The Study Of Relationship Of RA Outer Membrane Efflux Protein Gene Expression Levels And Drug Resistance

This paper has done a lot of studies of RA outer membrane efflux protein gene, containing bioinformatics analysis of RA OEP gene, cloning, prokaryotic expression, preparation of polyclonal antibody, crude extract of polyclonal antibody, the relationship of RA OEP gene expression levels and drug resistance. The results were as follows:1. Bioinformatics analysis of RA OEP geneRA OEP gene has an open reading frame(ORF), its fragment length is 1437bp. They have high similarity between RA OEP gene and Flavobacterium johnsoniae UW101 OEP gene. RA OEP gene codon usage bias analysis showed that it has not a strong codon bias and has no two or more rare codons strings. PHylogenetic analysis showed that RA OEP gene has the closest relationship with RA-YM OEP gene, and also belongs to the same branch. It has signal peptide, but no transmembrane region, It has the relative concentration of epitope. There were four different functional sites that protein kinase C pHospHorylation sites, casein kinase II pHospHorylation sites, N-myristoylation sites and N-glycosylation sites. Secondary structure prediction show that a helix accounted for the major part, the gene is located outside of the membrane.2. Cloning, prokaryotic expression and preparation of polyclonal antibody of RA OEP geneOEP gene sequence was designed appropriate primers for PCR amplification,1437bp Fragment length was amplified. The resulting pET-32a(+)/OEP was verified and transformed into BL21, then it was successfully expresses in the IPTG induced cells. A 64kD fusion protein OEP was expressed. The protein was purified and immunized the rabbits to raise polyclonal antibody. The collected antisera titer was up to 1:16 by agar diffusion assay. Western-Blot display prepared rabbit antiserum can produce specific response with purified recombinant protein.3. The study of relationship of RA outer membrane efflux protein gene expression levels and drug resistanceMIC value, which was 20μg/mL, was detected to show resistance of RA for tetracycline by double dilution method Additionally, the relationship between expression for OEP gene and RA resistance for tetracycline were analyzed using real time PCR, after RNA were extracted for reverse transcription in the range of the gradient 10μg/mL. The results showed that expression presented decrease after the first rise for OEP gene of RA under the 9μg/mL.