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Recombinant expression carrier containing schistosoma japonicum gene and application thereof

An expression vector and technology of schistosomiasis, applied in the field of bioengineering, can solve the problems that there are no research reports on thioredoxin peroxidase II of Schistosoma japonicum

Inactive Publication Date: 2010-03-17
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows for high levels of production of specific substances called schistostimulins (chemically related compounds) which are involved with protecting against diseases such as malaria or bacterial meningoencephalitis caused by Plasmodium falciparum paradoxus. These molecules have been shown to be effective at reducing symptoms associated with these conditions through their ability to stimulate antibodies produced during an infectious process on blood cells like neutrophils.

Problems solved by technology

This patented describes how certain substances called thromboxane B12 or sputterle bacteriovirus cause harmful organisms like Schispora sinensis fever. These viruses produce various types of chemical compounds known as redoxyradicans, which attack tissues throughout their lifecycle through complex processes involving interactions between molecules involved in metabolysis. Therapeutic agents target these damaging factors may help reduce symptoms associated with pathogenesis.

Method used

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  • Recombinant expression carrier containing schistosoma japonicum gene and application thereof
  • Recombinant expression carrier containing schistosoma japonicum gene and application thereof
  • Recombinant expression carrier containing schistosoma japonicum gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1 Construction of Prokaryotic Expression Vector of Schistosoma japonicum tpII Gene Coding Sequence

[0032] Find the sequence of Schistosoma japonicum tpII gene in Genebank, design primers, analyze its sequence characteristics with DNAstar, and find its largest open reading frame (SEQ ID NO: 2), which is the sequence part of its encoded protein, and design primers based on this part of the sequence as follows:

[0033] Sense: 5’-GGC GGATCC ATGAAGTGTTTAAATTCG-3' (SEQ ID NO: 3),

[0034] Anti-sense: 5’-GGC CTCGAG GTTTACAGAGGAAAAGTACG-3' (SEQ ID NO: 4),

[0035] The restriction enzymes BamH I and Xho I were introduced respectively (underlined). The TpII open reading frame was amplified using the Schistosoma japonicum female cDNA library as a template for PCR. The used prokaryotic expression vector pET28a(+) is a highly efficient expression vector expressing 6×His tag, and the vector is designed with a multi-cloning restriction site, which can insert the foreign gene f...

Embodiment 2

[0038] Example 2 Induced expression of Schistosoma japonicum tpII gene prokaryotic expression vector

[0039] Transform the plasmid SjtpII-pET28a(+) of the positive clone with correct sequencing into E. coli BL21(DE3) competent cells, spread it on the LB plate containing kanamycin, pick a single colony, and inoculate it after identification In the liquid LB medium, after reaching the logarithmic growth phase, add isopropyl-β-D-thiogalactoside (IPTG, final concentration of 1mM / L) to induce expression, and collect separately before induction and 1h after induction , 2h, 4h, 6h and 8h of the bacterial liquid, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE electrophoresis, 5% concentrated gel 80V voltage, 12% separation gel 120V voltage) analysis and verification.

[0040] The result is figure 2 Shown in figure 2 Middle, M: protein marker; 1: uninduced bacterial liquid; 2: 1h after induction; 3: 2h after induction; 4: 4h after induction; 5: 6h after inducti...

Embodiment 3

[0041] Example 3 Purification of prokaryotic expression vector expression product

[0042] Induce a small amount of recombinant bacteria (100ml), centrifuge at 4℃, 4000rpm for 15min, collect the bacterial pellet, wash with PBS, add 1× binding buffer to resuspend the bacterial, freeze-thaw repeatedly for 3 times, then ultrasonically break cell. The ultrasonic suspension was centrifuged at 4°C, 12000 rpm for 30 min, and the supernatant and precipitate were collected to identify the expression form. by image 3 It can be seen that the expression product of the recombinant plasmid appears in the precipitate after ultrasound, indicating that its expression form is an inclusion body. in image 3 Medium, 1: supernatant after sonication; 2: precipitation after sonication; 3: purified protein.

[0043] The fusion protein can be purified by the affinity of histidine and nickel ions in the recombinantly expressed fusion protein, and the purification steps are carried out in accordance with ...

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Abstract

The invention discloses a recombinant expression carrier containing a schistosoma japonicum gene which is a schistosoma japonicum thioredoxin peroxidase II gene. The invention also discloses a preparation method of the schistosoma japonicum thioredoxin peroxidase II and the application of the recombinant expression carrier. The recombinant expression carrier containing the schistosoma japonicum gene can highly express the schistosoma japonicum thioredoxin peroxidase II, and the highly expressed recombinant schistosoma japonicum thioredoxin peroxidase II can induce mouse to generate the protection function of resisting the infection of schistosoma japonicum at a certain level.

Description

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Claims

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Application Information

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Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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