Mucosal adjuvant and its preparation method and use

A mucosal adjuvant and plasmid technology, applied in the field of genetic engineering, can solve problems such as inability to effectively induce mucosal immune responses, and achieve the effects of preventing viral myocarditis, enhancing Th1 responses, and promoting activation

Inactive Publication Date: 2012-09-26
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The object of the present invention is to provide a kind of mucosal adjuvant and its preparation and application, described this kind of mucosal adjuvant cooperates other vaccines to carry out mucosal part (nasal drop, oral administration, via genital tract etc.) Technical problems due to the ineffectiveness of conventional vaccines in inducing mucosal immune responses

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  • Mucosal adjuvant and its preparation method and use
  • Mucosal adjuvant and its preparation method and use
  • Mucosal adjuvant and its preparation method and use

Examples

Experimental program
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Effect test

Embodiment 1

[0061] Example 1 Construction and in vitro expression of pcDNA3.1-pVP1 target antigen gene vaccine

[0062] 1) Obtain CVB3 VP1 gene from freshly cultured CVB3 by RT-PCR. According to the CVB3 Nancy strain VP1 cDNA sequence reported in the literature (Klump WM, et al. Complete nucleotide sequence of infectious Coxsackievirus B3 cDNA J Virol, 1990, 64(4): 1573), design VP1 upstream and downstream primers: KV1: 5'- CCCAAGCTTGCCACCATGGGCCCAGTGGAAGACGCG-3'; KV2: 5'-CGGGATCCTTACTAAAATGCGCCCGTATTTGTC-3'. And synthesize the upstream and downstream primers T7-1: 5'-CGATGAAGATCTCT-3'; T7-2: 5'-CGATGAAGATCTCT-3' in the cloning site in the pcDNA3.1 vector. The liquid phase viral RNA was extracted according to the RNAex Reagent&System Kit of Shanghai Huashun Biological Company. The cDNA was obtained by reverse transcription using the 2-N First Strand cDNA Synthesis Kit of Shanghai Sangon Biotechnology Co., Ltd. Carry out specific PCR with KV1, KV2 primer subsequently, the product is VP1...

Embodiment 2

[0064] Example 2 Construction and in vitro expression of pcDNA3.1-LTN mucosal adjuvant plasmid

[0065] 1) The LTN gene was obtained from ConA-activated mouse splenocytes by RT-PCR. Take the spleen of normal BALB / c mice 5×10 6 , to extract total cellular RNA. The cDNA was obtained by reverse transcription using the 2-N First Strand cDNA Synthesis Kit of Shanghai Sangon Biotechnology Co., Ltd. According to the gene sequence of LTN in NCBI GeneBank (NM_008510.1), design the upstream and downstream primers of LTN: LTN-1: 5'-5'CG GGATCC GCCACCATGAGACTTCTCCTCCT-3' has a BamH I restriction site added to its 5' end; LTN-2: 5'-CCG CTCGAG GGAGGCTGTTACCCAGT-3' has an Xho I restriction site added to its 3' end. Perform PCR with the above primers to obtain the chemokine Ltn coding gene (376bp) ( image 3 ). The RT-PCR product was purified and recovered with Golden Beads DNA Gel Purification Kit, digested with BamHI and Xho I, and ligated with pcDNA3.1 that had undergone the same d...

Embodiment 3

[0067] Example 3 In vitro chemotactic activity of pcDNA3.1-LTN mucosal adjuvant plasmid expression product

[0068] Concentrate the supernatant of stably transfected pcDNA3.1-LTN by 20-25 times with PEG20000, and add it into a 48-well chemotaxis cell (Neuroprobe, USA); 10 6 / ml) was added to the upper chamber and incubated for 4 hours. Take out the filter membrane, Wright Giemsa staining, count chemotactic cells and calculate the chemotaxis index (CI). The results showed that the supernatant of 293T cells stably transfected with pcDNA3.1-LTN could effectively chemoattract splenocytes to move across the membrane, CI=2.875; while the control pcDNA3.1 plasmid transfected supernatant or normal cell supernatant had no chemotactic activity, CI Figure 5 )

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Abstract

The invention provides a mucosal adjuvant. The mucosal adjuvant is prepared by copolymerization crosslinking compounding of oligo-chitosan and lymphotactin encoding plasmids. The invention also discloses a preparation method and a use of the mucosal adjuvant. When the mucosal adjuvant and a gene vaccine composed of chitosan or oligo-chitosan are synchronously dropped into a nasal cavity for immunization, coxsackievirus specific serum antibody and systemic (spleen and lymph glands) Th1-type immune responses are induced and especially, intestinal mucosa partially-reinforced specific-secretion-type sIgA and IFNgama+Th1 responses are induced, and thus a gene vaccine used with the mucosal adjuvant is obviously superior to an exposed gene vaccine and effectively prevents coxsackievirus-caused myocarditis. The mucosal adjuvant can be used as a novel mucosal vaccination adjuvant.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a mucosal adjuvant and its preparation and application, in particular to a mucosal adjuvant assembled with a chitosan oligosaccharide delivery system and its preparation and application. Background technique [0002] Pathogens such as human immunodeficiency virus (HIV), influenza virus, Mycobacterium tuberculosis, etc. that currently cause severe infectious diseases that are difficult to overcome all invade and infect the body through mucosal surfaces (genital tract, respiratory tract, gastrointestinal tract), because the body cannot Inducing an effective mucosal immune response cannot clear the mucosal infection pathogen at the first time and place of infection, so that the pathogen rapidly spreads into the blood, and then invades the whole body, causing body damage; at the same time, it develops into chronic infection, leading to serious diseases. [0003] For pat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/39A61K48/00A61K9/16A61K47/36A61P31/06A61P31/12
Inventor 徐薇熊思东岳艳
Owner FUDAN UNIV
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