大肠杆菌pks岛中插入岛的缺失以及插入岛生物学活性初步研究
发布时间：2019-03-07 19:17
【摘要】： 目的: 前期的研究表明在213株非五类肠道致病性大肠杆菌中检测到7株pks岛阳性菌株,在3株分子分型一致的pks岛阳性菌株中发现在pks岛中有两个新的插入岛(Insert1和Insert2),经PCR、测序和Southern blot证实,Insert1和Insert2大小分别约为90kb和13kb。功能预测Insert1主要与耶尔森杆菌素的合成、转运和调节有关,Insert2主要与磷酸戊糖通路和药物抗性有关。为初步探讨插入岛的基因功能,我们以其中的81号菌作为目标菌株,在其基因组中试图缺失Insert1和Insert2,并研究缺失后菌株的生化特性和细胞毒性。 方法: 采用一步缺失法-λRed同源重组系统构建缺失突变株的方法对大肠杆菌pks岛阳性菌株中两个插入片段(Insert1和Insert2)进行缺失突变,并采用PCR、测序对缺失突变株进行鉴定。对缺失成功的菌株采用细菌生化鉴定仪Walk Away 40 SI(Siemens)进行生化鉴定和药敏试验,同时,测定它们的生长速度和酸碱抗性。采用Hela细胞进行细胞毒性试验,当细胞汇合度达到80%时,细菌与细胞100:1共育2h,加入庆大霉素(200μg/mL)培养,采用吉姆萨染色纤维镜下观察细菌与细胞作用后2h、3h、5h的细胞数量和形态变化,同时,采用乳酸脱氢酶试验(LDH)检测细菌与细胞作用8h对细胞的毒性。 统计学分析采用SPSS14.0统计软件包,数据表示为均数±标准误,多组之间的均数比较采用单因素方差分析,两组之间的均数比较采用LSD检验,P0.05为差异有统计学意义。 结果: 1)利用PCR鉴定初筛到17个Insert2缺失疑似阳性菌株,经测序验证得到17、21号两个缺失株。没有得到Insert1缺失的菌株。 2) Insert2缺失前的81号菌与缺失后的17号菌除了卡那霉素抗性不同外,它们的生化鉴定、酸碱抗性、生长速度以及耐药性均没有区别。17号菌呈卡那霉素抗性阳性,而81号菌呈卡那霉素抗性阴性,说明同源重组成功将外源性的卡那霉素抗性基因整合到细菌染色体上。 3)吉姆萨染色后显微镜下比较81号菌与17号菌作用后的细胞数目和形态,显示81号菌作用的细胞贴壁细胞减少,细胞体积变大,细胞核变大。 4) LDH试验结果显示81号菌和17号菌在8小时的细胞死亡率分别为(13.2±9.5)%、(25.1±6.2)%,缺失株显著小于野生株(P0.05)。 结论: 1)利用一步缺失法成功缺失了大肠杆菌pks岛中插入的新岛Insert2。 2)缺失Insert2后的大肠杆菌(17号菌)生化特性与缺失前的大肠杆菌(81号菌)没有差别。因此,Insert2中没有与细菌生化特性相关的基因。 3)大肠杆菌pks岛中插入的Insert2缺失后对细胞的毒性降低,Insert2中可能含有以前没有预测到的新的细胞毒力基因。
[Abstract]:Objective: previous studies showed that 7 pks island positive strains were detected in 213 strains of non-five types of enteropathogenic Escherichia coli. Two new insertion islands (Insert1 and Insert2) were found in three pks island positive strains with identical molecular typing. PCR, sequencing and Southern blot confirmed that the sizes of Insert1 and Insert2 were about 90kb and 13kb. Functional prediction of Insert1 was mainly related to the synthesis, transport and regulation of Yersinia, and Insert2 was mainly related to pentose phosphate pathway and drug resistance. In order to explore the gene function of the inserted island, we used strain 81 as the target strain to try to delete Insert1 and Insert2, in its genome and to study the biochemical characteristics and cytotoxicity of the strain after deletion. Methods: two inserted fragments (Insert1 and Insert2) of Escherichia coli pks island positive strain were constructed by one-step deletion-位 Red homologous recombination system, and PCR, was used. The deletion mutants were identified by sequencing. Walk Away 40 SI (Siemens) was used for biochemical identification and drug sensitivity test. Meanwhile, the growth rate and acid-alkali resistance of the strains were measured. The cytotoxicity test of Hela cells was carried out. When the cell confluence reached 80%, the bacteria were co-cultured with 100 渭 g / mL for 2 h and cultured with gentamicin (200 渭 g / mL) for 2 h, 3 h, 2 h, 3 h, respectively, under the microscope of Giemsa staining, and the cells were incubated with Gentamycin (200 渭 g / mL) for 2 h and 3 h, respectively. The number and morphology of the cells were changed at 5 h, and the cytotoxicity of bacteria and cells to 8 h was detected by lactate dehydrogenase test (LDH). Statistical analysis using SPSS14.0 statistical software package, the data expressed as mean 卤standard error, multiple groups of mean comparison using one-way analysis of variance, the comparison between the two groups using LSD test, P0.05 is statistically significant. Results: 1) 17 suspected Insert2 deletion positive strains were identified by PCR, and 17 and 21 deletion strains were obtained by sequencing. No strain with Insert1 deletion was obtained. 2) there was no difference in biochemical identification, acid-base resistance, growth rate and resistance to kanamycin between strain 81 before deletion of Insert2 and strain 17 after deletion except that kanamycin resistance was different between strain 81 and strain 17 after deletion, and no difference was found between strain No. 17 and strain No. 17 in kanamycin resistance, but no significant difference was found in the resistance to kanamycin. The kanamycin resistance of strain 81 was negative, indicating that the exogenous kanamycin resistance gene was successfully integrated into the bacterial chromosome by homologous recombination. 3) comparing the number and morphology of the cells between strain 81 and strain 17 under microscope after Gimsa staining, the results showed that the number of adherent cells of strain 81 decreased, the cell volume became larger and the nucleus became larger. 4) LDH test showed that the cell death rates of strain 81 and strain 17 were (13.2 卤9.5)% and (25.1 卤6.2)% respectively at 8 hours, and the number of deleted strains was significantly lower than that of wild strains (P0.05). Conclusion: 1) the new island Insert2. inserted in Escherichia coli pks island was successfully deleted by one-step deletion method. 2) the biochemical characteristics of Escherichia coli (strain 17) after deletion of Insert2 were not different from those of E. coli (strain 81) before deletion. Therefore, there are no genes related to bacterial biochemical characteristics in Insert2. 3) the deletions of the inserted Insert2 in the pks island of Escherichia coli reduced the cytotoxicity to the cells, and the Insert2 might contain new cytotoxic genes that had not been predicted previously.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378
[Abstract]:Objective: previous studies showed that 7 pks island positive strains were detected in 213 strains of non-five types of enteropathogenic Escherichia coli. Two new insertion islands (Insert1 and Insert2) were found in three pks island positive strains with identical molecular typing. PCR, sequencing and Southern blot confirmed that the sizes of Insert1 and Insert2 were about 90kb and 13kb. Functional prediction of Insert1 was mainly related to the synthesis, transport and regulation of Yersinia, and Insert2 was mainly related to pentose phosphate pathway and drug resistance. In order to explore the gene function of the inserted island, we used strain 81 as the target strain to try to delete Insert1 and Insert2, in its genome and to study the biochemical characteristics and cytotoxicity of the strain after deletion. Methods: two inserted fragments (Insert1 and Insert2) of Escherichia coli pks island positive strain were constructed by one-step deletion-位 Red homologous recombination system, and PCR, was used. The deletion mutants were identified by sequencing. Walk Away 40 SI (Siemens) was used for biochemical identification and drug sensitivity test. Meanwhile, the growth rate and acid-alkali resistance of the strains were measured. The cytotoxicity test of Hela cells was carried out. When the cell confluence reached 80%, the bacteria were co-cultured with 100 渭 g / mL for 2 h and cultured with gentamicin (200 渭 g / mL) for 2 h, 3 h, 2 h, 3 h, respectively, under the microscope of Giemsa staining, and the cells were incubated with Gentamycin (200 渭 g / mL) for 2 h and 3 h, respectively. The number and morphology of the cells were changed at 5 h, and the cytotoxicity of bacteria and cells to 8 h was detected by lactate dehydrogenase test (LDH). Statistical analysis using SPSS14.0 statistical software package, the data expressed as mean 卤standard error, multiple groups of mean comparison using one-way analysis of variance, the comparison between the two groups using LSD test, P0.05 is statistically significant. Results: 1) 17 suspected Insert2 deletion positive strains were identified by PCR, and 17 and 21 deletion strains were obtained by sequencing. No strain with Insert1 deletion was obtained. 2) there was no difference in biochemical identification, acid-base resistance, growth rate and resistance to kanamycin between strain 81 before deletion of Insert2 and strain 17 after deletion except that kanamycin resistance was different between strain 81 and strain 17 after deletion, and no difference was found between strain No. 17 and strain No. 17 in kanamycin resistance, but no significant difference was found in the resistance to kanamycin. The kanamycin resistance of strain 81 was negative, indicating that the exogenous kanamycin resistance gene was successfully integrated into the bacterial chromosome by homologous recombination. 3) comparing the number and morphology of the cells between strain 81 and strain 17 under microscope after Gimsa staining, the results showed that the number of adherent cells of strain 81 decreased, the cell volume became larger and the nucleus became larger. 4) LDH test showed that the cell death rates of strain 81 and strain 17 were (13.2 卤9.5)% and (25.1 卤6.2)% respectively at 8 hours, and the number of deleted strains was significantly lower than that of wild strains (P0.05). Conclusion: 1) the new island Insert2. inserted in Escherichia coli pks island was successfully deleted by one-step deletion method. 2) the biochemical characteristics of Escherichia coli (strain 17) after deletion of Insert2 were not different from those of E. coli (strain 81) before deletion. Therefore, there are no genes related to bacterial biochemical characteristics in Insert2. 3) the deletions of the inserted Insert2 in the pks island of Escherichia coli reduced the cytotoxicity to the cells, and the Insert2 might contain new cytotoxic genes that had not been predicted previously.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378
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相关期刊论文 前2条
1 胡静,俞守义,阚飙,刘志华;缺失HPI毒力岛的EAggEC O42突变菌株的构建及鉴定[J];第一军医大学学报;2005年11期
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