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

A technology of DNA vaccine and antigen gene, which is applied in the field of polyvalent DNA vaccine of Schistosoma japonicum, can solve the problem of not being the most effective antigen form, and achieve the effects of scalable preparation, high separation efficiency, and convenience for storage and transportation

Inactive Publication Date: 2007-01-31
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
Comparison scheme
Effect test

example 1

[0064][Example 1] Cloning of Schistosoma japonicum antigen genes sjFABP, sj23, sjGAPDH, sj26, sjTPI, sj97

[0065] 1. Extraction of total RNA from adult worms

[0066] 1) Collect adults from the portal vein of rabbits infected for 42 days with normal saline aortic perfusion;

[0067] 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;

[0068] 3) Add 0.2ml chloroform, shake for 15S, and place at room temperature for 3min;

[0069] 4) Centrifuge at 12,000 rpm for 15 minutes at 4°C, and transfer the supernatant to another EP tube;

[0070] 5) Add 0.5ml of isopropanol, vortex and mix well, and let stand at room temperature for 10 minutes;

[0071] 6) Centrifuge at 12000rpm for 10min at 4°C to precipitate RNA and discard the supernatant;

[0072] 7) Add 1ml of 75% ethanol, centrifuge at 7500rpm for 5min at 4°C, and discard the supernatant;

[0073] 8) Dry the RNA in ...

example 2

[0091] (Example 2) Preparation of fusion antigen gene

[0092] 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.

[0093] 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)

[0094] Japanese blood

fluke resistance

Protogene

Reactant

volume

(μl)

PCR

Reaction conditions

sj23

10×PCR Buffer

MgCl 2 (25mM)

10mM dNTPs

pGEM-sj23

P1 (20μM)

P13 (20μM)

Taq DNA

Polymerase (2U / μl)

dd H 2 o

5.0

4.0

1.0

0.1

1.0

1.0

0.5

37.4

95℃5min→(94℃1min→45℃1min→72℃

1min) 35 cycles → 72°C 10min → 4°C storage

Japanese blood

...

example 3

[0107] [Example 3] Schistosoma japonicum DNA multivalent vaccine pVIVO 2 / sjFABP-23, pVIVO 2 Construction of / sjFABP-23 / sjGAPDH-26

[0108] 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 sj FABP-23 fusion antigen gene was double digested with Bam HI and EcoR I, and the sjFABP-23 gene was recovered. Digest pVIVO with Bam HI and EcoR I 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:

[0109] 1. Build pVIVO 2 Recombinant plasmid of sjFABP-23:

[0110] The sjFABP-23 fragment DNA was double digested with restriction endonucleases EcoRI and BamHI. The reaction system is: 10×(Y + Tango TM )buffer 5μl, plasmid 5μg, BamHI 10U, EcoRI 10U, and water up to 50μl. 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 antigen gene 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 contr...

Claims

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Application Information

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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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