Hybridoma cell strain CMOMP (chlamydophila major outer membrane protein)-5D7, monoclonal antibody secreted by same and application of monoclonal antibody

A CMOMP-5D7, hybridoma cell line technology, applied in the application, analytical materials, recombinant DNA technology and other directions, can solve the problem that the result judgment is easily affected by subjective factors, unfavorable to clinical popularization, low sensitivity, etc., and achieves stability after enzyme labeling , strong ability, good repeatability between batches

Active Publication Date: 2019-08-16
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Institute of Animal Husbandry and Veterinary Medicine of the Hubei Academy of Agricultural Sciences and the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences jointly developed a commercial IHA kit. The IHA method does not require high equipment and is simple and easy to implement, but the sensitivity is not high, and the result judgment is subject to subjective factors
Imported immunofluorescence detection kits and enzyme-linked immunosorbent assay kits are highly reliable, but they are expensive and not conducive to clinical popularization

Method used

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  • Hybridoma cell strain CMOMP (chlamydophila major outer membrane protein)-5D7, monoclonal antibody secreted by same and application of monoclonal antibody
  • Hybridoma cell strain CMOMP (chlamydophila major outer membrane protein)-5D7, monoclonal antibody secreted by same and application of monoclonal antibody
  • Hybridoma cell strain CMOMP (chlamydophila major outer membrane protein)-5D7, monoclonal antibody secreted by same and application of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Cloning of embodiment 1 MOMP gene

[0042] 1. Design of primers (shown in Table 1)

[0043] Table 1 - PCR Primers

[0044]

[0045]

[0046] The underlined part in the primer sequence in Table 1 is the corresponding enzyme cutting site, and the primers in Table 1 were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0047] 2. Amplification of MOMP gene

[0048] The MOMP gene (GenBank accession number JN411078.1) was amplified using the genomic DNA of Chlamydia abortus strain HB1043 as a template. The PCR amplification system is described in Table 2:

[0049] Table 2-PCR amplification system

[0050]

[0051] The amplification conditions of the MOMP gene sequence were: 94.0°C for 2min and then cycled. The cycle parameters were: 94.0°C for 15s, 58.0°C for 30s, 72.0°C for 1min, and after 35 cycles, 72.0°C for 10min. The amplified PCR product was analyzed by 0.8% agarose gel electrophoresis, and a 906bp DNA fragment (MOMP) was amplified by PCR (see S...

Embodiment 2

[0054] Prokaryotic expression of embodiment 2 recombinant plasmid pET28a-MOMP

[0055] 1. Plasmid transformation

[0056] The expression plasmid pET28a-MOMP (Kana-resistant) containing the MOMP gene of Chlamydia abortus was transformed into Escherichia coli BL21 competent cells.

[0057] The specific operation is as follows:

[0058] (1) Transfer 100 μL Escherichia coli BL21 competent cell suspension to a sterile 1.5ml EP tube, add 3 μL ligation product, swirl gently to mix the contents, and place on ice for 30 minutes.

[0059] (2) Put the centrifuge tube into a circulating water bath preheated to 42° C. for heat shock for 90 seconds.

[0060] (3) Quickly transfer the centrifuge tube to an ice bath to cool the cells for 1-2 minutes.

[0061] (4) Add 400 μL LB medium to each tube. Warm the medium to 37°C with a water bath, then transfer the centrifuge tube to a shaker at 37°C, and incubate for 45 minutes to recover the bacteria. In order to achieve effective conversion, t...

Embodiment 3

[0068] Example 3 Purification of pET28a-MOMP fusion protein

[0069] The pET28a-MOMP recombinant protein obtained in Example 2 mainly exists in the form of inclusion bodies and is purified using NiSepharose6Fast Flow (GE). The purification steps are as follows:

[0070] Centrifuge 200 mL of the bacterial solution 4 hours after induction at 12,000 r / min for 1 minute, discard the supernatant, resuspend the pellet with Buffer A (1 / 10), place it in a -80°C refrigerator, freeze and thaw repeatedly three times, and use ultra-high pressure wave Break it with a crushing instrument, centrifuge at 12000r / min for 30min, save the supernatant, wash the precipitate twice with Buffer A (1 / 10) and resuspend, add 20mL nickel column equilibration buffer (containing 8mol / L urea), place in Serve on ice.

[0071] Protein purification was performed using NiSepharose 6Fast Flow affinity chromatography column from GE Company. The specific operation steps are carried out according to the instruction...

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Abstract

The invention provides a hybridoma cell strain CMOMP (chlamydophila major outer membrane protein)-5D7 and a monoclonal antibody secreted by the same. The preservation number of the hybridoma cell strain CMOMP-5D7 is CCTCC NO: C2012141. The monoclonal antibody is high in immunological competence, high in titer which reaches to 1x105 and high in specificity. Shown by test results of a blocking ELISA(enzyme-linked immunosorbent assay) detection kit established by the aid of the monoclonal antibody and a method, the obtained monoclonal antibody can detect MOMP (major outer membrane protein) of Chlamydophila abortus, C.abortus, so that the monoclonal antibody is high in specificity, high in within-run repeatability and between-run repeatability and high in stability. Compared with a normal ELISA method, the method has the advantages that requirements on recombinant protein purification are lowered, and specificity in detection is improved. Compared with an indirect hemagglutination assay method, the method has the advantages that sensitivity in detection is improved, judging results are more objective, and the method can be used for detecting samples in large batches.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a hybridoma cell line CMOMP-5D7 and its secreted monoclonal antibody and application. Background technique [0002] Chlamydophila abortus (C. abortus) is a Gram-negative bacterium with a size of 0.2-1.0 μm, which belongs to the Chlamydiaceae genus Chlamydophila. Contact infectious diseases (Siarkou V, Lambropoulos AF, Chrisafi S. Subspecies variation in Greek strains of Chlamydophila abortus. Vet Microbiol. 2002. 85:145-157). The disease is widely distributed all over the world, causing huge economic losses to animal husbandry, and pregnant women can also be infected and cause abortion, thus affecting public health. Chlamydia major outer membrane protein (Major outer membrane protein, MOMP) accounts for about 60% of the outer membrane, is a relatively conserved functional protein, plays an important role in maintaining the structural integrity of Chlamydia, and MOMP...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C12N15/31C12N15/70C07K14/295C07K16/12G01N33/569G01N33/577G01N33/543C12R1/91
CPCC07K16/125C07K14/295C12N15/70G01N33/56927G01N33/577G01N33/54306C07K2317/35C07K2317/33
Inventor 刘威周丹娜袁芳艳田永祥刘泽文郭锐杨克礼段正赢高婷梁婉吴修竹
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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