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Schistosoma japonicum recombinant antigen, and preparation method and application thereof

A technology for recombinant antigens and schistosomiasis, applied in the field of bioengineering, can solve the problems of few reports and no research on protein characteristics, and achieve good application value

Inactive Publication Date: 2014-10-15
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

[0005] The glycolytic pathway widely exists in organisms such as Escherichia coli, yeast, bacteria, and human beings, but there are few reports on Schistosoma japonicum phosphoglycerate kinase at home and abroad, only the gene sequence was cloned, and the characteristics of the protein were not reported. more in-depth research
There are no reports at home and abroad on its enzymatic activity, protein localization, and evaluation of its protective effect as a vaccine candidate antigen.

Method used

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  • Schistosoma japonicum recombinant antigen, and preparation method and application thereof
  • Schistosoma japonicum recombinant antigen, and preparation method and application thereof
  • Schistosoma japonicum recombinant antigen, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Expression and Purification of Schistosoma japonicum Recombinant Antigen

[0042] 1. Method

[0043] 1.1 Construction of recombinant expression plasmids

[0044] According to the gene sequence of SiPGK with NCBI accession number FN322120.1, primers were designed, upstream primer: 5'-TAGGATCCATGGGTTTGTCGAAGCTCAG-3' (SEQ ID NO.3, containing BamH I restriction site); downstream primer: 5'-TCTCTCGAGTTAGTGAGCATCCGTAAG -3' (SEQ ID NO.4, containing Xho I restriction site). Using the cDNA of Schistosoma japonicum 42-day-old body as a template, PCR amplifies its cDNA fragment containing ORF, and the reaction composition is as follows:

[0045]

[0046]

[0047] PCR reaction conditions: 94°C for 5min, denaturation at 94°C for 30sec, annealing at 58°C for 90sec, extension at 72°C for 90sec, a total of 30 cycles; finally 72°C for 10min. After the PCR product was purified, it was connected to the pMD19-T vector, transformed into DH5α cells, and a single clone was ...

Embodiment 2

[0065] Example 2 Detection of Enzyme Activity of Schistosoma japonicum Recombinant Antigen rSjPGK

[0066] The reaction system for rSjPGK to catalyze 1,3-DPG to generate 3-PGA is: 0.1M triethanolamine (pH7.6), 5mM MgSO 4, 0.5mM EDTA, 1.0mM ATP, 0.2mM NADH, 6mM 3-PGA, 4U / mL GAPDH. Adjust the pH value of the assay buffer to 7.6, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0 (0.05M Tris-cl buffer), 9.5, 10.0, 10.5 (0.05M glycine-sodium hydroxide buffer). Then add other reagents in the reaction system, incubate at 25°C for 5 minutes, add the recombinant protein rSjPGK, measure the absorbance change at 340nm for 1 minute in each system, and repeat the experiment three times. Take the highest activity as 100%, calculate the ratio of the activity of other pH values ​​relative to the highest activity, take pH as the abscissa, and relative activity as the ordinate to make a reaction curve.

[0067] Adjust the temperature of the reaction system to eight different gradients of 10°C, 15°C, 20°C, 30°C, 3...

Embodiment 3

[0071] Example 3 Antigenic Detection of Schistosoma japonicum Recombinant Antigen rSjPGK

[0072] 1. Western Blotting analysis of recombinant protein antigenicity

[0073] Perform SDS-PAGE electrophoresis on the recombinant protein rSjPGK and Schistosoma japonicum 14d larval protein after Ni column purification, then transfer to NC membrane at 4°C with 130mA for 1h, and use anti-recombinant protein antiserum as the primary antibody Antigenicity analysis of recombinant proteins.

[0074] 2. Western Blot analysis of recombinant protein antigenicity results

[0075] The results of Western Blot analysis showed that the recombinant protein rSjPGK immune serum could recognize the recombinant protein and the natural worm protein in the 14d worm protein (as a natural protein of PGK, it is a positive control), indicating that the recombinant protein rSjPGK has good immunogenicity ( Figure 9 ). exist Figure 9 Middle, A: M: Marker; 1: recombinant protein rSjPGK as antigen; B: M: M...

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Abstract

The invention discloses a schistosoma japonicum recombinant antigen, and the recombinant antigen comprises an amino acid sequence, shown as SEQ ID NO. 1, of schistosoma japonicum phosphoglyceric kinase. The invention also discloses a preparation method of the schistosoma japonicum recombinant antigen and application of the schistosoma japonicum recombinant antigen to prepare vaccines or medicines for preventing or controlling schistosomiasis. In mouse immunization experiments, the schistosoma japonicum recombinant antigen is capable of inducing a mouse to generate specific IgG, IgG1 and IgG2a antibodies capable of resisting the recombinant antigen in vivo at a relatively high level, and in animal protection experiments, through induction of the schistosoma japonicum recombinant antigen, the insect reducing rate is 34.5% and the ovum reducing rate is 32.2%. The experiment results prove that the recombinant antigen is applicable as a vaccine candidate for resisting schistosomiasis and has extremely 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. However, there have been reports of praziquantel-resistant strains on Schistosoma mansoni in rece...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/70C12N1/21C07K16/40A61K38/45A61K39/00A61P33/12
CPCA61K39/00C07K16/40C12N9/1217C12N15/70C12Y207/02003Y02A50/30
Inventor 林矫矫黄莉妮洪炀傅志强张旻韩艳辉王馨茁李长健曹晓丹伍妙梨郭小勇韩倩刘艳涛马帅
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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