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Monoclonal antibody to toxoplasma gondii ROP (rhoptry protein) 18, cell strain secreting monoclonal antibody and application thereof

A monoclonal antibody, rod-shaped technology, applied in the field of immunodetection, can solve problems such as limiting molecular mechanism of action and clinical application research

Active Publication Date: 2018-12-25
ANHUI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although ROP18 has become a "star molecule" in the field of Toxoplasma gondii research, unfortunately, no commercial ROP18 monoclonal antibody has been released so far, which limits further research on its molecular mechanism of action and clinical application

Method used

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  • Monoclonal antibody to toxoplasma gondii ROP (rhoptry protein) 18, cell strain secreting monoclonal antibody and application thereof
  • Monoclonal antibody to toxoplasma gondii ROP (rhoptry protein) 18, cell strain secreting monoclonal antibody and application thereof
  • Monoclonal antibody to toxoplasma gondii ROP (rhoptry protein) 18, cell strain secreting monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Construction of pET28a-ROP18 recombinant plasmid

[0023] 1. Codon optimized ROP18, synthesized optimized gene ROP18u

[0024] Using Optigene from Shanghai Jierui Bioengineering Co., Ltd. TM The codon optimization analysis platform performs codon optimization on the ROP18 coding gene, and synthesizes the optimized full-length gene ROP18u.

[0025] 2. Double digestion and enzyme ligation of ROP18 gene and pET28a

[0026] ROP18 gene and pET28a were double-digested with restriction endonucleases BamH I and Sal I, and a double-digestion reaction system was prepared to react overnight in a water bath at 37°C.

[0027] Double enzyme digestion reaction system:

[0028]

[0029] Recover and purify by electrophoresis after digestion, use T4 DNA ligase to ligate the ROP18 gene and pET28a, keep the molar ratio of the insert fragment and the vector fragment between 3:1 and 7:1, prepare the enzyme linkage reaction system and react overnight at 16°C.

[0030] Enzyme...

Embodiment 2

[0050] Example 2: Expression and purification of immune proteins

[0051] 1. Induced expression of recombinant protein

[0052] ① Place the centrifuge tubes, micropipettes, pipette tips, test tube racks, alcohol lamps, lighters, LB medium, and kanamycin used in the test on the workbench for 30 minutes under the UV lamp.

[0053] ②Use a 1000μL micropipette to draw 3ml of LB into a 15ml centrifuge tube next to the alcohol lamp, and then use a 20μL micropipette to draw 3.5μL of kanamycin into the centrifuge tube.

[0054] ③Take out the strain from the -80°C refrigerator and place it on an ice box, use a 20 µL micropipette to pipette 20 µL of the strain into a centrifuge tube, and quickly put the strain back into the -80°C freezer. Mark the centrifuge tube and place it at 37°C with a 220rpm shaker for 13 hours overnight.

[0055] ④Take 2 mL of the small shaking bacteria solution, add it to 200 mL of LB medium (add 200 μL of kanamycin) and continue shaking the bacteria. 200 μL of ...

Embodiment 3

[0099] Example 3: Mice immunization and titer determination

[0100] (1) Immunization of mice: take two 2ml syringes, each draw Freund's adjuvant (complete adjuvant for the first immunization, incomplete adjuvant for the second and third immunization) and purified protein (according to 1:1 Equal volume) is connected with an infusion tube in the middle, mixed by pushing left and right, and fully emulsified (emulsification time is about 30 minutes). After the mice were fixed in the prone position, they were wiped with alcohol for disinfection, and the skin was pinched with the index finger and thumb, and the needle tip with the sharp side of the notch was pierced into the subcutaneous skin, and then the protein was pushed in. The immunization dose for each mouse was 50 μg, and the immunization was done every two weeks. The mice after the third immunization were given intraperitoneal injection of protein booster immunization without adjuvant, and 20 μg was injected.

[0101] (2)...

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Abstract

The invention discloses a monoclonal antibody to toxoplasma gondii ROP (rhoptry protein) 18, a cell strain secreting the monoclonal antibody and an application thereof. The cell strain of the monoclonal antibody of the toxoplasma gondii ROP18 is a hybrid tumor cell strain T2 and is preserved by the typical culture preservation center in China, the address is Wuhan University, Wuhan, China, the preservation number is CCTCC, NO: C2018115, and the preservation date is July, 20, 2018. The monoclonal antibody of the toxoplasma gondii ROP18 is secreted by the hybrid tumor cell strain T2 and can be used to prepare an antigen reagent for detecting the toxoplasma gondii ROP18; the mouse-derived cell strain T2 of the monoclonal antibody of the toxoplasma gondii ROP18 is prepared through a cell fusion technique, and the monoclonal antibody secreted by the cell strain is screened by indirect competitive elisa and preliminarily identified by epitope, which lays a foundation for further clinical application.

Description

technical field [0001] The invention relates to a monoclonal antibody of toxoplasma gondii rod protein ROP18, a cell line secreting the antibody and an application thereof, belonging to the technical field of immunoassay. Background technique [0002] Toxoplasma gondii is a kind of intracellular parasitic protozoa which is widely prevalent among humans and animals, and its infection seriously endangers human health and the development of animal husbandry. Although there is only one species of Toxoplasma gondii (T.gondii) in the genus Toxoplasma, there is rich genetic diversity in Toxoplasma gondii around the world Type1, Type2 and Type3 are the three classic genotypes prevalent in North America and Europe, It exhibits strong, medium and weak virulence characteristics in mice. Finding the effector molecules and their mechanism of action related to the virulence of Toxoplasma gondii genotype / strain is a research hotspot in this field in recent years. [0003] Toxoplasma gondi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/20G01N33/569C12R1/91
CPCC07K16/20C07K2317/35C07K2317/565G01N33/56905G01N2333/45
Inventor 余莉苏蕊沈继龙张发敏罗庆礼
Owner ANHUI MEDICAL UNIV
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