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Recombinant malignant malarial parasite hypoxanthine-guanine-xanthine phosphoribosyl transferase and its preparing method and use

A technology of xanthine phosphoribose and Plasmodium falciparum, applied in the field of DNA recombination technology and genetic engineering vaccines, can solve the problems of vaccines coming out, not being able to be used as effective anti-malarial immunogens, and not successfully expressing HGXPRT reports, etc.

Inactive Publication Date: 2009-08-05
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, for some unknown reason, no successful expression of HGXPRT has been reported
Therefore, it has been considered that HGXPRT cannot be used as an effective 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 malignant malarial parasite hypoxanthine-guanine-xanthine phosphoribosyl transferase and its preparing method and use
  • Recombinant malignant malarial parasite hypoxanthine-guanine-xanthine phosphoribosyl transferase and its preparing method and use
  • Recombinant malignant malarial parasite hypoxanthine-guanine-xanthine phosphoribosyl transferase and its preparing method and use

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Synthesis of Pfhgxprt gene

[0068] 1.1. Design of Pfhgxprt Totally Synthetic Gene

[0069] The sequence of the recombinant HGXPRT protein is derived from the amino acid sequence of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase HGXPRT. 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 to the 5' and 3' ends of the newly designed hgxprt 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 acid Asn to Gln (see SEQ ID NO: 2).

[0070]...

Embodiment 2

[0084] Secreted expression of Pfhgxprt gene in Pichia pastoris

[0085] 2.1: Construction of expression vector

[0086] Pfhgxprt gene was inserted into pPIC9 yeast expression plasmid (purchased from Invitrogen Company) through XhoI and EcoRI sites. In this way, the N-terminus of PfHGXPRT is fused to the C-terminus of the Saccharomyces cerevisiae α-factor signal peptide on the vector. Since the N-terminal of the PfHGXPRT 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.

[0087] 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 Pfhgxprt-containing fragment was excised with BamHI and SalI and inserted into the corresponding site of the pPIC9K expression plasmid (purchased from Invitrogen Co., L...

Embodiment 3

[0093] PfHGXPRT fermentation and purification

[0094] After optimizing the expression conditions, the expression level in the shake flask can reach 80mg / 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 =110. In the growth phase of glycerol addition, the cell growth increased linearly, and the cell density reached OD 600 =580. 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 1000mg / L.

[0095] The purification of PfHGXPRT expression product was carried out in three steps: the first step was to purify with Ni-NTA column. Since the C-terminus of PfHGXPRT contains 6...

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Abstract

The present invention provides the recombinant protein (HGXPRT) of malignant malarial parasite hypoxanthine-uanine-xanthine ribose phosphate transferase, the recombinant protein coding DNA sequence, vector containing the DNA sequence, host cell containing the vector, gene engineering process for preparing the recombinant protein, and the use of the recombinant protein. The recombinant protein HGXPRT has excellent immunogenicity and excellent enzymic kinetic characteristic, and can induce effective malarial parasite antagonizing immune response in the immunized individual and produce efficient enzymological activity.

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 Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (hypoxanthine-guanine-xanthine phosphoribosyltransferase, HGXPRT), a DNA sequence encoding the recombinant protein, comprising the DNA The vector of the sequence, the host cell containing the vector, the method for preparing the recombinant protein by genetic engineering, 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 co...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/63C12P21/02A61K38/45A61P33/06
CPCY02A50/30
Inventor 张冬梅潘卫庆
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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