猪CPB2基因可变剪接体的克隆及生物信息学研究

doi:10.11843/j.issn.0366-6964.2022.10.011

摘要/Abstract

摘要： 本研究旨在克隆猪CPB2基因的不同可变剪接体，预测对应蛋白的功能特性以及研究不同转录本表达特性。以健康状态和体重相同的3头12月龄巴马小型猪为试验动物，通过RT-PCR技术及基因平末端克隆技术扩增猪CPB2基因的CDS区及部分非编码区，利用生物信息学工具预测CPB2编码蛋白的基本生物学特征，利用RT-PCR技术检测CPB2基因在肝、心、肾、皮下脂肪、肠系膜脂肪、股二头肌和背最长肌中的表达特征。以实验室制备的代谢性疾病易感猪为试验动物，利用qRT-PCR技术检测CPB2基因在富营养饮食效应下肝中的表达特征。结果表明，本研究成功克隆出猪CPB2基因的3种可变剪接体（CPB2-1、CPB2-2和CPB2-3），其中CPB2-1与NCBI序列XM_001929146.4相同，CPB2-2和CPB2-3为新发现转录本。与CPB2-1相比，CPB2-2和CPB2-3发生了5'端可变剪接（alternative 5'splice site，A5SS）事件；CPB2-1编码423个氨基酸的酸性不稳定性蛋白，CPB2-2和CPB2-3编码同种281个氨基酸的碱性不稳定性蛋白；与CPB2-1相比，CPB2-2和CPB2-3缺少信号肽和激活肽，但具有相同羧肽酶活性结构域；CPB2基因3种转录本仅在肝和肾中表达，其他组织无表达；在富营养饮食诱导的代谢性疾病易感猪的肝中，CPB2-1（P<0.01）和CPB2-2（P<0.01）显著降低，CPB2-3未显著降低（P=0.14）。综上，本研究成功在肝中克隆了猪CPB2的3种可变剪接体，推测CPB2-1是行使纤溶与凝血等生理功能的正常转录本；新发现转录本CPB2-2和CPB2-3被留在肝和肾中可能具有羧肽酶活性和重要生理学功能；CPB2基因可能与代谢相关的慢性肝病存在一定联系。

关键词: 猪, CPB2, 可变剪接, 功能预测, 表达特征, 慢性肝病

Abstract: The objective of this study was to clone different alternative splices of pig CPB2 gene, so as to predict the functional properties of the corresponding proteins and study the expression characteristics of different transcripts. Three 12-month-old Bama miniature pigs with the same health status and body weight were used as experimental animal, the CDS region and part of non-coding region of CPB2 gene were amplified by RT-PCR and gene flat terminal cloning technology, while the basic biological characteristics of CPB2 coding protein were predicted by bioinformatics tools. The expression characteristics of CPB2 gene in liver, heart, kidney, subcutaneous fat, mesenteric fat, biceps femoris muscle and longissimus dorsi muscle were detected by RT-PCR. qRT-PCR was used to detect the expression characteristics of CPB2 gene in liver of metabolic disease susceptible pigs prepared in the laboratory under the effect of nutrient-rich diet.Three kinds of alternative splicing isoforms (CPB2-1, CPB2-2 and CPB2-3) of pig CPB2 gene were cloned successfully. CPB2-1 was identical with the NCBI sequence XM_001929144.6. CPB2-2 and CPB2-3 were newly discovered transcripts. Sequence alignment showed that alternative 5' splice site (A5SS) events occurred in CPB2-2 and CPB2-3 compared with CPB2-1. Bioinformatics showed that CPB2-1 encoded an acid unstable protein with 423 amino acids, and CPB2-2 and CPB2-3 encoded an alkaline unstable protein with 281 amino acids; Compared with CPB2-1, CPB2-2 and CPB2-3 lacked signal and activating peptides, but had the same carboxypeptidase activity domain. The three CPB2 transcripts were only expressed in liver and kidney, no expression was found in other tissues. In liver of metabolic disease susceptible pigs induced by nutrient-rich diet, CPB2-1 (P<0.01) and CPB2-2 (P<0.01) significantly reduced, CPB2-3 was not significantly reduced (P=0.14). In conclusion, three kinds of alternative splicing isoforms of pig CPB2 were successfully cloned in liver in this study, suggesting that CPB2-1 is a normal transcript of fibrinolysis and coagulation. Newly discovered transcripts of CPB2-2 and CPB2-3 may have carboxypeptidase activity and important physiological functions when retained in liver and kidney. CPB2 gene may be associated with metabolic related chronic liver disease.

Key words: pig, CPB2, alternative splicing, function prediction, expression characteristics, chronic liver disease

中图分类号:

S828.2

夏博策, 张凯艺, 苗佳坤, 杨宇, 彭焕祺, 王彦芳, 杨述林. 猪CPB2基因可变剪接体的克隆及生物信息学研究[J]. 畜牧兽医学报, 2022, 53(10): 3377-3390.

XIA Boce, ZHANG Kaiyi, MIAO Jiakun, YANG Yu, PENG Huanqi, WANG Yanfang, YANG Shulin. Cloning and Bioinformatics Study of Alternative Splicing Isoforms of Pig CPB2 Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3377-3390.

参考文献 33

[1]	REDLITZ A, TAN A K, EATON D L, et al.Plasma carboxypeptidases as regulators of the plasminogen system[J].J Clin Invest, 1995, 96(5):2534-2538.
[2]	EATON D L, MALLOY B E, TSAI S P, et al.Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma[J].J Biol Chem, 1991, 266(32):21833-21838.
[3]	SILLEN M, DECLERCK P J.Thrombin activatable fibrinolysis inhibitor (TAFI):An updated narrative review[J].Int J Mol Sci, 2021, 22(7):3670.
[4]	WILLEMSE J L, POLLA M, HENDRIKS D F.The intrinsic enzymatic activity of plasma procarboxypeptidase U (TAFI) can interfere with plasma carboxypeptidase N assays[J].Anal Biochem, 2006, 356(1):157-159.
[5]	FOLEY J H, KIM P, NESHEIM M E.Thrombin-activable fibrinolysis inhibitor zymogen does not play a significant role in the attenuation of fibrinolysis[J].J Biol Chem, 2008, 283(14):8863-8867.
[6]	DECLERCK P J.Thrombin activatable fibrinolysis inhibitor[J].Hamostaseologie, 2011, 31(3):165-173.
[7]	VERCAUTEREN E, GILS A, DECLERCK P J.Thrombin activatable fibrinolysis inhibitor:A putative target to enhance fibrinolysis[J].Semin Thromb Hemost, 2013, 39(4):365-372.
[8]	LEUNG L L K, MYLES T, NISHIMURA T, et al.Regulation of tissue inflammation by thrombin-activatable carboxypeptidase B (or TAFI)[J].Mol Immunol, 2008, 45(16):4080-4083.
[9]	SWAISGOOD C M, SCHMITT D, EATON D, et al.In vivo regulation of plasminogen function by plasma carboxypeptidase B[J].J Clin Invest, 2002, 110(9):1275-1282.
[10]	TE VELDE E A, WAGENAAR G T M, REIJERKERK A, et al.Impaired healing of cutaneous wounds and colonic anastomoses in mice lacking thrombin-activatable fibrinolysis inhibitor[J].J Thromb Haemost, 2003, 1(10):2087-2096.
[11]	NAGASHIMA M, YIN Z F, ZHAO L, et al.Thrombin-activatable fibrinolysis inhibitor (TAFI) deficiency is compatible with murine life[J].J Clin Invest, 2002, 109(1):101-110.
[12]	WANG X K, SMITH P L, HSU M Y, et al.Deficiency in thrombin-activatable fibrinolysis inhibitor (TAFI) protected mice from ferric chloride-induced vena cava thrombosis[J].J Thromb Thrombolysis, 2007, 23(1):41-49.
[13]	吴晓本.凝血酶激活的纤溶抑制物编码区基因CPB2多态性与2型糖尿病关系的研究[D].济南:山东大学, 2009.WU X B.Study on the association of genetic variations in thrombin activatable fibrinolysis inhibitor (TAFI) encoding region and the risk of type 2 diabetes Mellitus[D].Ji'nan:Shandong University, 2009.(in Chinese)
[14]	李敏, 孙玉倩, 范博, 等.血浆凝血酶激活的纤溶抑制物、视黄醇结合蛋白与2型糖尿病血管并发症的关系[J].牡丹江医学院学报, 2011, 32(4):44-46.LI M, SUN Y Q, FAN B, et al.Relationship between plasma thrombin activated fibrinolytic inhibitors, retinol binding protein and vascular complications in type 2 diabetes mellitus[J].Journal of Mudanjiang Medical University, 2011, 32(4):44-46.(in Chinese)
[15]	CAGLIANI R, FUMAGALLI M, RIVA S, et al.Polymorphisms in the CPB2 gene are maintained by balancing selection and result in haplotype-preferential splicing of exon 7[J].Mol Biol Evol, 2010, 27(8):1945-1954.
[16]	LIN J H H, NOVAKOVIC D, RIZZO C M, et al.The mRNA encoding TAFI is alternatively spliced in different cell types and produces intracellular forms of the protein lacking TAFIa activity[J].Thromb Haemost, 2013, 109(6):1033-1044.
[17]	ZHANG K Y, TAO C, XU J P, et al.CD8+ T cells involved in metabolic inflammation in visceral adipose tissue and liver of transgenic pigs[J].Front Immunol, 2021, 12:690069.
[18]	BERGET S M, MOORE C, SHARP P A.Spliced segments at the 5' terminus of adenovirus 2 late mRNA[J].Proc Natl Acad Sci U S A, 1977, 74(8):3171-3175.
[19]	CHOW L T, GELINAS R E, BROKER T R, et al.An amazing sequence arrangement at the 5' ends of adenovirus 2 messenger RNA[J].Cell, 1977, 12(1):1-8.
[20]	GRAVELEY B R.Alternative splicing:Increasing diversity in the proteomic world[J].Trends Genet, 2001, 17(2):100-107.
[21]	WANG E T, SANDBERG R, LUO S J, et al.Alternative isoform regulation in human tissue transcriptomes[J]. Nature, 2008, 456(7221):470-476.
[22]	杨阳, 张雪莲, 张万锋, 等.猪C1QTNF3基因可变剪接体的鉴定及介导miR-101调控成脂分化的研究[J].畜牧兽医学报, 2021, 52(11):3042-3052.YANG Y, ZHANG X L, ZHANG W F, et al.Study of alternative splicing of porcine C1QTNF3 gene and its regulation on adipogenesis by mediating miR-101[J].Acta Veterinaria et Zootechnica Sinica, 2021, 52(11):3042-3052.(in Chinese)
[23]	STAMM S, BEN-ARI S, RAFALSKA I, et al.Function of alternative splicing[J].Gene, 2005, 344:1-20.
[24]	KELEMEN O, CONVERTINI P, ZHANG Z Y, et al.Function of alternative splicing[J].Gene, 2013, 514(1):1-30.
[25]	ULE J, BLENCOWE B J.Alternative splicing regulatory networks:functions, mechanisms, and evolution[J].Mol Cell, 2019, 76(2):329-345.
[26]	贾浩, 张小白, 宋晓峰.人类胞内蛋白半衰期与其亚细胞定位的相关性研究[J].计算机与应用化学, 2011, 28(4):411-414.JIA H, ZHANG X B, SONG X F.Relationship between intracellular protein half-life and subcellular localization in human cells[J].Computers and Applied Chemistry, 2011, 28(4):411-414.(in Chinese)
[27]	BAJZAR L, MORSER J, NESHEIM M.TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades through the thrombin-thrombomodulin complex[J].J Biol Chem, 1996, 271(28):16603-16608.
[28]	BOFFA M B, BELL R, STEVENS W K, et al.Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor[J].J Biol Chem, 2000, 275(17):12868-12878.
[29]	BOFFA M B, REID T S, JOO E, et al.Characterization of the gene encoding human TAFI (thrombin-activable fibrinolysis inhibitor; plasma procarboxypeptidase B)[J].Biochemistry, 1999, 38(20):6547-6558.
[30]	张京华.凝血酶激活的纤溶抑制物(TAFI)、超敏C反应蛋白(hsCRP)和2型糖尿病微量白蛋白尿症的关系[D].沈阳:中国医科大学, 2020.ZHANG J H.The relationship between plasminogen inhibitor activated by hemomimetic enzyme and high sensitivity C-reactive protein and microalbuminuria in type2 diabetes mellitus[D].Shenyang:China Medical University, 2020.(in Chinese)
[31]	王胜江, 徐成伟.慢性肝病患者血浆中凝血酶激活的纤溶抑制物(TAFI)水平与慢性肝损伤的相关性[J].山东大学学报:医学版, 2012, 50(4):83-86.WANG S J, XU C W.TAFI levels in patients with chronic liver diseases:Correlation with chronic hepatic damage[J].Journal of Shandong University:Health Sciences, 2012, 50(4):83-86.(in Chinese)
[32]	SEJIMA H, SATOH S, DANSAKO H, et al.Molecular mechanism underlying the suppression of CPB2 expression caused by persistent hepatitis C virus RNA replication[J].Acta Med Okayama, 2016, 70(2):75-88.
[33]	YENER S, AKARSU M, DEMIR T, et al.Plasminogen activator inhibitor-1 and thrombin activatable fibrinolysis inhibitor levels in non-alcoholic steatohepatitis[J].J Endocrinol Invest, 2007, 30(10):810-819.