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Humanized anti-plasmodium falciparum antibody and application thereof

A technology of Plasmodium falciparum and antibodies, which can be used in the fields of antibodies, anti-infective drugs, and resistance to vector-borne diseases, which can solve problems such as inducing allergic reactions and affecting curative effects.

Inactive Publication Date: 2018-06-29
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, most of the obtained polyclonal antibodies and monoclonal antibodies against Plasmodium falciparum are mouse-derived antibodies or rabbit-derived antibodies. The immune reaction of heterologous protein produces human anti-mouse antibody or human anti-rabbit antibody, which not only affects the curative effect, but also induces allergic reaction, so it is difficult to be used in clinical practice

Method used

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  • Humanized anti-plasmodium falciparum antibody and application thereof
  • Humanized anti-plasmodium falciparum antibody and application thereof
  • Humanized anti-plasmodium falciparum antibody and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0174] Example 1. Construction of antibody library

[0175] Peripheral anticoagulation (5ml per person) of 10 patients infected with malignant protozoa was taken, lymphocytes were separated by Ficoll-Paque (Pharmacia, Uppsala, Sweden), and total RNA was extracted by kit (QIAGEN GmbH, Hilden, Germany); RNA was reverse transcribed into cDNA with Oligo(dT)16 using Gene-Amp RNA PCR kit (Perkin-Elmer Cetus, Norwalk, Conn), and immunoglobulin γ, κ was carried out with the upstream and downstream primers of the conserved sequence of human IgG light and heavy chain. , PCR amplification of λ chain gene; PCR products were purified by a kit (QIAGEN GmbH, Hilden, Germany), and then connected with human immunoglobulin expression vector pcDNA3.4 to form a light and heavy chain library.

[0176] Example 2 Recombinant expression of Plasmodium falciparum merozoite surface antigen 1 (MSP1)

[0177] MSP1 is the main target antigen for inducing host immune response, and it has high immunogenic a...

Embodiment 3

[0178] Example 3. Screening of human immunoglobulin G against Toxoplasma gondii

[0179] Take 10ng of antibody library DNA, transform 100µl JM109 Escherichia coli, spread the bacterial solution on Luria broth (10gsodii chloridum, 10g tryptone, 5g yeast extract / L, PH 7) plate (containing ampicillin 50µg / ml), culture at 37ºC for 7 hours, to be cloned (about 5х10 3 1 clone / 90mm diameter plate) when the diameter is about 0.3mm, cover the plate with a 82mm diameter nitrocellulose membrane (Armacia / Pharmacia). After the clones are completely transferred to the membrane, place the membrane in LB containing 1.0mM IPTG. On plates, expression was induced for 6 hours at 30ºC, followed by lysozyme, DNase, and bovine serum albumin (100 mM Tris–HCl [pH 7], 150 mM NaCl, 5 mM MgCl) 2 , 1.5% BSA, 1 mg of DNase, 40 mg lysozyme / ml) to lyse the membrane; after washing to remove residual bacterial debris on the membrane, it was blocked with bovine serum albumin (BSA), reacted with 400 µg of puri...

Embodiment 4

[0182] Example 4. Characterization of anti-Plasmodium falciparum merozoite surface protein MSP1 and anti-Plasmodium falciparum human antibody Pf-2

[0183] The plasmid of the positive clone Pf-2 was taken and sequenced, and the sequencing work was completed by Invitrogen; and the amino acid sequence was estimated by VectorNTI 10 software;

[0184] After the stable cell line of the positive clone Pf-2 was cultured in large quantities, the culture supernatant was collected for purification, and the protein content was measured with the DC Protein Assay (BioRad). Acrylamide gel electrophoresis (SDS-PAGE) was used to test its purity; the purified antibodies were tested for their affinity with Biocore3000 (Biocore AB, Uppsala Sweden), and then coated with purified recombinant antigen MSP1 protein at a concentration of 2.5 µg / ml, respectively. 1.25µg / ml, 0.625µg / ml, 0.3125µg / ml Pf-2 antibody was reacted with it, and the binding rate (KA) and dissociation rate (KD) were calculated b...

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Abstract

The invention belongs to the field of biotechnology and relates to a humanized anti-plasmodium falciparum antibody as well as a coding gene and application thereof. According to the invention, an anti-plasmodium falciparum human immunoglobulin gene library is constructed; the humanized anti-plasmodium falciparum antibody is screened from the library through ELISA, sequencing analysis and the like;expression, purification and identification are carried out to prove that the humanized anti-plasmodium falciparum antibody can specifically recognize and has high affinity to a plasmodium falciparummerozoite recombinant surface antigen MSP1, and also can be specifically recognized with plasmodium falciparum merozoite; the humanized anti-plasmodium falciparum antibody plays a role of preventingplasmodium falciparum from invading erythrocytes, and also plays roles of activating complements, causing immune response of a human body and the like so as to enhance a treatment effect and be applied to the human body safely and reliably; the humanized anti-plasmodium falciparum antibody can be used for preparing antibody drugs against malaria and antibody targeting drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a human-derived anti-Plasmodium falciparum antibody, in particular to a human-derived anti-Plasmodium falciparum antibody and its encoding gene and uses thereof. Background technique [0002] Statistics show that malaria is one of the most important public health problems in the world today, with 2.4 billion people living in malaria-endemic areas, 300 million to 500 million malaria patients each year, and more than 3 million deaths each year. In order to completely eliminate malaria, WHO / TDR proposed to control and eliminate malaria on a global scale in 2010. my country formulated the "China Malaria Elimination Action Plan (2010-2020)", which proposed the goal of eliminating malaria nationwide by 2020. However, the prevalence of malaria in my country is currently decreasing, and the number of local cases is less than 100 cases per year. However, due to the continuous increase of foreig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/20C12N15/63C12N1/21C12N1/19C12N5/10A61K39/395A61P33/06
CPCA61K2039/505C07K16/205C07K2317/24C07K2317/51C07K2317/515Y02A50/30
Inventor 程训佳橘裕司付永锋
Owner FUDAN UNIV
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