Entamoeba histolytica galactose/acetylgalactosamine (Gal/GalNAc) polypeptide fragment, and preparing method and application of polypeptide fragment

An amoebic galactose, acetamidosemi technology, applied in DNA/RNA fragments, chemical instruments and methods, peptides, etc., can solve the problem of unclear role of intermediate subunits, large molecular weight of full-length Igl, prone to cross-reaction, etc. It can improve the specificity and sensitivity, and overcome the missed detection and misdiagnosis.

Active Publication Date: 2011-08-31
SHANGHAI XINGYAO MED TECH DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While the anti-Igl mouse monoclonal antibody can inhibit the adhesion of trophoblasts to mammalian cells, cytotoxicity to mammalian cells, and phagocytosis of erythrocytes, but the role of the intermediate subunit in the whole lectin is still unclear
Although the recombinant protein of full-length Igl has been successfully prepared and can be recognized by the serum of amoeba patients, i

Method used

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  • Entamoeba histolytica galactose/acetylgalactosamine (Gal/GalNAc) polypeptide fragment, and preparing method and application of polypeptide fragment
  • Entamoeba histolytica galactose/acetylgalactosamine (Gal/GalNAc) polypeptide fragment, and preparing method and application of polypeptide fragment
  • Entamoeba histolytica galactose/acetylgalactosamine (Gal/GalNAc) polypeptide fragment, and preparing method and application of polypeptide fragment

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0043] Example 1 Culture of Entamoeba histolytica

[0044] Entamoeba histolytica HM-1: IMSS strain was aseptically cultured in BI-S-33 medium containing 15% adult bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin. Amoeba trophozoites were cultured in a 6ml glass culture tube with a cover, and the tube cap was tightly closed, and it was 5 o placed in a 36.5°C cell culture incubator.

Embodiment 2

[0045] Example 2 PCR reaction amplifies igl1 C-terminal gene

[0046] According to the kit instructions, use the RNeasy Total RNA kit to extract the total RNA of the aseptically cultured Entamoeba histolytica HM-1:IMSS strain. RT-PCR was performed using GeneAmp RNA PCR Kit to obtain cDNA. Using cDNA as a template, the C-terminal gene was amplified with primers EH150-S749-XHO (5’-CCCTCGAGACTGAACAAAGGCTAAAAGA-3’) and EH150-AS1088-XHO (5’-CCCTCGAGTTAAATGCCTTTAGCTCCATT-3). PCR execution program: 94°C 2min; 94°C 15sec, 55°C 30sec, 72°C 1min, a total of 30 cycles; 72°C 3min. After the reaction, each PCR product was subjected to 0.8% agarose gel electrophoresis. igl1 The C-terminal gene size is 1100bp (eg figure 1 shown).

Embodiment 3

[0047] Example 3 Construction of pET19b-Eh150-C plasmid

[0048] After purifying the PCR amplified product with QIAquick PCR Purification Kit, the PCR amplified product was digested with Xho I. After 0.8% agarose gel electrophoresis, DNA was purified with QIAEXII Agaose Gel Extraction Kit. The vector pET19b and the C-terminal gene fragment of the Igl gene were ligated with TAKARA DNA Ligation Kit overnight at 16°C. The ligation product was transformed into JM109 competent cells, cultured overnight at 37°C, and the plasmid was extracted. The results of 0.8% agarose gel electrophoresis showed that the recombinant gene fragments were successfully connected to the plasmid, and the size of the recombinant plasmid was 6.8kb, (such as figure 2 shown), sequencing identification confirmed that the insertion site of the target gene and the size of the insertion fragment were correct. Using Vector NTI 10 software, the amino acid sequence was deduced, and compared with the amino acid...

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Abstract

The present invention relates to the biotechnology field, and relates to entamoeba histolytica Gal/GalNAc polypeptide fragment and a preparing method and an application of the polypeptide fragment. The preparing method comprises the steps of: extracting total RNA from cultured entamoeba histolytica trophozoite, obtaining C terminal polypeptide fragment coding gene of Gal/GalNAc suppressible agglutinin of recombinant 150kDa through amplification, constructing expression vector, transferring to Escherichia coli for induction, realizing high expression of C terminal polypeptide fragment of the Gal/GalNAc suppressible agglutinin of the recombinant 150kDa in form of inclusion body, washing to obtain high-purity protein, and using glutathione reduction system for carrying out renaturation to obtain the C terminal polypeptide fragment of the Gal/GalNAc suppressible agglutinin of the recombinant 150kDa, wherein the polypeptide fragment possesses biological activity and solubility. The polypeptide fragment can be used for serology immunodiagnosis of amoebiasis, and overcomes detection miss and misdiagnosis of entamoeba histolytica cyst and trophozoite in stool examination in amoebiasis diagnosis, thereby obviously improving the specificity and sensitivity of the amoebiasis diagnosis.

Description

technical field [0001] The present invention relates to the field of biotechnology, and relates to Entamoeba histolytica galactose / acetylgalactosamine polypeptide fragments, in particular to a C-terminal polypeptide fragment of 150kDa Gal / GalNAc inhibitory lectin of Entamoeba histolytica And its preparation method and application. Background technique [0002] Entamoeba histolytica (E.h) is a pathogen that causes amebic colitis and extraintestinal abscess, and has the ability to invade host tissues or organs, adapt to host immune response and express pathogenic factors. About 50 million people in the world are infected with Entamoeba histolytica, and 100,000 patients die from amoebiasis every year, which is the second deadly protozoan disease after malaria. The average infection rate in my country is 0.949%, and the number of infected people is estimated to be 10.69 million (1992 data). [0003] Infectious cysts of E. histolytica are ingested orally, pass through the stoma...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/09C12N15/62C12N15/63A61K38/16A61P33/02G01N33/569
CPCY02A50/30
Inventor 程训佳橘裕司付永锋
Owner SHANGHAI XINGYAO MED TECH DEV CO LTD
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