Preparation method of anti-TMP (thrombopoietin mimic peptide) rabbit polyclonal antibody

A polyclonal antibody and white rabbit technology, applied in the field of genetic engineering pharmaceuticals, can solve the problems of immune side effects, low specificity and affinity, and high production costs, and achieve the effect of high affinity, high specificity, and high titer

Inactive Publication Date: 2019-02-15
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this fusion protein is that since Fc is a non-inert protein, it will bring immunological side effects
[0004] Reasonable immunization proced

Method used

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  • Preparation method of anti-TMP (thrombopoietin mimic peptide) rabbit polyclonal antibody
  • Preparation method of anti-TMP (thrombopoietin mimic peptide) rabbit polyclonal antibody
  • Preparation method of anti-TMP (thrombopoietin mimic peptide) rabbit polyclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation method of TMP rabbit polyclonal antibody serum

[0052] The thrombopoietin-mimetic peptide TMP was coupled with the keyhole limpet hemocyanin KLH to prepare KLH-TMP as an immune antigen. After adding Freund's complete adjuvant and emulsifying, New Zealand white rabbits were immunized in batches, and the 58th day after immunization The serum was collected, and the anti-TMP rabbit polyclonal antibody was isolated and purified therefrom, wherein the base sequence of the thrombopoietin mimetic peptide TMP is shown in SEQ ID NO: 1, and the amino acid sequence of the thrombopoietin mimetic peptide TMP is shown in SEQ ID NO :2 shown.

[0053] The specific steps are:

[0054] (1) The thrombopoietin mimetic peptide TMP is coupled with the keyhole limpet hemocyanin KLH, and the purity of the KLH-TMP fusion protein is greater than 95%;

[0055] (2) Take 2.5 mg of 1 mg / ml immune antigen KLH-TMP, add 2.5 ml of Freund's complete adjuvant, and emulsify.

[005...

Embodiment 2

[0061] The purification method of embodiment 2 antibody serum

[0062] ①Reagents related to antibody purification

[0063] Preparation of saturated ammonium sulfate solution: Dissolve 400g (NH4)2SO4 in 500ml of deionized water at 50-80°C, stir for 20 minutes after complete dissolution, and filter with filter paper. After cooling, the pH was adjusted to 7.4 with 15N NH4OH.

[0064] Naphthalene's reagent preparation: Weigh 11.5 grams of HgI2, 8 grams of KI, dilute to 50ml with deionized water, stir and dissolve fully, then add 50m of 20% NaOH solution.

[0065] ②Salting out purification method

[0066] (1) Take the rabbit serum collected by centrifugation and add an equal volume of normal saline, add the same amount of saturated (NH4)2SO4 as the diluted serum drop by drop under stirring, let it settle at 4°C for more than 3 hours, centrifuge at 4000g for 20 minutes, and discard the supernatant ;

[0067] (2) Dissolve the precipitate with physiological saline to X ml, then ad...

Embodiment 3

[0070] Example 3 Antibody Serum SDS-PAGE Gel Electrophoresis Analysis Method for Polyclonal Antibody Purity

[0071] ①SDS-PAGE related reagents

[0072] 1.0M Tris (pH 6.8): Dissolve 12.114g Tris in 80ml deionized water, adjust the pH to 6.8 with concentrated hydrochloric acid, and dilute to 100ml;

[0073] 1.5M TriS (pH 8.8): Dissolve 18.171g Tris in 80ml deionized water, adjust the pH to 8.8 with concentrated hydrochloric acid, and dilute to 100ml;

[0074] 10% SDS: Dissolve 10g SDS in 100ml deionized water, store at room temperature

[0075] 10% ammonium persulfate (APS): dissolve 1g ammonium persulfate in 10ml deionized water, store at 4°C

[0076] ② Antibody serum SDS-PAGE gel electrophoresis analysis method

[0077] (1) Configure 12% separating gel and 5% stacking gel;

[0078] (2) Take 0.1 μl and 1 μl of purified antibody, dilute to 20 μl with deionized water respectively, add 5 μl of 5× loading buffer, and denature in a metal bath at 100°C for 10 minutes;

[0079] ...

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Abstract

The invention relates to a preparation method of an anti-TMP (thrombopoietin mimic peptide) rabbit polyclonal antibody. The preparation method comprises the steps of coupling thrombopoietin mimic peptide (TMP) and keyhole limpet hemocyanin (KLH) to prepare KLH-TMP as an immunizing antigen, collecting blood from a purified antigen immunized New Zealand white rabbit to prepare antiserum, and separating and purifying the anti-TMP rabbit polyclonal antibody from the antiserum. The purity of the antibody serum can reach above 85% after purification by a salting-out method. The antibody prepared bythe method has the characteristics of high purity, specificity and affinity.

Description

technical field [0001] The invention belongs to the field of genetic engineering pharmacy, and specifically relates to the fields of microbiology and immunology, in particular to a preparation method of a polyclonal antibody. Background technique [0002] Thrombopoietin (TPO), also known as Megakaryocyte Growth Development Factor (MGDF), is a hormone-like substance that regulates platelet production in human blood, discovered by Kelemen et al. in 1958 . The human TPO gene is located on the long arm of chromosome 3 (q26-27), and the TPO protein encoded by it consists of 332 amino acids and contains two functional regions: the N-terminal consisting of 153 amino acids and the C-terminal consisting of 179 amino acids (Lin Fang, 2012). TPO can promote the proliferation, differentiation and maturation of megakaryocytes, and promote platelet production. It is the first discovered and most important regulator of platelet production. In 1997, through phage display technology, Cwir...

Claims

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Application Information

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IPC IPC(8): C07K16/22C07K16/06
CPCC07K16/06C07K16/22
Inventor 李红玉付秀峰茹毅李洋
Owner LANZHOU UNIVERSITY
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