禽伤寒沙门氏菌SG9R株asd基因缺失株平衡致死系统平台的构建

Construction of chromosome-plasmid balance lethal system based on Salmonella Gallinarum 9R asd deletion mutant

为研发以禽伤寒沙门氏菌(SG)SG9R弱毒疫苗株为载体表达外源基因的活菌疫苗,本研究利用Red同源重组系统对SG9R株基因组中编码天冬氨酸β-半醛脱氢酶的asd基因进行敲除,构建SG9R△asd缺失突变株。该突变株在缺乏外源DAP培养条件下溶菌死亡,而在添加DAP或导入表达链球菌asd+质粒p YA3342后才能够恢复增殖能力,以此建立了以asd营养基因为筛选标志的SG染色体-质粒平衡致死系统。并且进一步采用绿色荧光蛋白(GFP)基因作为报告基因,将其克隆于asd+质粒p YA3342中,电转化于SG9R△asd缺失突变株中,通过无DAP培养条件的筛选,获得了表达GFP外源基因的SG重组菌,经体外连续25次传代后,鉴定结果显示,GFP仍能够在重组SG中持续稳定的表达。本研究结果为以SG弱毒疫苗菌株作为活载体的基因工程疫苗的研制提供简便易行的操作平台。

Salmonella gallinarum 9R strain （SG9R） is an attenuated vaccine strain for efficiently preventing the fowl typhoid in chickens. In order to develop the vaccine of recombinant S.gallinarum expressing heterologous antigen gene, the asd gene deleted mutant of S.gallinarum SG9R strain （SG9RAasd） was generated using Red recombination system, as the result, the SG9R /hasd failed to grow in LB, which, however, recovered the ability of growth in LB when supplemented the diaminopimelic acid in medium or transformed the plasmid of pYA3342 expressing asd gene encoding 13-aspartic semialdehyde dehydrogenase. Then, the complemented plasmid of pYA3342 was transformed into the auxotrophic strain of SG9RAasd to set up a chromosome-plasmind balanced lethal system. Based on the platform of the balanced lethal system, the g/~ gene was cloned into pYA3342 and electroporated into asd SG9R to construct the recombinant bacteria, in which the GFP was stably expressed even through 25 serial passages in LB medium. Therefore, this balanced lethal system provided a useful platform for the development of live recombinant avirulent S.gallinarum vaccines as well as for other potential applications.