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Schistosoma japonicum katsurada recombinant antigen as well as preparation method and application thereof

A technology of recombinant antigen and schistosomiasis, applied in the field of bioengineering, to achieve the effect of good application value

Inactive Publication Date: 2014-03-26
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, the signal transduction pathway through which PGMRC1 binds progesterone has not been elucidated, but based on the amino acid sequence, it is predicted that tyrosine kinases, kinase binding, and SH2 and SH3 may be involved

Method used

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  • Schistosoma japonicum katsurada recombinant antigen as well as preparation method and application thereof
  • Schistosoma japonicum katsurada recombinant antigen as well as preparation method and application thereof
  • Schistosoma japonicum katsurada recombinant antigen as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Expression and Purification of Schistosoma japonicum Recombinant Antigen

[0038] 1. Method

[0039] 1.1 Construction of recombinant expression plasmids

[0040] According to the gene sequence of SjPGMRC2 with NCBI accession number FN317689.1, design primers, upstream primer: 5′-TC GAATTC TGCGTTCTACTTGGCTATTT-3' (SEQ ID NO.3, the underline is the EcoR I restriction site); downstream primer: 5'-CG CTCGAG AGCTAATTTTTCTTTGAGTG-3' (SEQ ID NO. 4, the Xho I restriction site is underlined). Using the cDNA of Schistosoma japonicum 28-day-old body as a template, PCR amplifies its cDNA fragment containing ORF, and the reaction composition is as follows:

[0041]

[0042]The reaction conditions were 95°C for 5min; then denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 2min, a total of 35 cycles, and finally 10min at 72°C. After the PCR product was purified, it was connected to the pMD19-T vector, transformed into DH5 cells, and singl...

Embodiment 2

[0060] Example 2 Antigenic Detection of Schistosoma japonicum Recombinant Antigen

[0061] 1. Western Blotting analysis of recombinant protein antigenicity

[0062] The recombinant protein pET32a(+)-PGMRC2 purified by Ni column was electrophoresed by SDS-PAGE, and then transferred to NC membrane at 4°C with 260mA for 1.5h, and the serum of BALB / c mice immunized with recombinant protein Sera from healthy BALB / c mice were used as primary antibodies to analyze the antigenicity of the recombinant protein.

[0063] 2. Western Blot analysis of recombinant protein antigenicity results

[0064] The results of Western Blot analysis showed that when the recombinant protein-immunized BALB / c mouse serum was used as the primary antibody, there was an obvious recognition band at 38kD, but when the serum of healthy BALB / c mice was used as the primary antibody, there was no obvious band at 38kD band, which shows that the recombinant protein pET32a(+)-SjPGMRC2 has good immunogenicity ( Fig...

Embodiment 3

[0065] Example 3 Immunoprophylaxis Experiment of Schistosoma japonicum Recombinant Antigen

[0066] 1. Method steps

[0067] 1.1 Animal immune protection experiment

[0068]The 6-week-old BALB / c mice were divided into three groups, namely the recombinant protein SjPGMRC2 immunized group, the 206 adjuvant control group and the PBS control group, with 15 mice in each group. Each time mice in the recombinant protein immunization group were immunized, each mouse was subcutaneously injected with 100 μL of the emulsion of recombinant protein SjPGMRC2 (20 μg) and 206 adjuvant. In the 206 adjuvant control group, each mouse was subcutaneously injected with 100 μL of the emulsion of 206 adjuvant and PBS. In the PBS control group, each mouse was subcutaneously injected with 100 μL PBS each time. A total of three immunizations were performed at intervals of 2 weeks. Seven days after each immunization, blood was collected from the orbit of each mouse, and serum was collected and stored ...

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Abstract

The invention discloses schistosoma japonicum katsurada recombinant antigen. The recombinant antigen comprises an amino acid sequence of schistosoma japonicum katsurada PGMRC2 protein indicated by SEQ ID No.1. The invention also discloses a preparation method of schistosoma japonicum katsurada antigen and application of the antigen in preparing a vaccine or a medicament for preventing or treating the schistosoma japonicum katsurada disease. By adopting the schistosoma japonicum katsurada recombinant antigen, the specificity IgG, IgG1 and IgG1a antibodies for resisting the recombinant antigen can be induced in a mice body, the worm reduction rates of 22.08 percent and 20.097 percent are respectively induced in two animal protection experiments, so that the recombinant antigen is suitable for being used as a candidate vaccine for resisting the schistosoma japonicum katsurada and has good application prospect.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a recombinant antigen of Schistosoma japonicum and its preparation method and application. Background technique [0002] Schistosomiasis japonicum is an important zoonotic parasitic disease. Although my country has made great achievements in the prevention and control of schistosomiasis, the prospect of comprehensive control of schistosomiasis transmission is still not optimistic due to the difficulty of eradicating the intermediate host snails and the serious phenomenon of repeated infection of schistosomiasis. Since its inception, praziquantel has been widely used in the clinical treatment and prevention of schistosomiasis due to its advantages of high efficiency, low toxicity, convenient use, and low price, and has become the only drug for the treatment of schistosomiasis. There are new drugs for the treatment of schistosomiasis. However, there have been reports of pr...

Claims

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Application Information

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IPC IPC(8): C07K14/705C12N15/12C12N15/63C12N1/21C12N5/10C12N15/70A61K39/00A61P33/12C07K16/18C07K14/54C07K14/57G01N33/68
CPCA61K38/00A61K39/00C07K14/721C07K16/2869C12N15/70Y02A50/30
Inventor 林矫矫吴秀娟傅志强洪炀张旻韩艳辉李学珍赵彬王飞魏梅梅王馨茁李长健曹晓丹
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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