版纳微型猪近交系CPN10克隆、亚细胞定位、组织表达和生物信息学分析

Cloning, Subcellular Localization, Tissue Expression and Bioinformatics Analysis of CPN10 Gene in Banna Mini-pig Inbred Line

【目的】以版纳微型猪近交系(BMI)为材料克隆CPN10基因,从亚细胞、m RNA水平及生物信息学方面初步探索其结构及功能特性,为该基因在猪—人异种器官移植免疫排斥中的作用研究奠定基础。【方法】采用RT-PCR方法获得CPN10基因c DNA序列,应用分子克隆技术构建目的基因和报告基因EGFP的重组真核表达载体pEGFP-C1-CPN10,将其转染猪肾细胞PK15进行瞬时表达,通过EGFP示踪CPN10在PK15细胞中的表达和定位。进一步应用荧光定量技术明确其mRNA的多组织表达特征。最后利用生物信息学分析CPN10蛋白质的结构特征。【结果】得到BMI CPN10 CDS 309 bp序列(GenBank登录号:KM098149),成功构建了BMI CPN10基因的绿色荧光蛋白融合表达载体pEGFP-C1-CPN10,转染PK15细胞且主要在细胞质中检测到绿色荧光蛋白表达。多组织荧光定量表达分析表明:BMI CPN10 mRNA在皮肤中具有最高表达量,在肝和肾上腺中的表达量较高,在其余各组织中呈中低度表达或几乎不表达。生物信息学分析表明:CPN10编码102个氨基酸,其蛋白分子量为10.93 ku,等电点为8.89,含1个CPN10保守结构域和5个磷酸化位点,二级结构以延伸链结构为主,无跨膜螺旋结构和信号肽。【结论】CPN10基因定位在细胞质中,在皮肤中高表达,该研究结果为进一步研究该基因在免疫排斥方面的功能奠定了理论基础。

[Purpose]In this study, the CPN10 gene was cloned from Banana mini-pig inbred line(BMI) and its structural and functional properties were preliminary explored from the subcellular,mRNA levels and bioinformatics, which laid the foundation for the research on the role of this gene in immune rejection of pig-human xenotransplantation.[ Methods] The cDNA sequence of BMI CPN10 was obtained by RT-PCR, and the recombinant eukaryotic expression vector pEGFP-C1-CPN10 of target gene and reporter gene EGFP was constructed by molecular cloning technology.Then, the recombinant vector was transfected transiently into porcine kidney cells(PK15) to confirm CPN10 localization in cells by tracing EGFP. Further, mRNA expression characteristics of multi-tissue were examined by qPCR techniques. Finally, structural characteristics of CPN10 protein were analyzed by bioinformatics.[Result]The CDS sequence of BMI CPN10 was 309 bp with GenBank accession number KM098149. The fusion expression vector pEGFP-C1-CPN10 of green fluorescence protein and CPN10 gene was successfully constructed and transfected into PK15 cells. The target proteins were mainly detected in the cytoplasm by tracing the green fluorescent protein. BMI CPN10 mRNA had the highest expression in the skin, higher expression in the liver and adrenal glands, and was moderate, low or hardly expressed in the remaining tissues. Bioinformatics analysis indicated the CPN10 encoded 102 amino acids with molecular weight of 10.93 ku and isoelectric point of 8.89, containing a conserved CPN10 domain and 5 phosphorylation sites. The secondary structure of the CPN10 protein is mainly extended chain structure without transmembrane helical structure and signal peptide.[ Conclusion] The CPN10 gene was located in the cytoplasm and highly expressed in the skin, which provided a theoretical basis for further study on the function of CPN10 gene in rejection of xenotransplantation.