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Mutant of schistosoma japonicum glutathione-S-transferase and application thereof

A technology of glutathione and transferase, applied in the field of enzyme genetic engineering, can solve the problems of not being able to block repeated infection of schistosomiasis, endangering human health, and generally low level of immune protection

Active Publication Date: 2015-05-20
NANJING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] Schistosomiasis is a zoonotic parasitic disease that seriously endangers human health. Schistosomiasis japonicum is mainly prevalent in my country, and it is widely prevalent along the Yangtze River in my country, as well as in Sichuan and Yunnan provinces. Acute infection cases occur from time to time. The disease has therapeutic effect, but can not block the recurrent infection of schistosomiasis, in addition, the only drug praziquantel for the treatment of schistosomiasis has been reported drug resistance, therefore, the screening of effective schistosomiasis vaccine and new drug report appears very important
[0003] The level of immune protection induced by current schistosomiasis vaccines is generally not high, and can only induce partial resistance of the host to schistosomiasis infection

Method used

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  • Mutant of schistosoma japonicum glutathione-S-transferase and application thereof
  • Mutant of schistosoma japonicum glutathione-S-transferase and application thereof
  • Mutant of schistosoma japonicum glutathione-S-transferase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Construction and protein purification of sj26GST and its variant Q67E prokaryotic expression vector

[0028] Using the plasmid pALEX as a template, the coding sequence of wild-type GST was amplified by PCR. The upstream primer used is: 5'-catgcc atgggc atg tcc cct atactaggt tattgg-3' introduces the NcoI restriction site (underline), and the downstream primer is: 5'-ctgtagcggccgctctaga ctcgag ttt tgg agg atg gtc gcc ac-3', introduce the restriction site of XhoI (underlined). After purification, the PCR product was digested with NcoI / XhoI and inserted into the corresponding site of pET21d. Perform DNA sequencing on the recombinant expression vector to analyze the correctness of its reading frame and coding sequence. The sequence of the mutant protein Q67E is consistent with the N / C terminal sequence constructed by the wild-type GST, and only specific site-specific mutations are carried out. This constructed GST has an additional LEHHHHHH added at the C-ter...

Embodiment 2

[0046]Example 2 Expression and purification of sj26GST and mutant enzyme Q67E of Schistosoma japonicum

[0047] The GST plasmid pET-21d-GST and its variant expression plasmid pET-21d-Q67E verified by sequencing were respectively transformed into Escherichia coli BL21 (DE3) host bacteria to induce expression. Pick positive transformants and shake culture overnight at 37°C and 200rpm in LB medium, then inoculate in LB medium (containing 100μg / ml ampicillin) and culture at 28°C and 250rpm until OD 600 is about 0.8, add IPTG with a final concentration of 0.2mM, and induce expression at 28°C for 6hr.

[0048] The fermentation broth was centrifuged at 6000rpm and 4°C for 10min, and the cells were resuspended in 20mL of buffer solution (50mM Na2HPO4-NaH2PO4, pH8.0, 150mM NaCl, 1mM PMSF), and the soluble protein was recovered by centrifugation after sonication.

[0049] The protein sample was loaded on a nickel metal chelate affinity chromatography column (1.6cm×5cm) equilibrated wit...

Embodiment 3

[0050] Example 3 Detection of Enzyme Activity of Schistosoma japonicum sj26GST and its variants

[0051] Add the enzyme solution to the 0.1M potassium phosphate solution at pH 6.5 containing 1mM 2,4-dinitrochlorobenzene (CDNB) and 1mM reduced glutathione (GSH), measure the change of its optical analysis value at 340nm, and repeat the experiment Three times (Habig, W.H. and Jakoby, W.B. Assays for differentiation of glutathione S-transferases. Meth. Enzymol. 1981, 77:398-405). Taking the highest activity of the wild-type sj26GST as 100%, the ratio of the activity of the mutant enzyme to the activity of the wild-type enzyme was calculated. ( figure 2 )

[0052] Determination of the apparent Michaelis constant of GST to GSH and CDNB: the reaction system was fixed at 1.0ml, the final concentration of CDNB in ​​the system was 1.0mmol / L, and the final concentration range of GSH was 40, 50, 66, 100, 200μmol / L; The final concentration of GSH in the system was 1.0mmol / L, and the fi...

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Abstract

The invention belongs to the technical field of genetic engineering of enzymes and in particular relates to a mutant of schistosoma japonicum glutathione-S-transferase and an application thereof. The invention provides a mutant enzyme schistosoma japonicum glutathione-S-transferase (sj26GST) for changing the schistosoma japonicum immunizing protection. Compared with a wild type, the single mutant enzyme obtained by mutating the glutathione-S-transferase from 26kDa of schistosoma japonicum (Schistosoma japonicum) has the advantages that the protectiveness of the mutant enzyme of sj26GST is improved, the IgG1 level of the mutant enzyme is obviously higher than that of a recombinant wild type sj26GST protein, the worm reduction rate is improved from the original 34 percent to 40 percent, and the egg reduction rate is improved from 37 percent to 40 percent. The recombinant antigen has good immunogenicity and is suitable to serve as an anti-schistosomiasis candidate vaccine.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering of enzymes, in particular to a variant of Schistosoma glutathione-S-transferase and its application Background technique [0002] Schistosomiasis is a zoonotic parasitic disease that seriously endangers human health. Schistosomiasis japonicum is mainly prevalent in my country, and it is widely prevalent along the Yangtze River in my country, as well as in Sichuan and Yunnan provinces. Acute infection cases occur from time to time. The disease has therapeutic effect, but can not block the recurrent infection of schistosomiasis, in addition, the only drug praziquantel for the treatment of schistosomiasis has been reported drug resistance, therefore, the screening of effective schistosomiasis vaccine and new drug report appears very important. [0003] The level of immune protection induced by current schistosomiasis vaccines is generally not high, and can only induce partial resistance of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70A61K39/00A61P33/12
CPCC12N9/1088C12Y205/01018A61K39/00Y02A50/30
Inventor 华子春李家璜
Owner NANJING UNIV
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