An Effective Cloning, Heterologous Expression and Physiological Activity in Lactococcus lactis NZ9000 of Catalase Gene from Escherichia coli DH5α
Journal of Advances in Microbiology,
In order to clone active Catalase gene from bacteria, we introduced a method of shotgun integrating specific screen from Escherichia coli DH 5a. Genome DNA was extracted from E. coli DH5 and partially digested with Sau3AI. Then some fragments more than 2.26kb were collected and ligated with T4 DNA ligase into the BamHI-cleaved plasmid pUC18 and transformed competent E. coli TG1 cells. The transforments were incubated anaerobically on brain heart infusion (BHI) containing tannic acid. The method could detect catalase activity and screen the catalase-positive clones. The catalase protein can be identified by SDS-PAGE. The results showed that the recombinant plasmid pUC18-kat was constructed successfully by PCR identification and restriction enzyme digestion. Opening the reading frame from DNA Sequence DH5a katE, the sequence length is 2262nt and coding protein are 753 Amino Acids, Molecular Weight is 84198.72 Daltons. This method is simple, and possess of popularization and application value. It would lay a foundation for cloning an active catalase later. Further, in this study, the fragment of 2,262 bp catalase gene katE was cloned into the expression vector pQE30 and transformed into Escherichia coli M15, and KatE protein was expressed after the induction with Isopropylthio-β-D-galactoside. The KatE protein was separated on SDS-PAGE and recovered using a His-tag affinity. New Zealand white rabbits were immunized with the purified protein to harvest polyclonal antibodies. Since L. lactis has no catalase, katE was inserted into Escherichia coli - L. lactis shuttle vector pMG36e and electro-transformed into L. lactis NZ9000. The expression of the KatE protein was confirmed by SDS-PAGE analysis and Western blot. Further experiment demonstrated that the expression of the KatE gene in L. lactis NZ9000 is able to produce active catalase that can provide efficient antioxidant activity. Additionally, to understand the import of catalase katE gene of Lactococcus lactis on the body’s physiological changes of immune function in mice, Enzyme-linked immunoassay (ELISA) was used on the blood of mice, to measure IgG, IgE, CD4 and CD8 levels, and find out whether there is a difference between Mice Groups eating the recombinant L. lactis NZ9000 and other groups i.e. (recombinant E. coli DH5α and L. lactis NZ9000; E. coli DH5α and saline). The experiments showed that recombinant L. lactis NZ9000 was significantly higher than the other on IgG concentrations; IgE, CD4 or CD8 levels are not significant. Mice with increased IgG levels of IgG after the intake of recombinant L. lactis NZ9000 helps to explain the recombinant L. lactis NZ9000 regulatory role in humoral immunity of mice; IgE level did not change, CD4 and CD8 levels were also not changed, thus suggesting that there is no significant effect of cellular immunity on the body in a short time.
- Lactococcus lactis NZ9000
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