Cloning, Expression And Characterization Of Nitrite Reductase Gene From Lactobacillus Plantarum From Pickled Radishes

This study chose a strain of lactic acid bacteria as material which was isolated before and has been demonstrated the capability to produce nir. The primer of the nir gene was designed, the gene was cloned and then was transformed into E. coli to build the expression vector which was induced to express to obtain the protein. Through optimizing the expression of fusion protein, the induced expression conditions were preliminarily screened. Extract the DNA of Lactobacillus plantarum, amplify nir gene by PCR, obtain recombinant plasmid pMD19-nir by TA clone, analyze the nucleotide sequence homology of nitrite reductase. Join into expression vector pET-32a(+) by double enzyme digestion to obtain recombinant expression plasmid pET-32a(+)-nir which was then transferred into E.coli BL(DE3). The results showed that the target protein was in the precipitation, which meant inclusion body was formed. The preliminary enzymatic properties were analyzed based on target protein which was purified. The main results of this study are as follows:(1) The primer was designed based on all gene sequences of Lactobacillus plantarum WCFS1 in Genbank. The nir gene was amplified through PCR technology, and the recombinant plasmid was obtained by TA clone. The results showed:the open reading frame bases were 1638bp, the highest homology with some other nir gene (JX434610.1) was 99% after contrasting by NCBI Blast.Which meant nir gene was cloned successfully.(2) The T vector and expression vector pET-32a(+) were double enzyme digested and then were combined to obtain recombinant expression vector which was then transformed into E.coli BL(DE3). After extraction of plasmid DNA of recombinant expression strain and after identification by PCR and double enzyme digestion, the cells were induced expression, and bodies were collected, then washed by pre-cooling PBS buffer, and ultrasonic fractured. SDS-PAGE electrophoresis of precipitation and supernatant was conducted. The results showed:The recombinant protein was appeared in precipitation indicated the formation of inclusion body and the molecular weight was 80kDa. Which meant the expression E.coli was successfully obtained, and the recombinant protein was successfully produced by inducing expression.(3) Because the recombinant protein formed inclusion bodies, a high concentration of urea of 8mol/L was necessary to dissolve the inclusion body. Using the characteristic that the recombinant protein had 6 his-tag, purified the protein using Ni2+affinity chromatography, wash out target protein from Ni2+ column when pH was declined to 4.3. After purification of target protein, the target protein was gradient dialyzed at 4mol/L, 2mol/L, Omol/L urea to renaturation, then protein were detected by SDS-PAGE. The refolding protein were detected the activity of the nitrite reductase enzyme at different pH and temperature. Detecting the purification results by electrophoresis:the relative concentration of target protein was increased since impurity protein was reduced significantly in the purified protein. The results showed that:when pH was fixed, the enzyme activity increased along with the rise of temperature in the range of 20-35 ℃, the highest peak value appeared at 35℃ which was 72.39U/mL, after that, the enzyme activity decreased with the rise of temperature; when the temperature was fixed, the enzyme activity increased at first and then decreased in the range of pH5.5-8.0, the highest peak value appeared at pH7.0, which is 101.99U/mL. The enzyme kinetic parameter Km was 59.17mmol/L, Vm was 166.67nmol/min·μL. To construct recombinant protein after inducing expression formed inclusion body, has a certain enzyme activity after purification.