Novel pharmaceutical protein DRACO and applications thereof in prevention and treatment of porcine reproductive and respiratory syndrome

A drug and protein technology, applied in the field of molecular biology, can solve the problems of weak cellular immunity, dispersing toxins, and short immune period, and achieve good antiviral effects

Inactive Publication Date: 2014-02-12
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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
However, the attenuated vaccine has the risk of strong virulence, recombination and potential infection

Method used

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  • Novel pharmaceutical protein DRACO and applications thereof in prevention and treatment of porcine reproductive and respiratory syndrome
  • Novel pharmaceutical protein DRACO and applications thereof in prevention and treatment of porcine reproductive and respiratory syndrome
  • Novel pharmaceutical protein DRACO and applications thereof in prevention and treatment of porcine reproductive and respiratory syndrome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 DRACO Gene Design Synthesis and Expression Analysis

[0047] 1. Design and synthesis of DRACO gene: According to NCBI (http: / / www.ncbi.nlm.nih.gov / ), the partial sequence of porcine protein kinase R (Protein Kinase R, PKR) (sequence number is AB104654.1) gene (as shown in SEQ ID NO.4) and the gene partial sequence of apoptosis protease activating factor 1 (apoptotic protease activating factor 1, Apaf-1, sequence number is AF013263.1) (as shown in SEQ ID NO.5) , N-terminal plus a transducing peptide sequence PTD, the amino acid sequence of PTD is shown in SEQ ID NO.3, and the DRACO protein coding gene is designed, and the sequence is shown in SEQ ID NO.1. The amino acid sequence of the DRACO protein encoded by the gene is SEQ ID NO.2. The designed DRACO sequence was sent to Sangon Bioengineering (Shanghai) Co., Ltd. (http: / / sangon.com / ) for synthesis.

[0048] 2. Construction of DRACO gene recombinant prokaryotic expression strain: the DRACO gene synthesize...

Embodiment 2

[0052] Example 2 Purification of DRACO protein

[0053] The DRACO protein was purified by His-tag through the column, and the BCA protein quantification kit (purchased from Thermo Scientific, catalog number: 23227) was used to determine the concentration of the DRACO protein.

[0054] 1. Prepare the protein sample, phosphate buffer, Binding buffer, Washing buffer, Elution buffer, 20% ethanol, distilled water, and install the Ni-NTA chromatography column on the iron stand.

[0055] 2. Use a syringe to add ddH2O 4 times the column volume (5mL) to wash the Ni-NTA chromatography column, equilibrate the chromatography column, and use a Binding buffer 4 times the volume of the column to balance the column. The Binding buffer plays an adhesion role, the purpose is For the next step, the filtered His-tagged protein adhered to Ni-NTA resin, and the filtered His-tagged protein was added to the chromatographic column equipped with Ni-NTA resin in the same way.

[0056] 3. Use 4 times ...

Embodiment 3

[0058] Example 3 DRACO Cytotoxicity Test

[0059]AlamarBlue (purchased from Invitrogen) is used as an indicator of living cell metabolism. Under the mitochondrial enzymatic reduction reaction, a measurable fluorescent metabolite will be produced, and the cell activity can be monitored by measuring its fluorescence intensity. Cultivate Marc-145 cells with DMEM medium containing 10% fetal bovine serum to 60-70%, discard the medium, add nutrient solution containing DRACO times dilution for 36 hours, set the PBS control group, and then add 10% ( V / V) ratio AlamarBlue continued to incubate for 3 hours, and read the fluorescence values ​​of excitation light at 540nm and emission light at 590nm with a multi-functional microplate reader to make a DRACO cytotoxicity map. See attached image 3 , taking the cell viability of the PBS control group as 100%, the fluorescence value of the cells treated with DRACO in multiple dilutions compared with the fluorescence value of the PBS contro...

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Abstract

The invention discloses a novel pharmaceutical protein DRACO and applications thereof in prevention and treatment of porcine reproductive and respiratory syndrome. Correlation sequences of pigs on NCBI are utilized to design the protein coding gene of DRACO which is provided with activity and capable of entering cells, then the DRACO protein is expressed and purified in a prokaryotic expression system, the antivirus activity of DRACO protein to HP-PRRSV (Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus) is researched at the Marc-145 cellular level by qRT-PCR (Quantitative Real-Time Polymerase Chain Reaction), Western-Blot, cellular supernatant TCID50 (Tissue Culture Infectious Dose 50) detection, IFA (Indirect Immunofluorescence Assay) and other methods, and the antiviral mechanism of DRACO in two processes of absorbing viruses and entering cells are further explained. The DRACO produced by the method disclosed by the invention has good antiviral effect to HP-PRRSV, and is expected to be a novel drug for preventing and treating porcine reproductive and respiratory syndrome.

Description

technical field [0001] The present invention relates to the field of molecular biology. More specifically, it relates to a new drug protein DRACO and its application in preventing and treating pig PRRS. 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 hyper...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N15/70C12N1/21A61K38/17A61P31/14
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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