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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 146bp的BPSS0180基因,诱导表达得到的His-BPSS0180融合蛋白大小约为45ku,且主要以包涵体形式存在。BPSS0180蛋白的分子式为C_1779H_2809N_545O_536S_7,分子质量为40.6ku,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30h。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。
The aim of the experiment was to clone and prokaryotic express the BPSS0180 gene of Burkholderia pseudomallei (B. pseudomallei), and analyze its expressed protein by bioinformatics analysis. A pair of primers was designed according to the BPSS0180 gene sequence of Burkholderia pseudomallei K96243 in GenBank. The BPSS0180 gene fragment was amplified by PCR from the Burkholderia pseudomallei strain hn-1. The resulting BPSS0180 gene was ligated into the pET-28a (+) vector to construct the recombinant plasmid pET-28a (+) - BPSS0180 and transformed into E. coli DH5α competent cells. The plasmid was digested with restriction endonucleases. After identification, the constructed pET-28a (+) - BPSS0180 recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells and induced by IPTG. The expressed product was analyzed by SDS-PAGE and Western blotting. Bioinformatic analysis of BPSS0180 gene sequence was performed using DNAMAN, ProtParam, SOPMA and Protscale. The results showed that 1 146 bp BPSS0180 gene was successfully cloned in this study. The size of His-BPSS0180 fusion protein induced by induction was about 45 ku and mainly existed as inclusion body. The molecular formula of BPSS0180 protein is C_1779H_2809N_545O_536S_7, the molecular mass is 40.6ku, the extinction coefficient is 40 575, and the hydrophobic index is 85.43. Its instability coefficient is 46.52, which belongs to the unstable protein. The theoretical isoelectric point (pI) is 5.54, which is acidic protein. The total average hydrophobicity (GRAVY) is -0.261, which is a hydrophilic protein. The secondary structure of the protein was mainly composed of α-helix (58.79%) and random coil (32.02%). The half-life of this protein was estimated to be 30 h in reticulocytes of mammals. The results provide a theoretical basis for further study of BPSS0180 gene in Burkholderia pseudomallei.