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An antibody that inhibits the growth of Plasmodium cynomolgus monkeys in vitro

A technology for Plasmodium and cynomolgus monkeys, which is applied in the field of antibodies that inhibit the growth of cynomolgus monkeys in vitro, can solve the problems of restricting research on Plasmodium vivax and long-term culture of Plasmodium vivax, and achieves the effect of broad application prospects.

Active Publication Date: 2022-04-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the variability and drug resistance of Plasmodium, the development of a multi-antigen vaccine to improve the efficacy of a single-antigen vaccine has become a key problem to be solved, and Plasmodium vivax cannot be cultured in vitro for a long time, which severely limits the ability to develop a multi-antigen vaccine. Plasmodium vivax research

Method used

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  • An antibody that inhibits the growth of Plasmodium cynomolgus monkeys in vitro
  • An antibody that inhibits the growth of Plasmodium cynomolgus monkeys in vitro
  • An antibody that inhibits the growth of Plasmodium cynomolgus monkeys in vitro

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Experimental program
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Effect test

Embodiment 1

[0026] Embodiment 1: Preparation of PvMSP8+1 specific antibody

[0027] Construction of recombinant vectors:

[0028] According to the sequence information of SEQ ID NO: 1, the PvMSP8+1 gene fragment was specifically synthesized and constructed into the pET32a(+) expression vector to obtain the pET32a-PvMSP8+1 recombinant plasmid.

[0029] Protein expression and purification:

[0030] Transform the pET32a-PvMSP8+1 recombinant plasmid into BL21(DE3) Escherichia coli expression cells, culture and sequence, and obtain the positive transformant BL21-PvMSP8+1 with correct sequencing, select a single clone and inoculate 5ml of LB medium containing ampicillin , cultivate overnight at 37°C, inoculate the bacterial solution in 500ml of fresh LB medium containing ampicillin, and cultivate to OD 600 When the concentration is 0.6-0.8, add IPTG to the reaction system at a final concentration of 1 mmol / L, and induce for 8 hours. The induced strain PvMSP8+1 was ultrasonically disrupted an...

Embodiment 2

[0033] Example 2: Specific recognition of PvMSP8+1-specific antibody to surface protein of Plasmodium cynomolgus Plasmodium merozoites

[0034] Specific recognition of PvMSP8+1-specific antibody to the merozoite surface protein of Plasmodium cynomolgus monkey by indirect immunofluorescence (IFA)

[0035] The schizont-enriched Plasmodium parasites were purified by Percoll gradient centrifugation, spotted onto multiwell glass slides, fixed in ice-cold acetone for 3 min, and air-dried. Nonspecific binding sites were blocked with PBS containing 5% nonfat dry milk for 30 min at 37 °C. Slides were incubated with 1:100 dilution of PvMSP8+1-specific antibody for 1 h at 37 °C. After washing 3 times with pre-cooled PBS, slides were stained with Alexa Fluor 546-conjugated goat anti-rabbit IgG secondary antibody, and nuclei were incubated with DAPI (Invitrogen) at 37°C for 30 minutes. Slides were mounted with coverslips in ProLong Gold Antifade reagent (Invitrogen) and photographed unde...

Embodiment 3

[0037] Example 3: PvMSP8+1 specific antibody inhibits the growth of Plasmodium cynomolgus monkey in vitro

[0038] In vitro inhibition test to detect the growth inhibitory effect of PvMSP8+1 specific antibody on Plasmodium cynomolgus monkey

[0039]Pre-synchronized cultures were adjusted to 1% hematocrit and 0.5% parasitaemia, then seeded in 96-well round-bottom culture dishes at a rate of 0.63 mL / well. Antibodies were prepared by serial dilution at 1 mg / mL (well 1). All antibodies were prepared by three-fold serial dilution in cynomolgi monkey (P. cynomolgi)-based medium to prepare 10 concentration points. Add approximately 7 µL of antibody to the parasites in duplicate. Forty-eight hours after incubation, each well was refreshed with 7 µL of complete growth medium. 96 h after incubation, aliquot 20 μL of culture from each well into a fresh round bottom plate, stain with 8 μM Hoechst 34580 and 150 nM MitoTracker Far Red molecular probe (mitochondrial red fluorescent probe)...

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Abstract

The invention discloses an antibody for inhibiting the growth of Plasmodium cynomolgus monkey in vitro, belonging to the fields of parasitology and immunology. The antibody is prepared from PvMSP8+1 recombinant protein, and the PvMSP8+1 recombinant protein is a fusion protein composed of Plasmodium vivax merozoite surface protein 8 (PvMSP8) and the C-terminal of 1 (PvMSP1‑19). In the in vitro growth inhibition assay, the PvMSP8+1 antibody showed a superior inhibitory effect than the PvMSP8 and PvMSP1‑19 single antigen antibodies. This provides a certain scientific basis for the development of multi-antigen malaria vaccines, and has broad application prospects.

Description

technical field [0001] The invention relates to an antibody for inhibiting the growth of Plasmodium cynomolgus monkey in vitro, which belongs to the field of parasitology and immunology. Background technique [0002] Malaria is a vector-borne infectious disease caused by Plasmodium infection caused by Anopheles mosquito bites or transfusion into the blood of a person with Plasmodium, which seriously affects human life and health. Malaria patients often have three stages of periodic chills, fever and sweating, followed by related complications such as anemia and splenomegaly. In severe cases, it will cause dangerous malaria and even lead to death. Severe physical and mental damage has also increased the global economic burden. Malaria is mainly caused by Plasmodium infection. There are 5 species of Plasmodium that can infect humans. Among them, Plasmodium vivax is the most widely distributed Plasmodium except in Africa, and its pathogenicity is relatively serious. Due to th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/70C12N1/21C07K16/20A61K39/395A61K39/015A61P33/06C12R1/19
CPCC07K14/445C12N15/70C07K16/205A61K39/015A61P33/06C07K2319/00A61K2039/505Y02A50/30
Inventor 程洋沈飞虎付海田雷瑶陆佳晨杨博徐琴雯
Owner JIANGNAN UNIV
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