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Oral recombined DNA vaccine for accelerating growth of animal, and application

A DNA vaccine and animal growth technology, applied in the field of animal genetic engineering, can solve the problems of unfavorable growth of large animals, affect the immune effect, and the inability of viruses to replicate in large quantities, so as to achieve the effect of increasing daily weight gain and feed utilization, and improving the immune effect

Inactive Publication Date: 2007-10-10
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this vaccine is that the recombinant vaccinia virus is used as a live carrier vaccine to directly immunize animals. After the initial immunization, antiviral bodies are produced in the animal body, and the virus cannot replicate in large quantities during the second immunization, which affects the immune effect; risks of
However, the application of the DNA vaccine in piglets has not been seen, and the production of the vaccine requires a large amount of plasmid extraction. During the extraction process, there may be risks of endotoxin poisoning in animals due to unclean removal of endotoxin. In addition, intramuscular injection is easy to make animals Generate greater stress, which is not conducive to the growth of large animals

Method used

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  • Oral recombined DNA vaccine for accelerating growth of animal, and application
  • Oral recombined DNA vaccine for accelerating growth of animal, and application
  • Oral recombined DNA vaccine for accelerating growth of animal, and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction of eukaryotic expression plasmid pGM-CSF

[0039] 1.1 Reverse transcription and PCR amplification

[0040] Using the RT-PCR kit (refer to the instruction manual of the RNA PCR Kit (AMV) Ver.3.0 kit of TaKaRa) to take 1-2 μg of the extracted total cellular RNA for reverse transcription, and react 10 μL total volume containing:

[0041] MgCl 2 2μL

[0042] OligodT primer (2.5pmol / μL) 0.5μL

[0043] dNTP Mixture (each 10mM) 1μL

[0044] RNase inhibitor (40U / μL) 0.25μL

[0045] Reverse transcriptase (5U / μL) 0.5μL

[0046] 10×RT Buffer 1μL

[0047] RNase Free dH 2 O to bring the total reaction volume to 10 µL.

[0048] Perform reverse transcription reaction under the following conditions: 30°C for 10min

[0049] 42℃30min

[0050] 99℃5min

[0051] 5℃5min

[0052] React for 1 cycle. The RT product was stored at -20°C for later use.

[0053] Add 10 μL of the reve...

Embodiment 2

[0082] Example 2 Construction of Fusion Expression Plasmid pGMCSF-SS

[0083] 2.1 Extraction and purification of plasmid pGM-CSF

[0084] Pick a single colony of newly activated pGM-CSF from the LB plate, inoculate it into 10 mL of LB liquid medium, cultivate overnight at 37°C and 240 rpm, dilute it into an appropriate volume of LB at a volume ratio of 1:250, and continue culturing for 12 hours. The bacteria were collected by centrifugation, and the pGM-CSF plasmid was extracted and purified (refer to the instruction manual of the plasmid extraction kit of Shanghai Huashun Biological Co., Ltd.).

[0085] 2.2 Amplification of GM-CSF coding gene

[0086] Referring to accompanying drawing 4, using Primer 5.0 primer design software, the GMCSF gene was obtained by PCR using the porcine eukaryotic expression plasmid pGM-CSF (Example 1) as a template. The upstream primer (P1) is: 5'-GTAT CTCAGAAGGATGTGGC-3', introduced the restriction endonuclease XhoI site (the sequence in the b...

Embodiment 3

[0101] Example 3 Preparation of Oral Recombinant DNA Vaccine Using Attenuated Salmonella Typhimurium as a Vector

[0102] 3.1 pGMCSF-SS plasmid transformation of attenuated Salmonella typhimurium

[0103] The same method as above was used to prepare fresh competent attenuated Salmonella typhimurium, and the fusion expression plasmid pGMCSF-SS was transformed into competent attenuated Salmonella typhimurium (the specific operation steps were the same as in Example 1.3).

[0104] The attenuated Salmonella typhimurium (Salmonella typhimurium) CSO22 / pGMCSF-SS containing the fusion expression plasmid was deposited in China Center for Type Culture Collection (CCTCC) on December 19, 2006, and the preservation number is CCTCC M206141.

[0105] 3.2 Identification of plasmids in recombinant Salmonella typhimurium

[0106] Pick the single colonies of positive clones obtained in step 3.1 and inoculate them in Amp-containing LB liquid medium and culture them overnight at 37°C, and extract...

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Abstract

This invention relates to preparation and application of orally administered recombinant DNA vaccine for accelerating animal growth, which Salmonella typhimurium CSO22 / pGMCSF-SS (CCTCC M206141) is containing fusion expression plasmid pGMCSF-SS. Salmonella typhimurium CSO22 / pGMCSF-SS is prepared by: cloning GMCSF, fusing with plasmid pcS / 2SS containing somatostatin gene to obtain fusion expression plasmid pGMCSF-SS, and transforming Salmonella typhimurium. This invention also discloses the application of Salmonella typhimurium CSO22 / pGMCSF-SS in orally administered recombinant DNA vaccine for accelerating animal growth.

Description

technical field [0001] The invention belongs to the technical field of animal genetic engineering, and in particular relates to an oral recombinant DNA vaccine for promoting animal growth and its application. Background technique [0002] Animal growth is the result of the comprehensive influence of factors such as genetics, nutrition, environment, and hormone regulation. Among them, strengthening breed improvement, providing full-price nutrition, and providing a suitable environment have become effective ways to improve animal production performance, but their effects are ultimately It is realized through the integration and regulation of neuroendocrine. At present, regulating animal growth by changing hormone secretion is one of the current research hotspots. With the rise and development of hormone immunology, hormone immunoneutralization technology (Hormone Immunoneutralization, HIN) came into being, that is, actively induce or pass...

Claims

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Application Information

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IPC IPC(8): C12N1/21A61K48/00C12R1/42
Inventor 杨利国舒邓群梁爱心
Owner HUAZHONG AGRI UNIV
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