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Schistosoma japonicum SjPPase recombinant antigen protein and preparation method and application thereof

A technology for recombining antigenic proteins and recombinant proteins, which is applied in the direction of recombinant DNA technology, the use of vectors to introduce foreign genetic material, animal/human peptides, etc., can solve the problems of unsustainable and normal development, cell autophagy, etc., and achieve high specificity, Wide application prospects, high purity effect

Inactive Publication Date: 2015-11-04
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, maintaining the activity of PPase is crucial for the elimination of PPi in the cells of Schistosoma japonicum during the developmental stage. The accumulation of PPi will not only lead to the failure of normal development, but also cause autophagy
In summary, SjPPase can be used as a potential drug target for Schistosoma japonicum, but the current research direction of this enzyme is focused on the discovery of the gene and the study of the transcription and expression levels of the gene in each stage
There are no related reports on the expression of Schistosoma japonicum inorganic pyrophosphatase by genetic engineering

Method used

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  • Schistosoma japonicum SjPPase recombinant antigen protein and preparation method and application thereof
  • Schistosoma japonicum SjPPase recombinant antigen protein and preparation method and application thereof
  • Schistosoma japonicum SjPPase recombinant antigen protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation of Schistosoma japonicum SjPPase Recombinant Antigen Protein

[0033] 1 material

[0034] 1.1 Adult cDNA of Schistosoma japonicum

[0035] The cDNA library of Schistosoma japonicum from the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention.

[0036] 1.2 Host strains Escherichia coli DH5α, BL21(DE3), and plasmid pET-28a(+) were from Tiangen Biochemical Technology Co., Ltd. and Novagen respectively.

[0037] 1.3 Main reagents and tool enzymes

[0038]

[0039] 1.4 Preparation of the main solution

[0040] 1.4.1 Ni-NTA affinity chromatography column bufferA and bufferB

[0041]

bufferA

bufferB

Tris-HCl(mM)PH7.4

20

20

NaCl(mM)

300

300

Imidazole (mM)

10

500

Glycerin (v / v)

10％

10％

[0042] 1.4.2 DEAE anion exchange chromatography column bufferA and bufferB

[0043]

bufferA

bufferB

Tris-HCl(mM)P...

Embodiment 2

[0084] Example 2 Enzymatic Activity Analysis of Schistosoma japonicum SjPPase Recombinant Protein

[0085] 1 material

[0086] 1.1 Schistosoma japonicum SjPPase recombinant protein

[0087] The SjPPase recombinant protein from Schistosoma japonicum purified in Example 1.

[0088] 1.2 Main reagents

[0089] Reagent name

Reagent source

BCA Protein Quantification Kit

Tiangen

K 4 PPi

Sinopharm

1-amino-2-naphthol-4-sulfonic acid

melodious

SDS

SIGMA

Ammonium molybdate

Sinopharm

other reagents

All domestic analytical reagents

[0090] 2 methods

[0091] The enzyme activity analysis of Schistosoma japonicum SjPPase recombinant protein is calculated by measuring the concentration of SjPPase recombinant protein catalyzing the hydrolysis of PPi to Pi within a certain catalytic time. Different temperature and pH conditions were measured to understand the effects of different conditions on the activ...

Embodiment 3

[0105] Example 3 Screening of Schistosoma japonicum SjPPase recombinant protein inhibitors

[0106] 1 material

[0107] 1.1 Schistosoma japonicum SjPPase recombinant protein

[0108] The SjPPase recombinant protein from Schistosoma japonicum purified in Example 1.

[0109] 1.2 Main reagents

[0110] Reagent name

Reagent source

BCA Protein Quantification Kit

Tiangen

K 4 PPi

Sinopharm

1-amino-2-naphthol-4-sulfonic acid

melodious

SDS

SIGMA

Ammonium molybdate

Sinopharm

[0111] IDP

SIGMA

MDP

SIGMA

NaF

Sinopharm

other reagents

All domestic analytical reagents

[0112] 2 methods

[0113] 1) The fixed reaction temperature is 37°C, pH is 8.0, the substrate PPi concentration is 1mM, the reaction time is 3min, and the inhibitors include substrate analogues IDP and MDP, Mg 2+ For the ion complexing agent NaF, the concentrations of the three inhibitors wer...

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PUM

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Abstract

The invention discloses a schistosoma japonicum inorganic pyrophosphatase (SjPPase) recombinant antigen protein which is the protein capable of catalyzing hydrolysis of inorganic pyrophosphate (PPi) to generate phosphate. (The amino acid sequence of the protein is shown as SEQ ID NO:1.) The invention further discloses a preparation method of the recombinant antigen protein. The preparation method comprises the following steps of: amplification of the schistosoma japonicum SjPPase coding gene sequence, construction and identification of recombinant plasmids, and inducible expression and purification of recombinant protein. In addition, the invention further discloses the application of the schistosoma japonicum SjPPase recombinant antigen protein in choosing SjPPase inhibitors.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a recombinant antigen protein of Schistosoma japonicum SjPPase; in addition, the invention also relates to a preparation method and application of the recombinant antigen protein. Background technique [0002] Schistosomiasis japonicum is a zoonotic parasitic disease caused by humans or mammals infected with Schistosoma japonicum, mainly distributed in southern China, the Philippines and Indonesia. Although there have been more than 50 years of schistosomiasis prevention and control history in China, schistosomiasis is still one of the most important public health problems in China. It is mainly prevalent in 110 counties and urban areas in 7 provinces of my country. There are still 790,000 patients and the population is at risk. 65 million. [0003] The developmental stages from juveniles to adults of Schistosoma japonicum are completed in mammalian hosts. During this proc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/11C12N15/70C12P21/02G01N33/68
Inventor 陈军虎吴群峰陈绅波王越张颋徐斌胡薇
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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