Saliva streptococcus 57.I urase gene and its urase

A technology of Streptococcus salivarius and urease, which is applied in the field of Streptococcus salivarius urease gene, and can solve problems such as cloning that has not yet been reported.

Inactive Publication Date: 2009-09-02
SHANGHAI NINTH PEOPLES HOSPITAL SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] But so far, the cloning of the complete sequence of Streptococcus salivarius urease gene U35248 has not been reported

Method used

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  • Saliva streptococcus 57.I urase gene and its urase
  • Saliva streptococcus 57.I urase gene and its urase
  • Saliva streptococcus 57.I urase gene and its urase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Extraction and Identification of Genomic DNA of Streptococcus salivarius 57.I

[0047] The purpose of the whole study is to obtain the clone of the active Streptococcus salivarius 57.I urease gene U35248. The first step is to obtain the template of the target gene, which is the whole genome DNA of the donor bacteria. Therefore, this experiment According to the principle of molecular cloning, the genomic DNA of Streptococcus salivarius 57.I will be extracted using a commercially available bacterial genome extraction kit as a template, so that in subsequent studies, PCR methods will be used to use genomic DNA as a template. The desired urease gene was cloned.

[0048] At the same time, because in the genome of prokaryotes, the DNA molecule encoding 16S ribosomal RNA is functionally stable and consists of a highly conserved region and a variable region. The mutation rate in the conserved region is extremely low, while the variable sequence of its characteristic region is d...

Embodiment 2

[0116] Segmental Cloning of 57.I Urease Gene U35248 from Streptococcus salivarius

[0117] Polymerase chain reaction (polymerase chain reaction, PCR) can be used to amplify the DNA segment between two pieces of known sequences, but the length of Streptococcus salivarius urease gene reaches 8Kb, if PCR method is directly used for one-time Amplification, it is difficult to ensure that the expected length can be achieved, and more mutations may appear during the amplification process. This experiment intends to adopt the method of segmental cloning. After analyzing the full-length sequence of Streptococcus salivarius urease gene U35248, it is found that there are two single restriction endonuclease cutting sites-Bam H I inside the gene. (located at 2038bp) and Xho I (located at 4812bp), so the design divides the urease gene into three sections, front, middle and back, and clones them respectively. There is a part of overlap between every adjacent two sections, and each The overl...

Embodiment 3

[0253] The product of segmental cloning is connected step by step into a complete urease gene

[0254] When initially segmenting the urease gene U35248 of Streptococcus salivarius 57.I, there was an overlapping region between the first segment and the second segment and the second segment and the third segment of the target gene segment. Each has a single enzyme cutting site. In the above experiments, the three fragments have been ligated into the same vector in the same direction. In this experiment, a single restriction site on the vector and a single restriction site inside the fragment will be used, and the method of double digestion will be used to target The gene and the vector were cut and then recombined, so that the three segmented clones that had been correctly sequenced in Experiment 3 were gradually connected into a complete urease gene.

[0255] 1.1 Materials and methods

[0256] 1.1.1 Main reagents

[0257] A small amount of DNA fragment quick gel recovery kit (...

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Abstract

The invention relates to a Streptococcus salivarius urease gene, in particular to a Streptococcus salivarius 57.I urease gene and urease thereof. A Streptococcus salivarius 57.I urease gene, its nucleotide sequence is described in SEQ ID NO.1. The nucleotide sequence of the gene MQO related to nickel ion binding and transport in this gene is described in SEQ ID NO.2. The advantages of the present invention are as follows: the clone of Streptococcus salivarius urease gene U35248 has been obtained by segmental cloning and step-by-step enzyme digestion and connection, and the length of the obtained target gene has reached 8Kb. Compared with the prior art, the combination of nickel ions and The transport-related gene MQO, so the ureolytic activity can be expressed without the addition of exogenous NiCl2. The obtained clones can be used in the research of caries prevention by alternative therapy in the future to construct alcalogenic effect bacteria that do not need to add exogenous NiCl2 and are more suitable for clinical application.

Description

technical field [0001] The invention relates to a Streptococcus salivarius urease gene, in particular to a Streptococcus salivarius 57.I urease gene and urease thereof. Background technique [0002] Caries is one of the most common bacterial infectious diseases that threaten human health, and it is also one of the main causes of human tooth pain and loss. According to the epidemiological survey data of oral health in my country in 1983 and 1995, caries prevalence is on the rise. Although there are many methods for the prevention of dental caries at present, a very ideal method has not been found so far. [0003] Replacement therapy is to permanently colonize human microbiota with a naturally occurring or laboratory-constructed harmless effector strain to replace and exclude the growth of a specific pathogenic microorganism, thereby preventing specific The method by which the disease occurs. In the early 20th century, foreign doctors tried to treat bacterial infectious dise...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52C12N1/20C12N9/00A61K48/00A61K38/43A61P1/02C12R1/46
Inventor 冯希平王艳谢幼华
Owner SHANGHAI NINTH PEOPLES HOSPITAL SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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