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首页> 外文期刊>Scandinavian journal of immunology. >Molecular cloning, expression, purification and immunological characterization of three low-molecular weight proteins encoded by genes in genomic regions of difference of mycobacterium tuberculosis.
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Molecular cloning, expression, purification and immunological characterization of three low-molecular weight proteins encoded by genes in genomic regions of difference of mycobacterium tuberculosis.

机译：结核分枝杆菌差异基因组区域中基因编码的三种低分子量蛋白质的分子克隆，表达，纯化和免疫学表征。

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The aim of this study was to clone, express and purify three major antigenic proteins, i.e. Rv3874, Rv3875 and Rv3619c, encoded by genes located in regions of difference of Mycobacterium tuberculosis and characterize them for immunogenicity in rabbits. The respective genes were amplified using gene-specific primers and genomic DNA of M. tuberculosis by polymerase chain reaction. The amplified DNA were cloned into pGEM-T Easy and subcloned into pGES-TH-1 vector for high-level expression in Escherichia coli and efficient purification. The results showed that the three fusion proteins, i.e. glutathione-S-transferase (GST)-Rv3874, GST-Rv3875 and GST-Rv3619c, were expressed at high levels and were purified (free of the GST fusion partner) to homogeneity using glutathione-Sepharose and Ni-NTA agarose affinity matrix after cleavage of the column-bound fusion proteins by thrombin protease. The purified recombinant Rv3874, Rv3875 and Rv3619c proteins were immunogenic and induced antigen-specific antibodies in rabbits. Testing of the rabbit sera with overlapping synthetic peptides showed that the antibodies were induced to several epitopes that were scattered throughout the sequence of each protein. These results show immunogenicity of all the proteins for inducing antigen-specific antibodies in rabbits and demonstrate the usefulness of pGES-TH-1 vector for obtaining purified recombinant proteins of M. tuberculosis for immunological characterization.
机译：这项研究的目的是克隆，表达和纯化三种主要抗原蛋白，即Rv3874，Rv3875和Rv3619c，它们由位于结核分枝杆菌不同区域的基因编码，并表征它们在兔中的免疫原性。使用基因特异性引物和结核分枝杆菌的基因组DNA通过聚合酶链反应扩增各个基因。将扩增的DNA克隆到pGEM-T Easy中，再亚克隆到pGES-TH-1载体中，以在大肠杆菌中高效表达并有效纯化。结果表明，三种融合蛋白，即谷胱甘肽-S-转移酶（GST）-Rv3874，GST-Rv3875和GST-Rv3619c，均以高水平表达，并使用谷胱甘肽-纯化（不含GST融合伴侣）至同质。凝血酶蛋白酶切割柱结合的融合蛋白后，琼脂糖和Ni-NTA琼脂糖亲和基质。纯化的重组Rv3874，Rv3875和Rv3619c蛋白具有免疫原性并在兔中诱导了抗原特异性抗体。用重叠的合成肽对兔血清进行的测试表明，该抗体被诱导为分散在每种蛋白质序列中的几个表位。这些结果显示了所有蛋白在兔中诱导抗原特异性抗体的免疫原性，并证明了pGES-TH-1载体对于获得结核分枝杆菌的纯化重组蛋白进行免疫学表征的有用性。

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