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Schistosoma Japonicum polymorphism antigen gene and application thereof as vaccine

A technology of schistosomiasis and schistosomiasis nucleic acid, applied in the field of biomedicine and parasites, can solve problems such as vaccines that do not affect diploid pathogens

Active Publication Date: 2013-01-16
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In diseases caused by haploid pathogens, such as influenza, AIDS, and malaria, the effect of antigenic polymorphism on vaccine protection has been widely reported, but how it affects diploid pathogen vaccines has not been reported
[0005] At present, there is still a lack of schistosomiasis vaccine with satisfactory effect in this field, so there is an urgent need to develop new vaccines and related substances to improve the immune protection effect of schistosomiasis

Method used

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  • Schistosoma Japonicum polymorphism antigen gene and application thereof as vaccine
  • Schistosoma Japonicum polymorphism antigen gene and application thereof as vaccine
  • Schistosoma Japonicum polymorphism antigen gene and application thereof as vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0134] Example 1. Schistosoma japonicum Sj29 allele transcription pattern

[0135] Schistosoma is a diploid organism with a pair of alleles. This example proves that a pair of alleles of Schistosoma japonicum Sj29 can be transcribed into mRNA, which is a pair of co-dominant alleles.

[0136] 1. Single adult genomic DNA and total RNA extraction

[0137] Under a dissecting microscope, use a dissecting needle and tweezers to separate the embracing male and female worms, and pick a single schistosome; use a scalpel to cut a single schistosome in half, and half of it is used to extract genomic DNA, and half is used to extract total RNA Genomic DNA was extracted with the Mammalian Genome Extraction Kit produced by Beijing Tiangen Biotechnology Co., Ltd.; the total RNA was extracted using the TRIZOL reagent produced by Invitrogen Company. The specific steps refer to the kit instructions. The extracted DNA and RNA are temporarily No, store at -80°C; synthesize eDNA according to the ...

Embodiment 2

[0147] Example 2. Single Nucleotide Polymorphism Analysis of Schistosoma japonicum Sj29 Coding Sequence

[0148] 1. Single adult total RNA extraction and cDNA synthesis are the same as in Example 1, RT-PCR amplification of the Sj29 coding sequence, the primers used are: primer P3: 5'-gggaattcatgaacagtggttttaaattc-3' (SEQ ID NO.43), primer P4: 5'-ggctcgagttatagaatgtatccactg-3' (SEQ ID NO. 44). After PCR amplification, 2 μl of the amplification product was taken for 2% agarose gel electrophoresis to detect the result.

[0149] 2. Digestion, Ligation and Transformation

[0150] After the PCR product was recovered, it was digested with BamHI and EcoRI. At the same time, the vector pBlu2KSM was digested with the same endonuclease, in a 37°C water bath, the recovered target fragment was mixed with the vector, and ligated by T4DNA ligase. Transform 5 μl of the ligation product into 20 μl of competent E. coli cells, and at the same time use the complete plasmid and vector self-liga...

Embodiment 3

[0158] Example 3. Expression and purification of Sj29 recombinant protein in Pichia pastoris

[0159] 1. Optimizing the codons of the Sj29 coding sequence

[0160] The coding sequences of Sj29-2, Sj29-4 and Sj29-20 were optimized according to the preferred codons of Pichia pastoris. Exclude the signal peptide of 26 amino acids at the N-terminus of the protein and the transmembrane region of 22 amino acids at the C-terminus; exclude possible glycosylation sites of the protein; exclude transcription termination sequences and commonly used restriction enzyme sites; add at the 5' end of DNA XhoI restriction site, 6×His coding sequence, stop codon and EcoRI endonuclease site are added at the 3' end. The optimized sequences were synthesized by conventional methods. The optimized sequences are respectively SEQ ID NO: 38 (optimized from Sj29-2), SEQ ID NO: 39 (optimized from Sj29-4), and SEQ ID NO: 40 (optimized from Sj29-20).

[0161] 2. Construction of expression vectors and line...

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Abstract

The invention relates to a Schistosoma Japonicum polymorphism antigen gene and an application of the gene as a vaccine, concretely, the invention discloses a sequence of 37 alleles of Schistosoma Japonicum envelope protein Sj29 and provides a method for expressing and purifying the Sj29 recombinant protein. The invention also discloses a method for producing a Sj29 univalent allele vaccine and a Sj29 multivalent allele vaccine. According to the invention, it is proved that Sj29-induced immune protection has allele specificity, and the multivalent allele vaccine can raises the protection effect of the vaccine. The invention can be used for developing novel schistosomiasis multivalent vaccines which can overcome antigenic polymorphism.

Description

technical field [0001] The invention belongs to the fields of biomedicine and parasites; more specifically, the invention relates to a polymorphic antigen gene of Schistosoma japonicum and its use as a vaccine, and provides a vaccine with better immune protection effect against the diploid pathogen Schistosoma japonicum new vaccines. Background technique [0002] The schistosome mantle covers the surface of the schistosomiasis, and is an important part of the body surface of the schistosome, constituting the "parasite-host" contact surface. Proteins on the envelope are important candidate antigens for schistosomiasis vaccines. For example, two schistosomiasis vaccine candidate molecules that have been extensively studied, schistosomiasis 23kD protein (Sj23 or Sm23) and schistosomiasis tetraspanin 2 (Sm-TSP-2) both localize On the schistosome capsule. [0003] Sm29 is a recently identified envelope protein of Schistosoma mansoni. The protein contains a signal peptide consi...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C07K14/435C12P21/02C12Q1/68A61K48/00A61K39/00A61K38/17A61P33/12
CPCY02A50/30
Inventor 潘卫庆徐新东
Owner TONGJI UNIV
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