WEI Qu-Hao, E-mail:
整合子在细菌耐药性的获得及传播中占据重要地位,对于整合反应检测方法的改良及反应机制的研究,可以加深我们对细菌耐药性产生和播散的理解,为遏制耐药菌株的产生和播散提供新的途径。
在细菌染色体上构建第1类整合子反应模型,用于评价整合酶介导的基因盒位点特异性重组。
采用PCR分别扩增含氯霉素耐药基因
构建的整合子模型片段经测序与预期一致,整合子模型片段成功插入大肠埃希菌JM109染色体中。转入高表达整合酶的质粒pHSint后,在链霉素平板上成功筛选出基因盒
在大肠埃希菌染色体上成功构建第1类整合酶介导基因盒位点特异性重组反应模型,为进一步揭示整合子捕获耐药性基因盒的反应机制奠定基础。
Integron plays important roles in acquisition and spread of antibiotic resistance among bacteria. The research on the improvement of detection methods and reaction mechanisms of integron integration reaction can deepen the understanding of the integron's contribution in antibiotic resistance acquisition and spread, and provide new ways in suppression of the emergence and spread of the resistant strains.
To construct class 1 integron reaction model on bacterial chromosome to evaluate integrase-mediated gene cassette site-specific recombination.
The CM fragment containing chloramphenicol resistance gene
The sequencing results of the constructed fragment of integron reaction model were identity with that of expected. The constructed fragment was successfully knocked into the chromosome of
Reaction model of class 1 integron integrase-mediated gene cassette site-specific recombination was successfully constructed on
随着抗菌药物在医疗、农业及水产养殖业的广泛使用,形成了抗菌药物的选择压力,造成耐药菌株不断出现,如耐碳青酶烯类肠杆菌、耐万古霉素肠球菌、耐甲氧西林金黄色葡萄球菌等检出率逐年升高[
整合子根据整合酶蛋白一级结构的不同进行分类,其中第1类整合子在临床菌株中分布最为广泛[
大肠埃希菌JM109工程菌株、质粒pACYC184由本实验室保存,质粒pHSint和pLACaadA5C由复旦大学附属华山医院检验医学科构建,本实验室保存[
PrimeSTAR HS DNA聚合酶、
寡核苷酸引物序列
Sequences of oligonucleotide primers
名称 | 序列 | 靶序列 |
USF800 | ATTGCAGGTAGGCATCGGAA | US片段上游 |
USF | AACTGACGTAGTACGCGAGA | US片段 |
USR | TATGTCGTTGGCAAAAGCGT | |
CMF | ACGCTTTTGCCAACGACATATTAAGGGCACCAATAAC | CM片段 |
CMR | CTGATGCCGCATAGTTAAGCCCTGTGACGGAAGATCAC | |
LacA5CF | GGCTTAACTATGCGGCATCAG | LacA5片段 |
LacA5CR | GTTGCAGGGCAGTTTGCCCCAATACGCAAACCGCCTC | |
PcSF | GCAAACTGCCCTGCAAC | PcS片段 |
PcSR | GACGATGCGTGGAGACC | |
DSF | GGTCTCCACGCATCGTCTCAGGAAAAGTAGTTCAACA | DS片段 |
DSR | CTAACCGCAGCCAGTTCTTC |
使用柱式细菌基因组DNA抽提试剂盒抽提大肠埃希菌JM109基因组DNA作为模板,使用PrimeSTAR HS DNA聚合酶,用引物USF和USR扩增插入位点上游500 bp同源臂,命名为US片段;以JM109基因组DNA为模板,用引物DSF和DSR扩增插入位点下游500 bp同源臂,命名为DS片段;以质粒pACYC184为模板,用引物CMF和CMR扩增
整合子反应模型的结构及整合反应示意图
The structure of the integration reaction model and the schematic diagram of the integration reaction
将上述US片段、CM片段、LacA5片段、PcS片段和DS片段等摩尔比混合作为模板,用引物USF和DSR,通过重叠延伸PCR扩增出上述5个片段的连接体。PCR反应体系:5×PrimeSTAR缓冲液10 μL,dNTPs (2.5 μmol/L) 6 μL,引物USF和DSR (10 μmol/L)各1 μL,混合模板(约200 ng/μL) 1 μL,PrimeSTAR HS DNA聚合酶(2.5 U/μL) 0.5 μL,纯水(HPLC级) 30.5 μL,共50 μL。PCR反应条件:94 ℃ 4 min;98 ℃ 10 s,55 ℃ 5 s,72 ℃ 4 min,共30个循环;72 ℃ 5 min。扩增的连接体片段命名为UCAPSD片段,即为打靶片段(
先将pKOBEG质粒(阿普霉素抗性)转化入大肠埃希菌JM109中,制备经30 mmol/L阿拉伯糖诱导的大肠埃希菌JM109电转化感受态细菌,通过电转化,将上述打靶片段电转入携带pKOBEG质粒的大肠埃希菌JM109中,用含25 μg/mL的氯霉素LB琼脂平板筛选插入成功菌株。
以上述氯霉素平板上筛选出的单克隆菌株基因组DNA为模板,用插入位点上游800 bp
将上述插入成功的模型菌株CLJM109Ⅰ制备成为感受态细菌,转入高表达第1类整合酶的质粒pHSint (氨苄西林抗性)。将携带pHSint质粒的CLJM109Ⅰ细菌单克隆接种至100 μg/mL氨苄西林、25 μg/mL氯霉素和0.1 mmol/L IPTG的LB液体培养基中,37 ℃、200 r/min振荡培养过夜后,涂布于含30 μg/mL链霉素的LB平板上,37 ℃培养过夜,筛选发生整合的菌株。
随机选取上述链霉素平板上生长的单克隆菌株,抽取细菌基因组DNA作为模板,用引物PcSF和PcSR进行PCR扩增,同时以含pUC19质粒的CLJM109Ⅰ菌株基因组DNA作为阴性对照。由于
PCR成功扩增出CM片段、LacA5片段、PcS片段和两侧同源臂,经重叠延伸PCR成功扩增出其融合片段,PCR产物长度约为4 000 bp,经测序验证,片段大小和序列与理论一致,UCAPSD打靶片段构建成功。
以氯霉素平板上筛选的待验证菌株CLJM109Ⅰ基因组DNA为模板,以引物USF800和CMR进行PCR扩增,成功扩增出长度约为1 700 bp的产物,而相应的对照大肠埃希菌JM109基因组DNA无PCR产物条带(
插入菌株验证PCR结果
Identification of knock-in strains by PCR
以链霉素平板中筛选出的14株待验证发生整合菌株基因组DNA为模板,使用引物PcSF和PcSR进行扩增,PCR鉴定结果如
整合菌株的PCR鉴定
Identification of integrated strains by PCR
整合菌株测序鉴定
Identification of integrated strains by sequencing
整合子因其在细菌耐药性产生和播散中的重要作用而受到研究者的关注,目前关于整合子的研究主要集中在整合子可变区基因盒分子流行病学研究、整合酶与重组位点
通过位点特异性重组对基因盒进行整合和剪切是整合子最重要的特征。研究者尝试采用不同的方法来评估整合子整合和剪切基因盒的效率,国外研究者通过接合试验和表型筛选的方法计算基因盒整合频率,该方法费时费力,需要2−3 d时间[
上述研究者构建的整合子模型均位于质粒上,由于质粒并非单拷贝,单个菌体中,所有质粒的整合和剪切并非同步进行,同时除了整合酶介导的位点特异性重组外,质粒之间的同源重组、质粒拷贝数的变化等均影响基因盒整合频率或剪切频率的检测。本研究中,我们采用同源重组的方法将构建的指示基因盒剪切与整合的模型片段成功插入大肠埃希菌JM109工程菌
本研究中插入细菌染色体的LacA5片段来源于前期的乳头状试验中所用的质粒pLACaadA5C,基因盒
近年来,整合子捕获与表达耐药性基因盒调控机制方面的研究虽然取得了一定的进展,但其具体的反应机制仍未明了。本研究的另一个目的是研究整合子捕获耐药性基因盒的具体反应过程,我们将第1类整合酶剪切与整合耐药性基因盒这一复杂的反应过程简化为耐药基因盒是否必须先剪切下来才能发生整合,将其反应过程分为“剪切-粘贴”模式(基因盒先剪切下来再整合入
本研究构建的染色体反应模型也有需要改进的地方,本模型中待整合的基因盒与
总之,我们通过同源重组的方法在大肠埃希菌染色体上成功构建了第1类整合子捕获耐药性基因盒反应模型,该反应模型排除了质粒多拷贝的干扰,一个细菌就是一个反应容器,代表一个分子的反应,为进一步揭示整合子捕获耐药性基因盒的反应机制奠定了基础。
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