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Schistosoma japonicum antigen protein rSjScP92 and application thereof

A technology of antigenic protein and schistosomiasis, which is applied in the direction of anti-infective drugs, antibody medical ingredients, medical preparations containing active ingredients, etc., can solve the problem of clearing child worms, etc., and achieve the effect of simple operation, high repeatability, and high sensitivity

Active Publication Date: 2022-07-29
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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  • Schistosoma japonicum antigen protein rSjScP92 and application thereof
  • Schistosoma japonicum antigen protein rSjScP92 and application thereof
  • Schistosoma japonicum antigen protein rSjScP92 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Cloning of Schistosoma japonicum SjScP15, SjScP57 and SjScP92 genes

[0077] According to the SjScP15, SjScP57 and SjScP92 gene sequences found by the inventors by predicting the Schistosoma japonicum genome, primers were designed and restriction sites were introduced. Since the 190-400 nucleotides at the 3′ end of the SjScP57-encoded gene are highly conserved, the amino acid sequence of the encoded protein is highly homologous to human and other mammalian tyrosine hydroxylases, and the immune epitope-induced antibody of the encoded protein is very easy Cause non-specific reaction, on the one hand, it may cause non-specific reaction of antibodies, thus affecting the specificity of diagnosis; on the other hand, if the host is immunized with this protein segment, the induced antibody may non-specifically affect the activity of host tyrosine protease, which is not conducive to vaccines 's research and development. Therefore, in the present invention, a non-conse...

Embodiment 2

[0092] Example 2 Expression and purification of recombinant proteins of Schistosoma japonicum rSjScP15, rSjScP57 and rSjScP92

[0093] The clones identified as positive by the above PCR were inoculated into 15 mL of LB liquid medium (containing 50 μg / ml ampicillin), cultured at 37°C at 200 rpm overnight, and 10 mL of the medium was transferred into LB medium (containing 50 μg / ml ampicillin) the next day. Penicillin) 1L, continue to cultivate to OD 600nm The value was 0.8, then IPTG with a final concentration of 1 mM was added for induction, expression was performed at 18°C ​​at 140 rpm for 16 hours, the cells were collected by centrifugation, and frozen at -80°C for use.

[0094] A small amount of bacteria before and after induction were resuspended in PBS buffer, added with SDS-PAGE loading buffer, mixed and boiled in a boiling water bath for 5 min to denature the protein.

[0095] 10 µl of the pre-induction and post-induction samples were added to each loading well for SDS-...

Embodiment 3

[0104] Example 3 Antigenicity detection of recombinant proteins of Schistosoma japonicum rSjScP15, rSjScP57 and rSjScP92

[0105] SDS-PAGE electrophoresis: take 100ng of recombinant protein for loading, and the electrophoresis conditions are: 100V for 20min, 120V for 1h.

[0106] Transfer membrane: 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.

[0107] Blocking: The PVDF membrane was blocked with 5% nonfat milk powder at room temperature for 2 h, and washed three times with TBST buffer.

[0108] Incubation with primary antibody: serum of BALB / c mice infected with S. japonicum for 42 days, serum of New Zealand white rabbits infected with S. japonicum for 42 days, and serum of patients with S. japonicum were added respectively. Company) as a positive control and healthy mouse serum as a negative control (1:500 dilution with blocking solution), incubated overnight at 4°C, a...

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Abstract

The invention provides a schistosoma japonicum antigen protein rSjScP92 and an application thereof. A series of genes which are highly expressed in schistosoma japonicum schistosoma are screened by using a schistosoma japonicum schistosoma whole genome expression profile chip, and antigen proteins coded by the genes SjScP15, SjScP57 and SjScP92 can be specifically recognized by serum of a schistosomiasis patient and show a relatively strong positive reaction. ELISA (enzyme-linked immunosorbent assay) detection shows that the antigen proteins have high sensitivity and specificity when being used for detecting the schistosoma japonicum and can be used for developing a schistosoma japonicum disease diagnostic reagent. After mice immunized by the schistosoma japonicum antigen proteins SjScP15, SjScP57 and SjScP92 are attacked by insects, the immune protection effect shows that the SjScP15, the SjScP57 and the SjScP92 are expected to be developed into schistosomiasis vaccines.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the antigenic protein rSjScP92 of Schistosoma japonicum and its application. Background technique [0002] Schistosoma, also known as Schistosoma, belongs to the class Flatworms. Schistosomiasis is an endemic parasitic disease caused by schistosomiasis parasitizing the human body. Among the six species of schistosomiasis that parasitize the human body, Schistosoma aegypti, Schistosoma mansoni and Schistosoma japonicum are the most widely distributed and the most harmful. Among them, Schistosoma japonicum is an endemic strain of Schistosoma japonicum in my country. They are harmful to human health, and it is necessary to conduct in-depth research on their prevention and treatment. [0003] Diagnosis is a central link in the field of schistosomiasis control. Accurate diagnostic techniques not only have important clinical significance for early detection and early treatment of schis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435G01N33/569G01N33/58G01N33/68A61K39/00A61P33/12
CPCC07K14/43559G01N33/56966G01N33/6893G01N33/581A61K39/0003A61P33/12G01N2333/43547Y02A50/30
Inventor 侯楠陈启军刘帅朴贤玉
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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