利用Cre-loxP系统构建变链菌无标记的htrA、clpP单基因缺陷株及htrA和clpP双基因缺陷株

发布时间：2018-01-02 10:29

  本文关键词：利用Cre-loxP系统构建变链菌无标记的htrA、clpP单基因缺陷株及htrA和clpP双基因缺陷株　出处：《天津医科大学》2011年硕士论文　论文类型：学位论文

  更多相关文章： 变异链球菌 htrA基因 clpP基因 Cre-loxP位点特异性重组系统 HtrA ClpP 无标记基因缺陷 突变

【摘要】：目的：将同源重组技术与Cre-lOxP位点特异性重组系统结合起来,在变链菌中构建htrA基因缺陷株和clpP基因缺陷株,并分别删除其抗生素抗性标记,获得无标记的单基因突变株,为构建无标记的基因缺陷株奠定基础。利用以Cre-loxP系统为基础的Cre-loxP*系统构建变链菌htrA和clpP双基因缺陷株,并删除抗生素抗性标记,获得无标记的双基因突变株,为构建无标记的多基因缺陷株提供一种新的方法。这三种缺陷株也将为进一步研究htrA基因和clpP基因在变链菌致龋过程中的作用提供实验菌株。 方法：1.设计引物PCR扩增卡那霉素(Km)抗性基因,使loxP位点位于Km抗性基因的两侧,构建出Km抗性基因盒(loxP-Km-lOxP).PCR扩增变链菌的htrA基因片段并克隆到pGEM-T-Easy TA载体后,双酶切以去除htrA基因的部分序列,并连入lOxP-Km-loxP,构建出htrA基因缺陷的同源重组载体pIB△htrA-Km。将该质粒线性化并电转化变链菌标准株,Km抗性筛选出发生同源重组的菌株,即htrA基因缺陷株。2.设计引物PCR扩增大观霉素(Sp)抗性基因,使loxP位点位于Sp抗性基因的两侧,构建出Sp抗性基因盒(loP-Sp-loxP)。PCR扩增变链菌的clpP基因片段并克隆到pGEM-T-Easy TA载体后,双酶切以去除clpP基因的部分序列,并连入lOxP-Sp-loxP,得到clpP基因缺陷的同源重组载体pIB△clpP-Sp。将该质粒线性化并电转化变链菌标准株,Sp抗性筛选出发生同源重组的菌株,即clpP基因缺陷株。3.以热敏质粒pCrePA分别电转化htrA基因缺陷株和clpP基因缺陷株,分别删除Km抗性标记和Sp抗性标记,改变温度培养以消除质粒pCrePA,分别获得无标记的htrA.clpP单基因缺陷株,并经PCR及DNA测序鉴定。4.按照上述方法构建出lOx71-Km-lox66和lox71-Sp-lox66,分别替换loxP-Km-loxP和loxP-Sp-loxP。利用上述方法构建无标记的htrA基因缺陷株,继而在该缺陷株中删除clpP基因及Sp抗性标记,获得无标记的htrA和clpP双基因缺陷株,并进行PCR分析及DNA测序鉴定。 结果：1.PCR扩增得到的Km抗性基因、Sp抗性基因、htrA基因、clpP基因片段经琼脂糖凝胶电泳检测,可见扩增产物为清晰的单一条带,未见非特异性扩增,分子量大小与预期大小一致。2.经酶切鉴定目的质粒各片段插入无误,含有目的质粒的大肠杆菌可分别在Km抗性、Sp抗性的LB培养基中生长良好,Km抗性的htrA基因缺陷株可在含Km的TSA和TPY培养基中生长良好,Sp抗性的clpP基因缺陷株可在含Sp的TSA和TPY培养基中生长良好,这都说明Km抗性和Sp抗性基因都正确表达。3.对无标记基因缺陷株的目的区域进行PCR扩增及DNA测序鉴定,结果显示htrA和clpP基因的部分序列已被删除,且无抗性标记。在单基因缺陷株中目的区域只保留一个34bp的loxP位点,在多基因缺陷株中目的区域只保留一个34bp的lox72位点。 结论：本研究利用Cre-loxP位点特异性重组系统和同源重组技术成功构建出变链菌的无标记的单基因缺陷株(htrA基因缺陷株、clpP基因缺陷株)以及htrA和clpP双基因缺陷株,为构建无标记的多基因缺陷株提供了一种新的有效的方法,并为进一步研究htrA和clpP基因的功能提供了实验菌株。
[Abstract]:Objective: homologous recombination and Cre-lOxP site-specific recombination system combined in S.mutans to construct htrA mutant and clpP mutant, and delete the antibiotic resistance marker, single gene markers of the mutant strain, for the construction of the unmarked gene mutant construction variable chain foundation. Strains htrA and clpP double mutant by Cre-loxP* system based on Cre-loxP system, and delete the antibiotic resistance marker gene, double labeled mutant, for the construction of multiple genetic defects without marking lines provide a new method. These three kinds of defects were also for the further study of htrA gene and clpP genes in the cariogenicity of role in the process of providing experimental strains.
Methods: 1. PCR primers amplified the kanamycin resistance gene (Km), on both sides of the loxP site in the Km resistance gene, construct the Km resistance gene cassette (loxP-Km-lOxP).PCR amplification of S.mutans htrA gene fragment was cloned into pGEM-T-Easy TA vector after double enzyme digestion to remove part of the sequence of htrA gene. And even into lOxP-Km-loxP, constructed the htrA gene defect of homologous recombinant vector pIB htrA-Km. the plasmid was linearized and electroporated into Streptococcus mutans standard strains, Km resistance were screened for homologous recombination strains, namely htrA gene deficient strain.2. amplified by PCR spectinomycin resistance gene (Sp), the loxP site on both sides located in the Sp resistance gene, construct the Sp resistance gene cassette (loP-Sp-loxP).PCR amplification of S.mutans clpP gene fragment was cloned into pGEM-T-Easy TA vector after double enzyme digestion to remove part of the sequence of clpP gene, and cloned into lOxP-Sp-loxP, clpP A defective gene homologous recombinant vector pIB clpP-Sp. the plasmid was linearized and electroporated into Streptococcus mutans standard strains, Sp resistance were screened for homologous recombination strains, namely clpP gene deficient strain.3. with sensitive plasmid pCrePA were electrotransformed into htrA mutant and clpP mutant, respectively, delete the Km resistance marker and Sp resistance mark, change the culture temperature to eliminate plasmid pCrePA, respectively obtaining marker free htrA.clpP single mutant, and by PCR and DNA sequencing of.4. according to the above method to construct lOx71-Km-lox66 and lox71-Sp-lox66, respectively, to replace loxP-Km-loxP and loxP-Sp-loxP. to construct the unmarked htrA mutant using the above method, and then delete the clpP gene and Sp resistance marker in the defects were obtained, labeled htrA and clpP double mutant, and the PCR analysis and DNA sequencing.
Results: Km gene was amplified by 1.PCR, Sp resistance gene, htrA gene, clpP gene fragment by agarose gel electrophoresis, the amplified product was a single band was clear, no non-specific amplification, plasmid molecular weight was consistent with the expected size of.2. by enzyme digestion. The fragment was inserted into the correct, containing purpose the plasmid of Escherichia coli respectively in Km resistance, Sp resistance in LB medium grew well, Km resistant htrA mutant in Km containing TSA and TPY medium grew well, Sp resistant clpP mutant in Sp containing TSA and TPY medium grew well, which are Km resistance and Sp resistance genes are correct expression of.3. was amplified by PCR and DNA sequencing of the region without marker gene mutant, results show that the partial sequence of htrA gene and clpP gene has been deleted, and no resistance markers in single gene mutant. Only one 34bp loxP site is retained in the target region, and only one 34bp lox72 site is retained in the target region of the polygenic strain.
Conclusion: This study uses the Cre-loxP site-specific recombination system and homologous recombination technique successfully constructed S.mutans unlabeled single mutant (htrA mutant, clpP mutant) and htrA and clpP double mutant, which provides a new and effective method for constructing gene marker the defect of strains, and provides experimental strains for further study of htrA and clpP genes.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

【参考文献】

相关期刊论文 前7条

1 徐赤宇;温海;王溪涛;朱红梅;顾菊林;徐红;;细胞芯片检测新生隐球菌菌株丝氨酸蛋白酶的表达[J];第二军医大学学报;2006年02期

2 张群;胥文春;尹一兵;朱兴华;李有强;张雪梅;;clpP基因缺陷对肺炎链球菌毒力表达降低的研究[J];第三军医大学学报;2009年04期

3 黄远帅;许颂霄;朱旦;阳强;刘明芳;陈淑惠;张雪梅;尹一兵;;HtrA基因缺陷对肺炎链球菌毒力影响的研究[J];第四军医大学学报;2006年17期

4 季星来,孙之荣;基于结构的丝氨酸蛋白酶超家族进化分析[J];电子学报;2001年S1期

5 张龙翔;;丝氨酸蛋白酶的蛋白质工程[J];生命的化学(中国生物化学会通讯);1992年02期

6 庞永奇,贾洪革,方荣祥,郭蔼光,陈晓英;利用不相容质粒共转化大肠杆菌对Cre重组酶体内重组活性的可视检测[J];微生物学报;2005年01期

7 陈晓翔;杨程德;顾越英;;纤溶酶功能研究进展[J];诊断学理论与实践;2005年05期

，

本文编号：1368831