Biological Characteristics Of Escherichia Coli S9922 And Prokaryotic Expression Of Specific Genes

Objective:In this study,the biological characteristics and resistance test to different environmental disinfectant of E.coli S9922 isolated from clinical calves encephalitis were studied.The genes of specific adhesion protein fli C3 and fim D of E.coli S9922 were cloned and recombinant plasmids p ET-32a-fli C3 and p ET-32a-fim D were constructed.The recombinant plasmids were transformed into E.coli BL21(DE3)for expression and purification,and then detected by WB.On the one hand,the results will provide more comprehensive information for the research of the bacterium and technical reference for clinical drug use and technical support for biological prevention and control.Meanwhile,it will provide experimental materials for the diagnosis and vaccine development of the disease.Methods:(1)Biochemical identification,phylogenetic grouping,drug sensitivity test,serotype identification,hemolytic assay and median lethal dose(LD₅₀)of E.coli S9922 were carried out.(2)Four kinds of environmental disinfectants commonly used in cattle farms were used to kill E.coli S9922 with different action time(5 min,10 min,20 min)and different action concentration(0.1%,0.5%,1%).The killing rate and the resistance of E.coli S9922 to common environmental disinfectants were determined.(3)The primers of fli C3 and fim D genes were designed by Primer5.0 software.The target genes were amplified by PCR.The recombinant plasmids p ET-32a-fli C3 and p ET-32a-fim D were constructed and transformed into E.coli BL21(DE3).IPTG was added to induce the expression.The expressed proteins were purified by Ni column,refolded with different concentrations of urea,and quantified after sucrose concentration.(4)The anti-his primary antibody and Goat anti rabbit Ig G secondary antibody were used to detect expressed protein by Western blot.Results:(1)E.coli S9922 V-P test was negative,and indoles test was positive.It could ferment mannitol,xylose,glucose,rhamnose and maltose,and all of them could produce acid but not gas.According to the three genes chu A,yja A and TSPE4.C2,the phylogenetic typing of E.coli S9922 was verified to belong to group A by PCR amplification.The serotype of E.coli S9922 was not detected in 27common serotypes O1,O2,O3,O8,O9,O11,O15,O29,O38,O44,051,053,O78,O80,O88,O93,O101,O104,O109,O114,O119,O126,O137,O138,O157,O158 and O161;No hemolytic circle was found on the blood plates of 5%rabbit and 5%sheep.E.coli S9922 was resistant to chloramphenicol,tetracycline,gentamicin,ciprofloxacin,ampicillin,kanamycin,levofloxacin,cefotaxime and penicillin,but not to polymyxin B and macromycin,and has multiple drug resistance characteristics;The calculated result of LD50 was 10^7.238 mg/kg.The results showed that E.coli S9922 was a strong virulent strain.(2)Under the action of 1%concentration of disinfectant,dilute glutaraldehyde solution,benzalkonium bromide solution,povidone iodine solution and sodium dichloroisocyanurate solution could kill the bacterium,but at 0.1%and 0.5%concentration,dilute glutaraldehyde solution and benzalkonium bromide solution had better disinfection effect than povidone iodine solution and sodium dichloroisocyanurate.(3)The length of fli C3 and fim D gene fragment were 942 bp and 1149 bp respectively.The constructed recombinant plasmid was identified by double enzyme digestion and induced by IPTG and SDS-PAGE.The size of fli C3 and fim D was 51.54 k Da and 59.13 k Da,respectively,which were consistent with the expected results.Both proteins were expressed in inclusion bodies.After purification,the concentration of fli C3 and fim D were 1.6 mg/ml and 1.9 mg/ml respectively.(4)Western blot results showed that the recombinant proteins fli C3 and fim D could be expressed,and their sizes were consistent with the expected results.Conclusion:Calf encephalitis Escherichia coli S9922 was an extraintestinal pathogenic E.coli,which belongs to group A in the systematic grouping.LD₅₀results showed that S9922 was a virulent strain.It was a pathogenic bacterium with complex antigen structure and resistant to multiple antibiotics and was multi drug resistant.It was sensitive to macromycin and polymyxin B,which can provide technical support for clinical medication.The environmental disinfectant purchased on the market can kill the bacteria in high concentration,but the disinfection effect of dilute glutaraldehyde solution and benzalkonium bromide solution is better in low concentration.The recombinant proteins fli C3 and fim D could be correctly expressed in the form of inclusion bodies in the precipitate.The results will provide comprehensive data for the study of bovine encephalitis Escherichia coli and technical services for the clinical drug use of this disease and the biological prevention and control of farm,as well as experimental materials for establishment of diagnostic methods and vaccine development of this disease.