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Application of antimicrobial peptide Protegrin-1 for preventing and controlling porcine reproductive and respiratory syndrome

A porcine blue ear disease and antibacterial peptide technology, applied in the field of medicine, can solve the problems of short immunization period, incomplete inactivation, and difficulty in PRRSV prevention and control, and achieve the effect of good antiviral effect.

Inactive Publication Date: 2014-03-12
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(5) Mixed infection. At present, clinical mixed infection, especially the mixed infection of circovirus, Haemophilus parasuis and PRRSV, makes the prevention and control of PRRSV even more difficult.
Among them, the inactivated vaccine has the following disadvantages: (1) It requires large doses of vaccination or the application of concentrated antigens, and the immunity period is short, often requiring booster vaccination; (2) It cannot cause local immunity, so that the effect of cellular immunity is weak; (3) It produces complete immunity It takes 2-3 weeks, which is not conducive to emergency vaccination and the reduction of vaccine costs; (4) There is a possibility of incomplete inactivation and loose virus
Whether antimicrobial peptide PG-1 has antiviral effect on PRRSV virus has not been reported so far

Method used

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  • Application of antimicrobial peptide Protegrin-1 for preventing and controlling porcine reproductive and respiratory syndrome
  • Application of antimicrobial peptide Protegrin-1 for preventing and controlling porcine reproductive and respiratory syndrome
  • Application of antimicrobial peptide Protegrin-1 for preventing and controlling porcine reproductive and respiratory syndrome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 PG-1 chemical synthesis and anti-E. coli activity experiment

[0037] 1. First search for the amino acid sequence of porcine antimicrobial peptide PG-1 through The Antimicrobial Peptide Database (APD) (http: / / aps.unmc.edu / AP / main.php), as shown in SEQ ID NO.1 As shown, the amino acid sequence was sent to Shanghai Gill Biochemical Co., Ltd. (http: / / glbetter.cn.1688.com / ) for synthesis, because the β-sheet structure of PG-1 is very important to its biological activity, and β- The formation of the folded structure depends on the two pairs of disulfide bonds in PG-1, so when synthesizing PG-1, we must ensure that the disulfide bonds are successfully synthesized and positioned accurately. The two pairs of disulfide bonds are the 6th and 15th Cys, the 8th and 13th Cys, and in order to enhance the biological activity of PG-1, its C-terminal is amidated and modified. HPLC 95% purity, synthesis 30mg. After synthesis, 0.01% glacial acetic acid sterilized water was dilut...

Embodiment 2

[0042] Example 2 PG-1 cytotoxicity test

[0043] AlamarBlue (purchased from Invitrogen) is used as an indicator of living cell metabolism. It produces measurable fluorescent metabolites under the enzymatic reduction reaction of mitochondria, and cell activity can be monitored by measuring its fluorescence intensity. Culture Marc-145 cells to 60-70% in DMEM medium containing 10% fetal bovine serum, discard the culture medium, add nutrient solution diluted with PG-1 times ratio for 36 hours, set PBS control group, then add 10 %(V / V) ratio AlamarBlue was cultured for 3 hours, and the fluorescence values ​​of 540nm excitation light and 590nm emission light were read using a multifunctional microplate reader to make PG-1 cytotoxicity chart (see attached figure 2 ). Taking the cell activity of the PBS control group as 100%, the fluorescence value of the cells treated with PG-1 diluted in multiples is compared with the fluorescence value of the PBS control group, which is the relative...

Embodiment 3

[0044] Example 3 PG-1 antiviral test

[0045] 1. When Marc-145 cells are cultured in a 6-well plate containing 10% fetal bovine serum in DMEM medium to a cell confluency of 70%, discard the culture medium, wash 3 times with PBS, and inoculate HP-PRRSV with MOI=0.1 , In the DMEM culture medium of 2% fetal bovine serum 37 ℃ continue to cultivate 5h.

[0046] 2. Wash with PBS 3 times, add 2% fetal bovine serum DMEM culture medium with a concentration of 20, 30, 40 mg / L PG-1 to the cells, culture for 36 hours at 37°C, and set a PBS control group, wash 3 times with PBS, and collect TCID for supernatant 50 Test, see attached image 3 ,by image 3 It can be seen that PG-1 can significantly reduce the virus production in the cell supernatant and inhibit the release of the virus into the cell supernatant; in addition, add 400 μL Trizol to each well to extract RNA and do qRT-PCR detection, see attached Figure 4 ,by Figure 4 It can be seen that at the transcription level, PG-1 can signific...

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Abstract

The invention belongs to the technical field of medicines, and in particular discloses application of an antimicrobial peptide Protegrin-1 for preventing and controlling porcine reproductive and respiratory syndrome. According to the invention, an antimicrobial peptide Protegrin-1 is chemically synthesized according to an amino acid sequence of reported Protegrin-1 firstly; then, antimicrobial activity of the antimicrobial peptide Protegrin-1 is proved through an escherichia coli resistant DH5 alpha experiment, and researches on the antivirus activity of the antimicrobial peptide to HP-PRRSV are made through qRT-PCR, Western-Blot, cell supernatant TCID50 detection, IFA and other methods; and further the antivirus mechanism of the antimicrobial peptide is explained in virus adsorption and entrance into cell processes. The antimicrobial peptide Protegrin-1 is expected to be used as a novel medicine for preventing and controlling porcine reproductive and respiratory syndrome.

Description

technical field [0001] The invention relates to the technical field of medicine, in particular to the application of antibacterial peptide Protegrin-1 in preventing and treating pig blue-ear disease. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), also known as porcine blue ear disease, is caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV). The disease first broke out in the United States in 1987, and then spread to Europe. The virus was isolated in my country in 1996. Currently, PRRSV is divided into two genotypes based on genome sequence and antigenicity differences, one is the European type represented by the Lelystad Virus (LV) strain, and the other is the American type represented by the ATCC VR-2332 strain . In my country, the American type of PRRSV is the main type, but it is reported that European type strains have also been isolated. In 2006, pig hypertherm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/10A61K9/19A61K9/20A61K9/48A61K9/16A61P31/14
CPCY02A50/30
Inventor 刘小红郭春和莫德林陈瑶生高进涛
Owner SUN YAT SEN UNIV
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