绿色荧光蛋白标记的非抗性示踪载体的构建及初步应用
摘要
随着微生物学研究的不断深入，研究者们迫切需要一种可以直接在活组织或动物消化道的任何时候随时被检测到的细胞标记物(cell marker)，从而可跟踪活的受体菌株中的分子事件。绿色荧光蛋白基因相对于其它报告蛋白基因在原位、实时的微生物生理生化研究中有很多优越性，受到越来越广泛的关注。本研究应用绿色荧光蛋白基因作为报告基因，构建了非抗生素抗性的示踪表达载体pW425t-GFP，并将其转化大肠杆菌X13。成功获得表达绿色荧光蛋白基因的大肠杆菌pW425t-GFP，在无抗生素压力选择情况下，连续培养10代，绿色荧光蛋白在重组菌中能获得稳定表达。最后将上述重组菌pW425t-GFP口服接种BALB／c小鼠，观察其在消化道中的分布。具体研究内容如下：
首先根据质粒PGFP uv的绿色荧光蛋白基因序列设计了一对引物，以载体质粒为模板，应用聚合酶链式反应(PCR)技术，扩增了938bp的含有LacZ启动子的GFP全基因。通过T-A克隆技术，将PCR产物克隆至pGEM-T-Vector中，转化至受体菌JM109中，通过质粒提取、限制性内切酶酶切、PCR、序列测定等方法鉴定构建出重组阳性克隆质粒pGEM-T-GFP。经DNASTAR软件分析核苷酸序列，结果显示该基因与Genbank上纪录的GFP基因序列同源性达到100％。
然后应用基因体外重组技术，将ThyA／红霉素双抗性表达载体pW425et中的红霉素抗性基因替换为含有强启动子的GFP基因，构建了非抗生素抗性的示踪表达载体pW425t-GFP，通过质粒提取、限制性内切酶酶切、PCR等方法鉴定构建出的重组阳性表达载体pW425t-GFP，并通过SDS-PAGE电泳检测绿色荧光蛋白基因的表达量。经荧光显微镜观察，结果显示该表达载体能稳定的表达出绿色荧光蛋白，并能发出稳定的绿色荧光。在无抗生素压力选择情况下，连续培养10代，绿色荧光蛋白在重组菌中能获得稳定表达。
将上述获得稳定表达绿色荧光蛋白的重组菌给小鼠口服，观察该重组菌在胃肠道中的分布。重组菌口服接种4周龄BALB／c小鼠，于接种后2，6，12，24，48和72h．……分别取胃肠内容物接种培养并计数，同时取胃、空肠、回肠和盲肠做冷冻切片，在荧光显微镜下观察重组菌的分布。结果表明重组菌pW425t-GFP在接种小鼠6h后，胃肠道的内容物中可发现pW425t-GFP。在42h时细菌数量达到最大，并且3d后胃肠道中仍存在一定数量(约10~4CFU／g)的重组菌。表明该重组菌可较长时间的定植于肠黏膜表面。在荧光显微镜下可清晰的观察到pW425t-GFP主要存在于肠道的内容物中，少数细菌粘附在各肠段的黏膜上。本研究为该示踪载体的进一步应用提供了理论基础和实验模型。
Along with the research of microbiology developed deeply, the researchers needed badly a cell marker that could be detected directly in the living tissue or animal alimentary tract at anytime.Then it could track the molecule occurrence of receptor;.Green fluorescent protein(GFP) owned much superiority and got more and more attention compared with the other proteinum gene that reported in situ, real time of the research in microbion physiology biochemistry.This research was applyed GFP to report gene and constructed the non-antibiotic expression vector pW425t-GFP, It was transferred into E.coli X13.We successely got GFP expressed in the E.coli under non-antibiotic circumstances.After continuous cultare 10 generation, the GFP was expressed stabliy in the recombinant germ. At last, BALB/c mice were Vaccinated orally by the above-mentioned recombinant pW425t-GFP,then its distribution could be observed in the alimentary tract.The concrete research content is as follow:
First of all,it was needed to design a pair of primers on the basis of the GFP gene of vector PGFPuv base on the plasmid vector as templet.Applying the technique of PCR and amplifing 938 bp which included the whole GFP gene of LacZ promoter,the PCR production was cloned and transferred into pGEM-T-Vector, then it was inverted to recipient germ JM109 through T-A cloning technology. The method of T-A cloning technique, restriction enzyme correspond restriction endonuclease,PCR, sequencing and so on were used to identify the recombinant masculine cloning plasmid pGEM-T-GFP. The nucleotide sequence could be analyzed by DNASTAR software, the consequence manifest that this gene has 100% homology with the GFP gene order in the Genebangk' record.
Using the gene recombinant in vitro technique replace the gene of ThyA/illotycin dipl-antibiotic resistance expression vector pW425et to the GFP gene that contain the strong promoter, constructed non-antibiotic resistance tagging expression vector pW425t-GFP, The methods just as extract plasmid, restriction enzyme correspond restriction endonuclease,PCR and so on can be used to construct recombinant masculine cloning plasmid pW425t-GFP.The expression vector was analysed through SDS-PAGE to detect the expressed quantity of GFP gene. Undergoing the observation in the fluorescence microscope ,the consequence manifest this expression vector was expressed GFP steadily,and sent out green fluorescence steadily. Continuous cultare 10 generation under non-antibiotic selective pressure circumstances, GFP could be expressed steadily in the recombination germ.
Took above-mentioned recombination germ of GFP which could be expressed steadily to mice orally,and observed the distribution of this recombinant germ in the alimentary tract. After 4 weeks age, BALB/c mice were vaccinated with recombinant germ orally,got the gastrointestinal contents and inoculated culture and counted number at 2,6,12,24,48,72h,meanwhile got gaster, jejunum、ileum and appendix to cryostat section,then cryostat section wasput under the fluorescence microscope to observe the distribution of the recombinant germ. The consequence manifest the recombinant germ pW425t-GFP could be detected in the gastrointestinal after 6h vaccination. The bacterial number was the most at 42h,and the recombinant germ still existed at a certain quantity number (about10~4CFU/g). Manifest this recombinant germ could permanent planting in intestinal mucosa surface long time. pW425t-GFP can be observed limpidly under the fluorescence microscope .It resided in the enteric chief content, minority of those bacterium adherenced on each enteric mucosa, even a few bacterium could enter the enteric mucosa proper layer.This research provide rationale and experimental model for the next tagging vector.
首先根据质粒PGFP uv的绿色荧光蛋白基因序列设计了一对引物，以载体质粒为模板，应用聚合酶链式反应(PCR)技术，扩增了938bp的含有LacZ启动子的GFP全基因。通过T-A克隆技术，将PCR产物克隆至pGEM-T-Vector中，转化至受体菌JM109中，通过质粒提取、限制性内切酶酶切、PCR、序列测定等方法鉴定构建出重组阳性克隆质粒pGEM-T-GFP。经DNASTAR软件分析核苷酸序列，结果显示该基因与Genbank上纪录的GFP基因序列同源性达到100％。
然后应用基因体外重组技术，将ThyA／红霉素双抗性表达载体pW425et中的红霉素抗性基因替换为含有强启动子的GFP基因，构建了非抗生素抗性的示踪表达载体pW425t-GFP，通过质粒提取、限制性内切酶酶切、PCR等方法鉴定构建出的重组阳性表达载体pW425t-GFP，并通过SDS-PAGE电泳检测绿色荧光蛋白基因的表达量。经荧光显微镜观察，结果显示该表达载体能稳定的表达出绿色荧光蛋白，并能发出稳定的绿色荧光。在无抗生素压力选择情况下，连续培养10代，绿色荧光蛋白在重组菌中能获得稳定表达。
将上述获得稳定表达绿色荧光蛋白的重组菌给小鼠口服，观察该重组菌在胃肠道中的分布。重组菌口服接种4周龄BALB／c小鼠，于接种后2，6，12，24，48和72h．……分别取胃肠内容物接种培养并计数，同时取胃、空肠、回肠和盲肠做冷冻切片，在荧光显微镜下观察重组菌的分布。结果表明重组菌pW425t-GFP在接种小鼠6h后，胃肠道的内容物中可发现pW425t-GFP。在42h时细菌数量达到最大，并且3d后胃肠道中仍存在一定数量(约10~4CFU／g)的重组菌。表明该重组菌可较长时间的定植于肠黏膜表面。在荧光显微镜下可清晰的观察到pW425t-GFP主要存在于肠道的内容物中，少数细菌粘附在各肠段的黏膜上。本研究为该示踪载体的进一步应用提供了理论基础和实验模型。
Along with the research of microbiology developed deeply, the researchers needed badly a cell marker that could be detected directly in the living tissue or animal alimentary tract at anytime.Then it could track the molecule occurrence of receptor;.Green fluorescent protein(GFP) owned much superiority and got more and more attention compared with the other proteinum gene that reported in situ, real time of the research in microbion physiology biochemistry.This research was applyed GFP to report gene and constructed the non-antibiotic expression vector pW425t-GFP, It was transferred into E.coli X13.We successely got GFP expressed in the E.coli under non-antibiotic circumstances.After continuous cultare 10 generation, the GFP was expressed stabliy in the recombinant germ. At last, BALB/c mice were Vaccinated orally by the above-mentioned recombinant pW425t-GFP,then its distribution could be observed in the alimentary tract.The concrete research content is as follow:
First of all,it was needed to design a pair of primers on the basis of the GFP gene of vector PGFPuv base on the plasmid vector as templet.Applying the technique of PCR and amplifing 938 bp which included the whole GFP gene of LacZ promoter,the PCR production was cloned and transferred into pGEM-T-Vector, then it was inverted to recipient germ JM109 through T-A cloning technology. The method of T-A cloning technique, restriction enzyme correspond restriction endonuclease,PCR, sequencing and so on were used to identify the recombinant masculine cloning plasmid pGEM-T-GFP. The nucleotide sequence could be analyzed by DNASTAR software, the consequence manifest that this gene has 100% homology with the GFP gene order in the Genebangk' record.
Using the gene recombinant in vitro technique replace the gene of ThyA/illotycin dipl-antibiotic resistance expression vector pW425et to the GFP gene that contain the strong promoter, constructed non-antibiotic resistance tagging expression vector pW425t-GFP, The methods just as extract plasmid, restriction enzyme correspond restriction endonuclease,PCR and so on can be used to construct recombinant masculine cloning plasmid pW425t-GFP.The expression vector was analysed through SDS-PAGE to detect the expressed quantity of GFP gene. Undergoing the observation in the fluorescence microscope ,the consequence manifest this expression vector was expressed GFP steadily,and sent out green fluorescence steadily. Continuous cultare 10 generation under non-antibiotic selective pressure circumstances, GFP could be expressed steadily in the recombination germ.
Took above-mentioned recombination germ of GFP which could be expressed steadily to mice orally,and observed the distribution of this recombinant germ in the alimentary tract. After 4 weeks age, BALB/c mice were vaccinated with recombinant germ orally,got the gastrointestinal contents and inoculated culture and counted number at 2,6,12,24,48,72h,meanwhile got gaster, jejunum、ileum and appendix to cryostat section,then cryostat section wasput under the fluorescence microscope to observe the distribution of the recombinant germ. The consequence manifest the recombinant germ pW425t-GFP could be detected in the gastrointestinal after 6h vaccination. The bacterial number was the most at 42h,and the recombinant germ still existed at a certain quantity number (about10~4CFU/g). Manifest this recombinant germ could permanent planting in intestinal mucosa surface long time. pW425t-GFP can be observed limpidly under the fluorescence microscope .It resided in the enteric chief content, minority of those bacterium adherenced on each enteric mucosa, even a few bacterium could enter the enteric mucosa proper layer.This research provide rationale and experimental model for the next tagging vector.
引文
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