| As the result of the drug abuse for many years,the number of tetracycline-resistant strains of R.anatipestifer has gradually increased.However,the current studies on tetracyclines resistance of R.anatipestifer mostly focus on the susceptibility test or epidemiological analysis.Only few studies on drug resistance genes and drug resistance mechanisms have been carried out,and the resistance mechanism of R.anatipestifer for tetracycline is rare.Assigning protein functions by comparative genome analysis and other methods,we found that the genome of R.anatipestifer serotype 1 strain CH3 may exist a functioning unit containing a tetracycline repressor protein Tet R(M949_RS06320),a type I secretion system Tol C family protein(M949_RS06325)and a efflux protein(M949_RS06330).Therefore,it is concluded that these three genes constitute a functional region related to tetracycline resistance.In this study,gene analysis and regulatory study of M949_RS06330 performed using bioinformatics software.Prokaryotic expression of M949_RS06330 and the monoclonal antibody of anti-M949_RS06330 were done using E.coli prokaryotic expression system and monoclonal antibody preparation techniques.To confirm the resistance function,expression of M949_RS06320,M949_RS06325 and M949_RS06330 gene under treatment with different doses of tigecycline or tetracycline was measured with quantitative real-time PCR.1.Bioinformatics analysis of M949_RS06330The published sequences of the R.anatipestifer genome in the Gen Bank database were searched for multiple sequences alignment and phylogenetic tree analysis of the M949_RS06330 homologous protein.The results showed that M949_RS06330 is closely related to homologous proteins in Flavobacteriaceae bacterium,Chryseobacterium,and homologous to Sphingobacterium mizutaii,Sphingobacterium lactis.Currently,31 complete R.anatipestifer genomes have been submitted to NCBI,BLAST analysis indicated that the M949_RS06330 homologous sequence was just observed in RA-CH-2,RA Yb2,RA-GD,RA DSM 15868,RA-YM,RA-CH-1,and CH3.Furthermore,the distribution of the homologous sequence of M949_RS06330 in different Riemerella strains collected from duck farms across the country was investigated.According to the survey,16 strains were found to exist this gene.After sequencing and comparison,the phylogenetic tree analysis showed that M949_RS06330 was well conserved among strains of type 2 /15serotypes,but dispersed in serum type 1 strains,which can be divided into two branches.Based on the above analysis,it can be concluded that M949_RS06330 may not be an intrinsic structural gene of R.anatipestifer,but an exogenous gene obtained during long-term evolution.2.Prokaryotic expression of a wild type I strain of Salmonella typhimurium CH3 M949_RS06330 and preparation of its monoclonal antibody The full-length gene M949_RS06330 was inserted into p ET28 a vector to construct p ET28a-M949_RS06330 recombinant plasmid and transformed into competent host strain BL21(DE3).Isopropylthiogalactopyranoside(IPTG)was added at 37°C for 6 hours to induce expression.The results showed that the protein expression was in line with the expected size of 40 k Da after SDS-PAGE detection and mainly in the supernatant.The p ET28a-M949_RS06330 recombinant protein was abundantly expressed and purified by column chromatography,and used as an immunogen in an equal volume of Freund's complete adjuvant.Eight-week-old BALB/C mice were injected.After three times of immunization and titer testing,the mice with the highest titer were selected for super-exemption and ultra-exemption.The mouse spleen cells were fused with SP2/0 hybridoma cells.A monoclonal cell line 1E11 secreting specific antibodies was screened by subcloning three times.Ascites was prepared for mass production of the monoclonal antibody.The subtypes of this antibody were identified,the light chain was k and the heavy chain was Ig G2 b.The ELISA test of the monoclonal antibody titer reached 1:2048000.The specificity of the monoclonal antibody was detected by Western blot.The results showed that the monoclonal antibody had good specificity.3.The regulation of tetracycline on M949_RS06330 Different doses of tigecycline or tetracycline(0.01?g/m L,0.05?g/m L,0.1?g/m L,and 0.2?g/m L)were added into LB medium,respectively,and CH3 bacteria solution without any drug was used as control.Firstly,the effect of tigecycline and tetracycline with different concentration gradients on gene expression of M949_RS06320,M949_RS06325,and M949_RS06330 was detected by using fluorescent quantitative PCR.The q-RT PCR-specific primers of M949_RS06320,M949_RS06325,and M949_RS06330 were designed.The 16 sr RNA of R.anatipestifer was used as the reference gene,and the results were calculated according to a normalized gene expression method(2-??CT).The results were statistically calculated using Graph Pad Prism 5 software.The results showed that tigecycline and tetracycline had obvious induction effects on the m RNA levels of the three genes.Addtionally,Western blot was used to detect the effects of different concentrationgradients of tigecycline and tetracycline on the protein expression of these three genes.The results showed that tigecycline and tetracycline had a significant induction effect on M949_RS06330,which was consistent with the results of fluorescent quantitative PCR. |
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