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Recombinant protein (rPgmp22) of gametophyte of plasmodium berghei as well as preparation method and application of recombinant protein (rPgmp22) to interruption of malaria transmission

A recombinant protein and Plasmodium technology, applied in the field of immunology, can solve the problem of not being able to achieve the effect of blocking transmission

Inactive Publication Date: 2017-08-18
中国医科大学
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the specific antibodies induced by these sexual stage vaccine candidate antigens of Plasmodium have not yet achieved a satisfactory transmission blocking effect.

Method used

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  • Recombinant protein (rPgmp22) of gametophyte of plasmodium berghei as well as preparation method and application of recombinant protein (rPgmp22) to interruption of malaria transmission
  • Recombinant protein (rPgmp22) of gametophyte of plasmodium berghei as well as preparation method and application of recombinant protein (rPgmp22) to interruption of malaria transmission
  • Recombinant protein (rPgmp22) of gametophyte of plasmodium berghei as well as preparation method and application of recombinant protein (rPgmp22) to interruption of malaria transmission

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Identification of candidate antigen Pgmp22 of malaria transmission blocking vaccine and preparation of its recombinant protein.

[0042] 1. Identify and predict the structural features and functions of Pgmp22 using molecular bioinformatics techniques.

[0043] The Pgmp22 (PBANKA_030590) gene is described in the Plasmodium database as a "conserved Plasmodium protein of unknown function". BioEdit analysis shows that Pgmp22 protein is relatively conserved among Plasmodium species figure 1 , it can be seen that amino acid residues with complete homology are shaded in black, and those with a homology greater than 50% are shaded in gray. SMART analysis showed that the Pgmp22 protein contained a signal peptide and two low-complexity regions. In order to express the full-length protein, we avoided the signal peptide and selected the B-cell epitope-rich region to obtain the full-length fragment, that is, the 19th fragment from the N-terminal Up to 218 amino acid residues, a to...

Embodiment 2

[0064] Preparation of immune serum and protein localization.

[0065] 1. Preparation of immune serum.

[0066] Ten BALB / c female mice aged 6-8 weeks were divided into 2 groups, 5 in each group. Group 1 was injected with the recombinant protein prepared in Example 1, and Group 2 was the PBS control group. Grind the purified recombinant protein (50 μg / mouse) and complete Freund’s adjuvant into a water-in-oil emulsion, and inject 100 μl / mouse subcutaneously to immunize mice. In the 3rd week and the 5th week, two booster immunizations were given respectively, 25 μg of the recombinant protein was ground with Freund’s incomplete adjuvant to form a water-in-oil emulsion, and 100 μl / mouse was injected subcutaneously to immunize the mice. The mouse serum was collected 10 days after the third immunization, and the specific antibody level of the mouse serum was detected by ELISA. Compared with the PBS immunization group, the antibody level was significantly increased (p Figure 5 , the ...

Embodiment 3

[0073] Construction of targeting vector and acquisition of Δpgmp22 malaria parasite.

[0074] 1. Construction of targeting vector for gene knockout.

[0075] The method of double-crossover homologous recombination was used to target knockout of Pgmp22 gene, and a gene knockout targeting vector was successfully constructed. According to the vector construction flow chart, see Figure 8 , Pgmp22 is the target gene, 5'UTR and 3'UTR are the homology arms on both sides of the target gene, and P1, P2 and P3 are designed primers for identifying the knockout genotype.

[0076] First, the PCR method was used to successfully amplify the 5'UTR fragment, the amplified length was 877bp, and the 5' ends of the upstream and downstream primers were respectively introduced with restriction endonuclease HindⅢ and PstI restriction sites; the primer sequence is as follows: Pgmp22-5U-F (SEQ ID NO: 5): CCCAAGCTTG CCATGGGTTT TGGGCCATGT TATAC; Pgmp22-5U-R (SEQ ID NO: 6): GGCTGCAGTT TCCCCTACCC TTTTA...

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Abstract

The invention belongs to the field of immunology and specifically relates to a recombinant protein (rPgmp22) of a gametophyte of plasmodium berghei as well as a preparation method and application of the recombinant protein (rPgmp22) to interruption of malaria transmission. The amino acid sequence of the recombinant protein (rPgmp22) of the gametophyte of the plasmodium berghei is SEQ ID NO: 1; the recombinant protein (rPgmp22) is an amino acid sequence with 95-100% of homology with the amino acid sequence limited by SEQ ID NO: 1, which is used for coding a protein with same function; or the recombinant protein (rPgmp22) is a derived pretein with the same activity, which is formed by increasing, deleting or replacing one or a plurality of amino acids for the amino acid sequence shown by SEQ ID NO: 1. The recombinant protein is a novel recombinant protein with a transmission interruption effect, so that the malaria transmission interruption effect can be further improved.

Description

technical field [0001] The invention belongs to the field of immunology, and in particular relates to a Plasmodium berghei gametocyte recombinant protein (rPgmp22), a preparation method thereof and an application in the process of blocking malaria transmission. Background technique [0002] Malaria is an infectious parasitic disease transmitted by Plasmodium via Anopheles mosquitoes, which seriously threatens human health. The latest WHO global malaria report pointed out that in 2015 alone, about 429,000 people died of malaria worldwide. In high-density malaria endemic areas, currently available anti-malarial methods are no longer sufficient to block the spread of malaria, and the increase and prevalence of drug-resistant mosquitoes and Plasmodium have further increased the difficulty of malaria control. Therefore, the International Expert Committee on Malaria Elimination (MalERA) believes that the key measure to control the spread of malaria is to develop new control method...

Claims

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Application Information

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IPC IPC(8): C07K14/445C12N15/30A61K39/015A61P33/06
CPCA61K39/015C07K14/445Y02A50/30
Inventor 曹雅明刘飞王庆辉冯辉王亚茹李莉杨帆洪民生
Owner 中国医科大学
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