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Monoclonal antibody for detecting porcine C-reactive protein (CRP)

A monoclonal antibody and reactive protein technology, which is applied in the field of immunoassay, can solve the problems of lack of effective detection methods and achieve the effects of simple and efficient preparation methods, reduced drug residues, and high affinity

Inactive Publication Date: 2015-05-20
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is a lack of effective detection methods for CRP using Western-blot

Method used

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  • Monoclonal antibody for detecting porcine C-reactive protein (CRP)
  • Monoclonal antibody for detecting porcine C-reactive protein (CRP)
  • Monoclonal antibody for detecting porcine C-reactive protein (CRP)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The amplification of embodiment 1 pig CRP gene and the construction of expression vector

[0047] 1. Design and synthesis of primers

[0048] According to the nucleotide sequence of the GenBank number (NM_213844.2) in the database in NCBI, a pair of primers containing the main coding region of the CRP gene were designed using DNAStar and Primer5.0 software. The upstream primer P1 has an NdeI restriction site, and the downstream primer P2 has a There is an Xhol restriction site, and the amplified fragment between the upstream and downstream primers includes 612 bases in the main coding region of the CRP gene.

[0049] Upstream primer P15′-cttcatatgcagacagacatgatcggaaaggcc-3′

[0050] (SEQ ID NO: 1)

[0051] Downstream primer P25'-tgactcgagttagggccacagctggggcttgacatacac-3' (SEQ ID NO: 2)

[0052] 2. Extraction of pig total RNA Take 50-100 mg of pig liver and add it to a 1.5 ml EP tube, first add 300 μL Trizol (Invitrogen), grind it with a grinding rod until it becomes ...

Embodiment 2

[0110] Example 2 Induced expression of recombinant protein and renaturation and purification of expressed protein

[0111] 1. Induced expression of recombinant protein and extraction of inclusion bodies

[0112] (1) Transform the expression plasmid pET21a-pigCRP into BL21(DE3), and after culturing for 12 hours, inoculate the monoclonal colony into 100mL Amp + In LB medium, shake slowly in a shaker for 8-10 hours, and then use it as a mother solution;

[0113] (2) Inoculate 1%-5% mother solution into 3mL culture medium, culture on a shaker at 37°C until OD600 reaches 0.4-0.6, set empty bacteria and empty plasmid controls, and rotate at 200rpm;

[0114] (3) The mother solution is transferred to 2L of Amp according to the inoculation volume of 1%. + In LB medium, 37°C, 180rpm, cultivate to OD value of 0.4-0.6, add IPTG to a final concentration of 1mM, 37°C, 160-180rpm, continue to cultivate for 5h;

[0115] (4) Harvest bacteria (the following steps are all carried out in ice),...

Embodiment 3

[0140] The preparation of embodiment 3 monoclonal antibody

[0141] 1. The porcine CRP protein obtained in Example 2 was used for the determination of antibody titer in immunized mice, and 4 SPF BALB / c female mice were immunized subcutaneously for the first time in the amount of 60 μg protein / mouse, numbered as: 1, 2 , 3, 4. After five subcutaneous booster immunizations (immunization dose: 30 μg protein / mouse), blood was collected from the orbits of the mice to measure the serum titer. Results (see image 3 ) No. 4 mouse had the highest serum antibody titer and was used for cell fusion experiments.

[0142] 2. Cell fusion experiment Take mouse splenocytes and SP2 / 0 cells, and use PEG method for fusion. The fused cells were selected and cultured with semi-solid medium (containing HAT).

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Abstract

The invention provides a monoclonal antibody for detecting porcine C-reactive protein (CRP), and preparation and application thereof. A gene engineering process is performed to obtain high-purity CRP recombinant protein; and the recombinant protein is utilized to screen out the hybridoma cell strain with the highest stability and highest antibody activity for secreting CRP protein antibody, wherein the collection number is CGMCC NO.9345. The monoclonal antibody generated by the hybridoma cell strain has the advantages of high specificity, high affinity and simple and efficient preparation method, and can monitor the CRP content in porcine serum, perform differential diagnosis on bacterial and virus diseases and perform auxiliary observation on treatment effects, thereby promoting the rational use of antibiotics, reducing the drug residues and ensuring the safety of animal food.

Description

technical field [0001] The invention relates to the field of immune detection, in particular to a monoclonal antibody which can be used to detect porcine C-reactive protein. Background technique [0002] C-reactive protein (C-reactive protein) is a 2+ Acute-phase reactive protein that, when present, reacts with capsular polysaccharide C in the pneumococcal cell wall to form a complex. In 1930, Tillett and Francis first found it in the serum of patients with acute lobar pneumonia. In 1941, Avery et al. determined that it was a protein, so it was called C-reactive protein (CRP). Later, CRP was detected in the serum of patients with non-infectious diseases and infectious diseases in the acute phase, so it was believed that CRP was a sign of a non-specific response to tissue damage. [0003] In my country, veterinary antibiotics are widely used and indispensable in veterinary clinics and animal feeding. In order to achieve the purpose of promoting growth and preventing and tr...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/18G01N33/68G01N33/577
Inventor 夏春樊淑华
Owner CHINA AGRI UNIV
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