猪α1干扰素基因的克隆与原核表达

Clone and Prokaryotic Expression of Porcine Interferon-α1 Gene

根据猪α1干扰素(pocine interferon-alpha1,poIFN-α1)的全基因序列(EU364896)设计合成1对特异引物,通过RT-PCR从三元杂交仔猪外周血淋巴细胞扩增出poIFN-α1全基因,将该基因克隆到pGEM-T载体,构建重组质粒pGEM-T-poIFN-α1,进行测序。以pGEM-T-poIFN-α1为模板,经PCR扩增带有酶切位点的poIFN-α1基因,将其定向克隆到原核表达载体pGEX-6P-1中,构建重组表达载体pGEX-6P-poIFNα1,转化大肠杆菌BL21(DE3)进行蛋白诱导表达和鉴定,并确定蛋白最佳表达条件。序列分析表明,克隆的poIFN-α1基因全长546bp,编码181个氨基酸,前23个氨基酸组成信号肽,无糖基化位点。SDS-PAGE和Western blotting证实重组表达菌经IPTG诱导后,可表达相对分子质量约46ku的融合蛋白(GST-poIFN-α1)。当以1.0mmol/L的IPTG于37℃下诱导表达9h时,蛋白表达量最高。poIFN-α1基因的克隆与表达为进一步研究其抗病毒活性,开发病毒治疗和疫苗免疫增强用生物制剂奠定了基础。

A pair of specific primer for porcine interferon-alphal （poIFN-α1） was designed and synthesized according to gene sequence （EU364896）, and polFN-α1 gene was amplified by RT-PCR from peripherals blood lymphocytes （PBLC） of ternary hybrid piglet. Then polFN-α1 complete gene was cloned into pGEM-T vector and sequenced. Moreover, polFN-α1 gene with enzyme sites was cloned into prokaryotic expression vector pGEX-6P-1 to construct recombinant expression plasmid pGEX-6P-polFN-α1 and transform it into E. coli BL21 （DE3）. Recombinant polFNα1 was expressed and identified with expression conditions confirmed. The analysis of sequence indicated that the cloned polFN-α1 lgene consisted of 546 bp encoding 181 amino acids with a signal peptide of 23 amino acids but without glycosylation site. The selected recombinant expressed approximately a 46 ku fusion protein with glutathione S-transferase （GST） and polFN-α1 which was demonstrated by SDS-PAGE and Western blotting. The amount of fusion protein reached the highest when the recombinants were induced for 9 hours on 37℃ with 1.0 mmol/L IPTG. Clone and expression of polFN-α1 gene laid a foundation for the study of antiviral activity and utilization of polFN-α1