Adjuvant of rabbit plague oral vaccine IL-2 and application

An oral vaccine, IL-2 technology, applied in the field of rabbit plague oral vaccine IL-2 adjuvant, can solve the problems of unsuitable expression of eukaryotic genomic DNA, difficulty in obtaining raw materials for vaccine preparation, difficulty in expressing soluble protein, etc., to avoid easy The problem of dispersing toxins, the simple and efficient way of immunization, the effect of enhancing humoral immunity and promoting the secretion of specific antibodies

Inactive Publication Date: 2017-09-26
SICHUAN AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of prokaryotic expression are as follows: (1) due to the lack of post-transcriptional processing mechanism, only cloned cDNA can be expressed, and eukaryotic genomic DNA is not suitable for expression; (2) due to the lack of appropriate post-translational processing mechanism, the expressed eukaryotic protein cannot form proper (3) The expressed protein often forms insoluble inclusion bodies, and complex renaturation treatment is required to make it active; (4) It is difficult to express a large amount of soluble protein, so it is necessary to Optimizing the conditions of protein expression, etc., is time-consuming and laborious; (5) The protein after prokaryotic expr

Method used

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  • Adjuvant of rabbit plague oral vaccine IL-2 and application
  • Adjuvant of rabbit plague oral vaccine IL-2 and application
  • Adjuvant of rabbit plague oral vaccine IL-2 and application

Examples

Experimental program
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Example Embodiment

[0040] Example 1 Cloning of rabbit IL-2 gene (accession number: NM_001163180.1) and VP60 gene

[0041] 1.1 Total RNA extraction (using healthy rabbit spleen as material for tissue RNA extraction) The specific operation steps are as follows:

[0042] (1) Use scissors to break the tissue (100 mg) under aseptic conditions, grind it to powder with liquid nitrogen under aseptic conditions, and add 1 mL of lysis solution.

[0043] (2) Put the ground tissue fluid into a centrifuge tube and let it stand at room temperature for 5 minutes.

[0044] (3) Centrifuge the tissue fluid after standing at 4°C at 12000r / min for 5min to remove lipoproteins, etc., suck the supernatant into a 2mL centrifuge tube.

[0045] (4) Add 200 μL of chloroform to the centrifuge tube, shake vigorously for 15 seconds, and let stand for 5 minutes. Centrifuge at 12000r / min for 15min at 4℃.

[0046] (5) The solution after centrifugation is divided into three layers (colorless water phase, white protein, organic phase), and...

Example Embodiment

[0090] Example 2 Construction and expression of eukaryotic expression vector of rabbit IL-2 and VP60

[0091] 1 Construction of recombinant vector

[0092] (1) T clone identifies the correct plasmid, and simultaneously digests the T-IL2 and pVAX1 vectors according to the digestion system in Table 5 below.

[0093] Table 5 Enzyme digestion system

[0094]

[0095] The digested fragments were recovered by gel and kept overnight at 16°C. The reaction system is as shown in Table 6 below:

[0096] Table 6T 4 Ligase ligation reaction system

[0097]

[0098] (2) Transformation and identification

[0099] For the specific operation procedure of transformation, refer to 1.5 (3) in Example 1. Replace the Amp-LB plate with Kan-LB, and replace the pMD19-T vector with the pVAX1 eukaryotic expression vector; the identification is the same as above, and the Amp is replaced with Kan.

[0100] The result is Figure 4 As shown, through colony PCR identification and specific restriction enzyme digestion, it...

Example Embodiment

[0121] Example 3 Construction of Attenuated Salmonella Oral Vaccine Adjuvant

[0122] 3.1 Preparation of Salmonella Competent

[0123] The attenuated Salmonella (SL7207) stored at a low temperature of -80°C was streaked into ordinary LB solid medium and cultured overnight at 37°C. A single colony with uniform morphology was picked and inoculated into 10mL LB liquid medium, and shaken at 37°C (160r / min) After culturing for 12-16 hours, the bacterial DNA is extracted for PCR identification and sent for sequencing. The correctly identified Salmonella (SL7207) was inoculated into 10% glycerol, aliquoted and stored at -20°C for later use.

[0124] Streak the resuscitated Salmonella on the LB plate. After incubating overnight at 37°C, pick a single uniform colony and inoculate it into 5mL LB broth. After shaking at 37°C (160r / min) for 12-16 hours, absorb 1mL bacterial solution Inoculate into 100mL LB broth, shake culture to OD at 37℃ (160r / min) 600 Centrifuge the bacterial solution at 4...

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Abstract

The invention discloses an adjuvant of a rabbit plague oral vaccine IL-2 and application. The adjuvant of the rabbit plague oral vaccine IL-2 is prepared by mixing recombinant bacteria SL7207-PVAX1-IL-2 and the recombinant bacteria SL7207-PVAX1-VP60. The presented recombinant strains SL7207-PVAX1-IL2 and SL7207-PVAX1-VP60 of attenuated salmonella typhimurium SL7207 were constructed correspondingly and the two recombinant bacteria are immunized in rabbits together, and IL-2 could exert the immune stimulating effect to enhance the immune effect of the oral vaccine. The adjuvant has high safety, high biosafety, simple and efficient immunization method, simple vaccine preparation and double adjuvant effects.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an IL-2 adjuvant for rabbit plague oral vaccine and its application. Background technique [0002] Rabbit haemorrhagic disease (RHD) is an acute and highly pathogenic infectious disease caused by rabbit haemorrhagic disease virus (RHDV), also known as "rabbit plague, rabbit haemorrhagic disease" . The disease is mainly characterized by solid organ bleeding, congestion and edema, tracheal hemorrhage, lung hemorrhage and swelling, and high fever (>40°C). Rabbit viral hemorrhagic disease was first reported in Jiangyin, Jiangsu, China, and Wuxi in 1984. In less than a year, about 140 million rabbits died of this disease, and the infected area was nearly 50,000 square kilometers. Rabbit viral hemorrhagic disease only infects rabbits, among which it is most harmful to domestic rabbits and European hares (Oryctolagus cuniculus). Experiments have shown that o...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N1/21A61K39/12A61K39/39A61P31/14C12R1/42
CPCA61K39/0275A61K39/12A61K39/39A61K2039/523A61K2039/53A61K2039/542A61K2039/552A61K2039/55533A61K2039/575C07K14/55C12N15/79C12N2770/16034A61K2300/00Y02A50/30
Inventor 耿毅邓钊宾余泽辉汪开毓陈德芳欧阳萍黄小丽杨泽晓
Owner SICHUAN AGRI UNIV
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