版纳微型猪近交系CD46基因克隆、序列及组织表达分析

Cloning,sequence and tissue expression analysis of CD46 gene in Banna mini-pig inbred line

根据猪NM_213888及EST序列,设计特异引物扩增BMI CD46基因,并进行克隆、测序和生物信息学分析。同时应用半定量RT-PCR技术对BMI 30个重要组织进行表达谱分析。获得了BMI CD46 1 092 bp的编码区序列(Gen Bank登录号:KJ513478),编码363个氨基酸。分析表明,CD46蛋白质分子量(Mw)为39.60 k D,等电点(p I)为5.39,存在4个保守域和1个跨膜结构域,N端有信号肽序列;其N末端疏水,C末端亲水;亚细胞定位显示,该蛋白位于细胞周质的概率是56.7%。活性位点分析表明,BMI CD46蛋白有6类活性位点。系统进化分析表明,BMI与牛的亲缘关系最近。BMI 30种组织表达分析表明,CD46基因在十二指肠中高表达;在睾丸、胸腺、甲状腺、附睾、肺、淋巴结、空肠、回肠、结肠、小脑及舌下腺中中度表达;在颌下腺、肝、肾上腺、盲肠、直肠、食管、垂体及脑干中低表达;在心、脾、肾、肌肉、胰脏、胃、皮肤、大脑、下丘脑及脊髓中不表达。该结果为进一步研究基因功能奠定基础。

Based on the NM_213888 and EST sequences of pig,we designed specific primers and amplified BMI CD46 for cloning,sequencing and bioinformatics analysis. Meanwhile,the expression of CD46 in 30 important tissues of BMI was analyzed by semi-quantitative RT-PCR. The research obtained 1 092 bp coding sequence（ Gen Bank accession number：KJ513478） of BMI CD46,which can encode a protein of 363 amino acids. Analysis showed that the molecular weight（ Mw）and isoelectric point（ p I） of predicted CD46 protein were 39.60 k D and 5.39; it contained four conserved domains and one transmembrane region; The N-terminal of the protein existed signal peptide sequences; hydrophobicity analysis indicated that the N-terminal of the protein was hydrophobic and the C-terminal of the protein was hydrophilic; It was a periplasm protein with a probability of 56.7%; The active site analysis showed there were six kinds of active sites in BMI CD46; Phylogenetic analysis demonstrated that BMI had the closest relationship with cattle. The analysis of tissue expression showed that the expression of CD46 examined in 30 tissues had obvious differences. It was highly abundant in the duodenum,moderately expressed in the testis,thymus,thyroid,epididymis,lung,lymph nodes,jejunum,ileum,colon,cerebellum and sublingual gland; and weakly expressed in the submandibular gland,liver,adrenal gland,cecum,rectum,esophagus,pituitary and brain stem; while it was undetected in the heart,spleen,kidneys,muscle,pancreas,stomach,skin,brain,hypothalamus and spinal cord. These results provided a useful foundation for further studies on CD46 function in swine.