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Application of isoindolone compound in preparation of medicine for preventing and treating osteolytic diseases

A subunit vaccine and genetic engineering technology are applied in the field of genetic engineering subunit vaccines for preventing new variant strains of infectious bursal virus of chickens and the preparation thereof, and can solve the problems of inability to control infection well, atrophy of bursal organs, There are no clinical symptoms and other problems, and the effect of preventing the immune effect is achieved.

Pending Publication Date: 2020-01-10
荆州市长新生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in recent years, more and more cases have been reported of novel variants of infectious bursal virus (Novel variant IBDV strains, nvIBDV). These variants have no obvious clinical symptoms after infecting chickens, but they can also cause a certain degree of disease. Immunosuppression and Pathologic Manifestations of Significant Atrophy of Bursal Organs in Pathological Anatomy
Infection with these new variants is poorly controlled with subunit vaccines targeting vvIBDV

Method used

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  • Application of isoindolone compound in preparation of medicine for preventing and treating osteolytic diseases
  • Application of isoindolone compound in preparation of medicine for preventing and treating osteolytic diseases
  • Application of isoindolone compound in preparation of medicine for preventing and treating osteolytic diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction of IBDV FJ-1812 strain VP2 protein gene cloning vector

[0033] 1. Extraction of RNA from disease materials

[0034] Main reagents: ultrapure total RNA extraction kit (Simgen product).

[0035] Operation process: Take about 100mg of chicken bursa tissue infected with the mutant strain of infectious bursal virus, and add 1ml of Trizol to a sterile 1.5mL Ep tube. Add 2 3mm stainless steel grinding balls. Place in a tissue grinder pre-cooled at -30°C and run at a vibration frequency of 50 Hz for 60 s. Add 200 μL of chloroform, shake vigorously to mix, and centrifuge at 12000 g for 15 min at 4°C. Take about 600 μL of the supernatant, add an equal volume of 70% ethanol, and mix well. Transfer 600 μL of the above mixture to a nucleic acid purification column, centrifuge at 12,000 g for 1 min at 4°C, and discard the filtrate. Then add the remaining mixture to the nucleic acid purification column, centrifuge at 12000g for 1min at 4°C, and discard the...

Embodiment 2

[0063] Example 2 IBDV FJ-1812 strain VP2 prokaryotic expression

[0064] 1. Construction and expression of E.coli BL21 / pET28a IBDV FJ-1812 VP2

[0065] The pTOPO-IBDV FJ-1812 VP2 plasmid was digested with a restriction endonuclease (BamHI / Xho I), and ligated with the expression plasmid pET28a digested with the same restriction endonuclease. The ligation product was named: pET28aIBDV FJ-1812 VP2.

[0066] The ligation product was used to transform Escherichia coli BL21(DE3), and transformants were screened. An engineering bacterium expressing IBDV FJ-1812 VP2 antigen protein was constructed. Name: E.coli BL21 / pET28a IBDV FJ-1812 VP2.

[0067] Induced expression experiments were carried out, the inducer was α-lactose, and the working concentration was: 0.03mol / L. Induced products were detected by SDS-PAGE electrophoresis. The results showed that E.coli BL21 / pET28a IBDV FJ-1812 VP2 had no obvious protein expression band.

[0068] 2. Optimization of expression codons of IBDV...

Embodiment 3

[0088] Example 3 Detection and Analysis of Escherichia coli BL21(DE3) / pET28a IBDV FJ-1812 VP2M3 Expression Protein

[0089] 1. Induced expression and molecular weight determination

[0090] The protein expression engineered bacteria constructed in this example were induced to express. The inducer is α-lactose, and the working concentration is: 0.03mol / L. Then, after ultrasonic centrifugation, the protein expression and dissolution properties were detected by SDS-PAGE electrophoresis. The results showed that the expression and dissolution properties of the target protein were better. The expression amount is about 7%-10% of the total protein, and the main expressed protein is stored in the supernatant after being centrifuged at 12000RPM for 10min, indicating that it is a soluble protein (see figure 1 ). Because the molecular weight of the expressed protein is very close to that of a self-protein of Escherichia coli, the molecular weight of the expressed protein is confirmed...

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Abstract

The invention discloses an application of an isoindolone compound in preparation of a medicine for preventing and treating osteolytic diseases. The isoindolone compound has a structure as shown in a formula (I) which is described in the specification. The isoindolone compound can remarkably inhibit the differentiation capacity of bone marrow-derived macrophages BMMs induced by RANKL to osteoclastsand the bone resorption capacity of the osteoclasts, and is concentration-dependent; a subsequent mechanism research finds that the isoindolone compound inhibits expression of a transcription factorNFATc1 by inhibiting activation of RANKL-induced MAPK and PI3K / AKT signal transduction pathways, so that expression of osteoclast related genes is reduced, and the capacity of inhibiting osteoclast differentiation and bone resorption is exerted. Besides, the isoindolone compound does not influence differentiation of osteoblasts, application of the isoindolone compound in prevention and treatment of osteolytic diseases is disclosed, and the isoindolone compound can be used for preventing and treating the osteolytic diseases together with a pharmaceutical composition prepared from a conventionalmedicinal carrier.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a genetic engineering subunit vaccine for preventing new mutant strains of chicken infectious bursal virus and a preparation method thereof. Background technique [0002] Infectious bursal disease (IBD) is a highly pathogenic infectious disease of chickens caused by infectious bursal disease virus (IBDV). The disease is characterized by the destruction of immature B lymphocytes in the central immune organ of chickens in the bursa of Fabricius, resulting in low immunity of infected chickens, increased susceptibility to other infectious diseases, and loss of immune response to various vaccines, severe Can cause death. IBDV is an RNA virus that mutates easily. When IBD first broke out in 1957, the virus was not very virulent. Due to the widespread use of attenuated live vaccines, the mutation of the virus was promoted. In 1989, a super virulent strain of infectious bu...

Claims

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Application Information

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IPC IPC(8): A61K39/12A61P31/14C12N15/40C07K14/08C12N15/70C12N1/21
CPCA61K39/12A61P31/14C07K14/005C12N15/70C12N2720/10022C12N2720/10034A61K2039/552
Inventor 荣俊匡红艳李国攀楚电峰蔡联燊徐保娟
Owner 荆州市长新生物技术有限公司
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