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 problem that there is no research report on heat shock protein 60 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

Problems solved by technology

[0004] So far, there is no research report about the application of Schist

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
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Construction of prokaryotic expression vector of Schistosoma japonicum hsp60 gene coding sequence

[0030] Find the sequence of Schistosoma japonicum hsp60 gene in Genebank to design primers, analyze its sequence characteristics with DNAstar, 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:

[0031] sense: 5'-CCG GGATCC ATGTTACGAGCTCTAAGTTCA-3' (SEQ ID NO: 3),

[0032] anti-sense: 5'-GCG CTCGAG CATCATGCCTCCCATTCCA-3' (SEQ ID NO: 4),

[0033] The restriction endonucleases BamH I and Xho I restriction sites (underlined parts) were respectively introduced. Amplification of the open reading frame of Hsp60 was carried out 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 multipl...

Embodiment 2

[0036] Example 2 Induced Expression of Schistosoma japonicum hsp60 Gene Prokaryotic Expression Vector

[0037]The plasmid Sjhsp60-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 and 6h of bacterial liquid, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE electrophoresis, 5% stacking gel 80V voltage, 12% separating gel 120V voltage) analysis and verification.

[0038] The result is as figure 2 shown in figure 2 Middle, M: protein marker; 1: Uninduced bacterial liquid; 2: 1 h after induction; 3: 2 h after induction; 4: 4 h after induction; 5: 6 h after induction. fro...

Embodiment 3

[0039] Embodiment 3 Purification of prokaryotic expression vector expression product

[0040] 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.

[0041] 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 heat shock protein 60 gene. The invention also discloses a preparation method of the schistosoma japonicum heat shock protein 60 and the application of the recombinant expression carrier. The recombinant expression carrier containing the schistosoma japonicum gene can highly express the schistosoma japonicum heat shock protein 60 gene, and the highly expressed recombinant schistosoma japonicum Hsp60 protein 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 one of the most widely distributed and most harmful neglected tropical diseases. Schistosomiasis is prevalent in my country, and its pathogen is Schistosoma japonicum. The life cycle of Schistosoma japonicum (Sj) is complex, with oncomelania as its intermediate host and human, buffalo, cattle and other mammals as its final host. In recent years, more and more attention has been paid to the research on the molecular mechanism of the life activities of schistosomiasis and its important functional molecules. Among them, heat shock proteins (Heatshock proteins, Hsp) are a kind of important functional molecules, which are the key to many life activities. participants. Heat shock proteins are a class of high-level basal expressed proteins,...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N1/21C12P21/02A61K48/00A61K39/00A61P33/12G01N33/53C12R1/19
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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