| Acinetobacter baumannii is widely found in nature because of its strong ability of acquisition of drug resistance and clone transmission.Therefore,along with extensive and irrationals use of antibiotics,the drug resistance of Acinetobacter baumannii gradually increased.Acinetobacter baumannii has become a very important opportunistic pathogen,the multidrug-resistant?MDR?,extensively drug-resistant?XDR?,and pandrug-resistant?PDR?Acinetobacter infections have become prevalent in the world,especially for the hospitalized patients with low immunity and serious illnesses.Although all countries in the world have paid great attention to the research and development of new antibiotics in recent years,the development speed of new antibiotics has slowed down significantly due to difficulties in research.The speed of development of new drugs is far behind that of evolution of bacterial strain.Due to the lack of effective drugs,the mortality rates of Acinetobacter baumannii infections increased.Clinical therapy faces severe challenge.Phage is a bactericidal virus that can be used to treat bacterial infections,which is called phage therapy or bacteriophage therapy.Because bacteriophage sterilization mechanism is completely different from antibiotics,in recent years its role as a natural killer of"super bacteria"has increasingly attracted the attention of researchers in various countries.Each phage has a strict host specificity compared to broad-spectrum antibiotics.In other words,the antibacterial spectrum of bacteriophage is very narrow.The virus always kills host bacteria that is sensitive to it,and has no effect on other bacteria.The specific recognition and adsorption of bacteriophage to bacteria is the key factor in determining the phage specificity and also play a key role in determining the success or failure of bacteriophage therapy.Therefore,there search focused on the interaction between phage receptor-binding protein?RBP?and phage receptor on the surface of host strain is very important.However,at present,most of the researches are mostly relate to E.coli phage.There is very little research on Acinetobacter phage.The bacteriophage ZZ1 was isolated and studied by our group.The phage can infect pandrug-resistant Acinetobacter baumannii clinical strains.The morphology of ZZ1 under electron microscope was very similar to that of bacteriophage T4.It belongs to Myoviridae with 6 long-tailed fibers and 6 short-tailed fibers.After the genomic analysis of ZZ1 genome,we have predicted the two kinds of phage receptor-binding protein gene,the long-tailed fiber protein gene ZZ1gp12[GeneID:18114118]and the short-tailed fiber protein gene ZZ1gp37[GeneID:13165127].In the study we will clone and express the two kinds of predicted genes,respectively.Moreover,the receptors corresponding to the two kinds of phage receptor-binding protein of ZZ1 were identified on the surface of Acinetobacter baumannii AB09V cell,respectively.Materials and Methods1.Strains,bacteriophages and plasmidThe Acinetobacter baumanni strain AB09V and the bacteriophage ZZ1 were collected by us.The plasmid pQE80L,the strain DH5?and the strain BL21 were purchased from Bioengineering Bioengineering?Shanghai?Co.,Ltd.2.Cloning and Expression of the two kinds of phage receptor-binding protein gene,ZZ1gp12 and ZZ1gp37.Primers were designed for the predicted genes,ZZ1gp12 and ZZ1gp37,and PCR experiments were performed using ZZ1 genomic DNA as a template.Double restriction enzyme digestion of the two amplification products and plasmid pQE80L were performed,respectively.Then the two digested products were ligated with the digested vector pQE80L,respectively,using T4 ligase to construct recombinant plasmids pQE80L-ZZ1gp37 and pQE80L-ZZ1gp12.The two recombinant plasmids were transformed into E.coli DH5?,respectively,and the two kinds of positive clones were obtained.Then the two kinds of recombinant plasmids were extracted,respectively,and the size of their inserts was analyzed by the double restriction enzyme digestion of recombinant plasmid electrophoresis,the correctness of the insertions of the two interest genes were verified by further sequencing.The two kinds of recombinant plasmids were introduced into the expression strain BL21,respectively,the two target proteins were also expressed after induction by IPTG,and were analyzed by SDS-PAGE and Western blotting.3.Purification and identification of the receptor-binding proteins of the bacteriophage ZZ1The two kinds of recombinant receptor-binding proteins of ZZ1 were purified using a nickel column.The interactions between the two kinds of recombinant proteins and phage ZZ1 neutralizing antibody was observed by Western blotting to identify the antigenicity of the recombinant proteins.The recombinant long-tailed fiber protein,the recombinant short-tailed fiber protein,and a mixture of the two recombinant proteins were interacted with the Acinetobacter baumannii AB09V strain,respectively.Then the competitive interference adsorption experiments were performed to observe the adsorption of the phage ZZ1 to the three kinds of pre-processed Acinetobacter baumannii AB09V strains,respectively.4.Identification of the receptors for the long tail fiber protein and the short tail fiber protein of phage ZZ1The Outer Membrane Protein?OMP?and the lipopolysaccharide?LPS?of the Acinetobacter baumannii AB09V strain were extracted with a bacterial outer membrane protein extraction kit and a bacterial lipopolysaccharide extraction kit,respectively.The extracted OMP and the extracted LPS were coated on the ELISA plates,respectively.Then the interaction between the two recombinant tail proteins of ZZ1 and the two kinds of coated ELISA plates were observed,respectively,with6×his tag monoclonal antibody,the enzyme-labeled secondary antibody,and substrate.The OD values were tested to determine the the receptors of the two recombinant tail fiber proteins.Moreover,the Acinetobacter baumannii strain AB09V was treated with proteinase K and periodate to destroy the protein and the LPS on surface of AB09V cells,respectively.Then the adsorption efficiency of bacteriophage ZZ1 against the two kinds of AB09V strains treated with different methods,respectively,were measured to further verified whether the interaction between the two kinds of recombinant tail proteins of ZZ1 on the two kinds of extracted and purified receptors?LPS and OMP?was consistent with the effect of the phage ZZ1 on the surface receptors of bacteria.Results1.After electrophoresis of the digested products of pQE80L-ZZ1gp37 and pQE80L-ZZ1gp12,the recombinant plasmids bands of 3900bp and 1500bp were observed.The result is in agreement with the predicted the two kinds of tail fiber gene,ZZ1gp37 and ZZ1gp12.The sequencing results further verified the correctness of the two recombinant plasmids SDS-PAGE showed that the electrophoresis bands of the long and short-tailed fiber proteins of recombinant ZZ1 were located at about 140kDa and about 55 kDa,respectively,which was also consistent with the predicted size?140.96 kb and 54.26 kb?of the two proteins,gp37and gp12.2.Western blotting showed that the neutralizing antibodies of bacteriophage ZZ1can specifically recognize the electrophoretic bands of the two kinds of recombinant receptor-binding proteins of ZZ1 after SDS-PAGE.Adsorption experiments showed that the purified bacteriophage ZZ1 recombinant long-tailed fiber protein gp37,the purified recombinant short-tailed fiber protein gp12 and the mixture of two kinds of proteins can significantly interfere with the adsorption of the phage ZZ1 against AB09V strain.3.ELISA showed that both the two kinds of recombinant proteins,gp37 and gp12,did not interact with OMP coated plates,the OD45050 values of gp37 group and gp12 group is 0.038±0.03and 0.054±0.03 respectively,but the OD45050 values of control group is 0.041±0.02.There were no significant difference between the two experiment groups and the control group?P>0.05?.However,the two kinds of recombinant proteins can combine with the LPS coated plates,the OD45050 values of gp37 group and gp12 group are 0.542±0.09 and 1.477±0.09 respectively,but the OD45050 values of the control group is 0.216±0.07,there is a significant difference between the two experiment groups and the control group?P<0.001?.Moreover,when LPS on the surface of AB09V of Acinetobacter baumannii was destroyed,the adsorption rate of ZZ1 to periodate treated AB09V decreased significantly,compared with the untreated AB09V,it decreased by 75%.However,when OMP on the surface of AB09V was destroyed,the adsorption rate of ZZ1 to the proteinase K treated bacterial cells remains the same as that to the untreated AB09V.There are no significant difference between the experiment group and the control group.Conclusions1.The two kinds of recombinant plasmid,pQE80L-ZZ1gp37 and pQE80L-ZZ1gp12,were constructed correctly and the two kinds of receptor-binding proteins of the phage ZZ1,gp37 and gp12,were expressed successfully.3.LPS is the receptor of the two kinds of the receptor-binding proteins of phage ZZ1,gp37 and gp12. |
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