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| Cloning, Expression and Subcellular Localization of fba Gene in Mycoplasma bovis Wuwei Strain |
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Abstract Abstract Mycoplasma bovis (Mb) is an important pathogenic mycoplasma, which can cause a variety of diseases if cow (Bos taurus) infected with the Mb. Fructose bisphosphate aldolase (FBA) is one of the pivotal enzymes that involves in the p rocess of glycolysis pathway, which provided the substrate for the dehydrogenation of glyceraldehyde 3-phosphate, and widely exists in the living nature. In order to study the distribution of FBA in Mb cells, In this study, a pair of specific primers were designed according to fba gene sequence of Mb PG45 reported in GenBank. Then the fba of Mb Wuwei stain was amplified by PCR, and it was cloned into pMD19-T vector. After finished sequencing and comparing in the NCBI with other strains of Mb isolating from China, the phylogenetic trees were constructed applying with DNA Star software. Based on the sequencing and gene optimization, the prokaryotic expression vector pET-fba was constructed and transformed into Escherichia coli Transetta (DE3). Then, The target protein was induced by isopropyl β-D-1-thiogalactopyr-Anoside (IPTG). After that, we purified recombinant protein rMbFBA using Ni-NTA His Bind to immunize New Zealand rabbit (Oryctolagus cuniculus), Which aimed at preparing the polyclonal antibodies against rMbFBA. Subsequently, the antibody lever was detected by enzyme linked immunosorbent assay (ELISA). What's more, subcellular localization of FBA in Mb was performed using Western bolt. In conclusion, The results showed that the open reading frame of fba gene contains 876 nucleotides (GenBank No. KX828563), encoding a protein with 292 amino acids protein. However, there were 2 TGA codes in the sequence, which encodes trptophan in mycoplasma, but it is translated as the terminator coden in Escherichia coli. So the overlap PCR was used to make the 2 TGA termination coden mutations successfully mutate into synonymous coden TGG. The phylogenetic tree analysis from fba gene sequence of Mb Wuwei strain has high homology with other Mb strains separating from China, which indicated that the fba gene is highly conserved. Flowing that, the optimized gene was successfully expressed in Escherichia coli Transetta(DE3) when induced with final concentration of 1 mmol/L IPTG for 10 h at 20 ℃, after detected by the technology of sodium dodecyl sulfonate-polyacrylamide geleletrophoresis (SDS-PAGE), the result display that the relative molecular weight was approximately 34 kD, which existed mainly in the form of soluble protein. However, the negative control in this non target strip. The ELISA titer of antiserum was approximately up to 1∶25 600. The results of Western bolt confirmed that the prokaryotic expression products of fba gene of Mb Wuwei strain has good immunogenicity, and the FBA of Mb distributed in both cytoplasm and the membrance equally. All the results of this research lay a foundation data for further study on the biological function of FBA of Mb.
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Received: 14 October 2016
Published: 13 January 2017
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