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Application of schistosomulum japonicum high-expression gene or coding protein thereof

A technology for encoding proteins and schistosomes, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve problems such as removing schistosomiasis, and achieve the effect of easy operation, even resistance to schistosomiasis infection, and stable results

Active Publication Date: 2020-09-25
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

The 0-14 days of juvenile worms are relatively delicate, which is the best stage for humoral immune attack, but because it takes 10-14 days for the antigens released to induce humoral immune response, the host's humoral immunity cannot catch up with the early stage to clear the juvenile worms

Method used

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  • Application of schistosomulum japonicum high-expression gene or coding protein thereof
  • Application of schistosomulum japonicum high-expression gene or coding protein thereof
  • Application of schistosomulum japonicum high-expression gene or coding protein thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 Cloning of Schistosoma japonicum SjScP25, SjScP37, SjScP41 genes

[0081] According to the sequences of SjScP25, SjScP37, and SjScP41 genes (the putative transcriptome data of S. japonicum CNUS0000104457.1, PG014672, PG010861) published in GenBank, primers were designed and restriction restriction sites were introduced. The primer sequences are as follows:

[0082] SjScP25:

[0083] PF: 5'- GGGGACAAGTTTGTACAAAAAAGCAGGCT CAAGCATCAGCACTGGGAGA-3' (SEQ ID NO: 7),

[0084] PR: 5'- GGGGACCACTTTGTACAAGAAAGCTGGGTCCTA TTCTCGTAAAGCAGCTGACA-3' (SEQ ID NO: 8);

[0085] SjScP37:

[0086] PF: 5'- GGGGACAAGTTTGTACAAAAAAGCAGGCTT CGTTGCGGCTAAAGATCTGC-3' (SEQ ID NO: 9),

[0087] PR: 5'- GGGGACCACTTTGTACAAGAAAGCTGGGTCCTA AACCCCGTGTGACTTTGCAT-3' (SEQ ID NO: 10);

[0088] SjScP41:

[0089] PF: 5'- GGGGACAAGTTTGTACAAAAAAGCAGGCTT GCAAGAATTAAGACGTTATGCTGA-3' (SEQ ID NO: 11),

[0090] PR: 5'- GGGGACCACTTTGTACAAGAAAGCTGGGTCCTA GTAATGAATACACTTCACTGG-3' (SEQ ID NO: 12). ...

Embodiment 2

[0096] Example 2 Expression and purification of Schistosoma japonicum SjScP25, SjScP37, SjScP41 recombinant proteins

[0097] The clones identified as positive by the above PCR were inoculated into 15 mL of LB liquid medium (containing 50 μg / ml ampicillin). Penicillin) 1L, continue to culture to OD 600nm The value was 0.8, and then IPTG with a final concentration of 1 mM was added for induction, expressed at 140 rpm at 18°C ​​for 16 hours, collected by centrifugation, and frozen at -80°C for future use.

[0098] Resuspend a small amount of pre-induction and post-induction bacteria in PBS buffer, add SDS-PAGE loading buffer, mix well, boil in a boiling water bath for 5 minutes to denature the protein.

[0099] 10 μl of the pre-induction and post-induction samples were added to each loading well for SDS-PAGE analysis (5% for stacking gel, 12% for separating gel).

[0100] The pDEST17-SjScP25, pDEST17-SjScP37, and pDEST17-SjScP41 recombinant plasmids were transformed into expre...

Embodiment 3

[0108] Example 3 Antigenic Detection of Schistosoma japonicum SjScP25, SjScP37, SjScP41 Recombinant Proteins

[0109] SDS-PAGE electrophoresis: 100ng of recombinant protein was loaded on the sample, and the electrophoresis conditions were: 100V for 20min, 120V for 1h.

[0110] Membrane transfer: The protein in the PAGE gel was transferred to the PVDF membrane by wet transfer method, and the electroporation conditions were: ice bath, 100V for 1h.

[0111] Blocking: the PVDF membrane was blocked with 5% skimmed milk powder at room temperature for 2 hours, and washed 3 times with TBST buffer.

[0112] Incubation with primary antibody: BALB / c mouse serum infected with Schistosoma japonicum for 42 days, serum of New Zealand white rabbits infected with Schistosoma japonicum for 42 days, and serum of patients with Schistosoma japonicum were added respectively, and mouse anti-His-tag antibody (Abimate Biomedical Co., Ltd. company) as a positive control, healthy mouse serum as a negat...

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Abstract

The invention relates to the technical field of biology and particularly discloses application of a schistosomulum japonicum high-expression gene or coding protein thereof. The invention provides application of schistosoma japonicum katsurada SjScP25 gene, SjScP37 gene or SjScP41 gene, or coding protein thereof or a biomaterial containing the genes in preparation of schistosoma japonicum katsuradavaccine or schistosoma japonicum katsurada medicines, and provides application of the schistosoma japonicum katsurada SjScP37 gene or coding protein thereof or a biomaterial containing the schistosoma japonicum katsurada SjScP37 gene in preparation of schistosoma japonicum katsurada detection reagents or kit or detection of schistosoma japonicum katsurada with nondiagnostic purpose. The schistosomulum japonicum high-expression gene serving as antigen to immunize mice to achieve a relatively good protection effect, can relieve, even resist schistosomiasis infection and can be applied to development of schistosomiasis vaccines. Furthermore, the schistosomulum japonicum high-expression gene or coding protein thereof have high sensitivity and specificity on schistosomiasis detection and can be used in development of schistosomulum japonicum diagnostic reagents.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of a high-expression gene of Schistosoma japonicum larvae or its encoded protein. Background technique [0002] Schistosoma japonicum Katsurada (1904) is Schistosoma japonicum Katsurada, also known as Schistosoma japonicum Katsurada. Schistosomiasis is an endemic parasitic disease caused by schistosomiasis in humans. There are three main types of schistosomes that parasitize humans: Schistosoma haematobium, Schistosoma mansoni, and Schistosoma japonicum. They are very harmful to human health, and it is necessary to conduct in-depth research on their prevention and treatment. [0003] Diagnosis is central to the field of schistosomiasis control. Accurate diagnostic techniques not only have important clinical significance for early detection and early treatment of schistosomiasis patients, but also provide criteria for judging the level of schistosomiasis endemic are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12A61K39/00A61P33/12G01N33/68
CPCA61K39/0003A61K2039/5156A61P33/12C07K14/43559G01N33/6893G01N2333/43547Y02A50/30
Inventor 侯楠陈启军
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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