Anti-silk fibroin polyclonal antibody and preparation method thereof

A technology of polyclonal antibody and silk fibroin, which is applied in the field of anti-silk fibroin polyclonal antibody and its preparation, can solve the problems of low specificity, no commercial products of silk fibroin, and inability to distinguish materials from surrounding collagen tissues, etc. Achieve the effect of simple operation, high purity and good specificity

Inactive Publication Date: 2014-09-24
SILK PLUG BEIJING BIOMEDICINE TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, both methods have disadvantages that cannot be ignored
The hematoxylin-eosin staining method stains the collagen tissue in the body at the same time as the silk fibroin, so this method is only suitable for the short-term implantation observation of the silk fibroin material. With the degradation of the material, it will be impossible to distinguish the material from the Peripheral collagen tissue
However, the isotope labeling method has the disadvantages of complicated operation and low specificity.
[0005] Antibody technology is an important part of the study of protein function and clinical testing. At present, there is no commercial product of silk fibroin antibody, which limits the in-depth analysis of the biological function of silk fibroin.

Method used

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preparation example Construction

[0026] In the preparation method of the anti-silk fibroin polyclonal antibody of the present invention, the method for obtaining the polypeptide whose amino acid sequence is shown in SEQ ID NO: 1 is prokaryotic system expression, eukaryotic system expression or in vitro synthesis.

[0027] In some embodiments, the method for obtaining the polypeptide whose amino acid sequence is shown in SEQ ID NO: 1 is in vitro synthesis.

[0028] In the preparation method of the anti-silk fibroin polyclonal antibody of the present invention, the immunization of animals specifically includes mixing the polypeptide with an adjuvant and injecting the animals several times for immunization.

[0029] The adjuvant is a non-specific immune enhancer. The mechanism of the adjuvant to enhance the immune response is to change the physical shape of the antigen, prolong the retention time of the antigen in the body; stimulate the ability of mononuclear phagocytes to present the antigen; stimulate lymphocy...

Embodiment 1

[0059] Embodiment 1: Preparation of anti-silk fibroin polyclonal antibody

[0060] (1) In vitro synthesis of a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 (CGYGAGAGAGYGA), the specific steps are as follows:

[0061] Removal of Fmoc groups and washing: add 20% piperidine / DMF solution to the resin, react for 30 minutes, and then wash the resin with DMF for 5 times;

[0062] Amino acid coupling (peptide bond formation): According to the amount of resin: the volume ratio of amino acid to be linked: HBTU: NMM is 1:3:3:6, mix and react in DMF solution for 3 hours, and then wash the resin 3 times with DMF; The desired peptide chain resin can be obtained by such a cycle, the resin is washed 3 times with methanol, the methanol is drained, and the resin is weighed for later use;

[0063] Peptide chain resin cleavage: with TFA:H 2 O solution with a volume ratio of 95:5 was used as a lysate, and 10 mL of lysate was added to every 1 g of resin, mixed and stirred fo...

Embodiment 2

[0077] Taking the slices of the animal experiment group in the body of the porous silk fibroin material as an example, the anti-silk fibroin polyclonal antibody prepared in Example 1 is used to measure the immunospecificity of silk fibroin. The specific method includes the following steps:

[0078] 1. Cut out the tissue block according to the conventional method, and put it into the fixative quickly, cut the sample tissue into a paraffin section, bake it in a 90-degree oven for half an hour, dewax it with xylene I and II for 5-10 minutes each, and then put it in 100% , 95%, 90%, 80%, 70% and other levels of alcohol solutions for 3-5 minutes each, and then put in distilled water for 3 minutes;

[0079] 2. 3% H 2 o 2 Incubate at room temperature for 5-10 minutes to eliminate the activity of endogenous peroxidase;

[0080] 3. Rinse with distilled water, then soak in PBS for 5 minutes, repeat this operation twice;

[0081] 4. Block with 5-10% normal rabbit serum (diluted in PBS...

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Abstract

The invention relates to the technical field of biology and in particular discloses an anti-silk fibroin polyclonal antibody and a preparation method thereof. The anti-silk fibroin polyclonal antibody is produced by an animal under the condtion that polypeptide with an amino acid sequence shown in SEQ ID NO:1 is taken as an immunogen. The anti-silk fibroin polyclonal antibody has the characteristics of good specificity and high purity, can be specifically conjugated with silk fibroin and is widely applicable to immunohistochemistry, immunoblotting and in vitro immunoassay. The preparation method of the anti-silk fibroin polyclonal antibody is simple in operation and is applicable to preparation of a large quantity of anti-silk fibroin polyclonal antibodies.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an anti-silk fibroin polyclonal antibody and a preparation method thereof. Background technique [0002] The defect or failure of cells and tissues caused by various diseases has become a major problem affecting human health, and promoting or guiding cell and tissue regeneration through foreign media has gradually become an effective method to solve the above problems. Selecting suitable materials as scaffolds for cell and tissue regeneration is a core issue in the field of regenerative medicine, and the performance of scaffold materials is of great significance for the treatment of tissue defects. [0003] Silk fibroin is a structural protein that widely exists in nature. It can be extracted from silk fiber of silkworm. It not only has sufficient source and simple extraction method, but also has good biocompatibility and biodegradability. Due to their excellent biocompatibility, me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K16/18C07K16/06
Inventor 林莎莎吕强吴昊
Owner SILK PLUG BEIJING BIOMEDICINE TECH
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