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Universal vaccine immunopotentiator

An immune enhancer, general-purpose technology, applied in the field of biotechnology genetic engineering, can solve problems such as few research and development reports

Active Publication Date: 2020-06-12
QINGDAO MINGQIN BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, researchers have developed a variety of new vaccines for humans and animals using VLPs, but there are few research reports on VLPs as immune enhancers for vaccines.

Method used

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  • Universal vaccine immunopotentiator

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Construction of Escherichia coli expression vector and expression strain

[0027] 1 Send the designed polypeptide encoding nucleotides (see the sequence table SEQ1 and SEQ3) to Shanghai Handsome Biotechnology Co., Ltd. for synthesis, and the two ends of the nucleotide fragments are respectively designed with BamH I (5' end) and HindIII (3' end) restrictions After the fragments were synthesized, they were respectively cloned into the pMD18T vector, and sequence determination confirmed that the inserted gene fragments were consistent with the designed sequence. The recombinant plasmids were named pMD18T-FN-LTB-FC and pMD18T-VP2 / VP6, respectively. Digest the plasmid with the corresponding restriction endonuclease. The expression vector of E. coli is the pRSETA plasmid from Invitrogen Company, which is also treated with the same restriction endonuclease. Digestion conditions: 10 μl reaction system, add 2 μl plasmid into the system , 5 activity units of restrictio...

Embodiment 3

[0031] Embodiment 3 Fermentation, purification and preparation of engineering bacteria

[0032] 1 Take the production strains pRSETA-FN-LTB-FC / BL21 (DE3, Plys) and pRSETA-VP2 / VP6 / BL21 (DE3, Plys) from fermentation, and inoculate them in 2mL LB liquid medium (containing 100 μg / mL ampicillin ), 37°C, 200rpm shaking culture for 12 hours to activate the strain. The inoculum was then placed in shake flasks at an inoculum size of 1:100, cultured with shaking at 37°C until OD600=3, and then inoculated into a fermenter at a ratio of 10%. The medium used for fermentation is a semi-synthetic medium prepared with distilled water. Calibrate dissolved oxygen and pH electrodes, turn on the tank to stir at 300 rpm, and sterilize the tank on-line. When the temperature of the culture solution in the tank drops to 37.0°C, calibrate the zero point of pH and dissolved oxygen (OD). The fermentation temperature was 37.0±0.1°C, the dissolved oxygen was controlled at about 40%, and the pH was contr...

Embodiment 4

[0036] Example 4 Safety Test of Universal Vaccine Immunopotentiator

[0037] 1 material

[0038] 1.1 Vaccine: The emulsified emulsion of the immune enhancer is provided by Qingdao Mingqin Biological Research and Development Center, and the batch numbers are 201701, 201702, and 201703.

[0039] 1.2 Experimental animals: 20-25g Balb / C mice were purchased from Beijing Huafukang.

[0040] 2 methods

[0041] Balb / C mice, 17. Each batch of vaccine immunized 5 mice, totally 3 batches, and immunized 15 mice. The immunization method was subcutaneous injection, and the control group was immunized with physiological saline emulsion in the same way. The health status of the mice was observed continuously for 10 days after immunization.

[0042] 3 results

[0043] The results are shown in Table 1. After immunization, the appetite, spirit and health status of all mice were normal, consistent with the control group, and no death occurred. It can be seen that the vaccine immune enhancer ...

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Abstract

The invention relates to preparation and application of a universal vaccine immunopotentiator. The universal vaccine immunopotentiator is prepared by the following steps of: using a gene recombinationtechnology and connecting a fusion protein coding gene of non-pathogenic escherichia coli flagellin Flic and a heat-labile enterotoxin B subunit (LTB) with a rotavirus VP2 / VP6 fusion protein viroid coding gene through a flexible linker; respectively cloning into a vector, transforming host bacteria, fermenting, purifying, renaturating and folding viroid particles, mixing preparations and the liketo obtain the universal vaccine immunopotentiator. The vaccine immunopotentiator can be mixed with conventional inactivated vaccines, live vaccines, nucleic acid vaccines, subunit vaccines, multi-epitope vaccines, protein engineering vaccines and other genetic engineering vaccine antigens and then emulsified with adjuvants to obtain immunogenicity enhanced vaccines. Animal experiments show that the vaccine immunopotentiator can effectively improve the humoral immunity, cellular immunity and mucosal immunity reaction levels of conventional vaccines.

Description

technical field [0001] The invention belongs to the field of genetic engineering of biotechnology, and mainly relates to the preparation and application of a universal vaccine immune enhancer. Specifically, using gene recombination technology, the fusion protein encoding gene of non-pathogenic Escherichia coli flagellin Flic and heat-labile enterotoxin B subunit (LTB) and the rotavirus VP2 / VP6 fusion protein virion-like particle encoding gene were respectively cloned Entering the vector, transforming the host bacteria, and undergoing fermentation, purification, renaturation and folding of virus-like particles, mixed preparations and other processes, a general-purpose vaccine immune enhancer and the application of the enhancer in vaccines are obtained. Background technique [0002] Vaccine adjuvants are one of the most important bottlenecks restricting the development of new vaccines. An ideal adjuvant can significantly reduce the dose of antigen used, effectively induce the...

Claims

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Application Information

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IPC IPC(8): A61K39/39A61P37/04C07K19/00C12N15/62C12N15/46C07K14/14C12N15/70C12N1/21
CPCA61K39/39A61P37/04C07K14/005C07K14/245C12N15/70C12N2720/12323C07K2319/00A61K2039/55516Y02A50/30
Inventor 李毅蒲勤李殿明季永超齐春梅田春辉党将将张晓丹任百亮张导春刘甜甜
Owner QINGDAO MINGQIN BIOLOGICAL TECH CO LTD
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