兔CCL28基因的克隆与序列分析

Molecular Cloning and Sequence Analysis of CCL28 Gene in Oryctolagus cuniculus

克隆并分析兔CCL28基因。基于电子延伸序列,设计1对克隆引物,兔盲肠黏膜组织提取总RNA,进行RT-PCR,将PCR产物与pMD19-T载体连接后转化E.coliJM109感受态细胞、检测阳性克隆、测序并进行序列分析。克隆的兔CCL28基因片段长为133 bp,编码由44个氨基酸残基组成的CCL28前体蛋白,克隆的兔CCL28基因与绵羊、野猪的同源性分别为76.7%、77.4%,推导的氨基酸序列与绵羊、野猪的同源性分别为56.8%、56.8%,结构特征与绵羊、野猪的相一致。并注册GenBank(Accession.EU727201)。

A pair of cloned primers were designed based on the in silico sequence information, total RNA was extracted from intestinum caecum mucosal tissue of Oryctolagus cuniculus and mRNA sequence was amplified by RT - PCR. The PCR products were ligated into the pMD19 - T vector, and then transformed into competent cells of E. coli JM109. The positive clone was identified and the sequence was sequenced and analyzed. The results indicated that CCL28 gene in Oryctolagus cuniculus consisted of 133 bp and encoded 44 amino acids. Identity analysis showed that the CCL28 nucleotide sequence in Oryctolagus cuniculus shared 76.7%, 77.4% homology with that of Ovis aries, Sus scrofa respectively, the predicted peptide shared 56.8%, 56.8% homology with that of Ovis aries, Sus scrofa respectively, revealing that the structural feature of CCL28 gene in Oryctolagus cuniculus is consistem with that of Ovis aries and Sus scrofa. The cloned CCL28 gene sequence has been submitted to GenBank （Accession. EU727201 ）.