铜绿假单胞菌重组质粒pGEX-OprF-I构建、鉴定及其在大肠埃希菌中的表达效率
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摘要
目的构建并鉴定铜绿假单胞菌重组质粒pGEX-OprF-I,观察其在大肠埃希菌中的表达效率。方法以铜绿假单胞菌PA01株的DNA为模板,PCR扩增OprF和OprI抗原编码基因,采用基因拼接法(gene SOEing)剪切OprF和OprI得到OprF-I融合基因,酶切后与表达载体pGEX-1λT连接,构建重组质粒pGEX-OprF-I并转化入大肠埃希菌中,抽提质粒进行双酶切和PCR鉴定。重组菌用异丙硫代-β-D-半乳糖苷(IPTG)诱导表达,采用SDS-PAGE和western blot分析和鉴定表达产物。结果基因拼接法获得1289 bp的OprF-I融合基因;其连接产物经双酶切和PCR鉴定证实该基因成功插入pGEX-1λT中。重组质粒转化DE3后经IPTG诱导,SDS-PAGE检测显示重组菌表达预期约68×10~3的融合蛋白,该蛋白约占菌体总量的18%。Western blot检测Pa感染的鼠血清可特异性识别该融合蛋白。结论成功构建并鉴定了铜绿假单胞菌重组质粒pGEX-OprF-I,转化大肠埃希菌后高效表达OprF-I融合蛋白,该蛋白具有反应原性。
Objectives To construct and identify the recombinant plasmid pGEX-OprF-I derived from Pseudomonas aeruginosa and to determine its expression efficiency in Escherichia coli. Methods DNA was extracted from Pseudomonas aeruginosa PAO1. Genes coding for the OprF and OprI antigens were amplified with PCR using DNA as a template. The OprF-I fusion gene obtained by gene SOEing was digested and ligated to the vector pGEX-1λT. The recombinant plasmid pGEX-OprF-I was constructed and transformed into Escherichia coli. The plasmid was extracted and identified using enzyme digestion and PCR. Expression of the recombinant strain was induced with isopropylthio-β-D-galactoside(IPTG), and the expressed product was analyzed and identified using SDS-PAGE and Western blotting. Results The 1289 bp OprF-I fusion gene was obtained by gene SOEing, the ligated product was successfully cloned into the vector pGEX-1λT according to restriction analysis and identified with PCR, and the recombinant plasmid pGEX-OprF-I was successfully constructed. The recombinant plasmid was transformed into DE3 and its expression was induced with IPTG. SDS-PAGE indicated that the expected 68×10~3 fusion protein was expressed by recombinant bacteria and accounted for about 18% of the total bacterial proteins. The fusion protein was specifically recognized by sera from mice infected with P. aeruginosa according to Western blotting. Conclusion The recombinant plasmid pGEX-OprF-I derived from P. aeruginosa was successfully constructed and identified, and the OprF-I fusion protein was highly expressed after transformation of E. coli. The protein had specific antigenicity.
Objectives To construct and identify the recombinant plasmid pGEX-OprF-I derived from Pseudomonas aeruginosa and to determine its expression efficiency in Escherichia coli. Methods DNA was extracted from Pseudomonas aeruginosa PAO1. Genes coding for the OprF and OprI antigens were amplified with PCR using DNA as a template. The OprF-I fusion gene obtained by gene SOEing was digested and ligated to the vector pGEX-1λT. The recombinant plasmid pGEX-OprF-I was constructed and transformed into Escherichia coli. The plasmid was extracted and identified using enzyme digestion and PCR. Expression of the recombinant strain was induced with isopropylthio-β-D-galactoside(IPTG), and the expressed product was analyzed and identified using SDS-PAGE and Western blotting. Results The 1289 bp OprF-I fusion gene was obtained by gene SOEing, the ligated product was successfully cloned into the vector pGEX-1λT according to restriction analysis and identified with PCR, and the recombinant plasmid pGEX-OprF-I was successfully constructed. The recombinant plasmid was transformed into DE3 and its expression was induced with IPTG. SDS-PAGE indicated that the expected 68×10~3 fusion protein was expressed by recombinant bacteria and accounted for about 18% of the total bacterial proteins. The fusion protein was specifically recognized by sera from mice infected with P. aeruginosa according to Western blotting. Conclusion The recombinant plasmid pGEX-OprF-I derived from P. aeruginosa was successfully constructed and identified, and the OprF-I fusion protein was highly expressed after transformation of E. coli. The protein had specific antigenicity.
引文
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[14] Horton RM,Cai ZL,Ho SN,et al.Gene splicing by overlap extension:tailor-made genes using the polymerase chain reaction[J].Biotechniques,1990,8(5):528-35.
[15] Rosano GL,Ceccarelli EA.Recombinant protein expression in Escherichia coli:advances and challenges[J].Front Microbiol,2014(5):172.
[16] Harper S,Speicher DW.Purification of proteins fused to glutathione S-transferase[J].Methods Mol Biol,2011(681):259-80.
[2] Maurice NM,Bedi B,Sadikot RT.Pseudomonas aeruginosa biofilms:host response and clinical implications in lung infections[J].Am J Res Cell Mol Biol,2018,58(4):428-39.
[3] Kizny Gordon AE,Mathers AJ,Eyl C,et al.The hospital water environment as a reservoir for carbapenem-resistant organisms causing hospital-acquired infections-a systematic review of the literature[J].Clin Infect Dis,2017,64(10):1435-44.
[4] Angeletti S,Cella E,Prosperi M,et al.Multi-drug resistant Pseudomonas aeruginosa nosocomial strains:Molecular epidemiology and evolution[J].Microb Pathol,2018,123:233-41.
[5] Tacconelli E,Carrara E,Savoldi A,et al.Discovery,research,and development of new antibiotics:the WHO priority list of antibiotic-resistant bacteria and tuberculosis[J].Lancet Infect Dis,2017,18(3):318-27.
[6] Gilleland HE,Gilleland LB,Fowler MR.Vaccine efficacies of elastase,exotoxin A,and outer-membrane protein F in preventing chronic pulmonary infection by Pseudomonas aeruginosa in a rat model[J].J Med Microbiol,1993,38(2):79-86.
[7] Finke M,Duchêne M,Eckhardt A,et al.Protection against experimental Pseudomonas aeruginosa infection by recombinant P.aeruginosa lipoprotein I expressed in Escherichia coli[J].Infect Immun,1990,58(7):2241-4.
[8] 刘潇,李文桂.铜绿假单胞菌重组Bb-OprI疫苗诱导小鼠保护力和脾细胞因子基因表达变化的研究[J].中国病原生物学杂志,2018,13(3):226-9.
[9] Price BM,Galloway DR,Baker NR,et al.Protection against Pseudomonas aeruginosa chronic lung infection in mice by genetic immunization against outer membrane protein(OprF) of P.aeruginosa[J].Infect Immun,2001(69):3510-5.
[10] Peluso L,Luca CD,Bozza S,et al.Protection against Pseudomonas aeruginosa lung infection in mice by recombinant OprF-pulsed dendritic cell immunization[J].BMC Microbiol,2010,10:9.
[11] Fruh R,Blum B,Mossmann H,et al.TH1 cells trigger tumor necrosis factor alpha-mediated hypersensitivity to Pseudomonas aeruginosa after adoptive transfer into SCID mice[J].Infect Immun,1995,63(3):1107-12.
[12] 陈春琳,刘祥,王成祥,等.铜绿假单胞菌外膜蛋白Ⅰ的原核表达、纯化及免疫保护作用研究[J].生物技术通报,2015,31(7):207-13.
[13] Gellatly SL,Hancock R EW.Pseudomonas aeruginosa:new insights into pathogenesis and host defenses[J].Fems Immunol Med Microbiol,2013,67(3):159-73.
[14] Horton RM,Cai ZL,Ho SN,et al.Gene splicing by overlap extension:tailor-made genes using the polymerase chain reaction[J].Biotechniques,1990,8(5):528-35.
[15] Rosano GL,Ceccarelli EA.Recombinant protein expression in Escherichia coli:advances and challenges[J].Front Microbiol,2014(5):172.
[16] Harper S,Speicher DW.Purification of proteins fused to glutathione S-transferase[J].Methods Mol Biol,2011(681):259-80.