Cloning, Expression, Protein Purification And Crystallization Of Drug Resistance Correlative Genes In Shigella Flexneri 2a

A study of positive multidrug ef?ux pump of bacteria is a relatively active area in study into the bacteria resistance. Multidrug ef?ux pump is a important mechanism that microorganism generate resistance to drug and other extra material. acrAB-tolC which is finished by control action of AcrA,AcrB and MarA is a general multidrug ef?ux pump.The objective of this research was to study the condition of crystallize by cloning marA,acrA and acrB of Shigella flexneri 2a, and expression, Purification of protein. Three pairs of primers were designed according to the genome sequence of Shigella flexneri 2a have reported, and amplified marA,acrA and acrB genes from the genome DNA by polymerase chain reaction(PCR). The amplified fragment was inserted into pMD18-T . Then the recombinant vector were inserted into pET-30a plasmid by two unique restriction sites of BamHⅠand Sa lⅠ. After confirmation of the correctness of open reading frame of target gene in positive recombinant plasmid by sequencing, the homology of marA,acrA gene sequence were 100%, and the homology of acrB gene sequence were 99.7% comparing with a reported nucleotide sequences. Three recombinant plasmids were transformed into the host strain of E.coli BL21(DE3) pLys and the target proteins were expressed by inducing with IPTG at 37℃. The expressed products of this marA, acrA and acrB genes were identified by SDS-PAGE. The results revealed that marA, acrA had been expressed successfully, The product were 21kDa for marA and 45 kDa for acrA . The results showed that acrA protein were solubly expressed in bacteria, and marA were expressed to form inclusion body . We renatured marA protein by lysing of isolated inclusion body using high concentration of urea, and then diluted in a buffer system containing oxidized/reduced glutathione. marA and acrA were purified by Ni-NTA His Bind Resin affinity chromatography under natural condition. obtained high purity of protein with 5 mg / mL through concentration .Screened the condition to crystallize of protein by the Hanging drop vapor diffusion method, and reference to the reagent compounds from Hampton Research. The results showed that many different crystals forms were observed after a week later, such as octahedron, diamond, and the needle-like crystal.This studies was the basis for studying the resistance mechanism of shigella flexneri, and provided structural basis for researching the interaction between the protein.