Oral DNA vaccine for promoting growth of animal by non-antibiotics resistance gene screening, and preparation and use thereof

A DNA vaccine and resistance screening technology, applied in the direction of recombinant DNA technology, DNA/RNA fragments, and microbial-based methods, can solve the problems of clinical drug-resistant strains spreading, increasing production costs, unfavorable vaccine promotion and application, etc., to achieve It is safe to use, promotes animal growth, and has good growth-promoting effects

Inactive Publication Date: 2009-04-15
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, the above-mentioned DNA vectors all contain resistance genes (Amp or Kan). During the process of cultivating bacteria, such resistant substances need to be added to screen recombinant bacteria, which not only increases production costs, but also may lead to the spread of clinical drug-resistant strains. It is not conducive to the popularization and application of vaccines, so the carrier carrying the target gene needs further research and improvement

Method used

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  • Oral DNA vaccine for promoting growth of animal by non-antibiotics resistance gene screening, and preparation and use thereof
  • Oral DNA vaccine for promoting growth of animal by non-antibiotics resistance gene screening, and preparation and use thereof
  • Oral DNA vaccine for promoting growth of animal by non-antibiotics resistance gene screening, and preparation and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Construction of non-resistance screening somatostatin eukaryotic expression plasmid pGS / 2SS

[0027] Using the original plasmid pGM-CSF / SS (patent application number: 200610125580.2, strain deposit number CCTCC NO: M206141) as a template, use primer 5.0 software to design primer pairs (synthesized by Shanghai Sangon Bioengineering Co., Ltd.), forward primer P1: 5'-CTCTAGA GCTAGC CTCAGAAGGA-3' (the underline is NheI enzyme); reverse primer: P2: 5'-TTTGTTCTACGT AAGCTT AAC-3' (the underline is HindIII enzyme), amplifies the somatostatin fusion expression gene GS / 2SS, and then ligates the purified and recovered GS / 2SS gene fragment to pMD18T-Simple Vector (purchased from Bao Biological Engineering Co., Ltd. (Dalian) Co., Ltd.), after identification and sequencing by NheI and HindIII double enzyme digestion, it was connected into the eukaryotic expression vector pVAX1 by NheI and HindIII double enzyme digestion to obtain the intermediate plasmid pVAX-GS / 2SS, wh...

Embodiment 2

[0079] Example 2: Preparation of DNA vaccine C500 / pGS / 2SS for non-resistance screening to promote animal growth

[0080] 2.1 pGS / 2SS plasmid extraction and purification

[0081] The plasmid mini-extraction kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used to extract the pGS / 2SS plasmid, and the specific operation steps refer to the instruction manual of the kit.

[0082] 2.2 Preparation of C500 Competent Cells

[0083] Refer to the literature (J. Sambrook, D.W. Russell, Molecular Cloning Experiment Guide [M]. Third Edition, Beijing: Science Press, 2002, 99-102) to prepare the Salmonella choleraesuis with the deletion of the asd gene by the power supply transformation method C500 competent cells and stored at -70°C.

[0084] 2.3 Electroconversion

[0085] Transfer the plasmid pGS / 2SS purified in 2.1 into C500 competent cells by electroporation, and the specific steps are as follows:

[0086] 1) Thaw electrocompetent cells on ice and incubate o...

Embodiment 3

[0095] Embodiment 3: the application of DNA vaccine of the present invention in promoting the growth of mice

[0096] 3.1 Vaccine immunization and sample collection

[0097] Use a sterile inoculation loop to pick a single colony of Salmonella choleraesuis C500 / pGS / 2SS containing the eukaryotic expression plasmid pGS / 2SS for non-resistance selection and inoculate it in LB liquid medium without any foreign substances, and at the same time inoculate C500 empty bacteria (Diaminopimelic acid (DAP) with a final concentration of 50 μg / mL needs to be added to the LB medium), shake culture at 37°C and 220rpm to OD6000.3-0.4, centrifuge at 3000g at 4°C for 10min, discard the supernatant, and use The method of plate counting uses sterilized PBS phosphate buffer (pH 7.4, the formula of every liter of PBS phosphate buffer is as follows: KH 2 PO 4 -0.2 g, Na 2 HPO 4 12H 2 O-2.9 grams, NaCl-8.0 grams, KCl-0.2 grams, add distilled water to 1L) adjust the bacterial concentration to 10 10...

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Abstract

The invention belongs to the preparation field of animal vaccines, and particularly relates to a non-antibiotically screened animal growth promotion oral DNA vaccine as well as a preparation method and applications thereof. A somatostatin with a GMCSF cell gene and a hepatitis b surface antigen fusion gene GS/2SS are cloned to a plasmid vector pVAX1 for obtaining a middle plasmid pVAX-GS/2SS; an aspartic acid Beta-galactose dehydrogenase gene (asd) is used for replacing a kanamycin (Kan) gene in the middle plasmid pVAX-GS/2SS to obtain an eukaryotic expression plasmid pGS/2SS of the non-antibiotically screened somatostatin; and then the plasmid pGS/2SS is converted into a pig cholera salmonella C500 lack of asd and crp genes for obtaining the non-antibiotically screened animal growth promotion oral DNA vaccine. The pig cholera salmonella (Salmonella enterica sv. Choleraesuis) C500/pGS/2SS which comprises the eukaryotic expression plasmid of the somatostatin is preserved in the China Center for Type culture collection (CCTCC) and the preservation number thereof is CCTCC NO: M208194. The method also discloses the preparation method of the vaccine and the applications thereof in the aspect of promoting the growth of the animals.

Description

technical field [0001] The invention belongs to the technical field of animal vaccine preparation, and in particular relates to the preparation and application of an oral DNA vaccine for promoting animal growth through non-resistance screening. technical background [0002] Somatostatin (SS) is a 14-peptide hormone released by the hypothalamus, which has a wide range of inhibitory effects in animals, especially on growth and lactation. Many studies have confirmed that active or passive immunization of animals with SS can increase GH levels and promote animal growth and lactation (Spencer, G.S.G, et al. A novel approach to growth promotion using auto-immunization against somatostatin. I. Effects ongrowth and hormone levels in lambs . Livest Prod Sci, 1983, 10:25-37). Due to the small molecular weight of somatostatin (1658kD) and weak immunogenicity, somatostatin should be coupled with protein carrier or recombined with protein gene during routine immunization to enhance the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/62C12N15/79A61K39/00A61P43/00C12R1/42
Inventor 杨利国梁爱心韩丽刘兴斌罗璇于雪王文慧张伟超
Owner HUAZHONG AGRI UNIV
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