大肠杆菌SLT-IIvB基因的克隆和表达

CLONING AND EXPRESSION OF SHIGA-LIKE TOXIN TYPE II VARIANT B GENE OF E.COLI

采用聚合酶链式反应 (PCR) ,从大肠杆菌O1 38基因组DNA中分别扩增出志贺菌样毒素II型变体B的结构序列和包括信号肽的全序列两段基因 ,定向克隆进表达载体质粒pYA3334(asd+)的EcoRI、BamHI位点之间 ,构建成重组表达质粒。在大肠杆菌X62 1 2(Δasd)筛选出重组质粒 pB0 和 pB1 后 ,经中间宿主X3730转化过渡 ,再导入ΔasdΔcyaΔcrp减毒鼠伤寒沙门氏菌X4550 ,构建成宿主和载体之间具有平衡致死结构的SLT IIvB重组菌。SDS PAGE和Western blot分析重组菌X4550 ( pB0 )在 7 6kD左右处有一条特异性蛋白条带。动物免疫试验表明该重组菌能刺激机体产生针对SLT IIvB和沙门氏菌的特异性抗体。这为进一步研制相应的抗猪水肿病及相应沙门氏菌病的双价活疫苗奠定了重要基础。

A structure sequence and a DNA fragment including the signal peptide sequence and structure sequence of Shiga\|like toxin II variant B subunit gene were amplified from \%E.coli\% strain O\-\{138\} by PCR. After digested with restriction endonuclease \%Eco\%RI and \%Bam\%HI, the two genes were orientally inserted into the polycloning site of expression vector pYA3334(asd\++) respectively. Recombinant plasmids pB\-0 and pB\-1 were constructed and amplified in E.coli X6212(asd\+-). pB\-0 and pB\-1 were then introduced into avirulent Salmonella typhimurium vaccine strain X4550 (asd\+-) by serial transformation through intermediate strain X3730 (asd\+-) to construct recombinant SLT\|IIvB strain. Results of nucleotide sequencing of the cloned fragments in pB\-0 and pB\-1 revealed that they were in correct ORF of SLT\|IIvB. The results of SDS\|PAGE and Western\|blot showed that 7.6 kD protein of SLT\|IIvB antigen was expressed at pretty high level in recombinant strain X4550(pB\-0). The results of mice immunization indicated X4550(pB\-0) could initiate the host to produce specific antibodies to SLT\|IIvB and LPS\|O antigen of X4550. So the recombinant strain X4550 (pB\-0) is worth considering as a candidate vaccine strain against porcine edema disease and Salmonella typhimurium infections.