Immunoenhancer as well as application thereof in vaccine preparation

An immune enhancer and variable region technology, applied in the development of therapeutic drugs, immune enhancer and its application in vaccine preparation, can solve the problems of weak stimulation of adaptive immune response, recombination, and low market acceptance

Active Publication Date: 2020-05-12
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing prevention and control of PRRSV mainly rely on attenuated live vaccines and inactivated vaccines. Existing evidence shows that the production of attenuated live vaccines can induce immune protection as soon as possible in the production practice, but the immunized animals shed the virus (vaccine strains) , long-term existence in immune...

Method used

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  • Immunoenhancer as well as application thereof in vaccine preparation
  • Immunoenhancer as well as application thereof in vaccine preparation
  • Immunoenhancer as well as application thereof in vaccine preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Preparation and Identification of Monoclonal Antibody 5D9

[0030] 1.1 Establishment of hybridoma cell lines

[0031] PRRSV virus SD16 (provided by the Immunobiology Laboratory of Northwest Agriculture and Forestry College of Veterinary Medicine) was used as an immunogen, mixed and emulsified with Freund's complete adjuvant (Sigma company) 1:1, and immunized 6-week-old female Balb / c mice (provided by provided by Xi'an Jiaotong University), subcutaneously injected into the abdomen at a dose of (3×10^7 PFU / bird), and booster immunization every 14 days. 7 days after the third immunization, the tail vein blood was collected and the antibody titer against the immunogen in the mouse serum was detected by IFA. The mouse with the best titer was injected into the tail vein for shock immunization, and the immunogen was mixed with normal saline 1:1. , the dose is (3×10^7 PFU / bird).

[0032] 1.2 Cell Fusion

[0033] (1) Spleen cells of the immunized mice were aseptica...

Embodiment 2

[0057] Example 2: Determination of neutralizing activity of monoclonal antibody 5D9

[0058] 2.1 Determination of neutralizing activity

[0059] The virus neutralization experiment was carried out using the monoclonal antibody 5D9 to detect its neutralizing activity. Spread PAM cells into 24-well plates, insert 0.01 MOI of different types of PRRSV SD16 virus, each virus was incubated with 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml of antibodies, and incubated at 37°C for 1 hour before changing After 36 hours, Western blot and qPCR tests were performed to confirm that it had neutralizing activity. For specific results, see figure 1 .

[0060] based on figure 1 The results show that the alveolar macrophages (PAMs), the natural target cells of PRRSV in the host, were cultured in vitro, and the purified monoclonal antibody 5D9 was used in accordance with the concentrations of 0.05, 0.1, 0.2, 0.4 μM / mL (micromoles per milliliter) and 0.01M PRRSV-SD16 virus was cultured at 37°C...

Embodiment 3

[0067] 3.1 Preparation of antigen and compounding with antibody (take PRRSV virus as an example below)

[0068] Use the PRRSV-SD16 strain to infect Marc145 cells, collect the virus culture supernatant 72 hours after the virus infection, centrifuge a sufficient amount of the virus culture supernatant at 12000g for 1 hour to remove cell debris, and use a 100kD molecular weight filter through the Labscale TFF filter Filter membrane (EMD Millipore Company) uses 100kD molecular weight filter membrane to concentrate 50 times, then purifies PRRSV-SD16 virion by liquid chromatography.

[0069] Treat the virus with β-propiolactone at a concentration of one-thousandth and place it at 4 degrees overnight to inactivate the virus. After placing it in a water bath at 37 degrees for 1 hour to degrade β-propiolactone, mix the virus with monoclonal antibody 5D9 (calculated by mass) ) were mixed at a ratio of 1:5, and placed at 37°C for 2 hours to form a complex.

[0070] 3.2 Preparation of va...

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Abstract

The invention adopts a monoclonal antibody 5D9 with neutralization activity to both PRRSV-I and PRRSV-II viruses to prepare an immunoenhancer. When a formed immune complex is combined with an adjuvantto immunize mice, T cells secreted by IFN-gamma are significantly increased, suggesting that combined immunization of the PRRSV specific antibody 5D9 and the normal oil in water adjuvant can enhanceCTL reaction in the process of inactivating virus immunity. Animal tests show that the immune protection rate of the immune complex prepared by the method can achieve a higher protection effect than that of a vaccine and a commercialized attenuated vaccine prepared only with a commercial adjuvant ISA 206.

Description

Technical field: [0001] The invention belongs to the field of biology, and in particular relates to an immune enhancer and its application in vaccine preparation, which can be used in the development of therapeutic drugs for porcine reproductive and respiratory syndrome. Background technique: [0002] Vaccines can be mainly divided into live attenuated vaccines, inactivated vaccines and protein subunit vaccines expressed by genetic engineering techniques. Among the above three types of vaccines, inactivated vaccines and protein subunit vaccines are the most safe, but because they cannot proliferate after being immunized into the body, it is difficult to effectively stimulate the host immune system, often requiring multiple immunizations, and stimulating the body's production of The immune response is mainly based on the humoral immune response (antibody), and it is difficult to stimulate the body to produce a cellular immune response (killer T cells, CTL). Therefore, for som...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13A61K39/42A61K39/12A61K39/39A61P31/14
CPCC07K16/10A61K39/42A61K39/12A61K39/39A61P31/14C07K2317/56C07K2317/76A61K2039/552A61K2039/5252A61K2039/55566A61K2039/505C12N2770/10034
Inventor 南雨辰周恩民武春燕
Owner NORTHWEST A & F UNIV
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