【作者】 张彦新；

【导师】 蒋云生；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 目的将肌酐水解酶基因克隆入乳酸菌NICE表达系统及乳酸菌pMG36e表达系统,构建能高效表达肌酐水解酶的乳酸工程菌,使之能产生肌酐水解酶,并测定比较其产酶活性。方法从斯氏假单胞菌A1501中提取基因组DNA,根据genbank上已知的固氮斯氏假单胞菌肌酐水解酶基因序列,设计引物PCR扩增肌酐水解酶基因片段,经测序鉴定后将其分别克隆入质粒pMG36e, PNZ8048构建成重组质粒pMG36e-Cr、PNZ8048-Cr,并分别转化大肠杆菌DH5α及MC1061,筛选鉴定,提取重组质粒PMG36e-Cr、PNZ8048-Cr,电穿孔转化乳酸菌L.lactis NZ9000,筛选鉴定。制备基因工程菌粗酶液,进行SDS-PAGE分析,并测定粗酶液肌酐水解酶的活性。结果1、以斯氏假单胞菌A1501基因组为模板PCR扩增的基因片段琼脂糖电泳大小约0.76kb,与Genbank肌酐水解酶基因片段大小符合。2、构建的含重组质粒pMD18-T-Crl, pMD18-T-Cr2的大肠杆菌DH5α进行菌液PCR扩出0.76Kb大小片段与目的片段大小符合。提取重组质粒pMD18-T-Crl以SpeⅠ和HindⅢ双酶切,pMD18-T-Cr2用SalⅠ和HindⅢ双酶切,两酶切产物电泳均示酶切片段大小为2.7Kb、0.76Kb与预期符合。基因测序结果比对示克隆片段序列与Genbank(序列号CP000304.1)肌酐水解酶基因序列完全一致。3、成功构建含pMG36e-Cr、PNZ8048-Cr的重组乳酸菌,提取重组质粒pMG36e-Cr、PNZ8048-Cr以质粒为模板PCR扩出0.76Kb大小片段与目的片段大小符合。PMG36e-Cr以SalⅠ和HindⅢ双酶切得片段大小为3.6Kb、0.76Kb与预期符合,PNZ8048-Cr以SpeⅠ和HindⅢ双酶切得片段大小为3.3Kb、0.76Kb与预期符合。4、粗酶液经不连续SDS-PAGE分析,重组乳酸菌表达重组蛋白的分子量约为27KD,与由肌酐水解酶253个氨基酸计算的理论分子量相符。经粗酶液活性测定,斯氏假单胞菌A1501粗酶液活性为0.32u/ml, L.lactis-pMG36e-Cr的基因重组乳酸菌酶活性0.24u／ml；L.lactis-PNZ8048-Cr的基因重组乳酸菌的酶活性为1.24u／ml,较斯氏假单胞菌A1501及L.lactis-pMG36e-Cr明显升高。结论利用乳酸菌NICE系统成功构建了高效表达肌酐水解酶的乳酸基因工程菌L.lactis-PNZ8048-Cr,比较原始菌及L.lactis-pMG36e-Cr表达肌酐水解酶活性明显提高,此工程菌的成功构建可以成为慢性肾衰肠道细菌疗法的重大进展,为进一步构建可以高效分解多种尿毒素的基因工程菌提供了可能。目的将尿酸氧化酶基因克隆入乳酸菌NICE表达系统及乳酸菌pMG36e表达系统,使之能产生尿酸氧化酶,并测定比较其产酶活性。方法从产朊假丝酵母菌中提取基因组DNA,根据genbank上已知的产朊假丝酵母菌尿酸氧化酶基因序列,设计引物PCR扩增尿酸氧化酶基因片段,经测序鉴定后将其分别克隆入质粒pMG36e,PNZ8048构建成重组质粒pMG36e-U、PNZ8048-U,并分别转化大肠杆菌DH5α及MC1061,筛选鉴定,提取重组质粒PMG36e-U、PNZ8048-U,电穿孔转化乳酸菌L.lactis NZ9000,筛选鉴定。制备基因工程菌粗酶液,进行SDS-PAGE分析,并测定粗酶液尿酸氧化酶的活性。结果1、以产朊假丝酵母菌基因组为模板PCR扩增的基因片段琼脂糖电泳大小约0.9kb,与Genbank尿酸氧化酶基因片段大小符合。2、构建的含重组质粒pMD18-T-U1, pMD18-T-U2的大肠杆菌DH5α进行菌液PCR扩出0.9Kb大小片段与目的片段大小符合。提取重组质粒pMD18-T-U1以NcoⅠ和SpeⅠ双酶切,PMD18-T-Cr2用SalⅠ和HindⅢ双酶切,两酶切产物电泳均示酶切片段大小为2.7Kb、0.9Kb与预期符合。重组菌经测序后序列比对示克隆片段序列与Genbank(序列号E12709)尿酸氧化酶基因序列完全一致。3、成功构建含pMG36e-U、PNZ8048-U的重组乳酸菌,提取重组质粒pMG36e-U、PNZ8048-U以质粒为模板PCR扩出0.9Kb大小片段与目的片段大小符合。PMG36e-U以SalⅠ和HindⅢ双酶切得片段大小为3.6Kb、0.9Kb与预期符合,PNZ8048-U以SpeⅠ和HindⅢ双酶切得片段大小为3.3Kb、0.9Kb与预期符合。4、粗酶液经不连续SDS-PAGE分析,重组乳酸菌表达重组蛋白的分子量约为34KD,与由尿酸氧化酶303个氨基酸计算的理论分子量相符。重组乳酸菌L.lactis-PNZ8048-U重组蛋白表达量明显高于L.lactis-pMG36e-U的基因重组乳酸菌。经粗酶液活性测定,产朊假丝酵母菌粗酶液活性为0.55u/ml, L.lactis-pMG36e-U的基因重组乳酸菌酶活性为0.29u/ml; L.lactis-PNZ8048-U的基因重组乳酸菌酶活性为1.92u／ml,较原始菌株及L.lactis-pMG36e-U明显升高。结论利用乳酸菌NICE系统成功构建了高效表达尿酸氧化酶的乳酸基因工程菌L.lactis-PNZ8048-U,比较产朊假丝酵母菌及L.lactis-pMG36e-U酶活性明显提高,此工程菌的成功构建可以成为慢性肾衰肠道细菌疗法的重大进展,为进一步构建可以高效分解多种尿毒素的基因工程菌提供了可能。更多还原

【Abstract】 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.更多还原