Anti-Pf332-DBL region monoclonal antibody capable of restraining invasion of plasmodiumfalciparum

A technology for Plasmodium falciparum, monoclonal antibodies, applied in the fields of parasitology and immunology

Inactive Publication Date: 2010-10-20
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports on the preparation of monoclonal antibodies against the DBL region of the Pf332 membrane protein of Plasmodium falciparum and the screening of monoclonal antibodies that can inhibit the parasites from invading red blood cells.

Method used

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  • Anti-Pf332-DBL region monoclonal antibody capable of restraining invasion of plasmodiumfalciparum
  • Anti-Pf332-DBL region monoclonal antibody capable of restraining invasion of plasmodiumfalciparum
  • Anti-Pf332-DBL region monoclonal antibody capable of restraining invasion of plasmodiumfalciparum

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Construction of recombinant expression vectors pQE-DBL and pGEX-4T-1-DBL

[0025] In this embodiment, according to the nucleotide sequence of the Pf332-DBL region, the following PCR primers were synthesized:

[0026] The upstream primer is: 5′-GCGAATTCAGCAACATCAACAACAAGG-3′;

[0027] The downstream primer is: 5′-GCGCGGCCGCTTAGGCGTACTTCTTCTCGA-3′

[0028] Using the Plasmodium falciparum cDNA as a template, carry out PCR amplification with the above primers to obtain the Pf332-DBL gene fragment, the nucleotide sequence of which is shown in SEQ ID No.3, and connect it to the His tag fusion expression vector pQE-70 , to construct the recombinant expression vector pQE-DBL to express the recombinant protein DBL-His containing His tag.

[0029] Using the Plasmodium falciparum cDNA as a template, the above primers were used for PCR amplification to obtain the Pf332-DBL gene fragment, which was connected to the GST tag fusion expression vector pGEX-4T-1 to construct a recombin...

Embodiment 2

[0032] Expression and Purification of Recombinant Antigens DBL-His and DBL-GST

[0033] The recombinant expression vectors pQE-DBL and pGEX-4T-1-DBL in Example 1 were respectively transformed into M15 and BL21(DE3) host bacteria, and after induction and expression were performed respectively, DBL-His was purified by affinity chromatography. and DBL-GST recombinant antigen for SDS-PAGE analysis (such as figure 1 ). Use anti-His tag monoclonal antibody or anti-GST tag monoclonal antibody and anti-Pf332-DBL mouse polyclonal antibody as the primary antibody to carry out Western blotting identification on the recombinant protein, and there are specific bands at 28kDa and 52kDa respectively (such as figure 2 ). The amino acid sequence of DBL-His is shown in SEQ ID No.4, and the amino acid sequence of DBL-GST is shown in SEQ ID No.5.

Embodiment 3

[0035] Preparation of Monoclonal Antibody Against Pf332 Membrane Protein DBL Region of Plasmodium falciparum

[0036] (1) Animal immunity

[0037] The immunization program adopts the method of long-term high-dose. Take 5 female BALB / c mice aged 6-8 weeks, and mix the immune antigen DBL-His purified in Example 2 with an equal amount of Freund's complete adjuvant for the first immunization, and inject it intraperitoneally after complete emulsification. Rats were immunized with 50 μg of antigen. Afterwards, the rats were immunized every 3 weeks with the same dose, and the adjuvant was replaced by Freund's incomplete adjuvant. Blood was collected from the tail vein 10-14 days after each immunization, and the serum was separated to detect the specific antibody titer by indirect ELISA method (the purified detection antigen DBL-GST coated ELISA plate in Example 2) until the antibody titer meets the requirements of cell fusion (1∶5×10 4 above). Three days before fusion, mice were...

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Abstract

The invention discloses an anti-Pf332-DBL area monoclonal antibody capable of restraining the invasion of plasmodiumfalciparum, which can be specifically combined with the DBL region functional polypeptide of plasmodiumfalciparum Pf332 membrane protein, and the polypeptide has the amino acid sequence shown as SEQ ID No.1 or 2. The anti-Pf332-DBL area monoclonal antibody is prepared by fusing splenocyte and Sp2 / 0 cells through a DBL recombination protein immunity mouse with a HIS mark, primarily selecting by using a DBL recombination protein with a GST mark, and carrying out positive selecting by using a functional polypeptide. The anti-Pf332-DBL area monoclonal antibody has stronger function for restraining the invasion of polypide, and the function is stronger than that of an MP antibody purified from Mlli human serum infected by plasmodiumfalciparum. A monoclonal antibody which can not be specifically combined with the functional polypeptide has weaker function for restraining the invasion of polypide or has no function for restraining the invasion of polypide. The DBL region functional polypeptide can be applied to the research and development of polypeptide medicaments for treating malaria and the preparation of preventing active polypeptide vaccine.

Description

Technical field: [0001] The invention relates to the fields of parasitology and immunology. More specifically, the present invention relates to an anti-Pf332-DBL region monoclonal antibody that can inhibit the invasion of Plasmodium falciparum and a functional polypeptide of the Pf332 membrane protein DBL region of Plasmodium falciparum Background technique: [0002] The emergence and spread of Plasmodium drug resistance has seriously exacerbated the global malaria epidemic. The annual death toll from malaria has risen from about 500,000 in the 1970s to about 1 million at present. The measures to prevent and control malaria with drugs are also facing serious difficulties and challenges. Therefore, the implementation of new malaria control measures has become a top priority. Vaccination has enabled many infectious diseases to be effectively controlled or even eradicated, so the development of effective malaria vaccines has become an important means to prevent Plasmodium infe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/445C07K16/20C12N5/20A61K39/015A61P33/06
CPCY02A50/30
Inventor 陈启军杜承姜宁尹继刚陆慧君
Owner JILIN UNIV
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