利用重組工程技術(shù)標記鼠疫菌染色體基因
發(fā)布時間：2019-05-15 05:44
【摘要】：背景：鼠疫是由鼠疫耶爾森氏菌(簡稱鼠疫菌)引起的自然疫源性烈性傳染病，可導致腺鼠疫和肺鼠疫。在人類歷史上因鼠疫而死亡的人數(shù)達數(shù)以億計。目前，鼠疫雖得到有效地控制，但鼠疫菌作為最為烈性的生物戰(zhàn)劑和生物恐怖劑為恐怖組織所利用的可能性仍然存在。隨著對多種微生物基因組測序工作的完成，經(jīng)典的遺傳學研究方式也在發(fā)生變化�；谥亟M工程技術(shù)的一步法突變和標記染色體基因已成為研究基因功能的重要手段。為加快研究鼠疫菌染色體基因及其產(chǎn)物的功能，我們將重組工程技術(shù)應(yīng)用于標記鼠疫菌染色體基因。 方法：將標簽序列合成于引物中，通過融合PCR的方法將標簽序列與卡那霉素或氯霉素抗性篩選基因連接構(gòu)建融合模塊，再將標簽和抗性基因的融合模塊克隆到pBluescript載體，獲得模板載體。根據(jù)染色體目的基因的末端序列及其下游序列設(shè)計帶短同源臂引物(40-50bp)，從模板載體上PCR擴增染色體目的基因的重組標簽盒。將標簽盒電轉(zhuǎn)化進入表達Red重組系統(tǒng)的鼠疫菌感受態(tài)細胞，經(jīng)同源重組表位標簽融合到目的基因的下游，由此產(chǎn)生的C末端標記的融合蛋白可經(jīng)標準的免疫學方法檢測。在本研究中，我們采用的表位標簽是Myc和6×His組和標簽，稱為MH標簽，所采用的抗性篩選標記是卡那霉素基因，標記的是鼠疫菌染色體rpoS基因(sigam因子)。重組標記克隆進行DNA序列分析，C末端標記的融合蛋白的表達通過針對His標簽的單克隆抗體進行Western blot驗證。 結(jié)果：Myc-His組合標簽和卡那霉素抗性篩選基因的PCR融合產(chǎn)物成功的克隆到pBluescript載體，獲得模板載體pbluescript-MH。以該質(zhì)粒作為模板，擴增標記標簽盒，，對鼠疫菌的rpoS基因進行了標記，利用針對組氨酸標簽的抗體能夠檢測到rpoS基因的表達。 結(jié)論：基于重組工程的基因標記技術(shù)易操作，檢測目的蛋白表達靈敏度高，利用標簽的特性可以為鼠疫菌及其他細菌基因功能研究提供一個有價值的研究手段。
[Abstract]:Background: plague is a natural epidemic severe infectious disease caused by Yersinia pestis (Yersinia pestis), which can lead to gland plague and pulmonary plague. Hundreds of millions of people have died from plague in human history. At present, although plague is effectively controlled, the possibility that plague bacteria can be used by terrorist organizations as the most powerful biological warfare agent and bioterrorism agent still exists. With the completion of genome sequencing of a variety of microorganisms, the classical genetic research methods are also changing. One-step mutation and marker chromosome gene based on recombinant engineering technology has become an important means to study gene function. In order to speed up the study of the function of Yersinia pestis chromosome gene and its products, we applied the recombinant engineering technique to label the chromosome gene of Yersinia pestis. Methods: the tag sequence was synthesized into primers, and the tag sequence was ligated with kanamycin or chloramphenicol resistance screening gene by fusion PCR to construct the fusion module, and then the fusion module of label and resistance gene was cloned into pBluescript vector. The template carrier is obtained. According to the terminal sequence of chromosome target gene and its downstream sequence, short homologue primers (40-50bp) were designed to amplify the recombinant label box of chromosome target gene from template vector by PCR. The label box was electrotransformed into Yersinia pestis cells expressing Red recombinant system and fused to the downstream of the target gene by homologous recombinant epitope tag. The resulting C-terminal labeled fusion protein could be detected by standard immunological method. In this study, the epitope tags used were Myc and 6 脳 His groups and tags, called MH tags. The resistance screening markers used were kanamycin gene and Yersinia pestis chromosome rpoS gene (sigam factor). The expression of C-terminal labeled fusion protein was confirmed by Western blot with monoclonal antibody against His tag. Results: the PCR fusion product of Myc-His combination tag and kanamycin resistance screening gene was successfully cloned into pBluescript vector, and the template vector pbluescript-MH. was obtained. Using the plasmid as template, the tag box was amplified to mark the rpoS gene of Yersinia pestis. The expression of rpoS gene could be detected by using the antibody against histidine label. Conclusion: the gene labeling technique based on recombinant engineering is easy to operate and has high sensitivity to detect the expression of the target protein. The characteristics of the tag can provide a valuable research method for the study of gene function of Yersinia pestis and other bacteria.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R346
本文編號：2477358
[Abstract]:Background: plague is a natural epidemic severe infectious disease caused by Yersinia pestis (Yersinia pestis), which can lead to gland plague and pulmonary plague. Hundreds of millions of people have died from plague in human history. At present, although plague is effectively controlled, the possibility that plague bacteria can be used by terrorist organizations as the most powerful biological warfare agent and bioterrorism agent still exists. With the completion of genome sequencing of a variety of microorganisms, the classical genetic research methods are also changing. One-step mutation and marker chromosome gene based on recombinant engineering technology has become an important means to study gene function. In order to speed up the study of the function of Yersinia pestis chromosome gene and its products, we applied the recombinant engineering technique to label the chromosome gene of Yersinia pestis. Methods: the tag sequence was synthesized into primers, and the tag sequence was ligated with kanamycin or chloramphenicol resistance screening gene by fusion PCR to construct the fusion module, and then the fusion module of label and resistance gene was cloned into pBluescript vector. The template carrier is obtained. According to the terminal sequence of chromosome target gene and its downstream sequence, short homologue primers (40-50bp) were designed to amplify the recombinant label box of chromosome target gene from template vector by PCR. The label box was electrotransformed into Yersinia pestis cells expressing Red recombinant system and fused to the downstream of the target gene by homologous recombinant epitope tag. The resulting C-terminal labeled fusion protein could be detected by standard immunological method. In this study, the epitope tags used were Myc and 6 脳 His groups and tags, called MH tags. The resistance screening markers used were kanamycin gene and Yersinia pestis chromosome rpoS gene (sigam factor). The expression of C-terminal labeled fusion protein was confirmed by Western blot with monoclonal antibody against His tag. Results: the PCR fusion product of Myc-His combination tag and kanamycin resistance screening gene was successfully cloned into pBluescript vector, and the template vector pbluescript-MH. was obtained. Using the plasmid as template, the tag box was amplified to mark the rpoS gene of Yersinia pestis. The expression of rpoS gene could be detected by using the antibody against histidine label. Conclusion: the gene labeling technique based on recombinant engineering is easy to operate and has high sensitivity to detect the expression of the target protein. The characteristics of the tag can provide a valuable research method for the study of gene function of Yersinia pestis and other bacteria.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R346
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