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  本文關(guān)鍵詞：利用Cre-loxP系統(tǒng)構(gòu)建變鏈菌無標(biāo)記的htrA、clpP單基因缺陷株及htrA和clpP雙基因缺陷株　出處：《天津醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 變異鏈球菌 htrA基因 clpP基因 Cre-loxP位點(diǎn)特異性重組系統(tǒng) HtrA ClpP 無標(biāo)記基因缺陷 突變

【摘要】：目的：將同源重組技術(shù)與Cre-lOxP位點(diǎn)特異性重組系統(tǒng)結(jié)合起來,在變鏈菌中構(gòu)建htrA基因缺陷株和clpP基因缺陷株,并分別刪除其抗生素抗性標(biāo)記,獲得無標(biāo)記的單基因突變株,為構(gòu)建無標(biāo)記的基因缺陷株奠定基礎(chǔ)。利用以Cre-loxP系統(tǒng)為基礎(chǔ)的Cre-loxP*系統(tǒng)構(gòu)建變鏈菌htrA和clpP雙基因缺陷株,并刪除抗生素抗性標(biāo)記,獲得無標(biāo)記的雙基因突變株,為構(gòu)建無標(biāo)記的多基因缺陷株提供一種新的方法。這三種缺陷株也將為進(jìn)一步研究htrA基因和clpP基因在變鏈菌致齲過程中的作用提供實(shí)驗(yàn)菌株。 方法：1.設(shè)計(jì)引物PCR擴(kuò)增卡那霉素(Km)抗性基因,使loxP位點(diǎn)位于Km抗性基因的兩側(cè),構(gòu)建出Km抗性基因盒(loxP-Km-lOxP).PCR擴(kuò)增變鏈菌的htrA基因片段并克隆到pGEM-T-Easy TA載體后,雙酶切以去除htrA基因的部分序列,并連入lOxP-Km-loxP,構(gòu)建出htrA基因缺陷的同源重組載體pIB△htrA-Km。將該質(zhì)粒線性化并電轉(zhuǎn)化變鏈菌標(biāo)準(zhǔn)株,Km抗性篩選出發(fā)生同源重組的菌株,即htrA基因缺陷株。2.設(shè)計(jì)引物PCR擴(kuò)增大觀霉素(Sp)抗性基因,使loxP位點(diǎn)位于Sp抗性基因的兩側(cè),構(gòu)建出Sp抗性基因盒(loP-Sp-loxP)。PCR擴(kuò)增變鏈菌的clpP基因片段并克隆到pGEM-T-Easy TA載體后,雙酶切以去除clpP基因的部分序列,并連入lOxP-Sp-loxP,得到clpP基因缺陷的同源重組載體pIB△clpP-Sp。將該質(zhì)粒線性化并電轉(zhuǎn)化變鏈菌標(biāo)準(zhǔn)株,Sp抗性篩選出發(fā)生同源重組的菌株,即clpP基因缺陷株。3.以熱敏質(zhì)粒pCrePA分別電轉(zhuǎn)化htrA基因缺陷株和clpP基因缺陷株,分別刪除Km抗性標(biāo)記和Sp抗性標(biāo)記,改變溫度培養(yǎng)以消除質(zhì)粒pCrePA,分別獲得無標(biāo)記的htrA.clpP單基因缺陷株,并經(jīng)PCR及DNA測序鑒定。4.按照上述方法構(gòu)建出lOx71-Km-lox66和lox71-Sp-lox66,分別替換loxP-Km-loxP和loxP-Sp-loxP。利用上述方法構(gòu)建無標(biāo)記的htrA基因缺陷株,繼而在該缺陷株中刪除clpP基因及Sp抗性標(biāo)記,獲得無標(biāo)記的htrA和clpP雙基因缺陷株,并進(jìn)行PCR分析及DNA測序鑒定。 結(jié)果：1.PCR擴(kuò)增得到的Km抗性基因、Sp抗性基因、htrA基因、clpP基因片段經(jīng)瓊脂糖凝膠電泳檢測,可見擴(kuò)增產(chǎn)物為清晰的單一條帶,未見非特異性擴(kuò)增,分子量大小與預(yù)期大小一致。2.經(jīng)酶切鑒定目的質(zhì)粒各片段插入無誤,含有目的質(zhì)粒的大腸桿菌可分別在Km抗性、Sp抗性的LB培養(yǎng)基中生長良好,Km抗性的htrA基因缺陷株可在含Km的TSA和TPY培養(yǎng)基中生長良好,Sp抗性的clpP基因缺陷株可在含Sp的TSA和TPY培養(yǎng)基中生長良好,這都說明Km抗性和Sp抗性基因都正確表達(dá)。3.對(duì)無標(biāo)記基因缺陷株的目的區(qū)域進(jìn)行PCR擴(kuò)增及DNA測序鑒定,結(jié)果顯示htrA和clpP基因的部分序列已被刪除,且無抗性標(biāo)記。在單基因缺陷株中目的區(qū)域只保留一個(gè)34bp的loxP位點(diǎn),在多基因缺陷株中目的區(qū)域只保留一個(gè)34bp的lox72位點(diǎn)。 結(jié)論：本研究利用Cre-loxP位點(diǎn)特異性重組系統(tǒng)和同源重組技術(shù)成功構(gòu)建出變鏈菌的無標(biāo)記的單基因缺陷株(htrA基因缺陷株、clpP基因缺陷株)以及htrA和clpP雙基因缺陷株,為構(gòu)建無標(biāo)記的多基因缺陷株提供了一種新的有效的方法,并為進(jìn)一步研究htrA和clpP基因的功能提供了實(shí)驗(yàn)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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346

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