2型猪链球菌N-乙酰氨基葡糖-6-磷酸脱乙酰酶基因的克隆表达及酶活性测定
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摘要
目的:克隆表达2型猪链球菌N-乙酰氨基葡糖-6-磷酸脱乙酰酶(NagA)的编码基因,并测定其酶活性。方法:根据GenBank中05ZYH33基因组序列设计引物,PCR扩增NagA(SSU05_1259)基因,将其克隆到pET28a载体中,构建重组质粒pET28a:NagA,转化至大肠杆菌BL21,筛选阳性转化子进行IPTG诱导表达,产物通过SDS-PAGE与质谱鉴定;利用Ni亲和层析柱对表达产物进行纯化,获得NagA重组蛋白后测定其酶活性。结果:在大肠杆菌中高效表达了NagA基因,重组表达的Nag A相对分子质量约为43×10~3,其酶促反应最适温度为37℃,最佳反应时间为30min,最适反应pH值为7.5,最佳底物浓度为13mmol/L。2型猪链球菌NagA的体外酶活为124U/mL,酶比活为78U/mg。结论:在原核系统中表达了NagA基因,获得的NagA蛋白具有良好的酶学活性。
Objective:To clone and prokaryotically express the gene encoding N-acetylglucosamine-6-phosphate deacetylase(NagA) of Streptococcus suis serotype 2 and to measure the enzymatic activity of the recombinant protein.Methods:The gene encoding NagA was amplified by PCR from the template S.suis 05ZYH33 genomic sequence,and was cloned into vector pET28 a to obtain the recombinant plasmid pET-28a:NagA,then it was transformed into E.coli BL21.The recombinant NagA protein was induced to express by IPTG,and analyzed with SDSPAGE and LC-MS/MS,purified by Ni affifnity chromatography,followed by enzymatic activity measurement.Results:NagA gene(SSU051259) was expressed in E.coli with Mr about 43×10~3.The optimum temperature,action time,pH and the substrate concentration of the purified NagA protein were 37℃,30 min,7.5 and 13 mmol/L,respectively.Enzymatic activity of NagA was 124 U/mL,specific activity was 78 U/mL.Conclusion:The NagA gene has been expressed in prokaryotic system,and the purified recombinant protein has the best enzymatic activity in optimum experimental condition.
Objective:To clone and prokaryotically express the gene encoding N-acetylglucosamine-6-phosphate deacetylase(NagA) of Streptococcus suis serotype 2 and to measure the enzymatic activity of the recombinant protein.Methods:The gene encoding NagA was amplified by PCR from the template S.suis 05ZYH33 genomic sequence,and was cloned into vector pET28 a to obtain the recombinant plasmid pET-28a:NagA,then it was transformed into E.coli BL21.The recombinant NagA protein was induced to express by IPTG,and analyzed with SDSPAGE and LC-MS/MS,purified by Ni affifnity chromatography,followed by enzymatic activity measurement.Results:NagA gene(SSU051259) was expressed in E.coli with Mr about 43×10~3.The optimum temperature,action time,pH and the substrate concentration of the purified NagA protein were 37℃,30 min,7.5 and 13 mmol/L,respectively.Enzymatic activity of NagA was 124 U/mL,specific activity was 78 U/mL.Conclusion:The NagA gene has been expressed in prokaryotic system,and the purified recombinant protein has the best enzymatic activity in optimum experimental condition.
引文
[1]Lun Z R,Wang Q P,Chen X G,et al.Streptococcus suis:an emerging zoonotic pathogen[J].Lancet Infect Dis,2007,7(3):201-209.
[2]Gottschalk M,Segura M,Xu J.Streptococcus suis infections in humans:the chinese experience and the situation in north America[J].Anim Health Res Rev,2007,8(1):29-45.
[3]Tang J Q,Wang C J,Feng Y J,et al.Streptococcal toxic shock syndrome caused by Streptococcus suis serotype 2[J].PLo S Med,2006,3:e151.
[4]Sriskandan S,Slater J D.Invasive disease and toxic shock due to zoonotic Streptococcus suis:an emerging infection in the east[J].PLo S Med,2006,3(5):e187.
[5]Thiebaut R,Leproust S,Chen G,et al.Effectiveness of prenatal treatment for congenital toxoplasmosis:a metaanalysis of individual patients,data[J].Lancet,2007,369(9556):115-122.
[6]Innes E A,Vermeulen A N.Vaccination as a control strategy Naginst the coccidial parasites Eimeria,Toxoplasma and Neospora[J].Parasitology,2006,133:S145-S168.
[7]Jiang W W,Baker H J,Smith B F.Mucosal immunization with Helicobacter,Cp G DNA,choleratoxin is protective[J].Infect Immun,2003,71(1):40-46.
[8]Maeda H.Role of microbial proteases in pathogenesis[J].Microbiol Immunol,1996,40(10):685-699.
[9]Barnhart M M,Lynem J,Chapman M R.Glc NAc-6-P levels modulate the expression of Curli fibers by Escherichia coli[J].J Bacteriol,2006,188:5212-5219.
[10]Park J T.Identification of a dedicated recycling pathway for anhydro-N-acetylmuramic acid and N-acetylglucosamine derived from Escherichia coli cell wall murein[J].J Bacteriol,2001,183:3842-3847.
[11]Jaeger T,Mayer C.N-acetylmuramic acid-6-phosphate lyases:role in cell wall metabolism,distribution,structure,and mechanism[J].Cell Mol Life Sci,2007,65:928-939.
[12]Plumbridge J A.Induction of the nag regulon of Escherichia coli by N-acetyl-D-glucosamine and glucosamine:role of the cyclic AMP-catabolite activator protein complex in expression of the regulon[J].J Bacteriol,1990,172:2728-2735.
[13]Freese E B,Cole R M,Klofat W,et al.Growth,sporulation,and enzyme defects of glucosamine mutants of Bacillus subtilis[J].J Bacteriol,1970,101:1046-1062.
[14]Mobley H L T,Doyle R J,Streips U N,et al.Transport and incorporation of N-acetyl-D-glucosamine in Bacillus subtilis[J].J Bacteriol,1982,150:8-15.
[15]Souza,J M,Plumbridge J A,Calcagno M L.N-acetylglucosamine-6-phosphate deacetylase from Escherichia coli:purification and molecular and kinetic characterization[J].Arch Biochem Biophys,1997,340:338-346.
[16]Shin H J,Kim M,Lee D S.Purification and characterization of N-acetylglucosamine-6-phosphate deacetylase from Thermus caldophilus[J].J Biosci Bioeng,1999,88(3):319-322.
[17]Yadav V,Panilaitis B,Shi H,et al.N-acetylglucosamine-6-phosphate deacetylase(nag A)is required for N-acetyl glucosamine assimilation in gluconacetobacter xylinus[J].PLo S One,2011,6(6):e18099.
[18]张凤玉,胡丹,龚秀芳,等.2型猪链球菌β-半乳糖苷酶基因的克隆表达及酶活性测定[J].中国生物工程杂志,2014,34(2):39-44.
[2]Gottschalk M,Segura M,Xu J.Streptococcus suis infections in humans:the chinese experience and the situation in north America[J].Anim Health Res Rev,2007,8(1):29-45.
[3]Tang J Q,Wang C J,Feng Y J,et al.Streptococcal toxic shock syndrome caused by Streptococcus suis serotype 2[J].PLo S Med,2006,3:e151.
[4]Sriskandan S,Slater J D.Invasive disease and toxic shock due to zoonotic Streptococcus suis:an emerging infection in the east[J].PLo S Med,2006,3(5):e187.
[5]Thiebaut R,Leproust S,Chen G,et al.Effectiveness of prenatal treatment for congenital toxoplasmosis:a metaanalysis of individual patients,data[J].Lancet,2007,369(9556):115-122.
[6]Innes E A,Vermeulen A N.Vaccination as a control strategy Naginst the coccidial parasites Eimeria,Toxoplasma and Neospora[J].Parasitology,2006,133:S145-S168.
[7]Jiang W W,Baker H J,Smith B F.Mucosal immunization with Helicobacter,Cp G DNA,choleratoxin is protective[J].Infect Immun,2003,71(1):40-46.
[8]Maeda H.Role of microbial proteases in pathogenesis[J].Microbiol Immunol,1996,40(10):685-699.
[9]Barnhart M M,Lynem J,Chapman M R.Glc NAc-6-P levels modulate the expression of Curli fibers by Escherichia coli[J].J Bacteriol,2006,188:5212-5219.
[10]Park J T.Identification of a dedicated recycling pathway for anhydro-N-acetylmuramic acid and N-acetylglucosamine derived from Escherichia coli cell wall murein[J].J Bacteriol,2001,183:3842-3847.
[11]Jaeger T,Mayer C.N-acetylmuramic acid-6-phosphate lyases:role in cell wall metabolism,distribution,structure,and mechanism[J].Cell Mol Life Sci,2007,65:928-939.
[12]Plumbridge J A.Induction of the nag regulon of Escherichia coli by N-acetyl-D-glucosamine and glucosamine:role of the cyclic AMP-catabolite activator protein complex in expression of the regulon[J].J Bacteriol,1990,172:2728-2735.
[13]Freese E B,Cole R M,Klofat W,et al.Growth,sporulation,and enzyme defects of glucosamine mutants of Bacillus subtilis[J].J Bacteriol,1970,101:1046-1062.
[14]Mobley H L T,Doyle R J,Streips U N,et al.Transport and incorporation of N-acetyl-D-glucosamine in Bacillus subtilis[J].J Bacteriol,1982,150:8-15.
[15]Souza,J M,Plumbridge J A,Calcagno M L.N-acetylglucosamine-6-phosphate deacetylase from Escherichia coli:purification and molecular and kinetic characterization[J].Arch Biochem Biophys,1997,340:338-346.
[16]Shin H J,Kim M,Lee D S.Purification and characterization of N-acetylglucosamine-6-phosphate deacetylase from Thermus caldophilus[J].J Biosci Bioeng,1999,88(3):319-322.
[17]Yadav V,Panilaitis B,Shi H,et al.N-acetylglucosamine-6-phosphate deacetylase(nag A)is required for N-acetyl glucosamine assimilation in gluconacetobacter xylinus[J].PLo S One,2011,6(6):e18099.
[18]张凤玉,胡丹,龚秀芳,等.2型猪链球菌β-半乳糖苷酶基因的克隆表达及酶活性测定[J].中国生物工程杂志,2014,34(2):39-44.