绿脓杆菌ATCC27853外毒素PE40基因的扩增与测序

Amplification and Sequencing of Exotoxin A Structural gene PE40 of Pseudomonas Aeruginosa ATCC27853

目的扩增绿脓杆菌ATCC27853外毒素基因PE40,并对PCR产物进行测序鉴定,为获得用于构建分子导向药物的毒素弹头基因PE40奠定基础。方法采用PCR技术,以绿脓杆菌ATCC27853基因组DNA为模板,扩增PE40基因并测序。用DNASTAR软件将测得的序列与GenBank中的国际标准产毒株PA103的PE40基因序列进行比较。结果扩增出目的基因片断,长度为1231bp。测序表明,ATCC27853与PA103核苷酸同源性为98%,产生了7个碱基的突变,突变碱基导致第364位的天冬酰胺变成丝氨酸,第506位的丝氨酸变成色氨酸,第515位的丝氨酸变成甘氨酸。但产生变化的3个氨基酸均不是文献报道中的重要活性位点。结论产生变化的3个氨基酸不会对PEA的酶活性及细胞毒性产生很大影响,绿脓杆菌ATCC27853的PE40基因可用作分子导向药物的弹头。

Objective： To analyse nucleotide sequence of exotoxin A structural gene PE40 of Pseudomonas aeruginosa ATCC27853. Method： The gene PE40 was amplified from genome DNA of Pseudomonas aeruginosa ATCC2785 by PCR technique. The PCR product was purified, sequenced and compared with a standard strain PA103 from GenBank by DNASTAR software. Result： An objective gene fragment with the length 1231bp was obtained. The nucleotide sequence analysis showed that ATCC27853 shared 98% alignment with PAl03. The mutations in amino acid sequence are： Asn-364 to Ser; Ser-506 to Tip and Ser-515 to Gly. Conclusion： The mutations have a little influence on the enzymatic activity and cytotoxicity, so the gene PF40 of Pseudomonas aeruginosa ATCC27853 will be usefull in the research of molecular targeted protein.