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Porcine neuronal interleukin B polypeptide, and preparation method and application of polyclonal antibody thereof

A polyclonal antibody, neurointermediate technology, applied in the fields of biochemistry and molecular immunology, can solve the problems of high cost, restricting NMB function research, and time-consuming

Active Publication Date: 2020-08-07
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of antibodies to detect porcine NMB protein on the market, and the preparation of porcine NMB monoclonal antibodies is complicated, costly, and time-consuming, the research on NMB in pigs mainly focuses on the gene level, which seriously restricts the development of NMB in pigs. functional study

Method used

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  • Porcine neuronal interleukin B polypeptide, and preparation method and application of polyclonal antibody thereof
  • Porcine neuronal interleukin B polypeptide, and preparation method and application of polyclonal antibody thereof
  • Porcine neuronal interleukin B polypeptide, and preparation method and application of polyclonal antibody thereof

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Embodiment 1

[0040] Embodiment 1, porcine NMB protein sequence analysis and design and synthesis of NMB polypeptide

[0041] Analysis of the porcine NMB gene sequence shows that the open reading frame (ORF) of the porcine NMB gene is 366bp, encoding 121aa.

[0042] Use the Protean module in the DNAstar software to analyze the hydrophilicity, surface accessibility, antigenic index and other indicators of the porcine NMB protein, and use the blastn in the NCBI blast to analyze the sequence homology, and screen out a suitable sequence DLPEPRSRAGKIRVHPRGNL is the fitted target sequence as antigen (30aa-49aa). In order to be able to couple with the carrier protein to increase the immunogenicity of the polypeptide, the C-terminus of the selected polypeptide sequence was modified to add cysteine ​​(Cys), so the final synthesized polypeptide sequence was DLPESRAGKIRVHPRGNLC. The purity of the synthesized polypeptide detected by high-performance liquid chromatography (HPLC) is 95.89%, and the mole...

Embodiment 2

[0043] Example 2. The C-terminal modified peptide of pig NMB is coupled to the carrier protein keyhole limpet hemocyanin

[0044] There are many kinds of carrier proteins used for coupling with polypeptides, the most commonly used are keyholelimpet hemacyanin (KLH), bovine serum albumin (bovine serum albumin, BSA), ovalbumin (ovalbumin, OVA). ) and bovine thyroglobulin (THY). Because KLH has higher antigenicity, it is the most commonly used polypeptide coupling carrier. Therefore, the present invention selects KLH as the carrier protein, and couples the C-terminal modified peptide of 5.0 mg of porcine NMB with 5.0 mg of the carrier protein KLH through the coupling agent Sulfo-SMCC to obtain the NMB modified polypeptide-KLH coupling protein, that is, completely antigen.

Embodiment 3

[0045] Embodiment 3, immunization experiment animal and preparation antiserum

[0046] Male New Zealand white rabbits of appropriate age were selected as immunized animals, and blood was collected from the rabbit's ear vein before the first immunization (all blood collection was before feeding in the morning), which was used as the control serum for subsequent ELISA detection.

[0047] For the first immunization, 1 mg of complete antigen was dissolved in 1 mL of PBS, and fully emulsified with an equal volume of Freund's complete adjuvant. The emulsified antigen was injected to immunize New Zealand white rabbits by multi-point subcutaneous injection on the back. A booster immunization was given 2 weeks after the first immunization. To boost immunization, take 1 mg of complete antigen and dissolve it in 1 mL of PBS solution, fully emulsify with an equal volume of Freund's incomplete adjuvant, and immunize experimental animals after emulsification. One week after the booster im...

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Abstract

The invention belongs to the field of biochemistry and molecular immunology, and relates to a porcine neuronal interleukin B polypeptide, and a preparation method and application of a polyclonal antibody thereof. The preparation method comprises the following steps: coupling C-terminal modified polypeptide of the porcine neuronal interleukin B polypeptide with a carrier protein keyhole limpet hemocyanin KLH to obtain an NMB modified polypeptide-KLH coupled protein; emulsifying the NMB modified polypeptide-KLH coupling protein, and immunizing New Zealand white rabbits; collecting and separatingto obtain serum containing a rabbit anti-pig NMB modified polypeptide antibody; and purifying the prepared NMB polyclonal antibody by adopting a Protein G purification column method. A polypeptide antigen and the polyclonal antibody have the characteristics of simple preparation method, low cost and high titer, and can specifically bind to an NMB protein in pig tissues.

Description

technical field [0001] The invention belongs to the fields of biochemistry and molecular immunology, and relates to a preparation method and application of a porcine neurointermediate B polypeptide and its antibody. The prepared antibody is mainly used for the detection of natural protein antigens, specifically a rabbit anti-porcine neurointermediate The preparation method of B polypeptide polyclonal antibody. Background technique [0002] Neuromedin B (Neuromedin B, NMB) is a neuropeptide isolated and purified from pig spinal cord in 1983, and it is a member of the mammalian bombesin (BN)-like peptide family. In vivo, NMB exerts a variety of biological effects by binding to receptors (NMBR or BB1) on the surface of cell membranes, including: stimulating smooth muscle contraction, regulating gastrointestinal motility, feeding and body temperature, regulating hormone secretion, mediating stress and fear Response, affect behavior and memory, promote cell growth, regulate repr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/575C07K19/00C07K16/26C07K16/06G01N33/74
CPCC07K14/575C07K14/795C07K16/26G01N33/74C07K2319/00C07K2317/10
Inventor 马志禹许艳刘德鹏杨瑞琪郭军培
Owner YANGZHOU UNIV
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