Dissertation
Dissertation > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Poultry > Goose

Cloning and Expression as Well as Bioinformatics Analysis of efaA Gene of Enterococcus Faecalis from Goose

Author ZengZuoRan
Tutor GaoYuan
School Inner Mongolia University for Nationalities
Course Preventive Veterinary Medicine
Keywords Enterococcus faecalis Goose efaA gene Cloning efaA protein Prokaryotic expression Bioinformatics analysis
CLC S858.33
Type Master's thesis
Year 2013
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Objective:To construct the prokaryotic expression vector of efaA gene of Enterococcus faecalis from goose and expressed in E.coli BL21which is the engineering bacteria. Detected the immunogenicity and bioinformatics analysis of efaA protein.Method:To extract the whole DNA from the standard strain and the isolates as the amplification temolet;According to the base sequence of endocarditis antigen of Enterococcus faecalis published in GenBank, used Primer Premier5and Oligo6software to design a couple of specific primers of efaA;It is successfully for amplificating the conditions of PCR, agarose gel electrophoresis are isolated. Isolated and recycled the fragment;Juncted the fragment and pMD18-T cloning vector then transformed into E.coli DH5α competence cell;Screened the positive clones;Then extracted the plasmid DNA, and the recombinant plasmid identificated by single and double restriction enzyme and PCR, then sent it to the company to be sequenced. Restriction enzyme the recombinant pMD18-T-efaA plasmid which was cloned successfully, and constructed the pET-28a-efaA recombined plasmid and transformed into E.coli BL21competence cell;Screened the positive strains of pET-28a-efaA/BL21which was successful cloned and analysised the best induced expression conditions of the concentration, the time and the temperature.Induced expression under the different concentration, time and temperature conditions, confirmed the optimum induced condition;By protein electrophoresis SDS-PAGE after the comfirmation identification, purification by nickel column, obtain purified efaA protein. The successful expression of the protein were analyzed by bioinformatics:Based on the analysis of efaA amino acid composition,molecular mass,titration curve and election point, predicted the primary structure of glycosylation sites and so on, according to the comprehensive analysis of its plasticity,hydrophilicity,antigenicity index,surface accessibility, β-turns and random coil, prediction for the potential antigen epitope.Result:efaA gene of Enterococcus faecalis from goose was successfully cloned. Nucleotide sequence of it is726bp, encoding242amino acids.pET-28a-efaA/BL21 expression vector was successfully constructed, the best induction conditions were confirmed:the final concenteation of IPTG was1.Ommol/L,the temperature wass28℃,time was4h, the expressed protein size was30KD. It was consistent with the expected size. SDS-PAGE and western blot identified that efaA protein was30KD,consistent with expectations. The amino acid which is deduced by efaA gene of Enterococcus Faecalis from goose was bioinformatic analysised, it had eight potential antigen epitope. Exon13~19、26~33、66~73、92~105、141~149、152~164、190~200、210~260amino acid of the protein of Enterococcus faecalis from goose,their hydrophilicity index>0、AI≥0、surface accessibility>1, and it had β-turns and random coil,it was the potential antigen epitope in B cell of efaA protein.Conclusion:It was successful to clone of efaA Gene of Enterococcus Faecalis from Goose,to express of the efaA protein,to determine the best conditions of the expression and to predict of efaA protein of potential antigen epitope in B cell.

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