Recombinant Plasmodium falciparum 175kD erythrocyte conjugated antigen functional region protein and its preparation and use

A recombinant protein and antigen-binding technology, which is applied in the field of DNA recombination technology and genetic engineering vaccines, can solve problems such as the advent of vaccines, the failure to express 175kD red blood cell-binding antigen, and the inability to implement and apply anti-malarial immunogens.

Inactive Publication Date: 2009-02-04
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although the 175kD erythrocyte-binding antigen (EBA-175) has been expressed in E. coli, the expressed EBA-175 is not immunogenic
In addition, for some unknown reason, although some people try to express EBA-175 in yeast and other eukaryotic cells, there is no report of the 175kD erythrocyte-binding antigen successfully expressed
Therefore, it has been considered that EBA-175 cannot be used as an immunogen against malaria
[0007] In summary, although biotechnology-based malaria vaccine research has been carried out for nearly 20 years, no vaccine with practical value has come out so far

Method used

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  • Recombinant Plasmodium falciparum 175kD erythrocyte conjugated antigen functional region protein and its preparation and use
  • Recombinant Plasmodium falciparum 175kD erythrocyte conjugated antigen functional region protein and its preparation and use
  • Recombinant Plasmodium falciparum 175kD erythrocyte conjugated antigen functional region protein and its preparation and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Synthesis of PfEBA-175IIF2 Gene

[0079] 1.1. Design of Pfeba-175IIf2 Totally Synthetic Gene

[0080] The sequence of the recombinant EBA-175IIF2 protein is derived from the amino acid sequence of the 175kD erythrocyte-binding antigen functional region EBA-175IIF2 of the Plasmodium falciparum 3D7 strain. The sequence was redesigned by selecting the codon usage frequency of Pichia pastoris. Use computer software "DNA Star", Omiga, etc. to analyze and exclude any sequences in the gene sequence that may be unfavorable to gene transcription and translation, such as transcription termination sequences, intron splice site sequences, long inverted repeat sequences Wait. In addition, XhoI and EcoRI restriction sites were added at the 5' and 3' ends of the newly designed eba-175IIf2 gene, respectively, so as to be connected with the expression vector. A potential glycosylation site in the antigen sequence was identified and excluded by converting the glycosylation site amino ...

Embodiment 2

[0097] Secretion and expression of Pfeba-175IIf2 gene in Pichia pastoris

[0098] 2.1: Construction of expression vector

[0099] Pfeba-175IIf2 gene was inserted into pPIC9 yeast expression plasmid (purchased from Invitrogen Company) through XhoI and EcoRI sites. In this way, the N-terminal of PfEBA-175IIF2 is fused with the C-terminal of the signal peptide of Saccharomyces cerevisiae α-factor on the vector. Because the N-terminal of the PfEBA-175IIF2 molecule contains three specific amino acid Glu-lys-Arg sequences of the signal peptide cutting point. Therefore, the target protein released by secretory expression does not contain a signal peptide sequence.

[0100] The basic elements of the pPIC9K vector are the same as pPIC9, but it has a kanamycin resistance gene, and G418 can be used for screening of high-copy inserts. In this way, the fragment containing Pfeba-175IIf2 was cut out with BamHI and SalI and inserted into the corresponding site of the pPIC9K expression plas...

Embodiment 3

[0106] Fermentation and purification of PfEBA-175IIF2

[0107] After optimizing the expression conditions, the expression level of shake flask can reach 20mg / L. Fermentation expression was performed in 15L tanks. During the whole fermentation cycle, the cells in the early stage grow exponentially, and the cell density reaches OD 600 =100. In the growth phase of glycerol addition, the cell growth increased linearly, and the cell density reached OD 600 =560. However, in the stage of methanol-induced expression, the total cell density remains basically unchanged, while the expression of the target protein begins 3-7 hours after the addition of methanol, and as time goes on, the expression yield increases rapidly and is continuously secreted into the fermentation broth. The amount can reach 300mg / L.

[0108] The purification of PfEBA-175IIF2 expression product was carried out in two steps: the first step was purification with Ni-NTA column. Since the C-terminus of PfEBA-175I...

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Abstract

The present invention is one kind of 175 kD recombinant protein (PfEBA175-IIF2) of plasmodium falciparum erythrocyte conjugated antigen functional region, its coding DNA sequence, vector containing the DNA sequence, host cell containing the vector, gene engineering process of preparing the recombinant protein, and the application of the recombinant protein in preparing malaria resisting vaccine and performing preclinical research on plasmodium invasion mechanism. The recombinant protein PfEBA175-IIF2 possesses excellent immunogenicity and good bionomics as well as the function of combining with sialic acid dependent erythrocyte, and can initiate effective plasmodium resisting immune response in immunized individuals.

Description

technical field [0001] The invention relates to the fields of DNA recombination technology and genetic engineering vaccines. More specifically, the present invention relates to a recombinant protein containing the 175kD erythrocyte-binding antigen functional region of Plasmodium falciparum, a DNA sequence encoding the recombinant protein, a vector containing the DNA sequence, a host cell containing the vector, and preparing the recombinant protein by genetic engineering. The method of the protein, and the application of the recombinant protein as a drug target protein and in the development of malaria vaccine. Background technique [0002] Malaria is one of the oldest infectious diseases of mankind, and it still seriously affects human health. According to the latest survey of the World Health Organization (WHO), about 40% of the world's population is still under the threat of malaria, distributed in more than 100 countries. At present, 300 to 500 million people suffer fro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/445C12N15/09C12N15/30C12N15/63A61K39/015A61P33/06
CPCY02A50/30
Inventor 潘卫庆张冬梅
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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