Improved DNA immunization with recombinase/transposase

A technology of recombinase and recombination site, applied in the direction of recombinant DNA technology, DNA/RNA vaccination, complete cell/virus/DNA/RNA components, etc.

Inactive Publication Date: 2008-02-13
人类多细胞治疗股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the development of safe and effective adjuvants is an ongoing challenge

Method used

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  • Improved DNA immunization with recombinase/transposase
  • Improved DNA immunization with recombinase/transposase
  • Improved DNA immunization with recombinase/transposase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Cloning of Example 1 Rabbit GMCSF

[0119] The peritoneal cavity of ZIKA rabbits was lavaged with 30-60 ml PBS / 2% FCS (Gibco; PET 10270098), and peritoneal macrophages were collected and washed twice. For cell counting, cells were plated in DMEM / 10% FCS in one well of a 24-well plate at a density of 1-3 x 10 5 cells / well, and with 1 μg / ml LPS E. coli O111:B4 (Sigma; L2630) in 5% CO 2 Stimulate for 5 hours at 37°C in an incubator. Harvest the cells with QIA Amp RNA-Mini-Mini-Kit (Qiagen) was used to isolate total RNA according to the manufacturer's instructions.

[0120] Using BD SMART RACE cDNA Amlification Kit (BD Biosciences), according to the manufacturer's protocol, with 3'-RACE CDS Primer A (5'-AAGCAGTGGTATCAACGCAGAGTAC(T) 30 VN-3') (SEQ ID NO: 3) was reverse transcribed for 3-6 μl of total RNA. With primer pair GMCSFup3 (5'-aaggctaaggtcctgaggagg-3') (SEQ ID NO: 4) and 10x universal primer A mix (5' CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3' (SEQ ID NO: ...

Embodiment 2

[0121] Example 2 Production of Anti-CD20 Antibodies in Rabbits by Gene Inoculation

[0122] Messenger RNA (mRNA) from LPS-stimulated rabbit peritoneal macrophages and lymphocytes was isolated and reverse transcribed. Rabbit GMCSF and Flt3L cDNAs were amplified by PCR. Next, the cDNAs are cloned into expression plasmids. A cDNA encoding the soluble portion of human CD20 was synthesized and cloned into the same expression plasmid. The final construct is shown in Figure 1. Plasmid DNA was purified and mixed with an equal amount of plasmid encoding C31 integrase. Plasmid DNA was coated on Helios gene gun bullets according to the manufacturer's instructions (approximately 1 μg / bullet). Rabbits were lightly anesthetized and each ear was shot on day 0 and day 14. Blood is collected and allowed to clot. Serum was collected by centrifugation. Anti-CD20 antibodies were detected by flow cytometry using human peripheral blood mononuclear cells. Comparing animals immunized with rab...

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Abstract

The present invention relates to improved methods to immunize / vaccinate or stimulate the immune system of animals, including humans, using vectors containing expression cassettes that encode for the DNA of one or more protein / peptide antigens and / or adjuvants, in particular, cytokines like GMCSF, Flt3L, interleukins, and the like, which can be encoded by DNA as well, also recombinase mediated integration. Adjuvants known to increase immune responses following DNA vaccination. In addition, the vectors contain one or more sites recognized by a recombinase / transposase, which catalyzes the insertion of the vector into the genome of transfected cells. Stable integration of the plasmid vector into the genome of transfected cells results in higher and longer-lasting expression of the encoded protein(s), and increases the immune response in the vaccinated animal. The present invention also relates to adjuvant compositions comprising the novel polypeptide, rabbit GMCSF, for boosting antibody production in rabbits.

Description

technical field [0001] The present invention relates to the field of DNA vaccines and adjuvants. In particular, the present invention relates to improved methods of immunizing / vaccinating or stimulating the immune system of animals, including humans, using vectors comprising expression cassettes encoding one or more protein / polypeptide antigens and / or adjuvants and recombinases that mediate the integration of DNA into the genome of animals. The present invention also relates to an adjuvant composition comprising the new polypeptide-rabbit GMCSF, which is used to increase the antibody production in rabbits. Background technique [0002] Immunization of animals with vaccines requires reproducible production of protein antigens, which is difficult and expensive. Immunization is performed with all or usually part of an organism, toxin or antigen. In order to maintain a certain level of immunity in the host, it is necessary to administer high or, in most cases, repeated doses ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/39C07K14/535C12N15/90
CPCA61K39/0008A61K2039/55522C07K2319/00C07K14/535A61K39/0011A61K48/00C07K16/2896A61K2039/53C12N15/90A61K38/00A61K2039/6031A61K38/43C07K2319/30A61P11/06A61P17/06A61P19/02A61P25/00A61P29/00A61P31/04A61P31/10A61P31/12A61P35/00A61P37/04A61P37/06A61P37/08A61K2300/00
Inventor 罗兰·比洛约瑟夫·普拉策
Owner 人类多细胞治疗股份有限公司
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