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Saliva streptococcus 57.I urase gene and its urase

A technology of Streptococcus salivarius and urease, applied in gene therapy, enzymes, enzymes, etc., can solve problems such as cloning that has not been reported yet

Inactive Publication Date: 2007-04-18
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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Extraction and Identification of Genomic DNA of Streptococcus salivarius 57.I

[0048] 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.

[0049] 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

[0117] Segmental Cloning of 57.I Urease Gene U35248 from Streptococcus salivarius

[0118] 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 products of segmental cloning were 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 connected 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 recove...

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Abstract

This invention relates to a urase gene of streptococcus salivarius, concretely, concerning about a 57.I urase gene of streptococcus salivarius and its urase. A 57.I urase gene of streptococcus salivarius , whose nucleotide sequence is what is described in SEQ ID NO.1. Nickel ion bonding in the gene and nucleotide sequence of transit-related gene MQO are what are described in SEQ ID NO.2. The advantage of this invention is that gaining the cloning of urase gene of streptococcus salivarius U35248 by the method of segment cloning and gradually enzyme-cutting connection ,and its object gene length proceeds to 8Kb, increasing Nickel ion bonding of the gene and transit-related gene MQO compared with existing technology , therefore, expressing ureolytic activity without adding exogenous NiCl2.Attained cloning can be used for future replacement therapy in anti-caries research building without adding exogenous NiCl2,more suitable for clinical application of production alkali effect germ.

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 Applications(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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