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Unmethylated CpG dinucleotide content detection method

A dinucleotide and methylase technology, applied in biochemical equipment and methods, microbial determination/inspection, measurement devices, etc., can solve the problems of high preparation cost, hindering clinical application and promotion, etc.

Inactive Publication Date: 2008-05-28
NAT INST FOR THE CONTROL OF PHARMA & BIOLOGICAL PROD
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Problems solved by technology

However, the current CpG-DNA research is all aimed at artificially synthesized oligonucleotides (ODNs) containing CpG motifs, and the high cost of preparation hinders the promotion of clinical applications.
[0005] Bacterial extracts are another source of CpG-DNA, but there is no report on CpG-DNA from bacteria as an adjuvant

Method used

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Embodiment Construction

[0024] The innovation and application significance of the present invention are described in detail below through specific examples and test results to help readers better understand the spirit and essence of the present invention, but it does not constitute a limitation to the implementation scope of the present invention.

[0025] 1. Preparation of BCG-CpG-DNA

[0026] Step 1, bacterial cell culture: dissolve the strain preserved at low temperature in liquid (BCG strain D2PB302 for the preparation of BCG in China, provided by the vaccine department of the China Institute for the Control of Pharmaceutical and Biological Products) at room temperature, and inoculate it in Potato Sutong medium. After culturing at 37°C for 15 days, transfer to improved liquid Sutong medium, and culture at 37°C for 14-20 days;

[0027] in,

[0028] The preparation method of potato Sutong culture medium can be:

[0029] Take and wash fresh potatoes (1), pierce them into cylinders with a piercer, ...

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Abstract

The invention provides a method for testing non-ethylating CpG content, which comprises adopting specific methylaseSssI to modify CpG dinucleotide aracytidine into 5-methylcytosine, then using nucleicacidase P1 and bacterial alkaline phosphatase to hydrolyze DNA into single deoxynucleoside, using a reversed-phase hplc method to test quantity of the 5-methylcytosinein DNA hydrolytic samples which are both modifying and un-modifying, and testing the CpG through differences of the 5-methylcytosine detectable amount between the modifying DNA hydrolytic sample and the un-modifying DNA hydrolytic samples.

Description

[0001] This application is a divisional application of a Chinese invention patent application with an application date of April 15, 2004, an application number of 200410033878.1, and an invention title of "An Immunological Adjuvant and a Vaccine Containing the Adjuvant". technical field [0002] The invention relates to a detection method for nucleotide content, in particular to a detection method for unmethylated CpG dinucleotide content. Background technique [0003] Adjuvant is an indispensable component in vaccines. In terms of immune adjuvant, although aluminum adjuvant has been used for nearly 80 years and its safety has been tested for a long time, it has no obvious effect on cellular immunity, especially for Vaccines based on cellular immunity, such as highly purified second-generation recombinant vaccines and third-generation DNA vaccines, have little effect, so people have been working on the research of new adjuvants. [0004] CpG-DNA stimulates innate immunity, p...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/00G01N30/60C12Q1/68A61K39/39
Inventor 王国治赵爱华贾淑珍乔来艳寇丽杰
Owner NAT INST FOR THE CONTROL OF PHARMA & BIOLOGICAL PROD
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