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Batroxobin and its preparing process and specific coding gene

A special encoding, batroxobin technology, applied in the field of batroxobin and its preparation, can solve the problems of slow progress in molecular biology research

Inactive Publication Date: 2009-12-02
JOINN LAB (SUZHOU) INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although researchers have a better understanding of the properties of batroxobin, the molecular biology research on the components of this snake venom has been progressing slowly

Method used

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  • Batroxobin and its preparing process and specific coding gene
  • Batroxobin and its preparing process and specific coding gene
  • Batroxobin and its preparing process and specific coding gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1, the synthesis of recombinant batroxobin gene BG0 and the detection of the effect of expressing batroxobin

[0050] 1. Acquisition of recombinant batroxobin gene BG0 and construction of expression vector

[0051] 1) Acquisition of recombinant batroxobin gene BG0

[0052] According to the amino acid sequence data of batroxobin (GENBANK Accession Number is X12747) published in Genebank, the genetic codes that both yeast and CHO cells can be preferred were selected, and the full-length gene sequence of recombinant batroxobin was artificially synthesized (with Nucleotide sequence of sequence 2). In order to allow the target gene to be inserted into the pPIC9 vector, a cutting enzyme XhoI was added to the 5'-end of the Batroxobin Gene (BG) (GENBANK Accession Number is X12747 from the 1st to 693rd nucleotides at the 5' end). Add TAA stop codon and EcoRI cutting point at the 3′-end, and add the codon AAA corresponding to the KEX2 protease recognition sequence Ly...

Embodiment 2

[0092] Example 2. Synthesis of batroxobin gene (IBG0) containing IL-2 signal peptide gene and detection of the effect of batroxobin expression.

[0093] 1. Synthesis, sequencing and construction of expression vector of batroxobin gene (IBG0) containing IL-2 signal peptide gene

[0094] Batroxobin gene BG0 was used as template (sequence 2 in the sequence listing) and IL-2 signal peptide gene (from GENBANK number NM_000586 the 5' end 295-354 nucleotides) by splicing overlap extension PCR (SOE-PCR) sequence) to connect, the specific method is:

[0095] Using the IL-2 signal peptide gene (from GENBANK No. 295-354 nucleotide sequence at the 5' end of NM_000586) as a template, use IL25 primer: 5'-CTGGATCCACGATGTATAGGATGCAACTGCTG-3' and IL23 primer: 5'-AGCGCTGTTGGTGACCAG- 3' is the primer for PCR, PCR reaction system: IL-2 signal peptide gene (7.5uM) 0.1μL, IL25 primer (15uM) and IL23 primer (15uM) each 0.5μL, dNTP (each 2.5mM) 2μL, KOD DNA Polymerase (5U / μL)0.1μL, 10*KOD buffer 2μ...

Embodiment 3

[0106] Embodiment 3, research on the glycosylation of batroxobin protein expressed by BG0 gene

[0107] 1. Acquisition of batroxobin glycosylation site mutation genes (BGm1, BGm2) and construction of expression vectors ( figure 2 )

[0108] The site-directed mutagenesis technique was used to mutate the BG0 gene, and Asn (from the 146th position of the amino terminal of sequence 1) was mutated into Gln (the motif of glycosylation is Asn-X-Thr), that is, the 5' end of sequence 2 in the sequence list The 448th-450th nucleotide (AAC) is mutated to CAA; the specific method is:

[0109] Using pPIC9-BG0 as a template, use a-Factor primer: 5′-TACTATTGCCAGCATTGCTGC-3′ and M13 primer: 5′- TTG GAACAGGTTAATGTTAGCACAG-3′( mutation codon ) as primers for PCR amplification, PCR reaction system: 0.1 μL (0.04ug) of pPIC9-BG0 plasmid, 0.5 μL of a-Factor primers (2.5 mM each) and 0.5 μL of M13 primers (15 uM each), 2 μL of dNTPs (2.5 mM each), KOD DNA Polymerase (5U / μL) 0.1μL, 10*KODbuffe...

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Abstract

The invention discloses batroxobin, its preparation method and special coding gene. The batroxobin coding gene is one of the following nucleotide sequences: 1) sequence 2 in the sequence listing; 2) sequence 4 in the sequence listing; 3) sequence 6 in the sequence listing; 4) sequence 8 in the sequence listing . The method for preparing batroxobin comprises introducing the coding gene described in any one of claims 1-3 into yeast or mammalian cell line for expression through a eukaryotic expression vector to obtain batroxobin protein. The method of the present invention has successfully expressed the batroxobin protein with high biological activity, and the yeast yield after purification reaches 10 μg / ml or 14 batroxobin units (14BU / ml) per milliliter of fermentation broth, and the specific activity is 1400BU / mg . This yield exceeds other published and reported expression levels and is suitable for large-scale production.

Description

technical field [0001] The invention relates to batroxobin, a preparation method thereof and a special coding gene. Background technique [0002] In 1963, Klobusitzi and Konig isolated a serine proteolytic enzyme, batroxobin, from the venom of the American lancehead Agkistrodon (Bothrops atrox). So far, the whole and partial amino acid sequences of more than 30 types of thrombin have been obtained, and their primary structures are very similar. Bleeding - coagulation process in animals. Among these snake venom-like coagulation proteases, the physical and chemical properties and clinical applications of batroxobin and Ancrod are well studied (Stocker K and Bariow G.H. Methods Enzymol. 1976, 45: 214-223). [0003] The specific action substrate of batroxobin is fibrinogen. Unlike thrombin, it only cuts the A chain of fibrinogen, and does not act on the B chain. When it hydrolyzes Arg in the A chain of plasma fibrinogen 16 -GLy 17 When the fibrinopeptide A is released, the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/57C12N9/64C12N1/19C12N15/85
Inventor 李招发于学玲黄金路方宏清陈惠鹏
Owner JOINN LAB (SUZHOU) INC
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