构建包含结核杆菌Ag85B基因的植物表达载体,并转化农杆菌LBA4404。以pcDNA3-Ag85B为模板,通过常规PCR克隆出Ag85B基因,定向克隆至含有玉米特异性启动子globulin-1的pCR2.1载体上,然后将globulin-1-Ag85B的融合片段酶切下来,并连接到经过加工修饰的含有抗除草剂基因bar的双元表达载体pCAMBIA1300上,将重组质粒转化至农杆菌LBA4404中。重组质粒双酶切可见0.8bp和10kb的两条特异性条带,与预期大小相一致。重组质粒测序表明克隆的Ag85B基因序列与Genbank上相一致,酶切从农杆菌中所抽提的质粒,片段大小与预期相一致。成功构建与转化了携带结核Ag85B基因的植物双元表达载体,为利用植物反应器生产口服结核疫苗奠定基础。
To construct the plant expression plasmid containing Mycobacterium tuberculosis Ag85B genes,and transform the recombinant plasmid into Agrobacterium tumerfaciens LBA4404.We used pcDNA3-Ag85B as template to amplify Ag85B gene by polymerase chain reaction(PCR),and cloned Ag85B gene into the vector pCR2.1.The fusion fragment of promoter globulin-1 and the target gene Ag85B which get from double enzymes digestion of the recombinant plasmid pCRGAg85B was inserted into the plasmid pCAMBIA1300 which contains the bar gene for herbicide resistance,transformed recombinant plasmid pCAMG-Ag85B into Agrobacterium tumerfaciens LBA4404.Restriction enzyme digestion demonstrated that the inserted gene fragments were 0.8 bp,DNA sequence analyzing revealed that the Ag85B gene sequences were totally consistent with the GenBank reported.We have successfully constructed expression plasmid containing Ag85B gene and transformed it into LBA4404,and lays a foundation for further study a better vaccine against MTB.
[1] Colditz G A,Brewer T F,Berkey C S,et al.Efficacy of BCG vaccine in the prevention of tuberculosis.Meta-analysis of the published literature[J].JAMA,1994,271(9):698-702.
[2] Fine P E.Variation in protection by BCG:implications of and for heterologous immunity[J].Lancet,1995,346(8986):1339-1345.
[3] Kariyone A,Tamura T,Kano H,et al.Immunogenicity of Peptide-25 of Ag85B in Th1 development:role of IFN-gamma[J].Int Immunol,2003,15(10):1183-1194.
[4] Dietrich J A C,Rappuoli R.Mucosal administration of Ag85B-ESAT-6 protects against infection with Mycobacterium tuberculosis and boosts prior bacillus Calmette-Guerin immunity[J].J Immunol,2006,1(177):6353-6360.
[5] Elvang T C J,Billeskov R.CD4 and CD8 T cell responses to the M.tuberculosis Ag85B-TB10.4 promoted by adjuvanted subunit,adenovector or heterologous prime boost vaccination[J].PLoS ONE,2009,4(4),e5139:1-13.
[6] Sinha R K,Verma I,Khuller G K,et al.Immunobiological properties of a 30 kDa secretory protein of Mycobacterium tuberculosis H37Ra[J].Vaccine,1997,15(6/ 7):689-699.
[7] Skinner M A B B,Wedlock D N.A DNA prime-Mycobacterium bovis BCG boost vaccination strategy for cattle induces protection against bovine tuberculosis[J].Infect Immun,2003,71(9):4901-4907.
[8] 张万江,邓喜玲,吴芳.结核杆菌Ag85B基因疫苗的免疫保护效果研究[J].生命科学研究,2006,10(2):128-133.
[9] Tregoning J S,Nixon P,Kuroda H,et al.Expression of tetanus toxin Fragment Cin tobacco chlorphast[J].Nucleic Acids Res,2003,31(4):1174-1179.
[10] Tacket C O,Mason H S,Loson G,et al.Human immune response to a novel Norwalk virus vaccine delivered in transgenic potatoes[J].Infect Dis,2000,182(1):302-305.
[11] Lou X M,Yao Q H,Zhang Z,et al.Expression of the human hepatitis B virus large surface antigen gene in transgenic tomato plants[J].Clin Vaccine Immunol,2007,14(4):464-469.
[12] Kumar G B,Ganapathi T R,Revathi C J,et al.Expression of hepatitis B surface in transgenic banana plants[J].Planta,2005,222(3):484-493.
[13] Nicholson L,Gonzalez-Melendi P,Dolleweerd C,et al.A recombinant multimeric immunoglobulin expressed in rice shows assembly-dependent subcellular localization in endosperm cells[J].Plant Biotech J,2005,3(1):115-127.
[14] Han M,Su T,Zu Y G,et al.Research advances on transgenic plant vaccines[J].Yi Chuan Xue Bao,2006,33(4):285-293.
[15] 及晓宇,于丽杰.植物生物反应器生产口服疫苗的研究进展[J].现代农业科技,2009,8;210-211.
[16] 李君武,王珊珊,宋东,等.玉米特异启动子驱动下结核Hsp65与Esat-6融合基因表达载体的构建及鉴定[J].华北农学报,2009,24(3):28-31.
[17] 李君武,黄清华,王珊.玉米特异启动子驱动下结核hsp65基因植物表达载体构建及鉴定[J].玉米科学,2009,4:550-554.
[18] Mei C,Wassom,J J,Widholm J M.Expression specificity of the globulin-1 promoter driven transgene(chitinase)in maize seed tissues[J].Maydica,2004,49(4):255-265.
[19] Hood E E B M,Beifuss K,Magallanes-Lundback M,et al.Criteria for high-level expression of a fungal laccase gene in transgenic maize[J].Plant Biotechnol J,2003,1(2):129-140.
