牛NF-κB1基因的原核表达与蛋白纯化

Prokaryotic expression and protein purification of gene bovine NF-κB1

NF-κB1是一类重要核转录因子,参与多种基因表达调控。NF-κB1能与奶牛乳腺中乳合成调控相关基因结合,发挥泌乳调节作用。研究应用PCR方法扩增奶牛NF-κB1基因编码序列,通过Eco RⅠ和XhoⅠ酶切位点将其定向插入p GEX-4T-1载体中,构建原核表达重组质粒p GEX-4T-1-NF-κB1,转化大肠埃希氏菌BL21(DE3)。经酶切和测序鉴定正确后,异丙基硫代-β-D-半乳糖苷(IPTG)大量诱导表达,8 mol·L^(-1)尿素裂解包涵体,裂解后上清经GST-Sepharose 4B亲和纯化,SDS-PAGE和Western blotting鉴定。结果表明,原核表达载体p GEX-4T-1-NF-κB1构建成功,SDS-PAGE证实NF-κB1融合蛋白存在于包涵体内,纯化后得到GST-NF-κB1融合蛋白,为进一步研究NF-κB1蛋白在奶牛泌乳中作用提供试验依据。

As an important nuclear transcription factor, NF-KB1 is involved in regulating the expression of lots of genes. Recent research found that NF-KB1 has important regulatory effect on milk synthesis by binding to many gens involved in the regulation of milk synthesis. Bovine NF-κB1 coding sequence was amplified by polymerase chain reaction （PCR） and was cloned into Eco R I and Xho I sites of pGEX-4T-1 vector to construct the recombinant plasmid pGEX-4T-1-NF-κB1. The recombinant plasmid was identified by restriction enzyme digestion and sequencing. Then it was transformed into Ecoli BL21 （DE3}, induced by IPTG and purified the inclusion bodies was cracked with 8 mol. L^-1 urea, and GST-NF-κB1 was purified by GST-Sepharose 4B beads, identified by SDS-PAGE and Western blotting analysis. Results showed that the prokaryotic expression vector pGEX-4T-1-NF-κB1 was successfully constructed. Western blotting and SDS-PAGE confirmed the recombinant protein GST-NF-KB1 were expressed and purified. This study provided the experimental basis for the further research on the protein function of bovine NF-κB1.