| Cfr protein is encoded by cfr gene methyltransferase, it can combine a variety of drug targets to mediate five kinds of drug resistance.cfr gene was found in Human and animal resources of bacteria in different countries and regions, which increase the rate year by year, its emergence and prevalence cause clinical medicine and public health’s potential hazards and high attention.Currently, ways to monitor cfr gene-positive bacteria is the ordinary PCR,but the results show that the bacteria can only be monitored whether they contain cfr gene,but can not determine whether the cfr gene express the Cfr protein,which is unable to further provide the basis for rational drugs use and risk assessment.Therefore, it’s quite important to establish the way to monitor Cfr Protein on the aspect of immunology.This study is based on preparing the recombinant protein Cfr monoclonal antibody;then make it to fight for fight, using Western blot method to detect clinical enterococcus, and provide a theoretical basis for the establishment of immunological methods for monitoring cfr gene-positive bacteria.In this study,we use the purified recombinant protein Cfr preserved in our laboratory as an immunogen, immune BALB/c mice. In the first immunization, Freund’s complete adjuvant and antigen 60μg(50μg-100μg available)were mixed in equal volumes to take the intrademal injection after fully emulsified; Month later, strengthened immunization to the mice, the amount of antigen injection volume remained the same, but instead of using Freund’s incomplete adjuvant and subcutaneously injection; Afterwards, boosted the immunization every two weeks,separation mouse spleen cells with SP2/0 cells fused after four times immunization, and cultured with the HAT selective medium (as mouse peritoneal macrophage the trophoblast); Making the purified recombinant Cfr protein as the original, detecting hybridoma and culturing supernatants by the indirect ELISA method, and cloning culture the selected Positive hybridoma; We got a strain. of effective hybridma (Cfr.-D9) at least three times’subcloning. Test shows this monoclonal antibodies with recombinant Cfr protein specificity, potency and high (ascites titer 1:243000). Subgroup identification results show that the monoclonal antibodies of subclass is IgG2a.To prepare monoclonal antibody as the first antibody, against 16 strains of clinical positive Enterococcus strains and 5 clinical negative Enterococcus analysed by means of Western blot. The results show that, McAbs can identify clinically positive Enterococcus strain specific in Cfr protein; also found were detected by protein content in 16 strains of Enterococcus, positive staining with coarse and fine, clinically positive Enterococcus strains that expressed in the same conditions, the amount of Cfr protein expressed in bacteria there are differences. In addition, also found that Cfr protein and the expression of the strains positive Enterococcus strains from different areas, different clones separated parts, different type has no direct relationship. But there is no research on the situation from the molecular mechanism.In summary, monoclonal antibody was successfully prepared; and the monoclonal antibody for anti, with Western blot method of 21 clinical strains of Enterococcus strains were detected, proved that the monoclonal antibody in experimental prepared have specific good.Preliminary experiment was established by using Western blot method to detect the expression of Cfr protein in clinical strains, to explore the establishment of immunological methods more to monitor clinical strains of Cfr protein provides theoretical basis.
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