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| Expression and activity identification of hlyA from Mycoplasma ovipneumoniae |
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Abstract Mycoplasma ovipneumoniae (MO) can lead to the atypical pneumonia for both sheep (Ovis aries) and goats (Capra hircus), which is wide- spreading and can cause serious harm. Based on the complete sequence of MO Y98 by our group, a potential hemolysin gene (hlyA) was obtained by using gene annotation and PCR method. In the present study, hlyA gene, which derived from MO Y98, was modified by using preference codons of Escherichia coli. And then prokaryotic expression vector (pET32a-hlyA) was constructed. pET32a-hlyA was transformed into E. coli BL21(DE3) polysS, and induced for the expression of HlyA. HlyA recombinant protein was visualized by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot method, and purified by using Ni2+ metal chelate column. The results showed that the hly gene (GenBank No.KT598390) of MO Y98 strain contained 375 bp, encoding a polypeptide of 125 amino acid residues. After optimization, the codon adaptation index was 0.91, the modified hlyA is suitable for expression in E.coli. Recombinant fusion proteins were mainly existed in the form of inclusion body. Expressed HlyA protein was about 31.6 kD, consistenting with the expected size. The results of Western blot analysis showed that supernatant of purified protein could react with the primary antibody, indicating that HlyA proved an efficacious immunological reactivity. When purified recombinant HlyA protein concentration was diluted to 0.125 μg/mL, the ability of dissolving red blood cells had declined. This research contributes to the study of hymolysin pathogenicity in MO.
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Received: 07 September 2015
Published: 05 February 2016
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