绿脓杆菌ATCC9027外毒素PE40基因克隆与系统发育分析
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摘要
目的为获得用于构建免疫毒素弹头的毒素蛋白基因,克隆绿脓杆菌外毒素PE40基因。方法 PCR扩增ATCC9027外毒素PE40基因,纯化PCR产物与T载体连接后转染感受态大肠杆菌JM109,蓝白斑筛选阳性克隆,HindⅢ酶切和测序鉴定阳性克隆。将ATCC9027的PE40基因进行blast搜索,以Genbank中所有的绿脓杆菌外毒素PE40基因为内群,以白喉毒素基因为外群进行系统发育分析。结果 PCR产物为1 098 bp。与PA103相比,ATCC9027外毒素PE40基因有14个碱基的突变,导致外毒素蛋白8个氨基酸的突变。系统发育树显示绿脓杆菌外毒素PE40基因形成单系群,ATCC9027和ATCC927853的亲缘关系最近。结论 ATCC9027外毒素关键位点的氨基酸未发生突变,获得了用于构建免疫毒素弹头的毒素蛋白基因PE40。
Objective To obtain the toxin gene PE40,which acts as the warhead of"biological missites".Methods Primers was designed according to the exotoxin gene of pseudomonas aeruginosa strain 103,and a HindIII site was introduced for development of expressing vector.PE40 gene was amplified from genome DNA of pseudomonas aeruginosa ATCC9027 by PCR,then the purified PCR product was cloned into the pMD18-T simple vector.The recombinant was identified by simple digestion(HindIII) and sequencing.Results The PCR product was 1 098 bp.Compared with Pseudomonas aeruginosa 103,PE40 gene of ATCC9027 produced 14 bp mutations.The mutations of 14 bp resulted in the mutations of eight amino acids,but the eight amino acids were not critical situs,and the mutations didn't affect enzymatic activity and cytotoxicity of the exotoxin.The phylogenetic tree indicated that PE40 gene matrix of pseudomonas aeruginosa exotoxin formed a monophyletic group.Pseudomonas aeruginosa ATCC9027 and ATCC27853 formed a branch,and had closest relationship.Conclusions PE40 gene to construct immunotoxin was obstained.
Objective To obtain the toxin gene PE40,which acts as the warhead of"biological missites".Methods Primers was designed according to the exotoxin gene of pseudomonas aeruginosa strain 103,and a HindIII site was introduced for development of expressing vector.PE40 gene was amplified from genome DNA of pseudomonas aeruginosa ATCC9027 by PCR,then the purified PCR product was cloned into the pMD18-T simple vector.The recombinant was identified by simple digestion(HindIII) and sequencing.Results The PCR product was 1 098 bp.Compared with Pseudomonas aeruginosa 103,PE40 gene of ATCC9027 produced 14 bp mutations.The mutations of 14 bp resulted in the mutations of eight amino acids,but the eight amino acids were not critical situs,and the mutations didn't affect enzymatic activity and cytotoxicity of the exotoxin.The phylogenetic tree indicated that PE40 gene matrix of pseudomonas aeruginosa exotoxin formed a monophyletic group.Pseudomonas aeruginosa ATCC9027 and ATCC27853 formed a branch,and had closest relationship.Conclusions PE40 gene to construct immunotoxin was obstained.
引文
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2 Kreitman RJ,Wilson WH,Bergeron K,et al.Efficacy of the anti-CD22 re-combinant immunotoxin BL22 in chemotherapy resistant hairy-cell leuke-mia[J].N Engl J Med,2002;345(4):241-7.
3 Hwang J,Fitzgerald DJ,Adhya S,et al.Functional domains of Pseudo-monas exotoxin identified by deletion analysis of the gene expressed in E.col[iJ].Cell,1987;48(1):129-36.
4 Posey JA,Khazaeli MB,Bookman MA,et al.A phaseⅠtrial of the sin-gle-chain immunotoxin SGN-10(BR96 sFv-PE40)in patients with ad-vanced solid tumors[J].Clin Cancer Res,2002;8(10):3092-9.
5 贾怡,李虹,陈文捷,等.新型重组免疫毒素DT390-mRantes治疗小鼠EAE的初步研究[J].细胞与分子免疫学杂志,2007;23(3):236-9.
6 IL-10(18-57)-PE40高效表达、纯化及细胞活性之研究[J].生物工程学报,2006;22(1):87-93.
7 Gray GL,Smith DH,Baldridge JS,et al.Cloning,nucleotide sequence,and expression in Escherichia coli of the exotoxin A structural gene ofPseudomonas aeruginosa[J].Proc Natl Acad Sci USA,1984;81(9):2645-9.
8 Iglewski BH,Kabat D.NAD-dependent inhibition of protein synthesis byPseudomonas aeruginosa toxin[J].Proc Natl Acad Sci USA,1975;72(6):2284-8.
9 Foss FM,Bacha P,Osann KE,et al.Biological correlates of acute hyper-sensitivity events with DAB(389)IL-2(denileukin diftitox,ONTAK)incutaneous T-cell lymphoma:decreased frequency and severity with steroidpremedication[J].Clin Lymphoma,2001;1(4):298-302.