Cloning Of Exotoxin A Gene From Pseudomonas Aeruginosa ATCC27853 Strain And Its Expression In Eukaryotic Cells

Objective Pseudomonas aeruginosa is a significant pathogen of animals and human beings.Pseudomonas aeruginosa infection in humans can occur at any site and organizations,causes endocarditis,gastroenteritis,empyema and septicemia.Young livestock and poultry often outbreak cyanomycosis,and often has polyinfection with E.coli or vaccination of Marek's disease vaccine for Chicken caused serious Pseudomonas aeruginosa infection,dead 70 to 80 percent in 1 to 2 days.Pseudomonas aeruginosa can produce a variety of toxin,and Pseudomonas aeruginosa exotoxin A(PEA)is a lethal exotoxin and the most important pathogenic factor.There is some research on the structure and mechanism of PEA.On the basis of this,people are reforming it to a protein having medical use with genetic engineering technology:PEA have being made to immunotoxin for the use of cell toxicity of which is cancer Drugs;PEA have being made to genetic engineering vaccine or vaccine adjuvant and vaccine vector for its good immunogenicity to prevent Pseudomonas aeruginosa infection and greatly enhance the immunogenicity;PEA have being used to gene therapy for it can carry a protein or DNA into the cells.The important clinical application of PEA has been stimulating the research on molecular structure and function of PEA.Pseudomonas strains,however,have large differences in toxicity,GeneBank has published only three full-length sequence of PEA(K01397,strain PA 103;AE004091,strain PA01;CP000438,strain UCBPP-PA14),Blast analysis showed that there are differences between their sequences of gene and amino acid.It can reveal the relationship between the structure and toxicity and immunogenicity to study differences in different strains on molecular structure and biological activity,so that the PEA is better used to immune toxins,vaccines and gene therapy.The purpose of present study is to clone the PEA full-length coding gene sequences of Pseudomonas strain ATCC27853 to provide material for comparative study of PEA and to construct the expression vector pcDNA3.1A/PEA,then transfer human embryonic kidney cells- HEK293T cells,the PEA gene with original signal peptide have expressed with secretary form in eucaryotic cells. This study will help us further study the relationship between the structure and cell toxicity and immunogenicity of PEA,so as to lay the foundation for the application on the immunotoxins,vaccines and gene therapy of the toxicity and immunogenicity of PEA.Methods This study designed a pairof specific primers on the basis of Pseudomonas strain PAl03 sequence published on the GeneBank,extracted genomic DNA from Pseudomonas aeruginosa ATCC27853 strains and amplified Pseudomonas aeruginosa exotoxin A(PEA)full-length coding genes by PCR.Construct the cloning vector pMD18-T/PEA using pMD18-T vector and sequence it after blue white spot screening, identificated by restriction enzyme Hindâ…¢and Xbaâ… and Kpnâ… .Compare the sequencing results with the sequence of PA103 strain.The PEA subcloned into the eukaryotic expression vector pcDNA3.1A after be sure that the cloned gene is the PEA full-length coding genes of Pseudomonas aeruginosa ATCC27853 strain then identificated it with Hindâ…¢and Xbaâ… and Apaâ… .In order to identificate the original signal peptide(prokaryote signal peptide)of the PEA whether express in eukaryotic cells,this study used eukaryotic expression vector pcDNA3.1A/PEA transfected human embryonic kidney cells-HEK293T cells,and pcDNA3.1A empty vector transfection HEK293T cells as a negative control.Through repeated tests established the reproducible calcium phosphate transfer methods for pcDNA3.1A/PEA transfer HEK 293T cells.Detected PEA expression with Western blot.Results Amplified PEA gene using genomic DNA template by PCR,then identified it whith the 1%agarose gel electrophoresis,get a specific fragment consistenting with expectations,namely 1914 bp.PCR product connected to the pMD18-T vector,and sent recombinant pMD18-T/PEA sequencing after identification by enzyme digestion.Be sure that the cloned gene is the PEA full-length coding genes of Pseudomonas aeruginosa ATCC27853 strain after comparing the sequencing results and the sequence of PA103 strain then subcloned PEA into the eukaryotic expression vector pcDNA3.1A.Identificate recombinant by enzyme digestion and the result of electrophoresis consistent with expectations.The results prove that pcDNA3.1A/PEA eukaryotic expression vector construct successfully.Transfered eukaryotic cells HEK 293T cells with recombinant pcDNA3.1A/PEA.Western blot results showed that there is an obvious hybrid zone about 130 kDa molecular weigh department,and empty vector had no hybrid zone in the corresponding position.Conclusion The PEA full-length coding gene was amplified from Pseudomonas strain ATCC27853 genomic DNA;sequence analysis found that the sequence of gene and amino acid for Pseudomonas strain ATCC27853 strain is different with PA103,and homology all reach 99 percent;Successfully constructed eukaryotic gene expression vector pcDNA3.1A/PEA,and transfered eukaryotic cells with it,achieved its expression with secretary form in eucaryotic cells,in order to further study the relationship between PEA structure and cell toxicity and immunogenicity,lay the foundation for the application of cytotoxicity and immunogenicity of PEA in the immunotoxin,vaccines and gene therapy.