSK protein, and subcellular localization analysis indicated that it was mainly distributed in the endoplasmic reticulum and nucleus. Conclusions In this study, the CDS region sequence of the CTSK gene in Guangxi Bama mini-pig is successfully cloned and analyzed, which provides a theoretical basis for further study on the function of this gene and the establishment of animal models of related diseases.
引文
[1] 魏巍, 孙文靖, 于旸,等. 人组织蛋白酶K基因研究进展 [J]. 国际遗传学杂志, 2012, 35(2): 82-85.Wei W, Sun WJ, Yu Y, et al. Advance in human cathepsin K gene [J]. Int J Genet, 2012, 35(2): 82-85.
[2] Abrahamson M, Alvarez-Fernandez M, Nathanson CM. Cystatins [J]. Biochem Soc Symp, 2003, (70): 179-199.
[3] Jedeszko C, Sloane BF. Cysteine cathepsins in human cancer [J]. Biol Chem, 2004, 385(11): 1017-1027.
[4] Gocheva V, Zeng W, Ke D, et al. Distinct roles for cysteine cathepsin genes in multistage tumorigenesis [J]. Genes Dev, 2006, 20(5): 543-556.
[5] Hou WS, Br?mme D, Zhao Y, et al. Characterization of novel cathepsin K mutations in the pro and mature polypeptide regions causing pycnodysostosis [J]. J Clin Invest, 1999, 103(5):731-738.
[6] Costa AG, Cusano NE, Silva BC, et al. Cathepsin K: its skeletal actions and role as a therapeutic target in osteoporosis [J]. Nat Rev Rheumatol, 2011, 7(8): 447-456.
[7] Szumilo J, Burdan F, Zinkiewicz K, et al. Expression of syndecan-1 and cathepsins D and K in advanced esophageal squamous cell carcinoma [J]. Folia Histochem Cytobiol, 2009, 47(4): 571-578.
[8] Kleer CG, Bloushtainqimron N, Chen YH, et al. Epithelial and stromal cathepsin K and CXCL14 expression in breast tumor progression [J]. Clinl Cancer Res, 2008, 14(17): 5357-5367.
[9] Yan X, Takahara M, Xie L, et al. Cathepsin K expression: a useful marker for the differential diagnosis of dermatofibroma and dermatofibrosarcoma protuberans [J]. Histopathology, 2010, 57(3): 486-488.
[10] Bühling F, Waldburg N, Gerber A, et al. Cathepsin K expression in human lung [J]. Adv Exp Med Biol, 2000, 477(477): 281-286.
[11] Troen BR. The regulation of cathepsin k gene expression [J]. Ann N Y Acad Sci, 2006, 1068(1): 165-172.
[12] Reghellin D, Poletti V, Tomassett S, et al. Cathepsin-K is a sensitive immunohistochemical marker for detection of micro-granulomas in hypersensitivity pneumonitis [J]. Sarcoidosis Vasc Diffuse Lung Dis, 2010, 27(1):57-63.
[13] 尹晓, 陈述林, 刘志芬,等. 肥胖症患者脂肪组织中组织蛋白酶K的表达及其功能初探[J]. 中国糖尿病杂志, 2008, 16(5):265-266.Yin X, Chen SL, Liu ZF, et al. Investigation of the expression and the function of cathepsin K(CTSK) gene in obesity subjects [J]. Chin J Diabetes, 2008, 16(5): 265-266.
[14] 移丽珍, 温宣, 薛洋. 组织蛋白酶K与口腔颌面部疾病 [J]. 口腔医学, 2015, 35(10): 881-884.Yi LZ, Wen X, Xue Y. Cathepsin K and stomatognathic diseases [J]. Stomatology, 2015, 35(10): 881-884.
[15] 张艳, 金蓉, 刘伟. Cathepsin K基因siRNA的筛选及其质粒载体的构建 [J]. 组织工程与重建外科杂志, 2009, 5(5): 256-259.Zhang Y, Jin R, Liu W. Experimental study on siRNA screening of cathepsin K gene and construction of its plasmid vector [J]. J Tissue Eng Reconstr Surg, 2009, 5(5): 256-259.
[16] 谢婉, 刘明君, 何剑雄,等. 陆川猪黑皮质激素受体4基因克隆及序列分析 [J]. 中国畜牧兽医, 2018, 45(2): 320-329.Xie W, Liu MJ, He JX, et al. Cloning and sequence analysis of MC4R gene in Luchuan pig [J]. Chin Anim HusbandryVet Med, 2018, 45(2): 320-329.
[17] Fontanesi L, Scotti E, Buttazzoni L, et al. A single nucleotide polymorphism in the porcine cathepsin K (CTSK) gene is associated with back fat thickness and production traits in Italian Duroc pigs [J]. Mol Biol Rep, 2010, 37(1): 491-495.
[18] 张艳, 柴岗, 刘伟,等. CathePsin K基因慢病毒质粒载体的构建 [J]. 组织工程与重建外科杂志, 2005, 1(3): 165-166.Zhang Y, Chai G, Liu W, et al. Construction of cathepsin K lentivirus plasmid [J]. J Tissue Eng Reconstr Surg, 2005, 1(3): 165-166.
[19] Yang X, Tao C, Shi S, et al. Clinical and animal research findings in pycnodysostosis and gene mutations of cathepsin K from 1996 to 2011 [J]. Orphanet J Rare Dis, 2011, 6(1): 20.
[20] 李菲, 李自军. 血清β-crosslaps、Cathe K水平变化在骨质疏松症诊断中的应用价值 [J]. 天津医科大学学报, 2017, 23(6): 534-536.Li F, Li ZJ. Application value of serum 13-crosslaps, Cathe K level in diagnosis of osteoporosis [J]. J Tianjin Med Univ, 2017, 23(6): 534-536.
[21] 唐特. 组织蛋白酶K在慢性肾脏病中的检测意义 [J]. 医学理论与实践, 2017, 30(11): 1582-1584Tang T. The significance of detection of cathepsins K in chronic kidney disease [J]. J Med Theory Pract, 2017, 30(11): 1582-1584.
[22] 袁琳, 类延娜, 李玉子,等. 组织蛋白酶K、胱抑素C与冠心病及其危险因素的相关性 [J]. 中国老年学杂志, 2015, 35(23): 6725-6727.Yuang L, Rei YN, Li Z, et al. Association of cathepsin K and cystatin C with coronary heart disease and its risk factors [J]. Chin J Geerontol, 2015, 35(23):6725-6727.
[23] Leusink FK, Koudounarakis E, Frank MH, et al. Cathepsin K associates with lymph node metastasis and poor prognosis in oral squamous cell carcinoma [J]. BMC Cancer, 2018, 18(1): 385.
[24] Asagiri M, Hirai T, Kunigami T, et al. Cathepsin K-dependent Toll-like receptor 9 signaling revealed in experimental arthritis[J]. Science, 2008, 319(5863): 624-627.
[25] Chiellini C, Costa M, Novelli SE, et al. Identification of cathepsin K as a novel marker of adiposity in white adipose tissue [J]. J Cell Physiol, 2003, 195(2):309-321.
[26] Xiao Y, Junfeng H, Tianhong L, et al. Cathepsin K in adipocyte differentiation and its potential role in the pathogenesis of obesity. [J]. J Clin Endocrinol Metab, 2006, 91(11):4520-4527.
[27] 吴延军, 夏攀洁, 严雪瑜,等. 高脂高糖饲料联合低剂量链脲佐菌素(STZ)诱导广西巴马小型猪2型糖尿病动物模型的建立 [J]. 基因组学与应用生物学, 2017(6): 2393-2398.Wu YJ, Xia PJ, Yan XY, et al. Combination of high fat/high carbohydrates diet and low-dose streptozotocin-(STZ) induced a model for type 2 diabetes in Guangxi Bama mini-pig [J]. Genom Appl Biol, 2017(6): 2393-2398.
[28] 李艳君, 宋少锐, 郭亚芬,等. 广西巴马小型猪动脉粥样硬化模型的制作 [J]. 中国畜牧兽医, 2015, 42(7): 1770-1776.Li YJ,Song SR,Guo YF, et al. Preparation of atherosclerosis model using Guangxi Bama mini-pig [J]. Chin Animal Husband Vet Med, 2015, 42(7): 1770-1776.
[2] Abrahamson M, Alvarez-Fernandez M, Nathanson CM. Cystatins [J]. Biochem Soc Symp, 2003, (70): 179-199.
[3] Jedeszko C, Sloane BF. Cysteine cathepsins in human cancer [J]. Biol Chem, 2004, 385(11): 1017-1027.
[4] Gocheva V, Zeng W, Ke D, et al. Distinct roles for cysteine cathepsin genes in multistage tumorigenesis [J]. Genes Dev, 2006, 20(5): 543-556.
[5] Hou WS, Br?mme D, Zhao Y, et al. Characterization of novel cathepsin K mutations in the pro and mature polypeptide regions causing pycnodysostosis [J]. J Clin Invest, 1999, 103(5):731-738.
[6] Costa AG, Cusano NE, Silva BC, et al. Cathepsin K: its skeletal actions and role as a therapeutic target in osteoporosis [J]. Nat Rev Rheumatol, 2011, 7(8): 447-456.
[7] Szumilo J, Burdan F, Zinkiewicz K, et al. Expression of syndecan-1 and cathepsins D and K in advanced esophageal squamous cell carcinoma [J]. Folia Histochem Cytobiol, 2009, 47(4): 571-578.
[8] Kleer CG, Bloushtainqimron N, Chen YH, et al. Epithelial and stromal cathepsin K and CXCL14 expression in breast tumor progression [J]. Clinl Cancer Res, 2008, 14(17): 5357-5367.
[9] Yan X, Takahara M, Xie L, et al. Cathepsin K expression: a useful marker for the differential diagnosis of dermatofibroma and dermatofibrosarcoma protuberans [J]. Histopathology, 2010, 57(3): 486-488.
[10] Bühling F, Waldburg N, Gerber A, et al. Cathepsin K expression in human lung [J]. Adv Exp Med Biol, 2000, 477(477): 281-286.
[11] Troen BR. The regulation of cathepsin k gene expression [J]. Ann N Y Acad Sci, 2006, 1068(1): 165-172.
[12] Reghellin D, Poletti V, Tomassett S, et al. Cathepsin-K is a sensitive immunohistochemical marker for detection of micro-granulomas in hypersensitivity pneumonitis [J]. Sarcoidosis Vasc Diffuse Lung Dis, 2010, 27(1):57-63.
[13] 尹晓, 陈述林, 刘志芬,等. 肥胖症患者脂肪组织中组织蛋白酶K的表达及其功能初探[J]. 中国糖尿病杂志, 2008, 16(5):265-266.Yin X, Chen SL, Liu ZF, et al. Investigation of the expression and the function of cathepsin K(CTSK) gene in obesity subjects [J]. Chin J Diabetes, 2008, 16(5): 265-266.
[14] 移丽珍, 温宣, 薛洋. 组织蛋白酶K与口腔颌面部疾病 [J]. 口腔医学, 2015, 35(10): 881-884.Yi LZ, Wen X, Xue Y. Cathepsin K and stomatognathic diseases [J]. Stomatology, 2015, 35(10): 881-884.
[15] 张艳, 金蓉, 刘伟. Cathepsin K基因siRNA的筛选及其质粒载体的构建 [J]. 组织工程与重建外科杂志, 2009, 5(5): 256-259.Zhang Y, Jin R, Liu W. Experimental study on siRNA screening of cathepsin K gene and construction of its plasmid vector [J]. J Tissue Eng Reconstr Surg, 2009, 5(5): 256-259.
[16] 谢婉, 刘明君, 何剑雄,等. 陆川猪黑皮质激素受体4基因克隆及序列分析 [J]. 中国畜牧兽医, 2018, 45(2): 320-329.Xie W, Liu MJ, He JX, et al. Cloning and sequence analysis of MC4R gene in Luchuan pig [J]. Chin Anim HusbandryVet Med, 2018, 45(2): 320-329.
[17] Fontanesi L, Scotti E, Buttazzoni L, et al. A single nucleotide polymorphism in the porcine cathepsin K (CTSK) gene is associated with back fat thickness and production traits in Italian Duroc pigs [J]. Mol Biol Rep, 2010, 37(1): 491-495.
[18] 张艳, 柴岗, 刘伟,等. CathePsin K基因慢病毒质粒载体的构建 [J]. 组织工程与重建外科杂志, 2005, 1(3): 165-166.Zhang Y, Chai G, Liu W, et al. Construction of cathepsin K lentivirus plasmid [J]. J Tissue Eng Reconstr Surg, 2005, 1(3): 165-166.
[19] Yang X, Tao C, Shi S, et al. Clinical and animal research findings in pycnodysostosis and gene mutations of cathepsin K from 1996 to 2011 [J]. Orphanet J Rare Dis, 2011, 6(1): 20.
[20] 李菲, 李自军. 血清β-crosslaps、Cathe K水平变化在骨质疏松症诊断中的应用价值 [J]. 天津医科大学学报, 2017, 23(6): 534-536.Li F, Li ZJ. Application value of serum 13-crosslaps, Cathe K level in diagnosis of osteoporosis [J]. J Tianjin Med Univ, 2017, 23(6): 534-536.
[21] 唐特. 组织蛋白酶K在慢性肾脏病中的检测意义 [J]. 医学理论与实践, 2017, 30(11): 1582-1584Tang T. The significance of detection of cathepsins K in chronic kidney disease [J]. J Med Theory Pract, 2017, 30(11): 1582-1584.
[22] 袁琳, 类延娜, 李玉子,等. 组织蛋白酶K、胱抑素C与冠心病及其危险因素的相关性 [J]. 中国老年学杂志, 2015, 35(23): 6725-6727.Yuang L, Rei YN, Li Z, et al. Association of cathepsin K and cystatin C with coronary heart disease and its risk factors [J]. Chin J Geerontol, 2015, 35(23):6725-6727.
[23] Leusink FK, Koudounarakis E, Frank MH, et al. Cathepsin K associates with lymph node metastasis and poor prognosis in oral squamous cell carcinoma [J]. BMC Cancer, 2018, 18(1): 385.
[24] Asagiri M, Hirai T, Kunigami T, et al. Cathepsin K-dependent Toll-like receptor 9 signaling revealed in experimental arthritis[J]. Science, 2008, 319(5863): 624-627.
[25] Chiellini C, Costa M, Novelli SE, et al. Identification of cathepsin K as a novel marker of adiposity in white adipose tissue [J]. J Cell Physiol, 2003, 195(2):309-321.
[26] Xiao Y, Junfeng H, Tianhong L, et al. Cathepsin K in adipocyte differentiation and its potential role in the pathogenesis of obesity. [J]. J Clin Endocrinol Metab, 2006, 91(11):4520-4527.
[27] 吴延军, 夏攀洁, 严雪瑜,等. 高脂高糖饲料联合低剂量链脲佐菌素(STZ)诱导广西巴马小型猪2型糖尿病动物模型的建立 [J]. 基因组学与应用生物学, 2017(6): 2393-2398.Wu YJ, Xia PJ, Yan XY, et al. Combination of high fat/high carbohydrates diet and low-dose streptozotocin-(STZ) induced a model for type 2 diabetes in Guangxi Bama mini-pig [J]. Genom Appl Biol, 2017(6): 2393-2398.
[28] 李艳君, 宋少锐, 郭亚芬,等. 广西巴马小型猪动脉粥样硬化模型的制作 [J]. 中国畜牧兽医, 2015, 42(7): 1770-1776.Li YJ,Song SR,Guo YF, et al. Preparation of atherosclerosis model using Guangxi Bama mini-pig [J]. Chin Animal Husband Vet Med, 2015, 42(7): 1770-1776.