关键词: 类鼻疽伯克霍尔德菌; BPSS0180基因; 克隆; 原核表达; 生物信息学分析

Abstract:

This study was aimed to clone and express the BPSS0180 gene of Burkholderia pseudomallei (B. pseudomallea), and perform bioinformatics analysis of its protein. A pair of primers was designed according to the BPSS0180 gene sequence information of B. pseudomallea K96243 strain in GenBank. BPSS0180 gene fragment was obtained by PCR amplification of B. pseudomallea hn-1 strain. The BPSS0180 gene fragment was ligated into the pET-28a(+) vector to construct the pET-28a(+)-BPSS0180 recombinant plasmid. The recombinant plasmid pET-28a(+)-BPSS0180 was transformed into E.coli DH5α competent cells, and the plasmids were identified by restriction enzyme digestion. Then, the recombinant plasmid pET-28a(+)-BPSS0180 was transformed into E.coli BL21 (DE3) competent cells. The expression was induced by IPTG. The expressed product was analyzed by SDS-PAGE and Western blotting. Bioinformatics analysis of BPSS0180 gene sequence was carried out using DNAMAN, ProtParam, SOPMA and Protscale. The results showed that the length of BPSS0180 gene was 1 146 bp; The expressed His-BPSS0180 fusion protein was about 45 ku, and was predominantly in the form of inclusion bodies; The molecular weight of the BPSS0180 protein was 40.6 ku (C₁₇₇₉H₂₈₀₉N₅₄₅O₅₃₆S₇); The extinction coefficient was 40 575; The hydrophobic index was 85.43; The instability coefficient was 46.52,which belonged to unstable protein;The theoretical isoelectric point (pI) was 5.54 and was acidic protein; The total average hydrophobicity (GRAVY) was -0.261,as hydrophilic protein; The secondary structure of the protein were mainly α-helix (58.79%) and random curl (32.02%), and its half-life of reticulocytes in mammals was predicted to be 30 h. This study provided a theoretical basis for further exploring the fuction of BPSS0180 gene of B. pseudomallei.

Key words: Burkholderia pseudomallei; BPSS0180 gene; cloning; prokaryotic expression; bioinformatics analysis