EST-based Analysis of Gene Expression in the Porcine Brain

Since pig is an important livestock species worldwide, its gene expressionhas been investigated intensively, but rarely in brain. In order to study gene expression profilesin the pig central nervous system, we sequenced and analyzed 43,122 high-quality 5'' end expressedsequence tags (ESTs) from porcine cerebellum, cortex cerebrum, and brain stem cDNA libraries,involving several different prenatal and postnatal developmental stages. The initial ESTs wereassembled into 16,101 clusters and compared to protein and nucleic acid databases in GenBank. Ofthese sequences, 30.6% clusters matched protein databases and represented function known sequences;75.1% had significant hits to nucleic acid databases and partial represented known function; 73.3%matched known porcine ESTs; and 21.5% had no matches to any known sequences in GenBank. We used thecategories defined by the Gene Ontology to survey gene expression in the porcine brain.

猪肌肉素基因的cDNA克隆与表达

从人肌肉素基因出发，在dbEST数据库中进行同源性搜索，找到七个有较高同源性的Expressed Sequence Tag（DY426490，CF787546，AJ660979，AJ664670，AJ663820，AJ680159，DN106254）。通过拼接和进一步RT-PCR实验验证，获得猪肌肉素基因全长cDNA序列，其全长651bp，开放阅读框为54～452bp，编码有132个氨基酸。同源性分析结果表明，与人、小鼠和大鼠的肌肉素基因cDNA编码区（CDS）同源性分别为87．2％、77．6％和77．9％。利用克隆出的猪肌肉素cDNA，构建表达载体pGEX-4T-1-musclin，并在BL21大肠杆菌中成功表达和纯化了分子量为38．59kD的融合蛋白GST-Musclin，并运用蛋白印迹技术进行鉴定。