牛支原体临洮分离株NOX-1的克隆表达、酶学活性及其亚细胞定位研究

PROKARYOTIC EXPRESSION OF MYCOPLASMA BOVIS LINTAO STRAIN NOX-1 GENE AND CHARACTERIZATION OF ITS ENZYME ACTIVITY AND IMMUNOGENICITY

为研究牛支原体(M. bovis)临洮分离株(LT strain)NOX-1基因特征表达产物的酶活性,及其在细胞中的具体存在部位,参照GenBank中M. bovis NOX-1HuBei株NOX-1基因序列(GenBank登录号:NC015725.1)设计引物,应用PCR扩增M.bovis临洮株的NOX-1基因,在完成测序后构建NOX-1基因原核表达载体pET-NOX-1,并转化大肠杆菌BL21(DE3)感受态细胞,经异丙基硫代半乳糖苷(IPTG)诱导后表达。表达产物纯化后进行酶活测定并免疫新西兰兔,制备多克隆抗体,继而应用Western blot及间接ELISA对NOX-1在M. bovis内的分布进行初步定位。结果显示,M. bovis临洮株NOX-1基因全长1365 bp,重组质粒经IPTG诱导在大肠杆菌BL21(DE3)中成功表达,重组蛋白His-NOX-1的分子量约为50 kDa;重组蛋白酶促反应最适酶促温度为30℃、pH为7.5,双倒数法求得重组蛋白Km和Vmax分别为256.41μmol/L、34.25μmol/(L·min);Western blot及间接ELISA结果表明His-NOX-1在M. bovis细胞膜上和细胞浆中均有分布且胞浆含量较高。本研究结果为进一步研究M. bovisNOX-1的生物学功能奠定了一定的基础。

In the present study, the primers were designed according to the Mycoplasma bovis NOX-1 gene sequence deposited in GenBank(accession No. NC015725.1) in order to amplify the corresponding gene from M. bovis Lintao strain using overlap PCR. Then the prokaryotic expression vector pET-NOX-1 was constructed and transformed into E. coli BL21(DE3). The recombinant rMbNOX-1 protein was expressed with induction of IPTG and purified for further research on its enzyme activity and immunogenicity. The results showed that the full-length of NOX-1 gene from M. bovis Lintao strain was 1365 bp and the molecular weight of the recombinant NOX-1 protein was about 50 kDa. Additionally, the recombinant NOX-1 protein was catalyzed via NADH oxidizatedion to NAD+. Some parameters were optimized, including temperature at 30℃ and pH at 7.5. The Michaelis constant(Km) and maximum of reaction velocity(Vmax) were determined to be 256.41 μmol/L and 34.25 μmol/(L·min), respectively. The results of Western blot and ELISA showed that His-NOX-1 in M. bovis was localized in membrane and cytoplasm. Overall, the results of this study would lay a solid foundation for the further study of M. bovis NOX-1.