Construction Of ELISA-type Systems Based On Macrolides Receptor Protein MphR(A And E) And Preliminary Application In Detection Of Macrolide Drugs Residue

Macrolide antibiotics has been extensively used for decades,widely used in human and animal therapy,and used as a growth-promoting agent in the farming industry. Long time and large-scale abuse of antibiotics will lead to antibiotic residues in food, feed and environment, causing bacterial resistance. In order to be effective and timely regulatory macrolide antibiotics, it is needed to develop a variety of macrolide antibiotic residue detection methods to bulid a efficient detection network to meet a variety of testing needs.The main detcting methods for macrolide antibiotic on the market including confirmation methods(chromatography / mass spectrometry), microbial growth inhibition, ELISA and so on. But each method has shortcomings, such as the confirmation methods have high requirements for instruments and operators, microbial growth inhibition methods need a long culture time, have many influence factors, it is difficult to achieve standardized testing, ELISA often can olny test one substance. So it is need to develop various methods to build a methods system. Using the combination-dissociation between macrolide antibiotics regulatory protein Mph R(A) or Mph R(E) and the specific double-stranded DNA sequences, can create ELISA-type systems which similar with ELISA. The ELISA-type systems can be used to analysis macrolide antibiotics, and the advantage of this system is can testing more macrolide antibiotics than ELISA method.At present, there are reports about in vitro or in vivo detecting systems based on Mph R(A) protein, but no system based on Mph R(E) protein reported. In order to study more the macrolide antibiotics detection systems which based on regulate protein-DNA interaction, this study use in vitro ELISA-type systems as study method, use Mph R(A) and it’s DNA sequence as model, comparative study Mph R(A)and Mph R(E) and the relate DNA sequences.In this study, Mph R(A) and Mp R(E) protein genes were synthesized and cloned into the expression vector p ET28b(+), to obtain recombinant expression plasmid of these two proteins. Expressing Mph R(A) and Mph R(E) protein in E. coli BL21(DE3), then build ELISA-type systems using purified proteins.The recognition specificity for macrolide antibiotics of ELISA-type systems based on Mph R(A)and Mph R(E) protein were compared respectively. The results show that most antibiotics have consistent response characteristics in these two systems. Dirithromycin response weaker in Mph R(A)system than in Mph R(E) system, other antibiotics tested response stronger in Mph R(A) system than in Mph R(E) system. 14-membered ring macrolide antibiotics have the strongest reactiveness in the two syetems, 15-membered ring macrolide antibiotics azithromycin have a relative high reactiveness and16-membered ring macrolide antibiotics have weak reactiveness. The IC50 value get from Mph R(A)system is lower than Mph R(E) system, which indicates that under the present reaction conditions,Mph R(A) protein is more suitable for the establishment of macrolide antibiotics in vitro assay systems than Mph R(E) protein.Our experiments confirmed that Mph R(A) and Mph R(E) can bind with DNA sequences which correspond to the two proteins, this indicates that the two protein have similar structure for DNA binding. Compare the three DNA sequences(1 relate to Mph R(A) and 2 relate to Mph R(E)), we found out a 22 bp pseudo-palindromic structure in each sequence. Palindrome is a significant feature of DNA sequences bind to Tet R family protein. Through our experiment, we think that the pseudo-palindromic structures are the core sequences of binding DNA.Using different DNA sequences build system, there are differences in the sensitivity of the system.A-DNA and B-DNA get similar system sensitivity, C-DNA resulting a higher sensitivity, and AC4-DNA resulting the highest sensitivity. The IC50 is 6.72±2.49 ng/m L(n=10)using A-DNA and B-DNA, the IC50 is 3.95±1.18 ng/m L(n=10)using C-DNA, and the IC50 is 2.33± 0.70 ng/m L(n=7)using AC4-DNA. The system needs a high protein concentration, and result high background when using pseudo-palindromic structure of B-DNA(B5-DNA) or C-DNA(C6-DNA), these two sequences not suitable for the ELISA-type system. According to the results of the ELISA-type system, we speculate that the affinity between protein and DNA can affects the system sensitivity, protein concentrations within a certain range, the lower the protein and DNA binding affinity, the higher the sensitivity of the system. Preliminary, we measured affinity constants of A-DNA, B-DNA, C-DNA, AC4-DNA with Mph R(A) protein using biolayer interferometry(BLI). The results showed that A-DNA, B-DNA,AC4-DNA have similar affinity constants with Mph R(A), the affinity constant get from C-DNA nearly an order of magnitude lower than the other three DNAs.Using Mph R(A) and A-DNA, adding protein and drugs step by step, we established a ELISA-type system for erythromycin residue testing in raw milk. The MRL of erythromycin for raw milk is 40ng/m L in China and EU, according to this MRL, the CCα value of the system is 43.28 ng/m L, CCβvalue is 46.44 ng/m L. The system basically meet the demand for raw milk samples, but as a practical use, the method needs further study to improve system stability.