抗噬菌体甲型副伤寒沙门菌突变菌的构建及其生物学特性

Construction of anti-phage Salmonella Paratyphi A mutant strains and its biological characteristics

目的:采用同源重组基因敲除方法构建3株甲型副伤寒沙门菌(副甲)抗噬菌体突变菌,并研究其生物学特性。方法:通过PCR和融合PCR方法获得了2、3、5号同源臂片段,经酶切后与pYG4载体连接,构建pYG4-2、pYG4-3和pYG4-5同源重组质粒。经PCR测序鉴定正确后,与野生型副甲菌进行同源重组,获得2、3、5号3株抗噬菌体突变菌。将3株突变菌和野生型副甲菌经不同种类和浓度的抗生素、不同pH值和温度及不同浓度的盐离子处理后,测量菌株的A595值,分析3株突变菌对抗生素、温度、pH值和盐离子的敏感性以及生长曲线。结果:PCR和融合PCR扩增出3个同源臂,构建的pYG4-2、pYG4-3和pYG4-5同源重组质粒经PCR测序表明构建成功;同源重组和抗噬菌体验证表明成功获得3株抗噬菌体突变菌。3株突变菌产生了卡那霉素抗性,且对链霉素表现出敏感性;2号菌表现出氨苄青霉素抗性,5号菌表现高温度耐受性;与野性型副甲菌比较,3株突变菌的生长曲线明显平缓,尤以2号突变菌最为突出。结论:成功构建抗噬菌体突变菌,并获得一些特殊的生物学特性。

Objective To construct three strains of anti-phage Salmonella Paratyphi A mutants by homologous recombination and knocking out genes in wild Salmonella Paratyphi A,and to analyze their biological characteristics.Methods The No.2,3,5 homology arm fragments obtained by PCR and fusion PCR were connected with pYG4 vector after digestion to construct the pYG4-2,pYG4-3,and pYG4-5 homologous recombination plasmids.Three strains of anti-phage mutants（No.2,3,5）were obtained after the plasmids were identificated by PCR sequencing and recombinated with wild Salmonella Paratyphi A.After the three mutant strains and wild Salmonella Paratyphi A were treated with different kinds and concentrations of antibiotics,different pH values and temperature and different concentrations of salt ions,respectively;the A595 values were measured,and their sensitivities to the antibiotics,temperature,pH values and salt ions and the growth curves were analyzed.Results The results of PCR and fusion PCR amplification suggested that three homology arms were obtained.The PCR sequencing showed that the homologous recombinant plasmids pYG4-2,pYG4-3,and pYG4-5were constructed successfully.The homologous recombination and anti-phage verification implied that three strains of anti-phage mutants were successfully gained.Three strains of anti-phage mutants gained kanamycin resistance and showed highly sensitivity to streptomycin;the No.2mutant strain showed ampicillin resistance and the No.5mutant strain performanced a high temperature tolerance.In addition,the growth curves of three mutants became obviously placid than that of wild Salmonella Paratyphi A,especially in the No.2mutant.Conclusion The antiphage mutants are successfully constructed and get some specially biological chatacteristics.