健康人粪便乳杆菌的分离、鉴定及遗传多态性的初步研究

发布时间：2018-09-07 19:15

【摘要】： 乳杆菌广泛存在于人和动物肠道、植物表面等生境中。近年来,人们从乳杆菌的形态学、生理学、分类学、遗传学、微生态学、生理功能及应用等诸多方面对其进行广泛而深入的研究。由于乳杆菌具有很多益生功能,国内外已有大量含有乳杆菌的益生菌食品、保健品、药品等产品问世,并受到消费者的普遍欢迎。为了更好的促进对乳杆菌的开发与利用,分离来源于人体肠道的乳杆菌更加具有实际应用价值,因为来源于人体的乳杆菌更加容易在人体肠道中定植。随着分子生物学的飞速发展及生物物理技术的应用,对微生物的鉴定方法已经从传统的形态学分析、生理生化试验发展到以PCR技术为基础的各种分子生物学方法。这些方法鉴定微生物不需要繁琐的微生物培养,通过设计的属及种的特异性引物或探针,可以很容易的将微生物鉴定到种的水平,利用分子标记技术甚至可以将微生物鉴定到株的水平。单纯的依靠传统生理生化鉴定方法对微生物进行鉴定,得出的结果不够可信,须与分子生物学方法相结合,才能对微生物进行准确的鉴定。本研究包括四方面内容: 1.乳杆菌的分离纯化以健康成人及学龄儿童的新鲜粪便为样品,经过稀释涂布于MRS培养基。通过形态学观察及H2O2酶、糖发酵等生理生化试验对所分得菌株进行初步鉴定,记录形态学特性、生理生化鉴定结果及糖发酵鉴定结果,得到乳杆菌56株。 2. 16S rDNA序列同源性分析(Sequence Homology Analysis)方法鉴定乳杆菌 16S rDNA序列同源性分析方法的具体步骤包括:乳杆菌基因组DNA的提取,PCR扩增、产物测序及同源性分析。将选择的20株试验菌株的测序结果与GenBank中该属内菌株的16S rDNA基因序列进行同源性分析,从而准确鉴定所分得菌株。最终得到嗜酸乳杆菌(Lactobacillus acidophilus)1株,卷曲乳杆菌(Lactobacillus crispatus)1株,食窦魏斯氏菌(Weissella. cibaria)1株,唾液乳杆菌唾液亚种(Lactobacillus salivarius subsp. salivarius)1株,黏液乳杆菌(Lactobacillus mucosae)2株,戊糖乳杆菌(Lactobacillus pentosus)1株,发酵乳杆菌(Lactobacillus fermentum)5株,植物乳杆菌(Lactobacillus plantarum)8株。 3.变性梯度凝胶电泳(Denaturing Gradient Gel Electrophoresis, DGGE)技术构建乳杆菌标准指纹图谱采用PCR-DGGE技术对试验菌株进行标准图谱的构建,选择基因组DNA的16S rDNA的V7-V8可变区序列进行PCR扩增,35~55%变形梯度凝胶电泳分离效果较好,观察指纹图谱发现,试验菌株大致分为7类。PCR-DGGE结果与鉴定结果不完全一致,说明DGGE方法也存在一定的局限性。 4.扩增片段长度多态性(Amplified Fragment Length Polymorphic DNA, AFLP)技术初步研究乳杆菌的遗传多态性从64对随机引物中筛选出来的1对多态性较好的引物用来分析乳杆菌属种间的遗传多态性。反复试验证明AFLP指纹图谱重复性好,准确度高,所得的聚丙烯酰胺凝胶电泳图显示出乳杆菌属内相同种不同菌株之间的遗传多态性,说明AFLP技术可作为乳杆菌同种不同菌株之间多态性研究的依据。本试验针对健康人的肠道菌群,采用传统生理生化方法与分子生物学方法相结合,对20株分离自健康人新鲜粪便中的乳杆菌进行鉴定到种的水平,通过变性梯度凝胶电泳(DGGE)方法构建了试验乳杆菌标准指纹图谱,并利用扩增片段长度多态性(AFLP)技术初步研究了不同乳杆菌菌株之间的遗传多态性。为多角度开发我国乳酸菌资源、益生菌菌种的筛选以及肠道微生态保健品的研发奠定基础。
[Abstract]:Lactobacillus is widely distributed in human and animal intestinal tract, plant surface and other habitats. In recent years, Lactobacillus has been extensively and thoroughly studied from morphology, physiology, taxonomy, genetics, microecology, physiological function and application. Lactobacillus has many beneficial functions, and a large number of Lactobacillus contain Lactobacillus at home and abroad. In order to promote the development and utilization of Lactobacillus, isolation of Lactobacillus from human intestinal tract has more practical value, because Lactobacillus from human intestine is easier to colonize in human intestine.
With the rapid development of molecular biology and the application of biophysical technology, the identification methods of microorganisms have developed from traditional morphological analysis, physiological and biochemical experiments to various molecular biology methods based on PCR technology. These methods do not require complicated microbial culture to identify microorganisms through the design of genera and species. Heterosexual primers or probes can easily identify microorganisms to the level of species, and molecular marker technology can even be used to identify microorganisms to the level of strains. Conduct accurate identification.
This study includes four aspects:
Isolation and purification of Lactobacillus 1.
Fifty-six strains of Lactobacillus were isolated from fresh feces of healthy adults and school-age children by diluting and coating on MRS medium.
Identification of lactobacillus by 2. 16S rDNA sequence homology analysis (Sequence Homology Analysis)
The specific steps of 16S rDNA sequence homology analysis include: genomic DNA extraction, PCR amplification, product sequencing and homology analysis of Lactobacillus spp. Lactobacillus acidophilus, Lactobacillus crispatus, Weissella. cibaria, Lactobacillus salivarius subsp. salivarius, Lactobacillus mucosae, Lactobacillus pentosus, fermentation 5 strains of Lactobacillus (Lactobacillus fermentum) and 8 strains of Lactobacillus plantarum (Lactobacillus plantarum).
3. Construction of Lactobacillus standard fingerprint by denaturing Gradient Gel Electrophoresis (DGGE)
The V7-V8 variable region sequence of 16S rDNA of genomic DNA was amplified by PCR-DGGE. 35-55% deformable gradient gel electrophoresis was used to isolate the V7-V8 variable region sequence of genomic DNA. Fingerprints showed that the tested strains could be divided into 7 groups. The results of PCR-DGGE were inconsistent with those of identification, indicating that DGGE method was also effective. There are some limitations.
4. Preliminary Study on Genetic Polymorphism of Lactobacillus by Amplified Fragment Length Polymorphic DNA (AFLP)
A pair of primers with good polymorphism was selected from 64 pairs of random primers to analyze the genetic polymorphism of Lactobacillus spp. AFLP fingerprint was proved to be repeatable and accurate by repeated experiments. The polyacrylamide gel electrophoresis map showed the genetic polymorphism of the same strain in Lactobacillus spp. It indicated that AFLP technique was effective. It can be used as a basis for studying polymorphism of different strains of Lactobacillus.
In this study, 20 strains of Lactobacillus spp. isolated from fresh feces of healthy people were identified to species level by traditional physiological and biochemical methods combined with molecular biological methods. The standard fingerprint of Lactobacillus spp. was constructed by denaturing gradient gel electrophoresis (DGGE) and the length of amplified fragments was used. The genetic polymorphism among different Lactobacillus strains was studied by AFLP technique, which laid a foundation for the development of lactic acid bacteria resources, the screening of probiotics and the development of intestinal microecological health products in China.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R371

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