目的:构建鲍曼不动杆菌5075株溶血素共调节蛋白(Hcp)基因的原核表达载体,完成蛋白的高效表达与纯化。方法:在Hcp蛋白生物信息分析的基础上,通过PCR方法扩增目的基因,对目的片段和pET-28a(+)质粒分别进行BamHⅠ和XhoⅠ位点的双酶切后连接并转化DH5α大肠埃希菌,于含抗卡那霉素的LB固体培养基上筛选,随机挑取单菌落进行菌落PCR鉴定及测序鉴定。将测序正确的pET-28a(+)-Hcp重组质粒再次转化E.coliBL21(DE3)中,探索合适的诱导条件。IPTG诱导Hcp蛋白分别在37℃和18℃进行表达,再采用BeyoMagTMHis标签蛋白纯化琼脂糖磁珠(NTA-Ni)纯化目的蛋白。结果:Hcp蛋白的生信分析显示,鲍曼不动杆菌Ab5075_Hcp基因编码167个氨基酸,蛋白质分子式为C833H1278N228O260S3,理论蛋白分子量大小为18.7kDa,蛋白等电点为6.52,不稳定指数为32.86,脂肪族指数为67.13,亲水性平均值为-0.625,为亲水稳定蛋白。无跨膜区和信号肽,序列高度保守。二级结构主要有无规则卷曲和延伸链。Hcp蛋白亚细胞定位于细胞核,有5个潜在的B细胞抗原表位。本研究成功构建Hcp基因原核表达载体,诱导表达后纯化获得大量可溶性Hcp蛋白,达到最适诱导条件为IPTG0.5mmol/L时,18℃下诱导15~16h。结论:鲍曼不动杆菌Ab5075_Hcp蛋白在预测值合理范围内且纯化效果较好,适合用于制备抗体以及候选疫苗的实验。
Objective: To construct a prokaryotic expression vector for the hemolysin co-regulated protein (Hcp) gene from Acinetobacter baumannii strain 5075, and to achieve high-efficiency expression and purification of the protein. Methods: Based on the bioinformatics analysis of Hcp protein, the target gene was amplified by PCR. The target fragment and pET-28a(+) plasmid were digested with BamHⅠ and XhoⅠ, ligated and transformed into E.coli DH5α, and screened on LB solid medium containing kanamycin. Single colonies were randomly selected for colony PCR identification and sequencing. The recombinant plasmid pET-28a(+)-Hcp was transformed into E.coli BL21 (DE3) again to explore the suitable induction conditions. The expression of Hcp protein was induced by IPTG at 37 ℃ and 18 ℃, respectively, and the target protein was purified by BeyoMagTM His tag protein purification agarose magnetic beads (NTA-Ni). Results: The bioinformatics analysis of Hcp protein showed that the Ab5075_Hcp gene from A. baumannii encodes 167 amino acids, with a protein formula of C833H1278N228O260S3, the theoretical protein molecular weight was 18.7 kDa, the protein isoelectric point was 6.52, the instability index was 32.86, the aliphatic index was 67.13, and the average hydrophilicity was -0.625, which was a hydrophilic stable protein. There was no transmembrane region and signal peptide, and the sequence is highly conserved. The secondary structure mainly had regular curl and extension chain. Hcp protein was located in the nucleus and had five potential B cell epitopes. In this study, the prokaryotic expression vector of Hcp gene was successfully constructed, and a large amount of soluble Hcp protein was purified after induction expression. The optimal induction conditions were IPTG 0.5 mmol/L and induction at 18 ℃ for 15-16 h. Conclusion: The Ab5075_Hcp protein of Acinetobacter baumannii is within a reasonable range of predicted values and has a good purification effect, which is suitable for the preparation of antibodies and candidate vaccines.
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