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Human streptococcus mutans genetic engineering vaccine for decayed tooth and preparation method thereof

A Streptococcus mutans and genetic engineering technology, applied in the field of human Streptococcus mutans genetically engineered dental caries vaccine and its preparation, to achieve high-efficiency immune response, good immune protection, and easy separation and purification

Inactive Publication Date: 2009-07-08
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing preventive measures such as fluoride, mechanical methods, antibacterial drugs, and sugar-restricted diets can only reduce the incidence of caries to a certain extent, and it is impossible to stop the prevalence of caries at all.

Method used

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  • Human streptococcus mutans genetic engineering vaccine for decayed tooth and preparation method thereof
  • Human streptococcus mutans genetic engineering vaccine for decayed tooth and preparation method thereof
  • Human streptococcus mutans genetic engineering vaccine for decayed tooth and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Example 1 Construction of expression vector of fusion gene GIR-LTB

[0106] 1. Primer design and PCR amplification

[0107] 1) The primers were designed as follows: According to the gene encoding GIR and the heat-labile enterotoxin B subunit LTB gene of S. Mutans (UA159 standard strain) found in GenBank, the software Primer Premier 5.0 was used to design and analyze primers. Design the linker (frame region) sequence at the 5' end of the upstream gene (GIR) and introduce the BamHI site, introduce the linker (frame region) sequence at the 3' end, and introduce the linker sequence at the 5' end of the downstream gene LTB (framed part), the XhoI site was introduced into the 3' end, and the GIR gene and the LTB gene were connected by the overlap extension PCR method. Primers were synthesized by Shanghai Yingjun Company.

[0108] GIR: Upstream primer P1: SEQ ID NO: 5

[0109] 5'- CAAGCACAAGTTAAT-3' (BamHI)

[0110] Downstream primer P2: SEQ ID NO: 6

[0111] 5'- A...

Embodiment 2

[0127] Example 2 Construction and expression of GLU-GIR-LTB fusion gene expression vector and recombinant expression engineering bacteria

[0128] 1. Primer design and PCR amplification

[0129] 1) The primers were designed as follows: according to the gene encoding GLU of S. Mutans (UA159 standard strain) found from GenBank, the primers were designed and analyzed using the software Primer Premier 5.0. The linker (frame region) sequence is designed at the 3' end of the GLU coding gene, and the upstream and downstream primers are respectively introduced into the NcoI and BamHI sites, and the sticky ends digested with BamHI can be combined with the fusion gene (GIR-LTB) Connect correctly. Primers were synthesized by Shanghai Yingjun Company.

[0130] GLU: upstream primer P1: SEQ ID NO: 9

[0131] 5'CCATGGATGAAATGGGCTATCAAGC-3'

[0132] Downstream primer P2: SEQ ID NO: 10

[0133] 5'- CCGAACTCGTTTCCAG-3'

[0134] 2) PCR amplification of the target gene

[0135] Strept...

Embodiment 3

[0148] Example 3 Preliminary Study on Immunogenicity of Fusion Protein

[0149] 1. Immunization of Mice

[0150] Purified target protein GLU-GIR-LTB was used to immunize 4-5 week-old Balb / c mice, 100ug / mouse / time, 100μL of antigen was mixed with the same amount of Freund's complete adjuvant, and injected subcutaneously into the abdomen and groin of mice. The immunization program is: 0, 1, 2 weeks, a total of 3 times, Freund's complete adjuvant is added for the 1st and 2nd time, no adjuvant is added for the 3rd time, inoculated in the abdomen and groin subcutaneous of mice, and the amount of antigen and adjuvant injected is about 0.2ml, once a week, 6 days after the third immunization, the tail was docked to collect blood, and ELISA was used to detect the change of serum specific antibody titer.

[0151] 2. Detection of specific immune response.

[0152] (1) Preparation of ELISA antigen-coated plates:

[0153] Dilute GLU antigen to 5 μg / ml with coating solution, coat ELIS...

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Abstract

The invention belongs to the biopharmaceutical field and relates to a method for constructing and preparing human streptococcus mutans gene engineering vaccine for tooth decay. In the method, the genes at a glucan binding domain at the end of glucosyltransferase C and at an N end immunodominance domain of glucan-binding protein B which are major protective antigens of streptococcus mutans of human primary cariogenic bacteria and the mucous membrane immunologic adjuvant heat-labile toxin B subunit are used for constructing fusion engineering bacteria by a gene recombination method, and fusion protein molecular vaccine with high purity is obtained by high density fermentation and a series of purifying procedures. The technology for preparing the vaccine is simple and easy for amplifying and has good repeatability; the obtained protein has high purity; primary animal experiments prove that the fusion protein can stimulate body to produce efficient immunity response; and the fusion protein is approved as good candidate antigens of gene engineering vaccine for tooth decay and has good application prospect.

Description

technical field [0001] The invention relates to the field of medical biotechnology, in particular to a human Streptococcus mutans genetically engineered dental caries vaccine and a preparation method thereof. Background technique [0002] Streptococcus mutans (S.Mutans) is the main cariogenic microorganism in humans, and its colonization in the oral cavity is closely related to the occurrence of caries. Caries is currently the most common disease in human beings. In 2003, WHO listed it as the third disease that needs to be prevented and treated after cardiovascular disease and cancer. Worldwide, 60% to 90% of children and adults have suffered from dental caries (The World Oral Health Report 2003). The third national oral health epidemiological survey in 2007 showed that the incidence of dental caries in my country was as high as 70%-80%. Because caries progresses slowly and is usually not life-threatening, it has not been taken seriously in developing countries. "Toothach...

Claims

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Application Information

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IPC IPC(8): A61K39/09A61P1/02
Inventor 邹全明刘涛郭鹰石云毛旭虎张卫军
Owner ARMY MEDICAL UNIV
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