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Schistosoma japonicum katsurada recombinant protein, preparation method, and application thereof

A technology of recombinant protein and schistosomiasis, applied in the field of bioengineering, to achieve good application value, broad application prospects, and good immunogenicity

Inactive Publication Date: 2017-04-05
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

However, so far, there have been no published reports on the SjC1qBP recombinant protein of Schistosoma japonicum as an anti-schistosomal drug

Method used

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  • Schistosoma japonicum katsurada recombinant protein, preparation method, and application thereof
  • Schistosoma japonicum katsurada recombinant protein, preparation method, and application thereof
  • Schistosoma japonicum katsurada recombinant protein, preparation method, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Expression and purification of Schistosoma japonicum recombinant protein

[0039] 1. Method

[0040] 1.1 Construction of recombinant expression plasmids

[0041] According to the gene sequence (FN315565) of SjC1qBP (abbreviated as SjC1BP) collected by NCBI, primers were designed, upstream primer (P1): 5'-CGCGAATTCTTACGGCGTGAAATGTCT-3' (SEQ ID NO.3) with EcoR I restriction site; P2): 5'-GCGCTCGAGCAGTCTTTAAGGATTGTGCA-3' (SEQ ID NO.4) with Xho I restriction site, used to amplify the SjC1qBP gene fragment. Using the cDNA of Schistosoma japonicum 42-day-old body as a template, PCR amplifies its cDNA fragment containing ORF, and the reaction system is composed as follows:

[0042]

[0043]

[0044] PCR reaction conditions: 94°C for 5min, denaturation at 94°C for 30sec, annealing at 56°C for 30sec, extension at 72°C for 90sec, a total of 30 cycles; finally 72°C for 15min. After the PCR product was purified, it was connected to the pMD19-T vector, transformed...

Embodiment 2

[0053] Example 2 Antigenic Detection of Schistosoma japonicum Recombinant Protein rSjC1qBP

[0054] 1. Western Blotting analysis of recombinant protein antigenicity

[0055] The recombinant protein rSjC1qBP purified by Ni column was subjected to SDS-PAGE electrophoresis, and then transferred to the NC membrane at 4°C with 130 mA for 75 min, and the antiserum against the recombinant protein was used as the primary antibody to analyze the antigenicity of the recombinant protein.

[0056] 2. Western Blot analysis of recombinant protein antigenicity results

[0057] The results of Western Blot analysis showed that the recombinant protein rSjC1qBP could be recognized by the recombinant protein immune serum and positive serum respectively, indicating that the recombinant protein rSjC1qBP had good immunogenicity ( Figure 4 ). exist Figure 4 Middle, M: Marker; A: The primary antibody is the mouse positive serum; B: The primary antibody is the normal mouse serum.

Embodiment 3

[0058] Example 3 Immunization experiment of Schistosoma japonicum recombinant protein rSjC1qBP (abbreviated as rSjC1BP)

[0059] 1. Method steps

[0060] 1.1 Animal immunization experiments

[0061] The 6-week-old BALB / c mice were divided into four groups, namely recombinant protein rSjC1qBP immunization group, 206 adjuvant control group, pET32-a(+) tagged protein group and PBS control group, with 10 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 rSjC1qBP (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 o...

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Abstract

The invention discloses a schistosoma japonicum katsurada recombinant protein, which is prepared through expression of a recombinant vector comprising a schistosoma japonicum katsurada C1qBP gene. The invention also discloses a preparation method of the schistosoma japonicum katsurada recombinant protein rSjC1qBP and an application of the recombinant protein in preparation of medicines for therapy of schistosomiasis. The schistosoma japonicum katsurada recombinant protein rSjC1qBP can induce generation of an anti-recombinant protein specific IgG antibody in bodies of mice at a relative high level during a mouse immune experiment, which means that the recombinant protein has the potential of development of a novel medicine for treatment of the schistosomiasis and has excellent application value. The recombinant protein rSjC1qBP can inhibit complement hemolysis and is more than 30% in hemolysis inhibition rate. The recombinant protein rSjC1qBP also has combination capability with a complement system C1q, so that the recombinant protein rSjC1qBP has a wide application prospect in therapy of complement activity abnormity diseases.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a recombinant protein of Schistosoma japonicum and its preparation method and application. Background technique [0002] Schistosomiasis japonicum is a widely distributed and serious zoonotic parasitic disease caused by Schistosoma japonicum. Oncomelania, the only intermediate host of Schistosoma japonicum, has a wide distribution area and complex breeding environment, making it difficult to eradicate; moreover, repeated infection of humans and animals is serious. Therefore, the prevention and control of schistosomiasis in my country is still a long-term, arduous and complicated task. [0003] Schistosoma cercariae burrow into the skin of the final host and transform into juvenile worms, migrate through the lungs and liver, develop into adults, reproduce and lay eggs in the host, and parasitize for a long time. The blood environment in which the schistosome resides provi...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C07K14/435C07K16/18A61K39/00A61P33/10
CPCY02A50/30
Inventor 傅志强贾秉光马帅宰金丽柴淑梅吕超韩倩张祖航陆珂洪炀李浩林矫矫
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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