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

Abstract

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.

CLC Number:

S828
S813.3

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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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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