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| Cloning and Prokaryotic Expression of pdhα and pdhβ Genes from Mycoplasma bovis and Its Subcellular Localization |
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Abstract Mycoplasma bovis (M.b) is one of the important pathogenic mycoplasmas. It can cause a variety of diseases if cow (Bos taurus) infected with the M.b. Pyruvate dehydrogenase complex E1 (PDHc E1) is a speed limit of enzymes that involve in biological sugar metabolic process, and widely exists in microbe, mammals and higher plants. It directly impact on pyruvate to acetyl coA. According to the gene sequences of M.b PG45 strain in GenBank, two pairs of primers were designed. PDHc E1 component subunit alpha (pdhα)(GenBank No.KU355295) and PDHc E1 component subunit beta (pdhβ) (GenBank No. KU3552956) genes of M.b Wuwei strain were amplified by PCR. After finished sequencing and point mutation, the genes were cloned into prokaryotic expression plasmid pET-28a(+), respectively. Then the recombinant plasmids pET-pdhα and pET-pdhβ were transformed into Escherichia coli BL21 (DE3), respectively. After induced expression by isopropyl β-D-1-thiogalactopyranoside (IPTG), the fusion protein recombinant PDHA (rPDHA) and recombinant PDHB (rPDHB) were purified and anti-serum against rPDHA or rPDHB was prepared respectively through immunizing New Zealand rabbit (Oryctolagus cuniculus). Subsequently, subcellular location of E1α and E1β in M.b was performed using Western blot and enzyme linked immunosorbent assay (ELISA). The results showed that the recombinant proteins successfully expressed in Escherichia coli BL21 (DE3) and their molecular weights were approximately 40 and 37 kD, respectively. Western blot and ELISA analysis indicated that the E1α and E1β of M.b equally distributed in both membrane and the cytoplasm. This study provides foundational data for further investigation of biological functions of M.b.
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Received: 23 August 2015
Published: 24 February 2016
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