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.
DING Ruipei
,
MU Ruxue
,
ZHANG Yu
,
LI Zilong
,
LI Liuyan
,
WANG Yilin
,
YE Ying
,
HE Maozhang
. Proteinbioinformatics analysis of the Hcp gene and construction of prokaryotic expression vector in Acinetobacter baumannii strain 5075[J]. Journal of Baotou Medical College, 2025
, 41(9)
: 33
-41
.
DOI: 10.16833/j.cnki.jbmc.2025.09.006
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