版纳微型猪近交系SLA-DOA基因克隆、mRNA表达及蛋白质功能生物信息学分析

Cloning,mRNA expression and protein functional bioinformatics analysis of SLA-DOA in Banna mini-pig inbred line

目的:克隆版纳微型猪近交系(Banna mini-pig inbred line,BMI)白细胞抗原DO座位alpha亚基(SLA-DOA)的编码序列,并对其19个重要组织mRNA表达作半定量分析。方法:RT-PCR方法获得SLA-DOA的全长cDNA序列;半定量RTPCR方法分析其在BMI重要组织的mRNA表达谱,并运用生物信息学对其核酸及蛋白序列进行分析,构建系统进化树。结果:BMI SLA-DOA cDNA长度为1 079 bp,编码250个氨基酸,蛋白质分子量为27.81 k D,等电点为6.48;位于猪7号染色体,由4个外显子和3个内含子构成;其mRNA高表达于淋巴结和胃,低表达于心脏、皮肤和十二指肠,在其他14种组织中不表达;SLA-DOA蛋白存在1个信号肽,1个跨膜螺旋,3个保守域,4个O连接的糖基化位点,18个磷酸化位点,位于细胞质的概率是94.1%;系统进化分析表明,在所选择的6个物种中,BMI(猪)与牛的亲缘关系最近。结论:本研究成功克隆了版纳微型猪近交系SLA-DOA基因并进行了生物信息学功能分析和多种组织表达分析,为版纳微型猪近交系的异种器官移植研究奠定基础。

Objective：To clone swine leukocyte antigen, class II, DO alpha （SLA-DOA） gene from Banna mini-pig inbred line （BMI） and detect its mRNA expression level in 19 important tissues. Methods： The complete cDNA sequence of SLA-DOA gene was cloned by RT-PCR method from BMI and the mRNA expression pattern in BMI important tissues was examined by semi-quantative RT- PCR method. Nueleotide and protein sequences of SLA-DOA were used to carry out bioinformaties analysis and construct the phylogenetie tree. Results： The eDNA length of BMI SLA-DOA was 1 079 bp, which encoded a protein of 250 amino acids with molecular weight （Mw） 27. 81 kD, and isoelectric point （pI） 6.48. Genome structure analysis showed it localized to Sus serofa chromosomes 7 and consisted of four exons and three introns. Semi-quantitative expression analysis showed that SLA-DOA gene expressed highly in the lymph nodes and stomaeh;weakly in the heart, skin and duodenum and none in other 14 tissues. Functional bioinformatics analysis indicated that SLA-DOA protein contained one signal peptide, one transmembrane region, three conserved domains, four O-GleNAe glyeosylation sites, 18 potential phosphorylation sites and to be located in the cytoplasm with 94. 1% certain- ty. Phylogenetie analysis demonstrated that BMI （pig） had the closest relationship with cattle. Conclusion：This study have successfully cloned the SLA-DOA gene of Banna mini-pig inbred line, performed bioinformatics analysis and tissue expression profile analysis. It will provide a basis for the studies of BMI xenotransplantation.