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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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  • Summary
  • 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 expression often needs to be emulsified during immunization. Although the emulsification process is simple, it is time-consuming, and the emulsified protein immunization method is mostly injection, which is easy to cause animals. Stress response, affecting immune effect
[0010] At present, the prevention of rabbit viral hemorrhagic disease (Rabbit haemorrhagic disease, RHD) is mainly through the injection of tissue inactivated vaccines. Although some effects have been achieved, some disadvantages have also been found with the application of inactivated vaccines. Difficulty, cumbersome preparation process and biological safety problems limit the prevention of this disease to a certain extent.

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
Comparison scheme
Effect test

Embodiment 1

[0040] Cloning of Example 1 Rabbit IL-2 Gene (Accession Number: NM_001163180.1) and VP60 Gene

[0041]1.1 Extraction of total RNA (extraction of tissue RNA from the spleen of healthy rabbits) The specific operation steps are as follows:

[0042] (1) Break up the tissue (100 mg) with scissors under sterile conditions, grind it to powder with liquid nitrogen under sterile conditions, and add 1 mL of lysate.

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

[0044] (3) After standing still, the interstitial fluid was centrifuged at 4°C and 12000r / min for 5min to remove lipoproteins, etc., and the supernatant was absorbed into a 2mL centrifuge tube.

[0045] (4) Add 200 μL of chloroform into the centrifuge tube, shake vigorously for 15 sec, and let stand for 5 min. Centrifuge at 12000r / min for 15min at 4°C.

[0046] (5) The solution after centrifugation is divided into upper, middle and lower layers (colorless a...

Embodiment 2

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

[0091] 1 Construction of recombinant vector

[0092] (1) T-cloning identified the correct plasmid, and simultaneously digested the T-IL2 and pVAX1 vectors according to the enzyme digestion system in Table 5 below.

[0093] Table 5 enzyme digestion system

[0094]

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

[0096] Form 6T 4 Ligase ligation reaction system

[0097]

[0098] (2) Transformation and identification

[0099] Refer to 1.5(3) in Example 1 for the specific operation process of the transformation, replace the Amp-LB plate with Kan-LB, replace the pMD19-T vector with the pVAX1 eukaryotic expression vector; identify the same as above, replace Amp with Kan.

[0100] The result is as Figure 4 As shown, through colony PCR identification and specific restriction endonuclease dige...

Embodiment 3

[0121] The construction of embodiment 3 attenuated salmonella oral vaccine adjuvant

[0122] 3.1 Preparation of Competent Salmonella

[0123] The attenuated Salmonella (SL7207) stored at -80°C was streaked and inoculated on ordinary LB solid medium, cultured overnight at 37°C, and a single colony with uniform shape was picked and inoculated in 10 mL of LB liquid medium, shaken at 37°C (160r / min) After culturing for 12-16 hours, the bacterial DNA was 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 future use.

[0124] Streak inoculate the revived Salmonella on LB plate, culture overnight at 37°C, pick a single uniform colony and inoculate it into 5mL LB broth, culture with shaking at 37°C (160r / min) for 12-16h, then draw 1mL of bacterial liquid Inoculate into 100mL LB broth, shake culture at 37°C (160r / min) until OD 600 0.5 to 0.6, centrifuge the bacter...

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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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