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Monoclonal antibody for anti-toxoplasma gondii rhoptry protein4(ROP4) and preparation method and application of monoclonal antibody

A monoclonal antibody, Toxoplasma gondii technology, applied in the biological field, can solve problems such as economic losses in animal husbandry, and achieve the effects of low cost, fast reaction, and convenient sample operation

Pending Publication Date: 2019-12-03
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, the outbreak of toxoplasmosis will also bring significant economic losses to the livestock industry.

Method used

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  • Monoclonal antibody for anti-toxoplasma gondii rhoptry protein4(ROP4) and preparation method and application of monoclonal antibody
  • Monoclonal antibody for anti-toxoplasma gondii rhoptry protein4(ROP4) and preparation method and application of monoclonal antibody
  • Monoclonal antibody for anti-toxoplasma gondii rhoptry protein4(ROP4) and preparation method and application of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1: Construction of pET32a-ROP4 recombinant plasmid

[0057] 1. Design of specific primers for ROP4 gene

[0058] According to the ROP4 gene sequence released in GenBank (accession number: EU047558.1), specific primers were designed for the Toxoplasma gondii ROP4 gene sequence, and an EcoRⅤ restriction site was introduced at the 5' end of the upstream primer ROP4F according to the cloning requirements , A HindⅢ restriction site was introduced at the 5' end of the downstream primer ROP4R. The gene fragment amplified by this set of primers is 1737bp long and encodes about 549 amino acids. The specific nucleotide sequences of the primers are as follows:

[0059] ROP4F: 5'-GATATCGAAAGAGGTAGGGTGCCCCAT-3' (SEQ ID NO.1)

[0060] ROP4R: 5'-AAGCTTTCACGTTTCCAGTGGTGGCAT-3' (SEQ ID NO.2)

[0061] 2. Synthesize the above primers, dilute them with sterilized double-distilled water to a final concentration of 50 pmol / μL, and store them at -20°C after aliquoting.

[0062]...

Embodiment 2

[0073] Example 2: Expression and purification of fusion protein

[0074] 1. Induced expression of pET32a-ROP4 recombinant plasmid

[0075] Take the plasmid of the positive clone and transform it into BL21, pick a single white colony from the cultured plate, inoculate them in 5 mL LB medium containing 100 μg / mL Amp+, and incubate at 37°C and 200 rpm for 6-8 hours until the bacteria appear slightly turbid. The bacterial solution was inoculated in 20 mL of 100 μg / mL Amp+LB medium at a ratio of 1:100, cultured at 37°C with shaking at 170 rpm, and in the logarithmic growth phase (OD600=0.5-1.0) was added with IPTG to a final concentration of 1 mM. Induced culture. Incubate at 37°C for 5 hours, and collect bacteria by centrifugation. Take 1mL of culture solution and centrifuge, add loading buffer and boil for 10min, then centrifuge to remove bacterial debris. The supernatant was taken for SDS-PAGE analysis, and the ROP4 fusion protein was expressed in the form of inclusion bodies...

Embodiment 3

[0078] Embodiment 3: Immunized mouse and serum titer determination

[0079] 1. Immunization of mice

[0080] Mix the purified ROP4 protein with an equal volume of complete Freund's adjuvant (IFC), and use an emulsifier to emulsify the mixture until dripping; select BALB / c mice aged 6 to 8 weeks for subcutaneous multi-point immunization, and inject Dose is 100μg / only. On the 15th and 29th days of immunization, the purified ROP4 protein was mixed with Freund's incomplete adjuvant in equal volumes, and the mice were immunized by subcutaneous multi-point injection, and the injection dose was 50 μg / mouse. One week after the third injection, blood was taken from the tail and the serum titer was determined; the antibody titer in the serum of BALB / C mice was detected by ELISA, and the serum of the blank control group was used as the negative control, and PBS was used as the blank control; the antibody titer was high When the ratio is 1:10000, it indicates that the antibody has suffi...

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Abstract

The invention relates to the field of biotechnology, in particular to a monoclonal antibody for anti-toxoplasma gondii rhoptry protein4(ROP4) and a preparation method and application of the monoclonalantibody. The monoclonal antibody for anti-ROP4 is produced by secretion of hybridoma cell strain 3B-7, and can be used for detecting toxoplasma gondii circulating antigen of serum and plasma of dogs, cats, pigs and chooks, and the lowest limit of the monoclonal antibody for toxoplasma gondii protein detection is 17.71 ng / mL. According to the monoclonal antibody for anti-toxoplasma gondii rhoptryprotein4(ROP4) and the preparation method and application of the monoclonal antibody, the murine hybridoma cell strain 3B-7 obtained through a cell fusion technique can stably secrete monoclonal antibody for anti-ROP4, it is discovered through determination that the monoclonal antibody has the advantages that the titer is high, and the specificity is high, and a foundation is laid for further application of the monoclonal antibody to detection of toxoplasma gondii pathogeny in clinic.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-toxoplasma rod protein 4 monoclonal antibody and a preparation method and application thereof. Background technique [0002] Toxoplasma is an intracellular parasitic opportunistic protozoa that can infect almost all warm-blooded animals and cause zoonotic diseases. Toxoplasma is a great threat to human health, especially pregnant women and those with weakened immunity. During pregnancy, pregnant women and pregnant animals infected with Toxoplasma gondii will have miscarriage or stillbirth. Toxoplasma gondii infection is an important factor causing death for HIV, organ transplantation, cancer patients and other immunocompromised persons. At the same time, the outbreak of toxoplasmosis will also bring significant economic losses to the animal husbandry. [0003] The serious consequences of toxoplasmosis infection urgently require us to pay attention to the resear...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/20C07K19/00C12N15/06G01N33/577G01N33/569A61K39/395A61P33/02C12R1/91
CPCC07K16/20G01N33/577G01N33/56905C12N15/02A61P33/02C07K2317/565C07K2319/00A61K2039/505Y02A50/30
Inventor 袁子国张秀香李贵峰杨子鹏杨晓颖谭杰兴王璐张锡锐
Owner SOUTH CHINA AGRI UNIV
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