Construction Of Gene Engineering L.lactis That Can Express Urotoxin Enzyme In High Level And Examination Of The Enzyme Activity

Objective Clone creatininase gene to NICE system of lactobacillus and PMG36e expression system. So that they could express creatininase. Testing and comparing its production activity.Methods Firstly, the genome DNA of Pseudomonas stutzeri A1501 was isolated. Then according to the creatininase gene sequence of Nitrogen-fixing Pseudomonas Steinmann in genbank, two pairs of primers were designed and the creatininase gene was cloned by PCR amplification with primers. After identification by sequencing, the creatininase gene fragment was digested with restriction endonucleases,and linked with the vector plasmid pMG36e and PNZ8048 digested with the same enzyme digestion to construct the recombinant expression vector pMG36e-Cr and PNZ8048-Cr, the recombinant plasmid pMG36e-Cr and PNZ8048-Cr was transformed into E.coli DH5αand MC1061 respectively. Then the recombinant Escherichia coli was screened and identified. Extract recombinant plasmids pMG36e-Cr and PNZ8048-Cr from DH5α,and transform the recombinant plasmids into Lactococcus lactis NZ9000 by electroportion respectively. Screen and identify the recombinant Lactococcus lactis NZ9000. The crude enzyme solution sample of the recombinant Lactococcus lactis NZ9000 was prepared and identified with SDS-PAGE analysis. Examine the creatininase activity of the crude enzyme solution.Results1. The gene fragment got from PCR amplification of Pseudomonas stutzeri A1501 genome, was 0.76kb, which was identical with the creatininase gene in Genebank.2. The gene fragment got from PCR amplification of the recombinant Escherichia coli (DH5 a) was 0.76Kb which was in accordance with the target fragment. The plasmid pMD18-T-Cr1 was digested with SpeⅠand HindⅢ, plasmid pMD18-T-Cr2 was digested with SalⅠand HindⅢ. Digestion products electrophoresis showed the fragment were 2.7Kb and 0.76Kb in line with expectations. The gene sequencing result was identical with creatininase gene in genebank (CP000304.1)3. The recombinant L.lactis-pMG36e-Cr and L.lactis-PNZ8048-Cr were constructed successfully. The fragment got from PCR amplification of pMG36e-Cr and PNZ8048-Cr was in accordance with target fragment. Plasmid PMG36e-Cr was digested with SalⅠand HindⅢ. Digestion products electrophoresis showed the fragment were 3.6Kb and 0.76Kb in line with expectations. Plasmid PNZ8048-Cr was digested with SpeⅠand HindⅢ. Digestion products electrophoresis showed the fragment were 3.3Kb and 0.76Kb in line with expectations.4. Discontinuous SDS-PAGE analysis of Crude enzyme solution showed:recombinant protein molecular weight was about 27KD, and in accordance with the theoretical molecular weight calculated from the 253 amino acid of creatininase. The crude enzyme activity assay showed, enzyme activity of Pseudomonas stutzeri A1501 was 0.32, L.lactis-pMG36e-Cr of recombinant lactic acid bacteria was 0.24u/ml; enzyme activity of L.lactis-PNZ8048-Cr was 1.24u/ml, significantly increased than the original strain and L.lactis-pMG36e-Cr.Conclusion Gene engineering L.lactis-PNZ8048-Cr that can express creatininase in high level was constructed successfully. Creatininase activity of L.lactis-PNZ8048-Cr was significantly increased than the original strain and L.lactis-pMG36e-Cr. The successful construction of this gene engineering bacteria can become significant progress in intestinal bacteria therapy of chronic renal failure, provide a basis for construction of gene engineering bacteria with multiple genes clone. Objective Clone uricase gene to NICE system of lactobacillus and PMG36e expression system, So that they could express uricase. Testing and comparing its production activity.Methods Firstly, the genome DNA of Candida utilis was isolated. Then according to the uricase gene sequence of Candida utilis in genbank, two pairs of primers were designed and the uricase gene was cloned by PCR amplification with primers. After identification by sequencing, the uricase gene fragment was digested with restriction endonucleases,and linked with the vector plasmid pMG36e and PNZ8048 digested with the same enzyme digestion to construct the recombinant expression vector pMG36e-U and PNZ8048-U, the recombinant plasmid pMG36e-U and PNZ8048-U was transformed into E. coli DH5 a and MC1061 respectively. Then the recombinant Escherichia coli was screened and identified. Extract recombinant plasmids pMG36e-U and PNZ8048-U from DH5α,and transform the recombinant plasmids into Lactococcus lactis NZ9000 by electroportion respectively. Screen and identify the recombinant Lactococcus lactis NZ9000. The crude enzyme solution sample of the recombinant Lactococcus lactis NZ9000 was prepared and identified with SDS-PAGE analysis. Examine the uricase activity of the crude enzyme solution. Results1. The gene fragment got from PCR amplification of Candida utilis genome was 0.9kb, which was identical with the uricase gene in Genebank.2. The gene fragment got from PCR amplification of the recombinant Escherichia coli (DH5 a) was 0.9Kb which was in accordance with the target fragment. The plasmid pMD18-T-U1 was digested with SpeⅠand NcoⅠ, plasmid pMD18-T-U2 was digested with SalⅠand HindⅢ. Digestion products electrophoresis showed the fragment were 2.7Kb and 0.9Kb in line with expectations. The gene sequencing result was identical with uricase gene in genebank (E12709)3. The recombinant L.lactis-pMG36e-U and L.lactis-PNZ8048-U were constructed successfully. The fragment got from PCR amplification of pMG36e-U and PNZ8048-U was in accordance with target fragment. Plasmid PMG36e-U was digested with SalⅠand HindⅢ. Digestion products electrophoresis showed the fragment were 3.6Kb and 0.76Kb in line with expectations. Plasmid PNZ8048-U was digested with SpeⅠand NcoⅠ,. Digestion products electrophoresis showed the fragment were 3.3Kb and 0.76Kb in line with expectations.4. Discontinuous SDS-PAGE analysis of Crude enzyme solution showed:recombinant protein molecular weight was about 34KD, and in accordance with the theoretical molecular weight calculated from the 303 amino acid of uricase. The crude enzyme activity assay showed, enzyme activity of Candida utilis was 0.55 u/ml, L.lactis-pMG36e-U of recombinant lactic acid bacteria was 0.29 u/ml; enzyme activity of L.lactis-PNZ8048-U was 1.92 u/ml, significantly increased than the original strain and L.lactis-pMG36e-U.Conclusion Gene engineering L.lactis-PNZ8048-U that can express uricase in high level was constructed successfully. Uricase activity of L.lactis-PNZ8048-U was significantly increased than the original strain and L.lactis-pMG36e-U. The successful construction of this gene engineering bacteria can become significant progress in intestinal bacteria therapy of chronic renal failure, provide a basis for construction of gene engineering bacteria with multiple genes clone.