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Fusion gene of Japan schistosome antigen gene and constituted DNA vaccin and preparing process

A DNA vaccine, schistosomiasis technology, applied in the field of schistosoma japonicum polyvalent DNA vaccine

Inactive Publication Date: 2011-08-10
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no endoplasmic reticulum in prokaryotic cells, and these chemical modifications cannot be carried out in prokaryotic expression. Therefore, the product of prokaryotic expression is not a true natural Schistosoma japonicum antigen, and naturally it is not the most effective form of antigen

Method used

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  • Fusion gene of Japan schistosome antigen gene and constituted DNA vaccin and preparing process
  • Fusion gene of Japan schistosome antigen gene and constituted DNA vaccin and preparing process
  • Fusion gene of Japan schistosome antigen gene and constituted DNA vaccin and preparing process

Examples

Experimental program
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Effect test

example 1

[0072] [Example 1] Cloning of Schistosoma japonicum antigen genes sjFABP, sj23, sjGAPDH, sj26, sjTPI, sj97

[0073] 1. Extraction of total RNA from adult worms

[0074] 1) Collect adults from the portal vein of rabbits infected for 42 days with normal saline aortic perfusion;

[0075] 2) Take 100mg of fresh adult worms, add 1ml Trizol, quickly grind on ice with a glass homogenizer to make a homogenate, and place it at room temperature for 5min;

[0076] 3) Add 0.2ml chloroform, shake for 15S, and place at room temperature for 3min;

[0077] 4) Centrifuge at 12,000 rpm for 15 minutes at 4°C, and transfer the supernatant to another EP tube;

[0078] 5) Add 0.5ml of isopropanol, vortex and mix well, and let stand at room temperature for 10 minutes;

[0079] 6) Centrifuge at 12000rpm for 10min at 4°C to precipitate RNA and discard the supernatant;

[0080] 7) Add 1ml of 75% ethanol, centrifuge at 7500rpm for 5min at 4°C, and discard the supernatant;

[0081] 8) Dry the RNA in...

example 2

[0104] (Example 2) Preparation of fusion antigen gene

[0105] 1. Construction of fusion PCR hinge region primers and upstream and downstream primers required for fusion genes of 30 Schistosoma japonicum antigen genes, as shown in Table 2.

[0106] 2. Fusion PCR to prepare fusion genes of 30 Schistosoma japonicum antigen genes (taking sj23-FABP as an example, and so on for other 29 kinds)

[0107] 1) The first step of PCR reaction

[0108]

[0109]

[0110]

[0111] 2) Overlap extension PCR reaction

[0112] After electrophoresis of the target gene sj23 and sjFABP fragments obtained from the above PCR products, the tape recovery kit (Omega Company) was used to recover the mixed target bands, and the overlap extension was performed under the following reaction conditions:

[0113]

[0114] 3) The third step PCR reaction

[0115] The above-mentioned overlap extension PCR reaction product was subjected to the third-step PCR reaction, and the fusion antigen gene sj2...

example 3

[0125] (Example 3) Schistosoma japonicum DINA multivalent vaccine pVIVO 2 / sjFABP-23, pVIVO 2 Construction of / sjFABP-23 / sjGAPDH-26

[0126] Fusion antigen gene fragment sjFABP-23, its 5' end contains BamHI restriction site ; Its 3' end contains an EcoRI restriction site . The above T vector containing the sjFABP-23 fusion antigen gene was double digested with BamHI and EcoRI, and the sjFABP-23 gene was recovered. Digest pVIVO with BamHI and EcoRI 2 , recovery of pVIVO 2 fragment. Insertion of sjFABP-23 gene into pVIVO 2 Between BamHI and EcoRI, build strategy see attached Figure 4 . The specific operation is as follows:

[0127] 1. Build pVIVO 2 Recombinant plasmid of sjFABP-23:

[0128] The sjFABP-23 fragment DNA was double digested with restriction endonucleases EcoRI and BamHI. The reaction system is: 10×(Y + Tango TM )buffer 5 μl, plasmid 5 μg, BamHI IOU; EcoRI 1OU, rehydrated to 50 μl. After mixing, react in a 37°C water bath for 8-9 hours or overnight...

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Abstract

The present invention discloses a fusion gene of schistosoma japonicum antigen genes, formed DNA vaccine and its preparation method. Said schistosoma japonicum DNA polyvalent vaccine is composed of schistosoma japonicum antigen genes (including sj23, sj FABP, sj26, sj GAPDSH, sj TPI and sj 97) and fusion gene of schistosoma japonicum antigen genes and eukaryotic expression vector with several insertion sites. The fusion gene of 30 schistosoma japonicum antigen genes is obtained by using 6 schistosoma japonicum antigen genes through 'fusion PCR' preparation process. Said fusion gene or antigengene can be inserted into eukaryotic expression vector so as to obtain the invented schistosoma japonicum DNA polyvalent vaccine.

Description

technical field [0001] The invention belongs to the field of biotechnology and pharmacy, and specifically relates to a fusion gene of Schistosoma japonicum antigen gene, a multivalent DNA vaccine of Schistosoma japonicum constituted, and a preparation method of the vaccine. Background technique [0002] Schistosomiasis is a zoonotic parasitic disease that seriously endangers the health of humans and livestock. According to WHO estimates, at least 600 million people in the world are threatened by schistosomiasis infection, and about 200 million people are infected. There are still 1.5 million patients in my country. Infection is also quite serious and is a major public health problem in developing countries, including China. Although the use of praziquantel chemotherapy has been safe and efficient for a long time, due to its high drug price, easy reinfection after treatment, and possible drug resistance of schistosomiasis strains, it has brought great difficulties to the cont...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/30A61K39/002C12N15/79C12Q1/68
CPCY02A50/30
Inventor 余龙江朱路鲁明波刘智李春艳
Owner HUAZHONG UNIV OF SCI & TECH
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