Hou Yi-ling, Hou Wan-ru, Ren Zheng-long, Hao Yan-zhe, Zhang Tian
College of Agriculture, Sichuan Agricultural University, Ya-an, China.
Int J Biol Sci. 2008;4(6):415-21. doi: 10.7150/ijbs.4.415. Epub 2008 Nov 14.
AlphaB-crystallin, a small heat-shock protein, has been shown to prevent the aggregation of other proteins under various stress conditions. Here we have cloned the cDNA and the genomic sequence of CRYAB gene from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology and Touchdown-PCR, respectively. The length of cDNA fragment cloned contains an open reading frame of 528bp encoding 175 amino acids and the length of the genomic sequence is 3189bp, containing three exons and two introns. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other four species studied, including Homo sapiens, Mus musculus, Rattus norvegicus and Bos taurus. The homologies for nucleotide sequences of Giant Panda CRYAB to that of these species are 93.9%, 91.5%, 91.5% and 95.3%, respectively, and the homologies for amino acid sequences are 98.3%, 97.1%,97.7% and 99.4%, respectively. Topology prediction shows that there are only four Casein kinase II phosphorylation sites in the CRYAB protein of the Giant Panda. The cDNA of CRYAB was transfected into E. coli, and the CRYAB fused with the N-terminally His-tagged protein gave rise to the accumulation of an expected 24KDa polypeptide, which accorded with the predicted protein. The expression product obtained could be used for purification and study of its function further.
αB-晶状体蛋白是一种小分子热休克蛋白，已被证明在各种应激条件下可防止其他蛋白质聚集。在此，我们分别使用RT-PCR技术和降落PCR从大熊猫（Ailuropoda melanoleuca）中克隆了CRYAB基因的cDNA和基因组序列。克隆的cDNA片段长度包含一个528bp的开放阅读框，编码175个氨基酸，基因组序列长度为3189bp，包含三个外显子和两个内含子。比对分析表明，核苷酸序列和推导的氨基酸序列与其他四个研究物种（包括智人、小家鼠、褐家鼠和牛）高度保守。大熊猫CRYAB核苷酸序列与这些物种的同源性分别为93.9%、91.5%、91.5%和95.3%，氨基酸序列的同源性分别为98.3%、97.1%、97.7%和99.4%。拓扑预测表明，大熊猫CRYAB蛋白中仅存在四个酪蛋白激酶II磷酸化位点。将CRYAB的cDNA转染到大肠杆菌中，与N端带有His标签的蛋白融合的CRYAB导致预期的24KDa多肽积累，这与预测的蛋白一致。获得的表达产物可用于进一步纯化和研究其功能。
