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Recombinant plasmid, recombinant malaria parasite and its application

A technology of recombining plasmids and malaria parasites, which is applied in the field of tumor cell immunotherapy, and can solve problems such as the inability to express multiple foreign genes at the same time, the inability to secrete genes, and the reverse insertion of target genes.

Active Publication Date: 2017-05-17
BLUE ELEGANT BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] CN 101838611A discloses a recombinant malaria parasite expressing foreign genes, the foreign genes in the recombinant malaria parasite include antigen gene, therapeutic gene, immunomodulator gene or peptide gene, wherein the plasmid used in this invention is pL0015 plasmid, pL0015 Plasmids mainly use single enzyme cutting (Bam HI) to insert the target gene, which is prone to reverse insertion of the target gene when inserting the target gene, which is not conducive to the construction of the plasmid vector, and the recombinant malaria parasites in this patent cannot be expressed at the same time Multiple exogenous genes, and the expressed genes cannot be secreted outside the body of Plasmodium parasites

Method used

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  • Recombinant plasmid, recombinant malaria parasite and its application
  • Recombinant plasmid, recombinant malaria parasite and its application
  • Recombinant plasmid, recombinant malaria parasite and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Preparation of recombinant plasmid pL0017-gRluc-X

[0058] 1. The basic molecular cloning technology used in this experiment:

[0059] 1) Extraction of plasmids.

[0060] Refer to the instructions of AxyPrep Plasmid DNA Mini Kit from Axygen Company (Axygen, #AP-MN-P-50).

[0061] 2) Gel DNA fragment extraction.

[0062] Follow the instructions of AxyPrep DNA Gel Recovery Kit from Axygen Company.

[0063] 3) Enzyme digestion of DNA and carrier identification.

[0064] The plasmid DNA was prepared into a suitable reaction system with the corresponding restriction endonuclease and its supporting buffer, and was bathed in 37°C water bath for 3-4 hours to identify the result of enzyme digestion.

[0065] The reaction system for enzyme digestion and recovery of DNA is as follows: 3 μl of 10× buffer, 20 μl of enzyme digestion carrier DNA, 0.5 μl of each enzyme E1 / E2 (choose a restriction enzyme according to the purpose of the experiment), 6 μl of ddH 2 O, the to...

Embodiment 2

[0114] The construction of embodiment 2 recombinant malaria parasites

[0115] 1. Large-dose extraction of pL0017-gRluc-mCherry plasmid, pL0017-gRluc-ibisi-mCherry plasmid, pL0017-gRluc-gpc3 plasmid, pL0017-gpc3:2F plasmid and pL0017-ibis1-gpc3:2F plasmid.

[0116] 1) Plasmid extraction

[0117] (1) Streak activation contains correctly constructed recombinant pL0017 plasmids, including pL0017-gRluc-mCherry plasmid, pL0017-gRluc-ibisi-mCherry plasmid, pL0017-gRluc-gpc3 plasmid, pL0017-gpc3:2F plasmid and pL0017-ibis1-gpc3 : the bacterial strain of 2F plasmid, culture 16h in the incubator;

[0118] (2) Randomly pick 2 single clones, transfer them to a 15ml centrifuge tube and culture them for 8-12 hours;

[0119] (3) According to the inoculum size of 1:500, inoculate the above-mentioned culture solution into a 1-liter Erlenmeyer flask containing 200 ml of ampicillin-resistant LB medium, shake and culture at 37°C for 12 hours, and collect the bacterial solution with QIAGEN’s la...

Embodiment 3

[0187] Embodiment 3 Immunological detection of murine Plasmodium yoelii immunized mice

[0188] Including CD8α+DC, Th1-related cytokines, and CTL response detection against GPC3 protein.

[0189] 1. Experimental materials: C57BL / 6 mice, Hepa1-6 cells; penicillin and streptomycin are products of Bio Basic Inc; pancreatin is a product of Amresco. Anti-CD11c-FITC, anti-CD8α-PE, anti-CD86-APC, anti-CD80-PerCP-Cy5.5 antibodies (purchased from eBioscience, San Diego, CA, USA), BD FACSAria flow cytometer, FlowJo analysis software (Tree Star , Inc.), mouse Th-related multifactor detection kit (BioLegend), BDTM ELISPOT MouseIFN-γ-Kit (BD Biosciences, #552569). Fortessa flow analyzer (BD), Legendplex analysis software (BioLegend); 1 × erythrocyte lysate (BD), 200 mesh cell filter, 1mL syringe, low-temperature refrigerated centrifuge (Germany eppendorf company), biochemical incubator (Shanghai No. Heng Instrument Company), tweezers, scissors, alcohol, capillary glass tube; DMEM and RPM...

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Abstract

The present invention relates to the field of cellular immunotherapy for tumors and, more particularly, to a recombinant plasmid for the construction of recombinant malaria parasites and their use, wherein the recombinant plasmid is a tumorigenin-specific antigen gene inserted into the pL0017 plasmid, Recombinant malaria parasite, comprising the recombinant plasmid. Compared with plasmid DNA and RNA vector, the recombinant malaria parasite can be multiplied by the proliferation of Plasmodium, which is beneficial to the increase of the antigen in vivo. Compared with the defective virus and bacterial carrier, it survives in the red blood cells of the body longer, not short-term by the body's immune system to clear, long-term effective expression of exogenous tumor antigen, is conducive to long-term antigen and immune stimulation. The recombinant malaria parasite is capable of activating the high expression of Th1-related cytokines in vivo and increasing the proportion of CD8a + DCs in total CD11c + DCs and further activating specific cytotoxic T lymphocyte responses against tumor antigens, which is beneficial to the antitumor effect of the vaccine.

Description

technical field [0001] The present invention relates to the field of tumor cell immunotherapy, in particular to a recombinant plasmid, a recombinant malaria parasite constructed by it and its application, specifically the construction of a recombinant malaria parasite based on the recombinant plasmid and its application in anti-hepatic tumor therapy . Background technique [0002] Hepatocellular carcinoma (HCC) accounts for 85% to 90% of primary liver cancers. It is one of the five most common cancers in the world, and its lethality ranks among the top three among all tumors. Data show that there are 782,500 new cases of liver cancer every year in the world, and 745,500 patients die of liver cancer every year. At present, the techniques used clinically for the treatment of liver cancer include surgical resection, orthotopic liver transplantation, hepatic arterial chemoembolization, and local radiofrequency therapy, etc. These methods have certain curative effects on early-...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N1/11A61K39/00A61K39/39A61K48/00A61P35/00C12R1/90
CPCA61K39/0011A61K39/39A61K48/0008A61K48/0058C12N15/79C07K14/4715C07K14/4725C07K14/4748A61K2039/55588C12N2800/60A61K2300/00Y02A50/30
Inventor 陈小平秦莉刘权
Owner BLUE ELEGANT BIOTECH CO LTD
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