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Echinococcus granulosusglutathione transferase gene and application thereof

A technology of Echinococcus granulosus and glutathione, applied in the field of bioengineering, can solve problems such as poor effect and inability to perform early diagnosis

Inactive Publication Date: 2011-08-31
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] The object of the present invention is to provide a kind of Echinococcus granulosus glutathione transferase gene and its application, and described this Echinococcus granulosus glutathione transferase gene and its application will solve the problems in the prior art. The effect of diagnosing cystic echinococcosis is not good, and the technical problems of early diagnosis cannot be carried out

Method used

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  • Echinococcus granulosusglutathione transferase gene and application thereof
  • Echinococcus granulosusglutathione transferase gene and application thereof
  • Echinococcus granulosusglutathione transferase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Construction of Echinococcus granulosus cDNA library

[0043] 1. Isolation of total RNA and purification of mRNA

[0044] Take about 1 mL of protoscole of Echinococcus granulosus frozen at -80°C, extract total RNA by Trizol method, and use quickprep from GE Healthcare TM The mRNA Purification Kit purifies mRNA.

[0045] 2. cDNA synthesis

[0046] (1) Synthesize the first strand of cDNA

[0047] In a 1.5mLependorf without RNA, add the following reaction system:

[0048] 10×first strand buffer 5μL

[0049] Methylated dNTP 3 μL

[0050] Linker-primer (1.4μg / μL) 2μL

[0051] DETP-treated water 14μL

[0052] RNase Block Ribonuclease

[0053] Inhibitor (40U / μL) 1μL

[0054] mRNA sample (5μg) 22μL

[0055] -------------------------------------------------- -----

[0056] 47μL

[0057] Mix the reaction, let it stand at room temperature for 10min, allow the primer to anneal to the template, then add 3μL Accu-Script-RT to make the total volume reach 50μL, ...

Embodiment 2c

[0119] The immune screening of embodiment 2cDNA library

[0120] 1. Processing of screened serum

[0121] Take 100 μL of mixed serum (10 parts, 10 μL each) from patients with cystic echinococcosis confirmed by surgery, add 1 mL of E.coli XL1-blue MRF' lysate diluted 1:10 with TBST, absorb at room temperature overnight, 5000 g Centrifuge for 10 min, transfer the supernatant to a new centrifuge tube, dilute with antibody diluent to make the final serum concentration 1:200, and store at 4°C.

[0122] 2. Immunoscreening of cDNA library

[0123] 2.1 Primary screening

[0124] 2.1.1 Bacterial culture

[0125] Pick up E.coli XL1-blue MRF' with a sterile pipette tip and inoculate it on LB culture plate (containing 15 μg / mL Tet) by streaking, and culture overnight at 37°C upside down. The next day, a single colony of E.coli XL1-blue MRF' was picked and cultured in 3 mL LB medium (containing 10 mM MgSO4, 0.2% maltose, 15 μg / mL Tet) with shaking at 200 rpm overnight at 37 °C. On the...

Embodiment 3

[0143] Example 3 Positive phage deletion and circularization into pbluescript recombinant plasmid

[0144] Pick a single colony of E.coli XL1-blue MRF' in LB medium and place it in 3mL LB medium (containing 10mM MgSO4, 0.2% maltose, 15μg / mL Tet), culture overnight at 37°C with shaking at 200rpm. On the next day, 100 μL of the culture solution was transferred to a new 3 mL LB medium, and cultured at 37° C. with shaking at 200 rpm for about 2 hours. The culture solution was centrifuged at 5000g for 5min to pellet the bacteria, and resuspended with 10mM MgSO4 to OD600=1.0. Add 200 μL E. coli XL1-blue MRF’ suspension, 50 μL SM buffer containing positive phage and 1 μL helper phage to the bacterial culture tube. Place the bacterial culture tube in a water bath at 37°C for 15 minutes, then add 3mL of LB medium, and culture with shaking at 37°C for 5h. Take out the bacterial culture tube, heat it at 65°C for 20min, then centrifuge at 3000g for 15min, transfer the supernatant to a n...

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Abstract

The invention belongs to the field of bioengineering and provides an Echinococcus granulosusglutathione transferase gene a base sequence of which is shown in SEQ ID NO: 1. The invention also provides a protein encoded by the Echinococcus granulosusglutathione transferase gene. The amino acid sequence of the protein encoded by the Echinococcus granulosusglutathione transferase gene is shown in SEQ ID NO: 2, or is the same as 1st-219th amino acid sequences as shown in SEQ ID NO: 3. The recombinant protein can be used for immunodiagnosis of cystic echinococcosis patients and can be used for coating a plate; and the serums of the patients having cystic echinococcosis, alveolitoid echinococcosis, cysticercosis and other parasite diseases and healthy people are detected by using an enzyme-linked immunosorbent assay (ELISA), and the obtained sensitivity and specificity are respectively 72.41% and 92.36%.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to an Echinococcus granulosus glutathione transferase gene and its application. Background technique [0002] Hydatid Disease, also known as Echinococcosis, is a disease caused by the mid-stage larvae of Echinococcus tapeworm parasitizing humans and animals. It is a zoonosis that seriously endangers the health of humans and animals. parasitic disease. There are many types of Echinococcus, and there are four species of Echinococcus that can infect humans: Echinococcus granulosus (Eg), Echinococcus multilocularis (Em), Echinococcus oligarthrus) and Echinococcus vogeli. These 4 species of Echinococcus can cause different types of echinococcosis. There are two types of echinococcosis in my country, namely echinococcosis granulosa, also known as cystic echinococcosis (CE) and multilocular echinococcosis (echinococcosis multilocularis), also known as alveolar type Echinococcosis (alveolar...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/10C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C07K19/00C07K16/40A61K39/00A61P33/00G01N33/53
Inventor 汪俊云朱慧慧高春花杨玥涛石锋
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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