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Research approach of IFN-alpha anti PCV2 and PRRSV induced by forsythiaside

A technology of forsythiaside and forsythiaside, which is applied in the field of antiviral research of alpha interferon induced by active ingredients of traditional Chinese medicine, can solve the problems of high infection rate, mixed infection, and no specific drugs in pigs, and achieves experimental data. Reliable, easily sourced results

Inactive Publication Date: 2009-03-11
BEIJING UNIV OF AGRI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some progress has been made in the research of PCV2 vaccine, it is still affected by many problems such as high-titer virus proliferation, immune efficacy evaluation, high infection rate in pig herds, mixed infection, etc., so far no vaccine has been successfully developed and applied.
Moreover, there is currently no specific drug for the prevention and treatment of PCV2 and PRSSV

Method used

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  • Research approach of IFN-alpha anti PCV2 and PRRSV induced by forsythiaside
  • Research approach of IFN-alpha anti PCV2 and PRRSV induced by forsythiaside
  • Research approach of IFN-alpha anti PCV2 and PRRSV induced by forsythiaside

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0009] Example 1 Anti-PCV2, PRSSV experiment of forsythiaside in vitro

[0010] 1 Toxicity determination of forsythiaside on PK15 and Marc-145 cells

[0011] 1.1 Screening of the serum concentration of PK15 and Marc-145 cells (purchased from the China Veterinary Drug Control Institute) The growth of the cells is too fast or too slow, which is not conducive to the drug and challenge experiments. This study compares 10%, 5%, and The influence of 2% fetal bovine serum on cell growth, the optimized serum concentration adapted to the experimental requirements.

[0012] 1.2 CPE method to determine the toxicity of forsythiaside to PK15 cells In 96-well cell culture plate, add 8×10 3 PK15 cells at a concentration of 0.1ml / well at 37°C with 5% CO 2 After culturing for 24 hours in the incubator, the cells were replaced with serum-free MEM basal medium, and the cells were synchronized for 12 hours. Use 5% MEM culture solution (add 50ml 5×MEM concentrate in 195ml three-distilled water,...

Embodiment 2

[0075] Example 2 Anti-induction of IFN-α by forsythiaside in vitro

[0076] 1 Sample preparation

[0077] Collect 80μg / ml, 40μg / ml, and 20μg / ml3 concentrations of forsythiaside for 1, 4, 8, 12, 18, 24, 48, and 72h of the cell supernatant and 100TCID of PK15 cells 50 Cell supernatants with different concentrations of PCV2 were stored at -80°C for later use.

[0078] 2 Preparation of standard curve and detection of samples

[0079] The IFN-α kit should be equilibrated to room temperature (20-25°C) before experimenting.

[0080] Add 100 μl of Standards and 100 μl of cell culture supernatant to corresponding reaction plate wells.

[0081]Gently mix for 30s, seal the plate well, and incubate at 37°C for 60min.

[0082] Plate washing: Shake off the liquid in the plate, wash the reaction plate with washing liquid (add 350 μl washing liquid to each well), and remove water droplets (pat dry on thick absorbent paper); wash 5 times repeatedly.

[0083] Add 100 μl 1x Biotin to each w...

Embodiment 3

[0098] Example 3 Quantitative determination of forsythiaside against PCV2 and PRRSV

[0099] 1 Establishment of cell models of PRRSV and PCV2

[0100] The PCV2 adapted to PK15 cells was blindly passed on Marc-145 for several generations, and the Marc-145 cells inoculated with PCV2 were treated with D-glucosamine, and PRRSV was inoculated after PCV2 was detected. The amount of inoculated PRRSV is appropriate because PRRSV is easy to multiply in Marc-145 cells and has cytopathic changes.

[0101] After the virus cell model was established, the concentration of the drug was 60, 40, and 20 μg / ml to test the simultaneous anti-PCV2 and PRRSV effects of forsythiaside.

[0102] 2 Extraction of PRRSV RNA and PCV2 DNA

[0103] 2.1 Extraction of PCV2DNA

[0104] The PK-15 cell culture for propagating PCV2 was repeatedly frozen and thawed 3 times, centrifuged at 12000r / min at 4°C for 10min, aspirated 0.3ml of supernatant, added an equal volume of tissue lysate and 10μl of proteinase K ...

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Abstract

The invention relates to an application of forsythiaside in anti-PCV2 and anti-PRRSV. The measured prevention effect of the forsythiaside in anti-PCV2 and anti-PRRSV is greater than the treatment effect and further greater than the direct killing effect. The IFN-Alpha secretion after being acted in cells is gradually increased along with the increase of the concentration of the forsythiaside within the range of the safe concentration of the forsythiaside, the virus proliferation is gradually decreased, while the virus proliferation and the amount of the IFN-Alpha show negative correlation. The forsythiaside can induce the IFN-Alpha to express and play the anti-virus role. The invention provides a new method for researching anti-virus traditional Chinese medicine, provides a drug for treating PCV2 and PRSSV and lays the foundation for the action mechanism of anti-PCV2 and anti-PRRSV in vitro of the forsythiaside. The application has the advantages that: materials are easy to obtain, cells are a cell system which can be passaged, the application has no individual difference of animals, and the obtained experimental data is comparatively reliable.

Description

technical field [0001] The invention relates to the field of veterinary medicine, and is a method for antiviral research on the induction of alpha interferon (interferon-alpha, IFN-alpha) by active ingredients of traditional Chinese medicine. Background technique [0002] Porcine postweaning multisystemic wasting syndrome (PMWS) is an infectious disease caused by porcine circovirus type 2 (PCV2) characterized by progressive emaciation, pale skin, respiratory symptoms, and swollen lymph nodes. Clinically, it mainly infects weaned piglets aged 6-15 weeks, and the morbidity and mortality are often between 4%-30% and 50%-90%. Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease characterized by abortion, stillbirth, fetal mummification, and respiratory disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). Both PRRSV and PCV2 can cause immunosuppression in infected pigs, and co-infection is very common clinically. Therefore, PRRS...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7028A61P31/12
Inventor 吴国娟杨明张中文
Owner BEIJING UNIV OF AGRI
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