猪LTβR基因克隆、结构功能预测、组织表达谱分析及真核表达载体构建

doi:10.11843/j.issn.0366-6964.2015.12.003

畜牧兽医学报 ›› 2015, Vol. 46 ›› Issue (12): 2135-2145.doi: 10.11843/j.issn.0366-6964.2015.12.003

猪LTβR基因克隆、结构功能预测、组织表达谱分析及真核表达载体构建

吴正常¹，戴超辉¹，殷学梅¹，孙寿永^1，2，包文斌^1，2*，吴圣龙^1，2*

（1.扬州大学动物科学与技术学院江苏省动物遗传繁育与分子设计重点实验室，扬州 225009；2.江苏省种猪繁育和健康养殖工程技术研究中心，扬州 225009）

收稿日期:2015-01-07 出版日期:2015-12-23 发布日期:2015-12-23
通讯作者: 包文斌，研究员，博导，主要从事猪遗传育种研究，E-mail：wbbao@yzu.edu.cn；吴圣龙，研究员，博导，主要从事猪遗传育种研究，E-mail：slwu@yzu.edu.cn
作者简介:吴正常(1987-)，男，江苏南京人，博士生，主要从事猪遗传育种研究，E-mail：wuzhengchang@126.com
基金资助:
转基因生物新品种培育科技重大专项（2014ZX08006-001B）；国家自然科学基金（31372285；31172183）；江苏高校优势学科建设工程资助项目(PAPD)

Gene Cloning，Prediction of Structure and Function，Analysis of Tissue Expression Profile and Construction of Eukaryotic Expression Vector of Pig LTβR Gene

WU Zheng-chang¹，DAI Chao-hui¹，YIN Xue-mei¹，SUN Shou-yong^1，2，BAO Wen-bin^1，2* ，WU Sheng-long^1，2*

(1.Key Laboratory for Animal Genetics，Breeding，Reproduction and Molecular Design of Jiangsu Province，College of Animal Science and Technology，Yangzhou University，Yangzhou 225009，China；2.Jiangsu Engineering Research Center for the Reproduction and Healthy Breeding of Boar，Yangzhou 225009，China)

Received:2015-01-07 Online:2015-12-23 Published:2015-12-23

摘要/Abstract

摘要：

为了进一步探究猪LTβR基因的生物学功能，本研究以猪淋巴组织cDNA为模板扩增LTβR基因CDS区，进一步利用生物信息学对猪LTβR蛋白结构与功能以及参与的GO和Pathway通路进行分析，利用Real-time PCR检测LTβR基因在大白猪不同组织中的表达水平，同时将LTβR基因扩增产物克隆至真核表达载体pEGFP-C1中，并分别转染猪HEK293和IPEC-J2小肠上皮细胞系，通过显微镜观察其表达情况。结果表明，克隆获得猪LTβR基因CDS区全长为1 209 bp，编码402个氨基酸，发现LTβR为脂溶亲水性的不稳定蛋白，存在1个跨膜结构（205~227 aa），不存在信号肽，亚细胞定位表明LTβR为非分泌型蛋白。LTβR蛋白具有2个糖基化位点，18个潜在的磷酸化位点，其中包括PKC、CKI、CDC2、GSK3、CDK5、INSR等10种保守的特异性蛋白质激酶的结合位点。该蛋白保守区域为2个TNFR superfamily，分别位于32~125和128~192位氨基酸，并发现C422T>A141V突变位于该区域内。KEGG和GO分析发现，LTβR基因共参与12项GO功能分类，同时还参与NF-kappa B信号通路、IgA 合成的肠道免疫网络等5项调控通路。定量检测发现，LTβR基因在大白猪肺中的表达水平极显著高于其他组织（P<0.01），在肾和脾等免疫器官，十二指肠和空肠等肠道组织中也均有较高的表达水平。细胞水平转染试验及显微镜观察发现其在猪HEK293和IPEC-J2两种细胞中均表达。本研究为猪LTβR基因及其介导的信号通路的功能分析提供了材料和依据，今后有必要在细胞水平上进一步验证分析猪LTβR基因及其介导的信号通路在猪抵抗细菌感染过程中发挥的重要作用，同时将C422T突变作为猪抗病育种的一个潜在遗传标记进行系统分析。

Abstract:

In order to further explore the biological function of pig LTβR gene，the coding sequence(CDS) of LTβR gene was amplified from the cDNA of porcine lymphoid tissue and the protein structure and function，GO function，regulatory pathways of porcine LTβR were further analyzed by bioinformatics tools.Real-time PCR was used to detect the expression level of porcine LTβR gene in different tissues of Yorkshire.Meanwhile LTβR gene was cloned into the eukaryotic expression vector pEGFP-C1，then transfected into HEK293 cell and small intestinal epithelial cell IPEC-J2，respectively.The expression of recombinant plasmid was detected in both cells through microscopic observation.The results showed that the pig LTβR cDNA full length was 1 209 bp encoding 402 amino acids.LTβR was a fat-soluble，hydrophilic and unstable protein，containing a transmembrane structure between 205 and 227 amino acids，meanwhile no signal peptide and subcellular localization indicated that LTβR protein was non-secretory.LTβR protein had 2 glycosylation sites，18 potential phosphorylation sites including 10 conservative binding sites of specific protein kinase such as PKC，CKI，CDC2，GSK3，CDK5，INSR，etc.Pig LTβR protein had 2 conservative domains named TNFR superfamily，which were located in the region between 32 and 125 amino acid，between 128 and 192 amino acid，respectively.Besides the mutation C422T>A141V occurred in the conservative domain.KEGG and GO analysis showed LTβR gene participated in 12 GO function classifications，5 regulatory pathways such as NF-kappa B signaling pathway，intestinal immune network for IgA production，etc.Real-time PCR analysis showed the expression level of LTβR gene in Yorkshire lung tissue was very significantly higher than other tissues(P<0.01).Moreover，LTβR gene was also highly expressed in both pig immune organs such as kidney，spleen and intestinal tissue such as duodenum，jejunum.Transfection experiments and microscopic observation showed the pEGFP-C1-LTβR recombinant plasmid was expressed in both HEK293 and IPEC-J2 cells.This study will provides materials and basis for studying the function of LTβR gene and LTβR-mediated signaling pathways，therefore it is necessary to further verify and analyze the regulatory mechanism and important role of LTβR gene and signaling pathways at cellular level，meanwhile we should systematically analyze the mutation C422T as a potential genetic marker for pig disease-resistant breeding.

中图分类号:

S828
S813.3

吴正常，戴超辉，殷学梅，孙寿永，包文斌，吴圣龙. 猪LTβR基因克隆、结构功能预测、组织表达谱分析及真核表达载体构建[J]. 畜牧兽医学报, 2015, 46(12): 2135-2145.

WU Zheng-chang，DAI Chao-hui，YIN Xue-mei，SUN Shou-yong，BAO Wen-bin，WU Sheng-long. Gene Cloning，Prediction of Structure and Function，Analysis of Tissue Expression Profile and Construction of Eukaryotic Expression Vector of Pig LTβR Gene[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(12): 2135-2145.

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参考文献

［1］BANKS T A，ROUSE B T，KERLEY M K，et al.Lymphotoxin-alpha-deficient mice.Effects on secondary lymphoid organ development and humoral immune responsiveness［J］.J Immunol，1995，155(4)：1685-1693.
［2］SHIMAZU T，TOHNO M，KATOH S，et al.Utilization of the porcine system to study Lymphotoxin-β regulation in intestinal lymphoid tissue［J］.Biochem Genet，2009，47(1-2)：126-136.
［3］LOCKSLEY R M，KILLEEN N，LENARDO M J.The TNF and TNF receptor superfamilies：integrating mammalian biology［J］.Cell，2001，104(4)：487-501.
［4］SHAKHOV A N，NEDOSPASOV S A.Expression profiling in knockout mice：lymphotoxin versus tumor necrosis factor in the maintenance of splenic microarchitecture［J］.Cytokine Growth Factor Rev，2001，12(1)：107-119.
［5］FÜTTERER A，MINK K，LUZ A，et al.The lymphotoxin β receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues［J］.Immunity，1998，9(1)：59-70.
［6］KANG W，KUDSK K A，SANO Y，et al.Effects of lymphotoxin β receptor blockade on intestinal mucosal immunity［J］.J Parenter Enteral Nutr，2007，31(5)：358-364.
［7］GUO H，FORTUNE M D，BURREN O S，et al.Integration of disease association and eQTL data using a Bayesian colocalisation approach highlights six candidate causal genes in immune-mediated diseases［J］.Hum Mol Genet，2015，24(12)：3305-3313.
［8］MARCHLER-BAUER A，LU S，ANDERSON J B，et al.CDD：a Conserved Domain Database for the functional annotation of proteins［J］.Nucleic Acids Res，2011，39(suppl 1)：D225-D229.
［9］BARBER R D，HARMER D W，COLEMAN R A，et al.GAPDH as a housekeeping gene：analysis of GAPDH mRNA expression in a panel of 72 human tissues［J］.Physiol Genomics，2005，21(3)：389-395.
［10］吴正常，殷学梅，夏日炜，等.猪杀菌/通透性增加蛋白基因siRNA载体构建及干扰效果评价［J］.畜牧兽医学报，2015，46(3)：491-496.
WU Z C，YIN X M，XIA R W，et al.Construction and evaluation of porcine bactericidal/permeability-increasing protein gene(BPI) siRNA expression vector［J］.Acta Veterinaria et Zootechnica Sinica，2015，46(3)：491-496.(in Chinese)
［11］LIVAK K J，SCHMITTGEN T D.Analysis of relative gene expression data using realtime quantitative PCR and the 2^-ΔΔCt method［J］.Methods，2001，25(4)：402-408.
［12］CROWE P D，VANARSDALE T L，WALTER B N，et al.A lymphotoxin-beta-specific receptor［J］.Science，1994， 264(5159)：707-710.
［13］BROWNING J L，DOUGAS I，NGAM-EK A，et al.Characterization of surface lymphotoxin forms.Use of specific monoclonal antibodies and soluble receptors［J］.J Immunol，1995， 154(1)：33-46.
［14］MAURI D N，EBNER R，MONTGOMERY R I，et al.LIGHT，a new member of the TNF superfamily，and lymphotoxin alpha are ligands for herpesvirus entry mediator［J］.Immunity，1998， 8(1)：21-30.
［15］KRAUSE P，ZAHNER S P，KIM G，et al.The tumor necrosis factor family member TNFSF14(LIGHT) is required for resolution of intestinal inflammation in mice［J］.Gastroenterology，2014，146(7)：1752-1762.
［16］BAKER S J，REDDY E P.Transducers of life and death：TNF receptor superfamily and associated proteins［J］.Oncogene，1996，12(1)：1-9.
［17］GRUSS H J，DOWER S K.Tumor necrosis factor ligand superfamily：involvement in the pathology of malignant lymphomas［J］.Blood，1995，85(12)：3378-3404.
［18］SMITH C A，FARRAH T，GOODWIN R G.The TNF receptor superfamily of cellular and viral proteins：activation，costimulation，and death［J］. Cell，1994，76(6)：959-962.
［19］SPAHN T W，EUGSTER H P，FONTANA A，et al.Role of lymphotoxin in experimental models of infectious diseases：potential benefits and risks of a therapeutic inhibition of the lymphotoxin-β receptor pathway［J］. Infect Immun，2005，73(11)：7077-7088.
［20］LUCAS R，TACCHINI-COTTIER F，GULER R，et al.A role for lymphotoxin β receptor in host defense against Mycobacterium bovis BCG infection［J］.Eur J Immunol，1999，29(12)：4002-4010
［21］EHLERS S，HÖLSCHER C，SCHEU S，et al.The lymphotoxin β receptor is critically involved in controlling infections with the intracellular pathogens Mycobacterium tuberculosis and Listeria monocytogenes［J］.J Immunol，2003，170(10)：5210-5218.
［22］SPAHN T W，MAASER C，ECKMANN L，et al.The lymphotoxin-β receptor is critical for control of murine Citrobacter rodentium-induced colitis［J］.Gastroenterology，2004，127(5)：1463-1473.
［23］BENEDICT C A，BANKS T A，SENDEROWICZ L，et al.Lymphotoxins and cytomegalovirus cooperatively induce interferon-β，establishing host-virus détente［J］.Immunity，2001，15(4)：617-626.
［24］刘志国.CRISPR/Cas9系统介导基因组编辑的研究进展［J］.畜牧兽医学报，2014，45(10)：1567-1583.
LIU Z G.Research progress on CRISPR/Cas9 mediated genome editing［J］.Acta Veterinaria et Zootechnica Sinica，2014，45(10)：1567-1583.(in Chinese)

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猪LTβR基因克隆、结构功能预测、组织表达谱分析及真核表达载体构建

Gene Cloning，Prediction of Structure and Function，Analysis of Tissue Expression Profile and Construction of Eukaryotic Expression Vector of Pig LTβR Gene

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