大腸桿菌pks島中插入島的缺失以及插入島生物學(xué)活性初步研究
發(fā)布時(shí)間：2019-03-07 19:17
【摘要】： 目的: 前期的研究表明在213株非五類腸道致病性大腸桿菌中檢測(cè)到7株pks島陽性菌株,在3株分子分型一致的pks島陽性菌株中發(fā)現(xiàn)在pks島中有兩個(gè)新的插入島(Insert1和Insert2),經(jīng)PCR、測(cè)序和Southern blot證實(shí),Insert1和Insert2大小分別約為90kb和13kb。功能預(yù)測(cè)Insert1主要與耶爾森桿菌素的合成、轉(zhuǎn)運(yùn)和調(diào)節(jié)有關(guān),Insert2主要與磷酸戊糖通路和藥物抗性有關(guān)。為初步探討插入島的基因功能,我們以其中的81號(hào)菌作為目標(biāo)菌株,在其基因組中試圖缺失Insert1和Insert2,并研究缺失后菌株的生化特性和細(xì)胞毒性。 方法: 采用一步缺失法-λRed同源重組系統(tǒng)構(gòu)建缺失突變株的方法對(duì)大腸桿菌pks島陽性菌株中兩個(gè)插入片段(Insert1和Insert2)進(jìn)行缺失突變,并采用PCR、測(cè)序?qū)θ笔蛔冎赀M(jìn)行鑒定。對(duì)缺失成功的菌株采用細(xì)菌生化鑒定儀Walk Away 40 SI(Siemens)進(jìn)行生化鑒定和藥敏試驗(yàn),同時(shí),測(cè)定它們的生長(zhǎng)速度和酸堿抗性。采用Hela細(xì)胞進(jìn)行細(xì)胞毒性試驗(yàn),當(dāng)細(xì)胞匯合度達(dá)到80%時(shí),細(xì)菌與細(xì)胞100:1共育2h,加入慶大霉素(200μg/mL)培養(yǎng),采用吉姆薩染色纖維鏡下觀察細(xì)菌與細(xì)胞作用后2h、3h、5h的細(xì)胞數(shù)量和形態(tài)變化,同時(shí),采用乳酸脫氫酶試驗(yàn)(LDH)檢測(cè)細(xì)菌與細(xì)胞作用8h對(duì)細(xì)胞的毒性。 統(tǒng)計(jì)學(xué)分析采用SPSS14.0統(tǒng)計(jì)軟件包,數(shù)據(jù)表示為均數(shù)±標(biāo)準(zhǔn)誤,多組之間的均數(shù)比較采用單因素方差分析,兩組之間的均數(shù)比較采用LSD檢驗(yàn),P0.05為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果: 1)利用PCR鑒定初篩到17個(gè)Insert2缺失疑似陽性菌株,經(jīng)測(cè)序驗(yàn)證得到17、21號(hào)兩個(gè)缺失株。沒有得到Insert1缺失的菌株。 2) Insert2缺失前的81號(hào)菌與缺失后的17號(hào)菌除了卡那霉素抗性不同外,它們的生化鑒定、酸堿抗性、生長(zhǎng)速度以及耐藥性均沒有區(qū)別。17號(hào)菌呈卡那霉素抗性陽性,而81號(hào)菌呈卡那霉素抗性陰性,說明同源重組成功將外源性的卡那霉素抗性基因整合到細(xì)菌染色體上。 3)吉姆薩染色后顯微鏡下比較81號(hào)菌與17號(hào)菌作用后的細(xì)胞數(shù)目和形態(tài),顯示81號(hào)菌作用的細(xì)胞貼壁細(xì)胞減少,細(xì)胞體積變大,細(xì)胞核變大。 4) LDH試驗(yàn)結(jié)果顯示81號(hào)菌和17號(hào)菌在8小時(shí)的細(xì)胞死亡率分別為(13.2±9.5)%、(25.1±6.2)%,缺失株顯著小于野生株(P0.05)。 結(jié)論: 1)利用一步缺失法成功缺失了大腸桿菌pks島中插入的新島Insert2。 2)缺失Insert2后的大腸桿菌(17號(hào)菌)生化特性與缺失前的大腸桿菌(81號(hào)菌)沒有差別。因此,Insert2中沒有與細(xì)菌生化特性相關(guān)的基因。 3)大腸桿菌pks島中插入的Insert2缺失后對(duì)細(xì)胞的毒性降低,Insert2中可能含有以前沒有預(yù)測(cè)到的新的細(xì)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 鹵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 鹵9.5)% and (25.1 鹵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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：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 鹵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 鹵9.5)% and (25.1 鹵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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R378
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 胡靜,俞守義,闞飆,劉志華;缺失HPI毒力島的EAggEC O42突變菌株的構(gòu)建及鑒定[J];第一軍醫(yī)大學(xué)學(xué)報(bào);2005年11期
2 王恒，
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