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Hybridoma cell strain, and anti-chlamydia abortus monoclonal antibody secreted by hybridoma cell strain

A technology of hybridoma cell line and Chlamydia abortus, which is applied in the field of immunology, can solve the problems of inaccurate test results, loss, misdiagnosis and other problems, and achieve the effects of strong specificity and fluorescence characteristics, stable ability and high titer

Active Publication Date: 2018-09-04
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the quality of antigen and antibody detection kits circulating in the market is uneven, which leads to inaccurate test results and even misdiagnosis of the disease due to inaccurate test results, delaying the disease and possibly causing huge economic losses.

Method used

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  • Hybridoma cell strain, and anti-chlamydia abortus monoclonal antibody secreted by hybridoma cell strain
  • Hybridoma cell strain, and anti-chlamydia abortus monoclonal antibody secreted by hybridoma cell strain
  • Hybridoma cell strain, and anti-chlamydia abortus monoclonal antibody secreted by hybridoma cell strain

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0054] Preparation and cloning of Chlamydia abortus OmpA gene:

[0055] Dilute the freeze-dried powder of Chlamydia swine abortus CP12 strain with 1 ml of sterilized physiological saline, inoculate it into 6-day-old SPF chicken embryos, and carry out the reproduction of the virus strain. Collect the yolk sac membrane of chicken embryos that died on the 3rd to 6th day, add 1mL of PBS to grind into a suspension; centrifuge at 3000rpm for 10min, discard the precipitate and keep the supernatant, which is the chlamydia bacterial liquid, and extract chlamydia according to the steps of the bacterial DNA extraction kit Genome. With reference to the OmpA gene sequence of the Chlamydia abortus S26 / 3 strain registered in GenBank: CR848038, OmpA gene primers (as follows P1, P2) were designed and synthesized:

[0056] P1(F): 5'—ATG AAA AAA CTC TTG AAA TCG G—3'(SEQ ID No.3),

[0057] P2(R): 5'—TTA GAA TCT GAA TTG AGC ATT CAT—3' (SEQ ID No. 4).

[0058] Primers were synthesized by Beijing...

Embodiment 2

[0095] Embodiment 2 The establishment of the direct immunofluorescence detection method of monoclonal antibody (CA1-MM01H)

[0096] 1. Reagent preparation:

[0097] Preparation of carbonate solution: 0.5mol / L, carbonate solution of pH9.5, 3.7g of sodium bicarbonate, 0.6g of sodium carbonate (anhydrous), 100mL of distilled water; preparation of PBS eluent: pH7.2, 0.01mol / L Phosphate Buffer Saline (PBS), Disodium Hydrogen Phosphate (Na 2 HPO 4 2H 2 O) 27g, sodium chloride (NaCl) 8.5g, sodium dihydrogen phosphate (NaH 2 PO 4 2H 2 O) 0.39g, add distillation to 1000mL. Fluorescein isothiocyanate (FITC): purchased from Sigma Company, SepHadex G200 dextran gel: purchased from Beijing Suolaibao Company.

[0098] 2. Fluorescence-labeled monoclonal antibody method

[0099] According to the measured protein content, weigh FITC (F7250, Sigma Company) with 1 / 20 mass of the protein content and dissolve it in the prepared carbonate solution of 0.5mol / L, pH9.5, stir gently when dissol...

Embodiment 3

[0114] Embodiment 3 Stability and specificity detection of antibody secreted by hybridoma cells (CA1-MM01H)

[0115] 1. Stability detection of antibodies secreted by hybridoma cells

[0116] 1) Subculture the hybridoma cells, collect the cell culture supernatant one by one, detect the antibody titer by indirect ELISA every 5 generations, and observe whether the antibody titer is stable;

[0117] 2) The hybridoma cells were cryopreserved in liquid nitrogen for 6 months, then recovered and subcultured continuously, and the antibody titer of the supernatant of the cell culture medium was detected by indirect ELISA method for every 5 generations.

[0118] 2. Specific detection of antibodies secreted by hybridoma cells

[0119] PEDV, PPV, PRV, CSFV, PRRS, PCV pathogen solution and Chlamydia swine abortus CP12 bacterial solution were used as antigens respectively, and the enzyme plate was coated with the same conditions, and the primary antibody was positive hybridoma cell culture ...

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Abstract

The invention belongs to the field of immunology, and more specifically discloses a hybridoma cell strain, and an anti-chlamydia abortus monoclonal antibody secreted by the hybridoma cell strain. According to a preparation method, purified prokaryotically expressed chlamydia abortus major outer membrane protein (MOMP) is used for immunization of mouse as an antigen, indirect ELISA is adopted for screening of positive clones, and the hybridoma cell CA1-MM 01 capable of secreting the monoclonal antibodies aiming at chlamydia abortus major outer membrane protein is obtained. The identification ofthe monoclonal antibody in the aspects of the stability of the hybridoma cell after continuous continuous passage culture and cryopreservation, and the subtype and titer of the monoclonal antibody iscarried out, it is shown by results that the capacity of the hybridoma cell in secretion of antibodies is excellent and stable, the monoclonal antibody titer is high. It is shown that the obtained monoclonal antibody possesses extremely high specificity and combination capacity with chlamydia abortus.

Description

technical field [0001] The invention belongs to the field of immunology, and in particular relates to a hybridoma cell line and the monoclonal antibody against chlamydia abortus secreted by it. Background technique [0002] Chlamydia abortus (C. abortus) is an obligate intracellular parasitic Gram-negative microorganism between bacteria and viruses, which can cause various animal and human infections, and is an important human and animal infection. Comorbid pathogens. Chlamydia abortus is the most common pathogen of infectious abortion in small ruminants (sheep and goats). It can also cause abortion in cattle, pigs and other livestock. Pregnant women can suffer from abortion or other diseases after infection. Abortion chlamydiosis poses a threat to the intensive breeding industry, causing huge economic losses to animal husbandry production in many countries and regions, and also has an impact on public health. [0003] Chlamydia detection and diagnosis technology is mainly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/12G01N33/577G01N33/569
CPCC07K16/125G01N33/56927G01N33/577
Inventor 何诚郭永霞
Owner CHINA AGRI UNIV
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