类鼻疽伯克霍尔德菌BPSS0180基因的克隆、原核表达及生物信息学分析

试验旨在对类鼻疽伯克霍尔德菌（Burkholderia pseudomallei，B.pseudomallea）的BPSS0180基因进行克隆和原核表达，并对其表达蛋白进行生物信息学分析。参照GenBank中类鼻疽伯克霍尔德菌K96243标准株BPSS0180基因序列设计1对引物，对类鼻疽伯克霍尔德菌hn-1株进行PCR扩增获得BPSS0180基因片段。将得到的BPSS0180基因连接到pET-28a （+）载体，构建pET-28a （+）-BPSS0180重组质粒，转化至大肠杆菌DH5α感受态细胞中，提取质粒进行酶切鉴定。鉴定正确后，将构建成功的pET-28a （+）-BPSS0180重组质粒转化到大肠杆菌BL21（DE3）感受态细胞中，经IPTG诱导表达，表达产物用SDS-PAGE和Western blotting进行分析。应用DNAMAN、ProtParam、SOPMA和Protscale对BPSS0180基因序列进行生物信息学分析。结果显示，本试验成功克隆了1 146 bp的BPSS0180基因，诱导表达得到的His-BPSS0180融合蛋白大小约为45 ku，且主要以包涵体形式存在。BPSS0180蛋白的分子式为C₁₇₇₉H₂₈₀₉N₅₄₅O₅₃₆S₇，分子质量为40.6 ku，消光系数为40 575，疏水指数为85.43。其不稳定系数为46.52，属于不稳定蛋白；理论等电点（pI）为5.54，为酸性蛋白；总平均疏水性（GRAVY）是-0.261，为亲水性蛋白。该蛋白的二级结构以α-螺旋（58.79%）和无规卷曲（32.02%）为主，预测其在哺乳动物网织红细胞的半衰期为30 h。本试验结果为进一步探究类鼻疽伯克霍尔德菌的BPSS0180基因提供了一定的理论依据。

Cloning,Prokaryotic Expression and Bioinformatics Analysis of BPSS0180 Gene of Burkholderia pseudomallei

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.