Rockfishes chitinase A gene, expression vector containing the same, recombinant strain and use thereof

A technology of chitinase and expression vector, which is applied in the field of genetic engineering to achieve the effects of biotransformation, environmental protection, and gene pool enrichment

Inactive Publication Date: 2009-05-06
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The seed cultivation of grouper is the key to the sustainable development of aquaculture production, but at present there is no bait a...

Method used

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  • Rockfishes chitinase A gene, expression vector containing the same, recombinant strain and use thereof
  • Rockfishes chitinase A gene, expression vector containing the same, recombinant strain and use thereof
  • Rockfishes chitinase A gene, expression vector containing the same, recombinant strain and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Construction of grouper stomach cDNA library and acquisition of chitinase A gene ESTs

[0057] According to the instruction manual of CloneMiner cDNA library construction kit (Invitrogen), the grouper gastric cDNA library was constructed.

[0058] Transform Escherichia coli DH10B (commercially available) with the above-mentioned grouper stomach cDNA library, and transform the solution in LB (Kan + ) plate, cultured overnight at 37°C, picked a single colony to LB (Kan + ) culture medium at 37°C overnight, and use the universal primer M13 to carry out bacterial liquid PCR identification (the electrophoresis identification of the bacterial liquid PCR amplification product is as follows: figure 1 After that), the bacterial solution containing the insert plasmid was picked and sequenced. After the obtained ESTs sequences were searched by Genebank Blastx for homology, 42 ESTs sequences related to the chitinase gene were obtained.

Embodiment 2

[0059] Example 2 Synthesis of the 5' end fragment of grouper chitinase A gene

[0060] In this embodiment, the ESTs sequence related to the chitinase gene obtained in Example 1 is subjected to 5' RACE to obtain the 5' end fragment of the target gene, specifically using the terminal deoxyribonucleic acid transferase (TDT enzyme) method 5' RACE, this test is a routine practice in the art, and its operation can be performed with reference to the 5'Racer Kit Protocol.

[0061] According to the chitinase gene-related ESTs sequence obtained in Example 1, two downstream primers P1 and P2 were designed for nested PCR.

[0062] Primer P1: 5'-GAGCAGGCTTGTCCAGTTC, the nucleotide sequence of which is shown in SEQ ID NO: 4;

[0063] Primer P2: 5'-GGGGCACCATTGCTCTTC, the nucleotide sequence of which is shown in SEQ ID NO:5.

[0064] The first round of PCR:

[0065] The nucleotide sequence from the 689th position to the 5' end of the ESTs sequence related to the chitinase gene obtained in...

Embodiment 3

[0073] Example 3 The full-length sequence of grouper chitinase A gene

[0074] Through biological software analysis, the sequenced 42 chitinase-related ESTs sequences obtained in Example 1 and the 5' end sequence fragments obtained in Example 2 were spliced ​​to finally obtain the complete grouper chitinase gene. The long sequence, whose nucleotide sequence is shown as SEQ ID NO: 1, has a length of 1658bp, and is named grouper chitinase A gene.

[0075] According to the amino acid sequence analysis of the grouper chitinase A gene, it is deduced that the 53-1483 nucleotides of the nucleotide sequence encode 456 amino acids, and its amino acid sequence is shown in SEQ ID NO: 3, 1484 -The 1486th nucleotide is the terminator sequence TAA. Amino acid sequence analysis showed that the peptide chain of 456 amino acids was the mature peptide of grouper chitinase A, which was a fragment of grouper chitinase A precursor, which was processed This fragment is produced after the signal p...

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Abstract

The invention discloses a chitinase A gene of a grouper, an expression vector containing the gene, a recombinant and application of the gene to preparing fusion protein and additives of fish feed. A nucleotide sequence of the chitinase A gene of the grouper is expressed as SEQ ID NO:1, nucleotides in site 53 to 1,483 in the sequence code 456 amino acids, the nucleotide sequence is a sequence of mature peptide of the chitinase A gene of the grouper, and the amino acid sequence is expressed as SEQ ID NO:3. The chitinase A gene of the grouper not only enriches gene libraries of the grouper, but also provides novel research strategies for cultivation research for seeds of the grouper. The chitinase A gene of the grouper, the expression vector containing the gene, and the recombinant obtained after transformation can be used for preparing the fusion protein and the additives of the fish feed, can effectively promote growth of young fish, can improve immune function of the young fish at the same time, and have higher economic value.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a grouper chitinase A gene, an expression vector containing the gene, a recombinant strain and the preparation of a fusion protein and a fish feed additive by the grouper chitinase A gene in the application. Background technique [0002] Chitinase (chitinase, CHI) is a hydrolyzing polysaccharide enzyme, which cuts off the β(1-4) N-acetyl-D-glucose unit of chitin, and its amino acid sequence is characterized by containing several necessary Conserved regions include: glyco-18 region (containing the active site), chitin binding region and a hinge region. [0003] Chitinase exists widely in the biological world and is the second most abundant polysaccharide in nature. A variety of chitinases have been found in bacteria, fungi, and plants, and many have been used in transgenic research. Chitinases are also found in insects, crustaceans, amphibians and verteb...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/42C12N15/70C12N15/81C12N1/21C12N1/19C07K19/00A23K1/165C12R1/19C12R1/84A23K20/189A23K50/80
Inventor 李文笙冯少珍林浩然
Owner SUN YAT SEN UNIV
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