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ª‡-N-acetyl galactosamine enzyme and encode gene and applications thereof

A technology that encodes genes and genes, which is applied in the fields of application, genetic engineering, and plant genetic improvement, etc., and can solve problems such as incomplete enzymatic hydrolysis and inability to meet the requirements of blood type transformation

Inactive Publication Date: 2008-05-21
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

Some of these glycoside hydrolases come from higher animals, yeast, Aspergillus niger, etc. These enzymes are all lysosomal enzymes, the optimum pH is between 3.5 and 4.5, and the specific activity of the enzyme generally does not exceed 50U / mg, and the reaction requires a high concentration of enzymes Protein (>3mg / mL) and low pH value, incomplete enzymatic hydrolysis, cannot meet the requirements of blood type conversion

Method used

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  • ª‡-N-acetyl galactosamine enzyme and encode gene and applications thereof
  • ª‡-N-acetyl galactosamine enzyme and encode gene and applications thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Embodiment 1, cloning of α-N-acetylgalactosaminidase α NAGA coding gene

[0049] Inoculate the Chryseobacterium meningosepticum (Chryseobacterium meningosepticum) ZYP01 CGMCCNo.2198 strain into the culture medium (5g / L yeast extract, 5g / L peptone, 5g / L NaCl, 3g / L K2HPO4, the rest is water. pH7.2) , 200rpm, and cultivate overnight at 30°C. Genomic DNA was extracted according to the instructions of the Promega Genomic DNA Extraction Kit. Using the extracted genomic DNA as a template, PCR was performed using an upstream primer (sequence: 5'-ATGGGCGCCTTAATTCCC-3') and a downstream primer (sequence: 5'-TTAGTAGTCGTCATTTATTGC-3'). The PCR conditions were as follows: pre-denaturation at 95°C for 5 min; then denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 90 s, a total of 30 cycles; finally, extension at 72°C for 10 min. The PCR product was detected by agarose gel electrophoresis, and a band with a molecular weight of about 1335 bp was obta...

Embodiment 2

[0051] Embodiment 2, the expression of αNAGA

[0052] 1. Construction of the expression vector pET-22b-αNAGA containing the gene encoding αNAGA

[0053] Using pTE-αNAGA as a template, with an upstream primer (sequence: 5'-ATCATATGCCAAAAAAGTAAGAATTGC-3' (with Nde I restriction site)), and a downstream primer (sequence: 5'-TGCTCGAGGTAGTCGTCATTTATTGC-3' (with Xho I Restriction site) PCR amplified nucleotide sequence is the αNAGA gene fragment from the 52-1332th deoxyribonucleotide at the 5' end of sequence 1 in the sequence listing. PCR conditions are: first 95 ℃ pre-denatured for 5min; Denaturation at 95°C for 30s, annealing at 43°C for 30s, extension at 72°C for 90s, a total of 30 cycles; final extension at 72°C for 10min. The PCR product was detected by agarose gel electrophoresis, and a band with a molecular weight of about 1300bp was obtained (in Figure 1, Lane 2).

[0054] According to the operation guidelines of TAKARA company, the PCR product was connected to the pMD-18...

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Abstract

The invention discloses an α-N-acetylgalactosaminidase and its encoding gene and application. The α-N-acetylgalactosaminidase is a protein of the following (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing; A protein derived from (a) having α-N-acetylgalactosaminidase activity through the substitution and / or deletion and / or addition of one or several amino acid residues in the amino acid sequence. The α-N-acetylgalactosaminidase can change the blood type of human red blood cells from A to O, and from AB to B. αNAGA can be used to prepare kits for blood group conversion; αNAGA can also be used to prepare universal red blood cells.

Description

technical field [0001] The invention relates to an α-N-acetylgalactosaminidase, its coding gene and application. 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 was 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 transfusions caused by subjective and objective factors such as blood type identification errors and recognition errors has remained high....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/14C12N15/56G01N33/80A61K35/14G01N33/68
Inventor 章扬培郁成雨徐华
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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