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Alpha-galactosidase and expression and purification method thereof

A technology of galactosidase and expression vector, which is applied in the field of bacterial α-galactosidase and its expression and purification, to achieve the effects of low cost, broad market prospects, and simple expression and purification methods

Active Publication Date: 2010-09-29
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This tool enzyme has the function of enzymatically decomposing B-type red blood cells, but the specific activity of the enzyme is only 0.3U / mg

Method used

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  • Alpha-galactosidase and expression and purification method thereof
  • Alpha-galactosidase and expression and purification method thereof
  • Alpha-galactosidase and expression and purification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, cloning of α-galactosidase α-gal coding gene

[0047] Genomic DNA of Bacteriodes fragilis ATCC25285 (purchased from American TypeCulture Collection (ATCC)) was extracted using the Genomic DNA Extraction Kit of Promega Company according to the instructions. Using the extracted genomic DNA as a template, use an upstream primer (sequence: 5′-GC GGATCCATGAAGACAATCCTACTCTT-3′) and a downstream primer (sequence:

[0048] 5'GGAAGCTTTCGCTCTGAAATCTTCACGT-3') for PCR amplification. The PCR conditions were as follows: pre-denaturation at 95°C for 5 min; then denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 120 s, a total of 30 cycles; finally, extension at 72°C for 10 min. After the reaction, the PCR product was detected by 1% agarose gel electrophoresis, and the results were as follows: figure 1 As shown (M: DL2000 DNA Marker, l: full-length gene of α-gal), a band with a molecular weight of about 1788bp was obtained, which was c...

Embodiment 2

[0051] Example 2, Expression and purification of α-galactosidase α-gal

[0052] 1. Construction of the expression vector pET-22b(+)-α-gal containing the coding sequence of the α-galactosidase α-gal gene

[0053] Using pGEM-α-gal as a template, use upstream primer (sequence: 5'-GGGGATCCTGATGACGACGACAAGGAGCGTGTTTATGACATT-3' (with BamH I restriction site), and downstream primer (sequence: 5'-GGAAGCTTTCGCTCTGAAATCTTCACGT-3' (with Hind III restriction site)) PCR amplification of α-galactosidase α-gal gene coding sequence. PCR conditions are: first 95 ° C pre-denaturation 5min; then 95 ° C denaturation 30s, 56 ° C annealing 30s, 72 ° C extension 120s, A total of 30 cycles; the last extension was 10min at 72°C. After the reaction, the PCR product was detected by 1% agarose gel electrophoresis, and the detection results were as follows: figure 2 As shown (M: DL2000DNA Marker, l: α-gal gene coding sequence), a band with a fraction of about 1700bp was obtained, which was consistent wi...

Embodiment 3

[0065] Example 3. Human erythrocytes are converted from type B to type O with α-galactosidase α-gal

[0066] Human B-type erythrocytes (provided by the Blood Transfusion Department of 307 Hospital) were washed twice with raw saline, and human B-type erythrocytes were washed twice with enzymatic hydrolysis buffer (isotonic citrate disodium hydrogen phosphate buffer, pH6.8), Take 0.2mL of packed B-type red blood cells, add 50ug of α-galactosidase expressed and purified in Example 2, make up 0.5mL with enzymolysis buffer, incubate at 26°C for 2 hours, and shake continuously to make it evenly enzymolyzed . Blood types were detected with anti-B blood typing reagent (Changchun Bode Biotechnology Co., Ltd.) and primary-side cross-match test.

[0067] 1. Detection of red blood cell blood type with blood typing reagent

[0068] Take 40ul of erythrocytes before or after enzymolysis, wash them twice with normal saline, make a 5% cell suspension, and add 20ul of anti-B and anti-A antibo...

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Abstract

The invention discloses an alpha-galactosidase and an expression and purification method thereof. The alpha-galactosidase is expressed by SEQ ID NO:1 in a sequence table, is obtained by encoding a recombinant genome DNA sequence SEQ ID NO:3 of bacteroides fragilis (Bacteroides fragilis) ATCC25285 and is a protein with effect of converting a red cell blood group of the human body from B-type into O-type and from AB-type into A-type. The alpha-galactosidase of the invention has the advantages of wide application, simple and convenient expression and purification method, low cost, important clinical and scientific research significance and wide market prospect.

Description

technical field [0001] The invention relates to an enzyme in the field of biotechnology and a purification method thereof, in particular to a bacterial alpha-galactosidase (α-galactosidase) and an expression and purification method thereof. Background technique [0002] In 1900, Landsteiner first discovered the ABO blood group system and proposed the same type of blood transfusion, which laid the foundation for modern transfusion medicine. Landsteiner won the 1930 Nobel Prize for this. In 1960, Witkins proved that the antigenic determinant of ABH is sugar, which deepened people's understanding of blood types. Over the past two decades, through the use of advanced analysis methods in other disciplines, blood transfusion medicine has made great progress, and has played a huge role in ensuring the safety of people's lives. However, blood transfusion safety and blood type bias are still two major problems before us. For a long time, the incidence of erroneous blood transfusion...

Claims

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Application Information

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IPC IPC(8): C12N9/40C12N15/56C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21
Inventor 宫锋王玲燕高红伟鲍国强金泗虎李素波季守平檀英霞虞立霞王颖丽
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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