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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 anti-phage mutants of Salmonella paratyphi A (Paramyxovirus) by homologous recombination gene knockout method and to study their biological characteristics. Methods: The homologous arms fragments 2,3,5 were obtained by PCR and fusion PCR. After digested with pYG4 vector, the homologous recombination plasmids pYG4-2, pYG4-3 and pYG4-5 were constructed. After identification by PCR sequencing, homologous recombination with wild-type parathyroid was performed to obtain 3 anti-phage mutants of No. 2,3,5. Three strains of mutant and wild-type Paratuberculosis were treated with different kinds and concentrations of antibiotics, different pH and temperature and different concentrations of salt ions, the A595 value of the strains was measured, and the antibiotic, temperature, pH Sensitivity of values and salt ions as well as growth curves. Results: Three homologous arms were amplified by PCR and fusion PCR. The constructed recombinant plasmids pYG4-2, pYG4-3 and pYG4-5 were successfully constructed by PCR. Homologous recombination and anti-phage identification showed that 3 Strain anti-phage mutants. Three strains of mutants developed kanamycin resistance and showed sensitivity to streptomycin; strain 2 showed ampicillin resistance and strain 5 showed high temperature tolerance; compared with wild-type Paratelly, , The growth curve of three strains of mutants was obviously gentle, especially the No. 2 mutant was the most prominent. Conclusion: The successful construction of anti-phage mutants, and access to some special biological characteristics.