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Recombinant expression vector containing Schistosoma japonicum gene and its application

An expression vector, the technology of schistosomiasis thioredoxin, 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: 2011-12-14
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

[0006] So far, there is no research report about the application of Schistosoma japonicum thioredoxin peroxidase II of the present invention as a vaccine

Method used

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  • Recombinant expression vector containing Schistosoma japonicum gene and its application
  • Recombinant expression vector containing Schistosoma japonicum gene and its application
  • Recombinant expression vector containing Schistosoma japonicum gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Construction of Schistosoma japonicum tpII gene coding sequence prokaryotic expression vector

[0032] Find the sequence of Schistosoma japonicum tpII gene in Genebank to design primers, analyze its sequence characteristics with DNAstar, and find its largest open reading frame (SEQ ID NO: 2), that is, the sequence part of its coding 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 endonucleases BamH I and Xho I restriction sites (underlined parts) were respectively introduced. The TpII open reading frame was amplified by PCR using the cDNA library of female Schistosoma japonicum as a template. The prokaryotic expression vector pET28a(+) used is a high-efficiency expression vector expressing 6×His tags. The vector is designed with multiple cloning restriction site...

Embodiment 2

[0038] Example 2 Induced Expression of Schistosoma japonicum tpII Gene Prokaryotic Expression Vector

[0039]The plasmid SjtpII-pET28a(+) of the positive clone with correct sequencing was transformed into Escherichia coli BL21(DE3) competent cells, spread on the LB plate containing kanamycin, picked a single colony, and inoculated in In the liquid LB medium, after reaching the logarithmic growth phase, add isopropyl-β-D-thiogalactopyranoside (IPTG, the final concentration is 1mM / L) to induce expression, collect before induction and 1h after induction , 2h, 4h, 6h and 8h of the bacterial solution were analyzed and verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE electrophoresis, 5% stacking gel 80V voltage, 12% separating gel 120V voltage).

[0040] The result is as figure 2 shown in figure 2 Middle, M: protein marker; 1: Uninduced bacterial fluid; 2: 1 h after induction; 3: 2 h after induction; 4: 4 h after induction; 5: 6 h after induction; ...

Embodiment 3

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

[0042] Induce a small amount of recombinant bacteria (100ml), centrifuge at 4°C, 4000rpm for 15min, collect the bacterial pellet, wash with PBS, add 1× binding buffer (binding buffer) to resuspend the bacterial cells, freeze and thaw 3 times, and then ultrasonically disrupt cell. The sonicated suspension was centrifuged at 4° C., 12,000 rpm for 30 min, and the supernatant and precipitate were collected to identify its expression form. Depend on image 3 It can be seen that the expression product of the recombinant plasmid appears in the precipitate after ultrasonication, indicating that its expression form is inclusion body. exist image 3 Middle, 1: Supernatant after sonication; 2: Precipitation after sonication; 3: Protein after purification.

[0043] The fusion protein can be purified by utilizing the affinity of histidine and nickel ions in the recombinantly expressed fusion protein,...

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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

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a recombinant expression vector containing Schistosoma japonicum gene and its application. Background technique [0002] Schistosomiasis is a zoonotic parasitic disease widely prevalent in tropical and subtropical regions. The pathogen caused by it is Schistosoma japonicum. my country is mainly affected by Schistosoma japonicum. The life cycle of Schistosoma japonicum is complex, including the alternation of sexual generations in the final host and asexual generations in the intermediate host. Its final hosts mainly include various mammals such as humans, cattle, sheep, pigs, and rabbits, and the worms are adsorbed on the inner walls of their portal veins and mesenteric veins. The blood of the host is in an environment of aerobic respiration, and to achieve long-term parasitism, the worm must have a set of mechanisms to overcome the damage caused by oxygen free radicals, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N1/21C12N9/08A61K48/00A61K39/00A61P33/12G01N33/573C12R1/19
CPCY02A50/30
Inventor 朱传刚汪勇沛张磊林矫矫陆珂
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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