商品长白杂交猪IgA H链CH1-CH3区基因的克隆与表达

Cloning and expression of CH1-CH3 domain gene of IgA heavy chain from an outbred Landrace pig

在猪IgA重链CH1-CH3区设计一对引物,用RT-PCR方法从地方杂交品种长白猪肠系膜淋巴结组织中扩增出预期大小的片断,插入pGEM-Teasy载体,测序并与约克夏猪、人及其他动物的IgA进行序列比较。随后,将该序列酶切后引入到pQE-30表达载体相应位点,转化JM109大肠杆菌,经IPTG诱导表达后,进行SDS-PAGE分析。结果显示,本研究克隆了猪IgA重链恒定区部分CH1亚区及完整的CH2-CH3亚区基因序列,全长822bp,编码274个氨基酸,该序列与GenBank上登录的约克夏猪参考序列核苷酸序列同源率为99.8%,有2处核苷酸变异,氨基酸序列的同源率为100%。但与人及其他动物显示从84%～51%不等的同源性;在大肠杆菌表达出约31ku的蛋白条带,表达量约占菌体总蛋白的32.3%。本试验为今后的IgA免疫功能研究及基因工程化检测试剂开发打下了基础。

According to the sequence of IgA gene from GenBank, a pair of primers containing Kpn Ⅰ and HindⅢ restriction enzyme-clearage sites was designed to clone the CH1-CH3 domain of IgA gene from an outbred swine （Landrace） mesenteric lymph node by RT-PCR. The DNA fragment yielded in the PCR was inserted into the pGEM-T Easy vector. After that, the amplified fragment was digested with Kpn Ⅰand HindⅢ, and ligated to an expression vector named pQE-30. The result revealed the IgA gene in partly CH1 domain and completely CH2-CH3 domain of swine was composed of 822 bp nucleotides encoding 274 amino acid residues, which corresponds to the predicted result. Two nucleotides changes were found in the IgA CH1-CH3 gene of swine, and the homology ratios of nucleotides and amino acids were 99. 8% and 100%, respectively, compared with the sequence of IgA gene of swine （Yorkshire） in GenBank. By sequencing and contrasting to other animal IgA CH1-CH3 region sequences, it demonstrated 84% -51% homology for other animals and 79. 1% homology to human IgA2. SDSPAGE revealed that a 31ku objective protein was expressed