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Anti-PEDF monoclonal antibody, and preparation method and application thereof

A monoclonal antibody, diabetes technology, applied in the direction of antibody, anti-growth factor immunoglobulin, anti-animal/human immunoglobulin, etc., can solve the problem of amputation of patients

Active Publication Date: 2014-09-17
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no effective treatment at home and abroad, resulting in 72,000 amputations every year

Method used

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  • Anti-PEDF monoclonal antibody, and preparation method and application thereof
  • Anti-PEDF monoclonal antibody, and preparation method and application thereof
  • Anti-PEDF monoclonal antibody, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Preparation and Identification of PEDF Monoclonal Antibody

[0021] 1. Preparation of PEDF monoclonal antibody

[0022] 1. PEDF recombinant protein preparation

[0023] Select pET30a(+) (purchased from Novagen) as the carrier, design primers according to the sequence of human PEDF gene, PCR amplify the cDNA of PEDF gene, select EcoRI and HindIII restriction endonucleases (purchased from Takara) to digest and purify PEDF The cDNA was ligated with pET30a(+) which had been cleaved with the same enzyme, to obtain the ligation product pET30a(+)-PEDF. The pET30a(+)-PEDF plasmid was transformed into Escherichia coli BL-21?DE3, and the bacteria were collected after IPTG induction, resuspended with Binding buffer, and soluble recombinant protein was obtained after ultrasonic centrifugation. Then the protein was fully combined with Ni-NTA resin (purchased from Novagen) at 4°C, then loaded on the column, washed with Washing buffer to remove impurities, and finally elut...

Embodiment 2

[0050] Example 2 Application of PEDF Monoclonal Antibody

[0051] 1. Establishment of animal model of diabetes

[0052] Forty 8-week-old C57BL / 6J mice (provided by Animal Center of Sun Yat-Sen University) were divided into normal group and diabetic group, 20 mice in each group, and fed with normal diet and high-fat diet (provided by Guangdong Animal Center) for 4 weeks respectively. The post-diabetes group received continuous intraperitoneal injection of small doses of streptozotocin (STZ) (dose 70 mg / kg) for 7 days. ) experimental test to determine the success of the diabetes model.

[0053] 2. Establishment of peripheral trauma model and therapeutic effect of PEDF monoclonal antibody

[0054] The mice in the above normal group were randomly divided into two groups, GST group and PEDF group. After the back of the mouse was depilated and disinfected with depilatory cream, two circular marks with a diameter of 6 mm were made with a biopsy punch, and the full-thickness skin w...

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Abstract

The invention discloses an anti-PEDF monoclonal antibody, and a preparation method and application thereof. The preparation method comprises the following steps: immunizing a BALB / c mouse with PEDF protein, fusing splenocyte and myeloma cell of the immune mouse, screening and cloning to obtain a hybridoma strain capable of secreting the anti-PEDF monoclonal antibody, and finally acquiring the required monoclonal antibody. The monoclonal antibody disclosed by the invention can be used for preparing medicines for treating PEDF-increase wound healing diseases.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an anti-PEDF monoclonal antibody and its preparation method and application. Background technique [0002] Diabetic microvascular complications are the main cause of disability and death in diabetic patients. More and more studies have shown that due to the inhibition of angiogenesis (angiogensis), the local wound healing (wound healing) of diabetic patients is impaired. Research estimates that 15% of diabetic patients have diabetic foot (diabetic foot). At present, there is no effective treatment at home and abroad, which leads to amputation of 72,000 patients every year. In addition to repeated trauma and bacterial infection, impaired wound healing is an important factor in the etiology of diabetic foot. Under certain conditions, such as the development of diabetes, angiogenesis is blocked, and the wound healing process is disrupted or delayed, and chronic wounds develop, whi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/22A61K39/395A61P3/10A61P17/02
Inventor 高国全杨霞齐炜炜戴智育
Owner SUN YAT SEN UNIV