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					                                                                          P39




                                                 P39




   P39                                           S19


                                                                         P39



                       P39
   +
pSK -P39                      pGEX4T1 pSK+                             P39
 pGEX4T1-P39                    P39                                          pGEX4T1-P39
       IPTG    pGEX4T1-P39                                      BL21



   P39           pSK                        PCR
                             pGEX4T1-P39                  P39


                                                    P39
               BL21                   pGEX4T1
                                      P39            P39




                                            5
                                                 - E. Coli
                                            6
                                                - PCR: Polymerase Chain Reaction.
                                            7
                                                - IPTG: Isopropyl -    - D - thiogalactopyranoside.
                                            8
                                              - GST Sepharose: Glutathione S Transferase
                                                 Sepharase.
                                            9
                                              - Fusion Protein.
                                                                         P39




                                       pGEX4T1
P39
                                                                 S19
                                  E.coli


                 BL21         E.coli
                                   BL21




                        P39




                                  P39                                      P39




                                                          P39


           P39

                                                 P39
                                                                E.Coli
             S19
E.Coli DH5         E.Coli                                                        E.coli
                                       BL21


       +
 pSK
pGEX4T1             P39                          1
                                                     Brucellergene.
                                                 2
                                                     Denoel.
                                                                        P39




XhoI


                PCR           P39
          PCR
                              DNA                                       CTAB/NaCl




                       Taq DNA Polymerase
                  PCR                                 TE
                                                  K              SDS


                                                                                     CTAB/NaCl


                                    DNA
                                                                                            DNA


                                                                               DNA
      PCR                                                        TBE
                                                                                      DNA
                       EDTA
                                                           P39




                                     PCR                               BamHI


pSK                   P39                         1
                                                    - CTAB: Cetyl trimethyl ammonium bromide.
                                                  2
                                     pGEX4T1         TE: Tris 10mM , EDTA 1 mM , pH 8.
                                                  3
                                                     CTAB 10% , NaCl 0.7 M.
                                                  4
                  XhoI BamHI                PCR      TBE: Tris base 890mM. Boric acid 890mM ,
                                                      EDTA 25mM.
                                                  5
                                                    -Forward: 5 GCC GGA TCC ATG GGC GCC
                                                      TGT TGC CAA 3
                                                      Reverse: 5 C CGC CTC GAG TTA TTT TGC
6
    -Roche.                                           GGC TTC AA 3
                                                                            P39




                                                XhoI BamHI
                                                                    P39
                         SDS-PAGE                              T4 DNA ligase


                            DNA                            pGEX4T1-P39 pSK+-P39
                                                         DH5
                     DNA                                                                 BL21
                                         P39
                                   PCR                               PCR
                                                                                    pSK+-P39
                                                              PCR
P39                                       pSK

                                                                      P39
IPTG                        P39                       pGEX4T1-P39


           P39



                                                    IPTG




                                                                                  IPTG




                                                          SDS-PAGE


                                                        GST




                                                1
                                                    MWG.
4                                               2
    SDS PAGE: SDS- Poly Acrylamide gel              Sanger.
                                                3
     electrophoresis.                               OD: Optimal density.
                          P39




        1      2




bp     P39         bp




                        P39
     GST-P39
                                                              P39




              Th1




 L7/L12
                            P39




                    P39




            P39


      P39


                                                 Rev1               S19




                      P39




P39
                                  13
                                     - Malta fever.
pET                               14
                                     - Mediterranean fever.
                                  15
                                     - Undulant fever.
                                                         P39




                               E.coli




E.coli                         P39
                                               .



 1.Collier    L, Balows A, Susana M.
 Microbiology and Microbial Infections. 9th
 ed. vol:2. London: Arnold pub; 1998. p.
 250-275.
2. Refai M. Incidence and control of
brucellosis in the near east region. Vet      pGEX
Microb 2000; 90: 81 111.
3. Corbel MG. Brocellosis: an overview
emerging. Infect Dis 1997 ; 3: 213
221.
4. Schuring GG, Sriragana N, Corbel MG.                          Lac
Brucellosis Vaccines: past , present and
future. Vet Microb2002; 90: 479 496.
5.Denoel PA, et al. Charactization ,
occurrence and molecular cloning of a 39
kilodalton brucella abortus cytoplasmic
protein immunodominant in cattle. Infect
Immune 1997; 65(2): 495-502.
  L7/L12
            Balb/c                      P39


7.Collier L, Balows A, Susana M.
Microbiology and Microbial Infections. 9th
ed. vol: 3. London: Arnold pub; 1998. p.
165-210.
8.Tibor A, Jacques I,Guilloteau L,et al.
Effect of P39 Gene deletion in live
Brucella vaccine strain on residual
                                                               pGEX4T1
virulence and protective activity in mice.
Infect Immune 1998; 66(11):5561-5564.                    P39
9.Kreuzer H, Massey A. Recombinant
DNA and Biotechnology. Second ed.
Washington: ASM press; 2001.p. 369-410

                                                   Lac          P39
                                                                       P39




Expression of recombinant P39 protein from Brucella Abortus in
                       Escherichia coli

                Abtahi H , Hatef Samanian A , Rafati S , Behzadian Nejad G


Abstract

        Introduction: Brucellosis, caused by Brucella spp., is an important zoonotic disease
that causes abortion and infertility in cattle, and undulant fever in human. At present, live
attenuated strain 19 of Brucella Abortus is used to immunize domestic animals, but this
vaccine has several disadvantages. Therefore, the development of more effective and safer
vaccines is necessary for disease control in animals and of more importance in human. So
research on development of subunit vaccine seems appropriate, and regarding this, P39
preplasmic protein have shown to be highly immunogenic.
        Materials and methods: In this experimental study, we amplified P39 gene by
Polymerase chain reaction (PCR) method and sub cloned to prokaryotic expression vector
pGEX4T1. Escherichia coli BL21 was transformed with pGEX4T1-P39 and gene expression
was induced by IPTG. Then it was purified by GST Sepharose kit. The concentration of P39
was assayed by Bradford method.
        Results: The recombinant plasmid (pSK-P39) was sequenced. The sequencing result
was confirmed by Sanger method and was same as P39 gene. The concentration of purified
protein was 200µg/ml.
        Conclusion: The vectors that contain fusion protein can produce the recombinant
P39 protein.
        Key words: Brucella Abortus , P39 gene , P39 protein , Gene expression ,
Recombinant , E. coli

				
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