Molecular cloning of virB12 gene in pET28a vector(1).pdf

511Asian Pacific Journal of Tropical Medicine(2012)511-513Document heading doi:Molecular cloning of virB12 gene of Brucella melitensis 16M strain in pET28a vectorShiva Mirkalantari1,Nour Amirmozafari1*,Bahram Kazemi2,Gholamreza Irajian11Tehran University of Medical Sciences,School of Medicine,Microbiology Department,Tehran,Iran2Shahid Beheshti University of Medical Sciences,Department of Biotechnology,Tehran,Iran Contents lists available at ScienceDirectAsian Pacific Journal of Tropical Medicinejournal homepage: INFO ABSTRACTArticle history:RReceived 7 November 2011Received in revised form 27 January 2012Accepted 15 March 2012Available online 20 July 2012Keywords:BrucellaCloningvirB12pET28apJE1.2 *Corresponding author:Nour Amirmozafari,Tehran University of Medical Sciences,School of Medicine,Microbiology Department,Tehran,Iran.Tel:+982188058649 Fax:+982188058649 E-mail:A1.Introduction Brucellosis is a widespread zoonotic infectious disease that is acquired by humans primarily through contact with infected abortion-related animal tissues and contaminated dairy products.It can have diverse clinical manifestations with symptoms that overlap with other diseases1-3.Owing to the fact that bacteriological methods are not sensitive enough for brocellusis detection4,5,serological tests are often the preferred method for diagnosis of Brucellosis in both humans and animals6.Since the available serological tests detect circulating antibodies to bacterial lipopolysaccharide,these tests suffer from extensive cross reaction with other Gram-negative bacteria;therefore,interest in finding alternative and more specific bacterial antigen to detect brucellosis is on the rise7-10.Several proteins from Brucella melitensis(B.melitensis)were found to induce an antibody response in infected animals and humans11-13.It was recently reported that VirB12 protein,a surface-localized protein of Brucella spp.elicit antibody responses in both experimentally and naturally infected animals2,14.The aim of this study was cloning of virB12 gene in pET28a expression vector for production of recombinant protein to be used as antigenic component for future serological test development.2.Materials and methods2.1.Bacterial strains,plasmids B.melitensis 16M strain was procured from Pasteur institute of Iran.Escherichia coli(E.coli)strains DH5毩 and BL21(DE3)were received from Novagene Co.,Cloning vector pJET1.2 and expression vector pET28a were obtained from Fermentas and Invitrogen Co.,respectively.2.2.Culture of bacteria B.melitensis was cultured in Brucella broth and E.coli was grown in Broth and agar Luria-Bertani medium.When antibiotic selection was necessary,the above media were supplemented with appropriate concentrations of antibiotic.2.3.Genomic DNA extraction Genomic DNA of B.melitensis 16M strain was extracted by Bioneer AccuPrep Genomic DNA Extraction kit.Quality Objective:To clone the virB12 gene in pET28a expression vector for production of recombinant protein to be used as antigenic component for future serological test development.Methods:Brucella melitensis(B.melitensis)16M strain was cultured and bacterial DNA was extracted by Bioneer AccuPrep Genomic DNA Extraction Kit.Oligonucleotide primer pair was designed based on Brucella virB12 gene sequence with BamHI and HindIII restriction site at 5 end of the forward and reverse primers,respectively.DNA amplification was performed using PrimSTAR HS DNA polymerase and the PCR product was purified by DNA AccuPrepGel Purification Kit.Purified DNA was cloned into pJET1.2 cloning vector.VirB12 gene fragment was excised from pJET1.2 using BamHI/HindIII and subsequently subcloned into pET28a(+).Results:Brucella virB12 gene was successfully cloned in pJET1.2 and then in pET28a(+)plasmids.PCR and restriction enzyme digestion confirms the procedure.Conclusion:We cloned and expressed the Brucella virB12 gene which could be used as antigenic component for specific serological assay development.Shiva Mirkalantari et al./Asian Pacific Journal of Tropical Medicine(2012)511-513512and purity of the extracted DNA were assessed using agarose gel electrophoresis and spectrophotometerically.2.4.Primer design A pair of oligonucleotide was designed based on the virB12 gene sequence of B.melitensis 16M strain obtained from Gene Bank(Accession no.AF226278)with BamHI and HindIII restriction site at the 5 end of the forward and reverse primers,respectively(Table 1).Table1Primers designed for amplification of the virB12 gene of B.melitensis.virB12 PrimerForward virB12AGTGGATCCATGCGCACATTGGTTATGGTCGCReverse virB12CACAAGCTTGATATCCACGCGCCTGTTCAGGThe attached restriction enzyme sites are underlined.2.5.PCR amplification The PCR reaction mixture included 2 毺L of bacterial genomic DNA(containing 100 ng),150 nM dNTPs,and 1 PCR buffer(containing of MgCl2),1.25 units of PrimSTAR HS DNA polymerase and 40 picomoles each of the forward and reverse primers in 50 毺L final volume.PCR amplification was performed by the following parameters:denaturing at94 曟 for 40 s,annealing at 58 曟 for 30 s and extension at 72 曟 for 30 s in an Eppendorf master gradient thermocycler.PCR product was electrophoresed on 1.5%(w/v)agarose gel,stained with ethidium bromide and visualized under UV transilluminator.2.6.Gene cloning DNA band was sliced under long wave UV and recovered by Bioneer AccuPrepGel Purification Kit.Recovered DNA was ligated into pJET1.2 cloning vector at 22 曟 for 1 h with T4 DNA ligase(Fermentas Co.).The ligation product was transformed into E.coli DH5毩 strain competent cells and dispersed onto LB agar plates containing 100 毺g/mL ampicillin.After 16-18 h incubation at 37 曟,colonies on the agar plate that contained recombinant plasmids were detected.For confirmation,PCR amplifications were performed on these colonies using primers specific for virB12 gene and pJET vector and colonies containing the recombinant plasmid were selected.Recombinant plasmid was extracted by Bioneer AccuPrep Plasmid Extraction Kit and digested by BamHI and HindIII restriction enzymes(Fermentas Co.).Following electrophoresis,the DNA digested bands were purified by Bioneer AccuPrepGel Purification Kit.Fragments of BamHI and HindIII digestes were subcloned in HindIII and BamHI digested pET28a(+)expression vector and transformed into E.coli DH5毩 competent cells and spread onto LB agar plates containing 30 毺g/mL kanamycin.Then,the recombinant plasmid pET28a-virB12 was confirmed by PCR,restriction enzymes digestion and sequencing which was performed by a commercial company using universal forward and reverse T7 promoter and terminator primers(TAG Copenhage A/S Symbion,Denmark).3.Results Brucella virB12 gene was amplified from genomic DNA of B.melitensis 16M strain.The PCR products analyzed on 1.5%(w/v)agarose gel displayed a single band with the correct size(513 bp)pertaining to the amplification of the virB12 gene(Figure 1).The PCR product was cloned in pJET1.2 plasmid.Colonies containing the pJETvirB12 recombinant plasmid were confirmed by PCR using VirB12 gene and pJET 1.2 primers.Recombinant plasmid pJET-virB12 was successfully digested by BamHI and Hind III restriction enzymes.The digested band(virB12 gene)was extracted and subcloned into pET28a expression vector.Presence of the inserted gene was confirmed by PCR method,using primers designed according to the sequence of the pET28a and virB12 gene and digestion of pET28a-virB12 recombinant plasmid by BamHI and HindIII(Figure 2).Negative control was pET28a vector alone which displayed a lower molecular weight band relative to the recombinant plasmid.Finally,the integrity and orientation of virB12 in construct were confirmed by DNA sequencing.Results show a 513 bp fragment corresponding to the B.melitensis virB12 gene was successfully cloned and transformed to the bacterial expression vector pET28a.500 bp1 2 3Figure 1.Electrophoresis of the amplified virB12 gene on 1.5%(w/v)agarose gel.Lane 1:100 bp Plus DNA ladder.Lane 2,3:Single expected band of virB12 gene(approximately 513 bp).500 bp1 2 3 4Figure 2.Analysis of enzymatic digestion of recombinant plasmid on 1.5%(w/v)agarose gel.Lane 1:DNA ladder.Lane 2:virB12-pET28a recombinant plasmid.Lane 3:Mono digestion of virB12-pET28a recombinant plasmid with BamHI.Lane 4:Double digestion of virB12-pET28a recombinant plasmid with BamHI and HindIII.Shiva Mirkalantari et al./Asian Pacific Journal of Tropical Medicine(2012)511-5135134.Discussion Brucellosis is an infectious disease that is endemic in most developing countries15,16.Rapid and accurate diagnosis of Brucellosis has an important role in effective treatment of patients and improvement of public hygiene.The routine serological tests that are often used are based on detection of anti-lipopolysaccharide antibodies.In these serological tests,cross reaction occur between Brucella and many other Gram negative bacteria7.Elucidation of an antigenic component for diagnosis and vaccination of brucellosis infection serves as a valuable tool.There are several studies on different proteins of Brucella as antigenic component including the OMP 31 kDa,28 kDa and 26 kDa periplasmic proteins from B.melitensis4,9,13.The proteins that are located on the surface of bacteria have traditionally been used as useful antigens for diagnostic and vaccine component.For this reason,in the present study,cloning of the virB12 gene of Brucella that is located on bacterial surface was designed.Previous study indicated that natural hosts infected with Brucella produce an antibody response to the protein encoded by the virB12 gene2.VirB12 protein is a part of the type IV secretion system that is encoded by the virB locus and is located on the surface of Brucella.Brucella cell surface proteins have shown to elicit an immune response related to the protection of the host which can also potentially be used for diagnostic purpose2,14.In this study,the virB12 gene is cloned using DH5毩 E.coli as a host which doesnt contain the T7 RNA polymerase and the expression vector pET28a was used that is a powerful system for cloning and expression of recombinant proteins in E.coli.The nucleotide sequence of virB12 gene cloned in this study was consistent with the sequence of virB12 gene as published in the GeneBank.Cloning site in the pET28a also contained His Tag sequences for detection and purification.In conclusion,we were able to clone the virB12 gene of B.melitensis in expression vector for protein expression in order to be used in future study as antigenic component.Conflict of interest statement We declare that we have no conflict of interest.Acknowledgment This study was supported by grant from Tehran University of Medical Sciences.References 1 Clavijo E,Daz R,Anguita A,Diaz R,Anguita A,Garcia A,et al.Comparison of a dipstick assay for detection of brucella-specific immunoglobulin M antibodies with other tests for serodiagnosis of human brucellosis.Clin Diagn Lab Immunol 2003;10:612-615.2 Hortensia 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