猪LGALS1基因的原核表达及序列分析

Prokaryotic expression and sequence analysis of LGALS1 gene in pigs

目的原核表达猪源LGALS1并进行序列分析。方法提取猪肺泡巨噬细胞总RNA,RT-PCR法扩增LGALS1的cDNA序列,与pMD18-T载体连接,构建重组克隆质粒pMD18-T-LGALS1并测序,应用生物分析软件对基因序列进行同源性比对及系统进化树分析;将构建正确的质粒pMD18-T-LGALS1亚克隆至原核表达载体pET-30a(+),构建重组表达质粒pET-30a-LGALS1,转化大肠埃希菌BL21(DE3)感受态细胞,经IPTG诱导表达、Ni2+亲和层析纯化后,进行Western blot分析。结果PCR扩增获得了全长为408 bp的cDNA序列,与GenBank中登录的LGALS1编码区序列(NM001001867.1)相似性达99%,核苷酸序列同源性分析表明,克隆基因序列与猪源LGALS1同源性最高;质粒pET-30a-LGALS1经PCR及双酶切鉴定证明构建正确;表达的LGALS1相对分子质量约21000,经纯化后,可与LGALS1 Antibody发生反应,具有反应原性。结论成功表达了猪源LGALS1。

Objective To express LGALS1 in prokaryotic cells and analyze its sequence.Methods The total RNA of porcine alveolar macrophages was extracted,with which the cDNA sequence of LGALS1 was amplified by RT-PCR and inserted into vector pMD18-T.The constructed recombinant plasmid pMD18-T-LGALS1 was sequenced,and the homology of target gene sequence was analyzed by bioanalytical software,based on which a phylogenetic tree was plotted.The correctly constructed plasmid pMD18-T-LGALS1 was subcloned into prokaryotic expression vector pET-30 a(+),and the obtained recombinant plasmid pET-30 a-LGALS1 was transformed to E.coli BL21(DE3)and induced with IPTG.The expressed product was purified by nickel ion affinity chromatography and Western blot.Results A 408 bp cDNA sequence was obtained by PCR amplification,of which the similarity was 99%to the LGALS1 coding region sequence(NM001001867.1)registered in GenBank.Nucleotide sequence analysis showed the highest homology of the cloned gene sequence to porcine LGALS1 gene.Plasmid pET-30 a-LGALS1 was confirmed to be correctly constructed by PCR and restriction analysis.The expressed LGALS1,with a relative molecular mass of about 21000,showed reaction with LGALS1 antibody,indicating a reactogenicity.Conclusion Porcine LGALS1 was successfully expressed.