Sequence Analysis Of Nitrite Reductase Gene Of Lactobacillus Plantarum

Lactobacillus plantarum have the ability to degrade nitrite.According to the conserved amino acid sequences of the known fourtypes of nitrite reductase from Lactobacillus plantarum, we have usedthe protein analysis software and bio-informatics tools to find out theproducts of eight genes in Lactobacillus plantarum which might beable to degrade nitrite. The eight genes were cloned from Lactobacillusplantarum genome DNA by PCR and we successfully constructed theprokaryotic expression vectors pET-32a(+)-NiRs. Then the recombinantplasmids were transformed into E.coli BL21(DE3). The activity of the eightproteins were detected in accordance with the GMS15032.1.3v.A kitinstructions from GENMED. The results showed that HP protein, GRprotein and ORD protein of Lactobacillus plantarum could degradenitrite. The activity reached15.48unit/mg,2.49unit/mg,3.32unit/mg(unit=μmol nitrite/minute) respectively.Anlysising by bioinformatics, we found that the tertiary structureof HP, GR and ORD proteins all contained Alpha helix, Random coil andExtended strand. The HP protein tertiary structure based on Alpha helix,which accounted for the proportion of53.76%. The GR protein tertiarystructure based on Random coil, which accounted for the proportion of50.45%. The ORD protein tertiary structure based on Random coil, whichaccounted for the proportion of51.37%. We analyzed the signalpeptide and transmembrane region of the three proteins, whichsuggested that there was no signal peptide and transmembrane regionin the three proteins. So they were not trans-membrane proteins andthey couldnt be secretted into the extracellula. The localizationprediction of the three proteins in cell suggested that HP protein, GRprotein and ORD protein were existent in the cytoplasm of the greatpossibility. Then we designed the primers for NrfA gene from E.coli. TheE.coli NrfA gene was cloned from the E.coli genome DNA by PCR andinserted into the vector pET-28a(+). The prokaryotic expression vectorpET-28a(+)-NrfA was constructed successfully. E.coli NrfA protein wasexpressed by IPTG induction and purified. Polyclonal antibody againstNrfA protein was generated by immunizing rabbit with the routinemethod. The titer of rabbit anti-NrfA polyclonal antibody obtained afterimmunization and purification was about1:102400. After WesternBlotting, the polyclonal antibody against E.coli NrfA protein with a highaffinity and specificity was obtained. In the last, we applied thepolyclonal antibody of E. coli NrfA protein in detecting related proteinof Lactobacillus plantarum and the result was consistent with theexpected value.