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Recombinant virus and application thereof in preparation of medicine for preventing or treating renal fibrosis

A kidney fibrosis and recombinant virus technology, applied in the field of genetic engineering, can solve the problems of not showing additive effect, unable to completely block the overexpression of TGFβ1, and unable to completely block the progress of renal failure

Inactive Publication Date: 2018-11-27
SHANGHAI SIXTH PEOPLES HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there are currently no drugs specifically targeting renal fibrosis in clinical practice.
In basic medical research, researchers often pay more attention to TGFβ1 neutralizing antibodies or small molecule inhibitors. However, neutralizing antibodies are difficult to reach the target site due to their large molecular weight; and small molecule inhibitors are mostly cytotoxic , and have serious toxic side effects on the body
Studies have shown that angiotensin II (Ang II) inhibitors, namely ACEI / ARB, can reduce the level of urinary TGFβ1, but the combination of the two does not show an additive effect, and cannot completely block the overexpression of TGFβ1, so it can only delay rather than completely arresting the progression of renal failure
In addition, clinical studies have shown that the clinical use of specific human monoclonal antibodies against TGFβ1 in patients with renal fibrosis failed to delay the progression of renal fibrosis

Method used

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  • Recombinant virus and application thereof in preparation of medicine for preventing or treating renal fibrosis
  • Recombinant virus and application thereof in preparation of medicine for preventing or treating renal fibrosis
  • Recombinant virus and application thereof in preparation of medicine for preventing or treating renal fibrosis

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Effect test

Embodiment 1

[0036] The Specimen Bank of Shanghai Sixth People's Hospital collected renal specimens from patients who signed the informed consent form and were pathologically diagnosed as renal fibrosis between 2015 and 2017. Immunohistochemical staining was performed on paraffin sections of renal biopsies , the specific steps are as follows: perform paraffin section, dewax to water with xylene and 95% alcohol; then, rinse with distilled water, and then remove endogenous peroxidase with 3% hydrogen peroxide aqueous solution (37°C for 30min); Then, wash with double distilled water and TBST, and then use sodium citrate solution for microwave repair; after the solution is cooled, normal sheep serum is blocked for 20 minutes, and primary antibody is added, incubated at 37°C for one hour, and overnight at 4°C; the next day , After the slices were equilibrated at room temperature, washed three times with TBST, added secondary antibody and incubated at 37°C for one hour, after color development by...

Embodiment 2

[0039] Construction of YY1 Overexpression Recombinant Virus

[0040] see Figure 4 , the inventors used pHBAAV-CMV-MCS-3flag-T2A-ZsGreen, an adeno-associated virus vector, and used two restriction enzymes, BamH I and Kpn I, to digest with CutSmart buffer at 37°C for 1 hour. The gel was cut and recovered, and the purified linear vector was further obtained. Meanwhile, YY1 (SEQ ID NO.1) was amplified by PCR, the forward primer used was 5'-CCATAGAAGACACCGGGATCCGCCACCATGGCCTCGGGCGACACC-3'(SEQ ID NO.4), and the reverse primer used was 5'-GTAGTCGTTAATTAAGGTACCCTGGTTGTTTTTGGCCTTAGCATGT- 3' (SEQ ID NO.5); after PCR, the DNA was recovered to obtain a purified CDS fragment of linear YY1. Next, ligate the prepared linear vector with the CDS fragment of the linear YY1 (LigationHigh enzyme, 16°C for 1h), and then perform transformation, which specifically includes the following steps: add 10ul of the ligation product to Beijing Quanshijin Biotechnology Co., Ltd. Stable3 competent cells ...

Embodiment 3

[0043] Kidney-specific knockout of YY1 experiment

[0044] 4-week-old C57BL6 / J YY1 flox / flox Male rats were fed with a high-fat diet for 6 weeks and injected intraperitoneally with streptozotocin (STZ, 40 mg / kg), and injected every other day. Fasting blood glucose was measured after a total of three times. Male rats greater than 16.7 mmol / L were randomly divided into two groups. group, 12 rats in each group; one week later, the kidneys were injected with AAV-cre virus in situ: after the mice were anesthetized with pentobarbital sodium, the abdomen was opened, and the left kidney was carefully separated, and three points were taken from the lower border of the left renal cortex. Injection (10ul per point 1*10 12 AAV-cre virus); the control group was treated in the same way, the wound was sutured after injection of the same dose of AAV-GFP control virus, and the mice were sacrificed after continuing to feed for 12 weeks. During the feeding period of the mice, the body weight a...

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Abstract

The invention relates to the technical field of gene engineering, and particularly relates to a recombinant virus and application thereof in preparation of a medicine for preventing or treating renalfibrosis. According to the technical scheme, the progress of renal fibrosis is retarded since TGF beta1 is negatively regulated based on a transcriptional factor YY1, a key factor causing reduction ofthe transcriptional level of TGF beta1 and target gene thereof is found out, and a series of recombinant viruses containing an SEQ ID NO.1 sequence, especially the transcriptional factor YY1 overexpressed recombinant virus (AAV-YY1 virus), are provided, which can be used for preparing a medicine for preventing or treating renal fibrosis. Therefore, the target gene sequence, recombinant virus andmedicine provided by the invention have a wide clinical application prospect in the aspect of preventing or treating renal fibrosis.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant virus and its application in the preparation of medicines for preventing or treating renal fibrosis. Background technique [0002] Renal fibrosis and sclerosis are one of the common causes of death in patients with end-stage renal failure, and various diseases can cause renal fibrosis, including but not limited to diabetic nephropathy, nephritis and renal tumors. The main pathological changes of renal fibrosis are the thickening of capillary basement membrane and the increase of basement membrane matrix, which eventually lead to diffuse or nodular glomerulosclerosis; the pathogenesis is accompanied by complex metabolic disorders Its pathogenesis is complex, including the interaction of hemodynamics, advanced glycation end products, oxidative stress, polyol pathway, protein kinase C, and activation of various cytokines. [0003] In recent years, a large ...

Claims

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Application Information

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IPC IPC(8): C12N15/864A61K38/17A61K48/00A61P13/12
CPCA61K38/1709A61K48/005A61P13/12C12N15/86C12N2750/14143C12N2800/107
Inventor 刘军力高攀杨柳贾伟平
Owner SHANGHAI SIXTH PEOPLES HOSPITAL
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