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Recombinant chicken osteocalcin maturation protein monoclonal antibody and application thereof

A technology of monoclonal antibody and chicken osteocalcin, which is applied in the field of bioengineering, can solve the problems of difficult acquisition, restriction of osteocalcin research and utilization, unfavorable preparation of BGP antibody, etc., and achieve good specificity

Inactive Publication Date: 2011-08-17
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the natural protein of BGP has a small molecular weight and is not easy to obtain, which restricts the research and utilization of osteocalcin to a certain extent, is not conducive to the preparation of BGP antibodies, and also restricts the application of BGP and the study of physiological characteristics.

Method used

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  • Recombinant chicken osteocalcin maturation protein monoclonal antibody and application thereof
  • Recombinant chicken osteocalcin maturation protein monoclonal antibody and application thereof
  • Recombinant chicken osteocalcin maturation protein monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The preparation of embodiment 1 recombinant chicken osteocalcin mature protein (rchBGP)

[0036] 1.1 Acquisition of the target gene and the acquisition of the target gene cloning vector

[0037] Chicken tibia was taken, stored in liquid nitrogen, and total RNA was extracted by Trizol method. Select OD 260 / 280 Perform reverse transcription above 1.8. RNA 2μg, Oligo(dT18) 1μL, with Nase-free H 2 Make up O to 10 μL, bathe in 75°C water for 5 minutes, and ice-bath for 2 minutes; add 5×Buffer 4 μL, dNTP 2 μL, RNase inhibitor 0.5 μL, M-MLV reverse transcriptase 0.5 μL, RNase-freeH 2O 3μL, 42°C for 1h, react at 95°C for 5min, and store at 4°C for later use. According to the chicken BGP sequence (U10578) reported by GenBank, the specific primers P1 and P2 of the mature peptide gene were designed and inserted into the EcoR I and Xhol I restriction sites respectively, and the 5' end primer P1: 5'CAG GAATTC CACTACGCCCAGGAC-3' (SEQ ID NO.2), 3' end primer P2: 5'-ATA CTCGAG A...

Embodiment 2c

[0050] The preparation of embodiment 2chBGP monoclonal antibody

[0051] For 6 female BALB / C mice aged 6 weeks, 0.5 mL of 200 μg / mL expressed protein solution was mixed and emulsified with an equal volume of complete Freund's adjuvant, and injected subcutaneously at multiple points, 0.2 mL per point. After 3 weeks, 0.5 mL of 200 μg / mL expressed protein solution and incomplete Freund's adjuvant were injected subcutaneously at multiple points, 0.3 mL per point. After 3 weeks, the dose and method of booster immunization were the same as before. One week later, ELISA was used to detect the serum antibody titer. After another week, 1000 μg / mL expression protein solution was injected intramuscularly without adjuvant. After 3 days, the spleen was taken for fusion.

[0052] The myeloma cells SP2 / 0 were resuscitated two weeks before the fusion. In order to ensure the sensitivity of the myeloma used to the HAT screening medium, 8-azaguanine (8-AG) was added to the medium for screening ...

Embodiment 3

[0056] Example 3 Monoclonal Antibody Antigen Recognition Site Analysis

[0057] Antigen recognition sites of monoclonal antibodies were identified by ELISA using the method of measuring additive index. Coat the ELISA plate with the optimum antigen concentration, add any pairwise monoclonal antibody ascitic fluid after washing. Incubate at 37°C for 1h, add HRP-goat anti-mouse IgG after washing, incubate at 37°C for h, develop color after washing, measure OD respectively 450 value, and each sample was replicated 3 times. According to the formula AI=[2A12 / (A1+A2)-1]×100%, calculate the AI ​​values ​​of the 6 monoclonal antibodies after being superimposed in pairs. AI50% antigenic epitopes are different. It has been identified that 3D10 and 3D6 belong to similar antigen recognition sites; 3H4 and 4E4 belong to similar antigen recognition sites; triple F1 and second F1 belong to similar antigen recognition sites.

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Abstract

The invention belongs to the technical field of biological engineering, and relates to a recombinant chicken osteocalcin maturation protein monoclonal antibody and application thereof. The recombinant chicken osteocalcin maturation protein monoclonal antibody is secreted by hybridoma cell 3H4 with the preservation number of CGMCC No.4502. The preservation number of the hybridoma cell 3H4 which ispreserved in the china general microbiological culture collection center at December 23rd, 2010 is China general microbiological culture collection (CGMCC) No. 4502. The hybridoma cell system 3H4 canstably secrete the recombinant chicken osteocalcin maturation protein monoclonal antibody, wherein the antibody is good in specificity and can be combined with the specificity of the chicken osteocalcin protein to assay the chicken osteocalcin protein. The chicken osteocalcin assay reagent prepared by the antibody and particularly the chicken osteocalcin enzyme-linked immuno sorbent assay (ELISA)kit can be used for assaying the content of the chicken serum osteocalcin, thereby being good for monitoring the bone growth condition, preparing the reagent for diagnosing the chicken bone disease, and diagnosing the other diseases which can cause the abnormal change of the BGP.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a recombinant chicken osteocalcin mature protein monoclonal antibody and its application. Background technique [0002] Osteocalcin (BGP) is mainly synthesized and secreted by osteoblasts in bones and teeth. It is the most abundant non-collagenous bone matrix protein in the body. It is hydrolyzed by the liver and kidney, and finally eliminated by the kidney. The BGP gene is first translated into a proosteocalcin of 88 amino acid residues. After the proosteocalcin removes the signal peptide, the γ-carboxylation recognition site binds to the vitamin K-dependent carboxylase and undergoes carboxylation reaction. Inactive glutamate residues are converted to active γ-carboxyglutamate, after which the propeptide is removed to mineralize bone. Chicken osteocalcin (chBGP) is composed of 49 amino acids, which is characterized by three carboxylated glutamic acid residues at positions ...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/18G01N33/577C12R1/91
Inventor 侯加法江莎周振雷邓益峰
Owner NANJING AGRICULTURAL UNIVERSITY
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