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Sulpho-oligodeoxynucleotide with immunostimulation activities and application thereof

An immunostimulatory activity, deoxynucleotide technology, applied in the direction of organic active ingredients, medical preparations containing active ingredients, allergic diseases, etc. Undisclosed issues such as nucleotides, to achieve good immune stimulation activity, tumor growth inhibition, good tumor growth effect

Active Publication Date: 2011-02-23
苏州博特龙免疫技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, some CpG-ODNs and related applications are also disclosed in patent documents CN 1271733A, CN 1526718A, US 2005 / 0267057A1 or WO 99 / 56755, but in the above documents, there is no disclosure about having two or more than two copy of the nucleotide of the 5'-NTCGTT-3' motif (N does not represent A or G), nor does it disclose any information about the cross-immunity of thiooligonucleotides containing this motif to humans and mice Technical Implications for Stimulatory Activity

Method used

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  • Sulpho-oligodeoxynucleotide with immunostimulation activities and application thereof
  • Sulpho-oligodeoxynucleotide with immunostimulation activities and application thereof
  • Sulpho-oligodeoxynucleotide with immunostimulation activities and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1. Human immunostimulatory activity of oligodeoxynucleotides with 5'-NTCGTPy-3' (wherein N represents A, G, C or T, Py represents pyrimidine C or T) motif

[0062] In order to elucidate the CpG motif for activating human immune cells, the inventors conducted mutation studies on the remaining four nucleotides in the CpG motif except for the core nucleotide CpG. To this end, the inventors designed a set of CpG-ODNs, as shown in Table 1, each sequence is 12 nucleotides in length and contains two identical copies of the CpG motif. These CpG-ODNs are used to stimulate human peripheral blood mononuclear cells (PBMC) cultured in vitro, and their activity on human immune cells can be evaluated by measuring the IgM content in the culture supernatant.

[0063] The CpG-ODN used in this example was designed by the present inventors, entrusted Shanghai Sangong Biotechnology Co., Ltd. to artificially synthesize it, and carried out full-chain thio modification, purified by pol...

Embodiment 2

[0069] Example 2. Immunostimulatory activity of oligodeoxynucleotides with 5'-NA / TCGTT-3' (wherein N represents A, G, C or T) motifs on mice

[0070] In order to elucidate the CpG motif that activates mouse immune cells, the inventors conducted mutation studies on the remaining four nucleotides in the CpG motif except the core nucleotide CpG. See Table 1 for the CpG-ODN sequences used in this example. Mouse spleen mononuclear cells (SMMC) were stimulated with these CpG-ODNs, and their activity on mouse immune cells could be evaluated by measuring the IgM content in the culture supernatant. The Balb / c mice used were female, 6-8 weeks old, purchased from the Experimental Animal Center of the Chinese Academy of Medical Sciences.

[0071] Take the spleen of Balb / c mice under sterile conditions, and prepare SMMC suspension with RPMI 1640 medium; after counting, use RPMI 1640 medium to adjust the cell concentration to 1×10 6 / ml, inoculated into 96-well cell plate, 200 μl per well...

Embodiment 3

[0074] Example 3. The immunostimulatory activity of oligodeoxynucleotides of the present invention on human immune cells

[0075] According to the CpG motifs found in Examples 1 and 2 that have cross-immunostimulatory activity to humans and mice, the inventors designed and synthesized a set of CpG-ODNs, as shown in Table 2, with a length of 15 to 35 nucleotides , containing two or more than two copies of 5'-TTCGTT-3' motifs (numbered T1-T22) and 5'-CTCGTT-3' motifs (numbered C1 and C2), respectively.

[0076]The CpG-ODN in Table 2 except the control sequence T7C and 1826 are respectively cited from the literature "Chinese Journal of Microbiology and Immunology" 2001, 21 (5), pages 471-475, "The Journal of Immunology" 1998, volume 160 , pages 870-876, all the rest are designed by the inventor. The reference sequence used in this example and the sequence designed by the inventors were entrusted to Shanghai Sangon Biotechnology Co., Ltd. to synthesize, modify the whole chain by ...

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Abstract

The invention relates to sulpho-oligodeoxynucleotide with immunostimulation activities. The sequence of the sulpho-oligodeoxynucleotide has two or more than two copied 5'-NTCGTT-3' elements having 15 to 35 nucleotides respectively, wherein CpG is non-methylated and N does not represent A or G. In vitro, the sulpho-oligodeoxynucleotide has high immunostimulation activities for the immunocytes of human and a mouse, and the immunostimulation activities of the sulpho-oligodeoxynucleotide in a human body can be evaluated according to the result of the immunostimulation activities in mouse. Used asa vaccine adjuvant, the sulpho-oligodeoxynucleotide can obviously strengthen the immune response of the mouse to a genetic engineering hepatitis B vaccine and make the immune response deflected to a TH1 direction, and thus, it is proved that the sulpho-oligodeoxynucleotide can be used for preventing and treating allergy and infection. Meanwhile, the sulpho-oligodeoxynucleotide has high tumor growth restraining activities in the mouse body.

Description

[0001] This application is a divisional application of the application with the filing date of January 23, 2008, the application number of 200810004736.0, and the title of the invention "thio-oligodeoxynucleotides with immunostimulatory activity and their applications". technical field [0002] The invention relates to the field of biology, in particular to a thiooligo-deoxynucleotide with immunostimulatory activity and its application. Background technique [0003] In the 1980s, Japanese scholars discovered that the DNA component of Bacillus Calmette-Guerin (BCG) had anti-tumor activity in humans and animals. Later, scientists who studied antisense nucleic acid therapy discovered that artificially synthesized thiooligo-deoxynucleotides had non-toxic properties. Specific immunostimulatory activity. Krieg AM and others revealed the essence of these phenomena in "Nature" 1995, volume 374, pages 546-549, and found that the immunostimulatory active components in nucleic acids ar...

Claims

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Application Information

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IPC IPC(8): A61P35/00C12N15/117A61K39/39A61K31/7125A61P31/00A61P37/08
Inventor 许洪林
Owner 苏州博特龙免疫技术有限公司
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