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Recombined T4 bacteriophage of expressing cholecystokinin gene

A bacteriophage, residue technology, applied in active immunization technology and its application field

Inactive Publication Date: 2005-10-26
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But there is no report of expressing CCK or its active part (such as CCK-33 peptide, CCK-8 peptide) with phage

Method used

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  • Recombined T4 bacteriophage of expressing cholecystokinin gene
  • Recombined T4 bacteriophage of expressing cholecystokinin gene
  • Recombined T4 bacteriophage of expressing cholecystokinin gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1. Construction of the recombinant plasmid containing cck-33 gene (1cck)

[0058] According to the base sequence of the chicken cck gene and the high-frequency codons of Escherichia coli, the cck-33 gene was designed and synthesized.

[0059] Specific primers P1 (5'-CATGGATCCGGTTCTACTGGCCGCT-3') / P2 (5'-CATGAGCTCCCAAAATCCATCCAGCC-3') were designed according to the artificially synthesized cck-33 gene, and the PCR method was used to amplify the artificially synthesized cck-33 gene as a template. Increase cck-33 gene fragment. The PCR reaction system (200 μL) for amplifying the cck-33 gene fragment is composed as follows: 20 μL 10×PCR buffer, 16 μL dNTPs, 2 μL Taq enzyme, 1.5 μL primer P1, 1.5 μL primer P2, 0.5 μL artificially synthesized cck-33 gene template , and finally add water to a total volume of 200 μL. The PCR temperature cycling program is as follows: cycle 1: 94°C for 3 min; cycle 2-31: 94°C for 40 sec, 56°C for 40 sec, and 72°C for 60 sec; cycle 32...

Embodiment 2

[0061] Embodiment 2. Contain the construction of the recombinant plasmid of cck-33 gene 2 concatemers (2cck)

[0062] The recombinant plasmid pRSETA-1CCK was digested with two groups of enzymes at 37°C: the first group was digested with BamHI and HindIII to recover smaller fragments; the second group was digested with BglII, HindIII and CIAP to recover larger fragments. Then, under the action of T4 ligase, the recovered large fragments and small fragments are ligated. Escherichia coli DH5α was transformed with the ligation product. via Amp r Through resistance screening, recombinants containing 2cck were obtained. Then, the recombinants containing 2cck gene were screened by PCR with specific primers P1 / P2. The PCR method refers to "Example 1". For the positive bacteria screened by PCR, pick a single colony and inoculate it in 3mL LA medium, culture at 37°C with shaking at 200r / m for 16-20h, extract the plasmid, and then digest it with restriction endonuclease and determine...

Embodiment 3

[0063] Embodiment 3. Contain the construction of the recombinant plasmid of cck-33 gene 4 concatemers (4cck)

[0064] The recombinant plasmid pRSETA-2CCK was digested with two groups of enzymes at 37°C: the first group was digested with BamHI and HindIII to recover smaller fragments; the second group was digested with BglII, HinIII and CIAP to recover larger fragments. Then, under the action of T4 ligase, the recovered large fragments and small fragments are ligated. Escherichia coli DH5α was transformed with the ligation product. via Amp r Through resistance screening, recombinants containing 4cck were obtained. Then use specific primer P3 (5'-GATAAGGATCGATGGGGATCC-3') / P4 (5'-ATGGTACCAGCTGCAGATCT-3') to carry out PCR screening to the recombinant containing 4cck. The PCR method refers to "Example 1". PCR products were detected by electrophoresis, and specific bands were observed ( figure 2 ). For the positive bacteria screened by PCR, pick a single colony and inoculate ...

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Abstract

The present invention relates to a recombined T4 bacteriophage for expressing cholecystokinin gene, the recombined T4 bacteriophage has chicken cholecystokinin 33 peptide gene or nucleotide acid in series. This invention offers chicken cholecystokinin 33 peptide or amino acid residue sequence in series, utilizes recombined T4 bacteriophage to carry active immunity technology and application for animal.

Description

technical field [0001] The present invention relates to the characteristics and application of recombinant T4 phage expressing cholecystokinin gene. The present invention further relates to the active immunization technology and application of recombinant T4 phage to animals such as pigs, rabbits, cattle, chickens, ducks and geese. Background technique [0002] Cholecystokinin (CCK) is an effective factor regulating animal feed intake. Cholecystokinin, also known as cholecystokinin or trypsin, is a polypeptide hormone secreted by I cells of the small intestinal mucosa. The precursor of CCK is 130 amino acids, which are gradually decomposed into small molecules of CCK. The CCKs discovered so far include CCK-83, CCK-58, CCK-39, CCK-33, CCK-12, CCK-8 and CCK-4. Its distinct sites are mainly at the carboxy-terminus, but extend in length at the amino-terminus. The postprandial plasma mainly contains macromolecular CCK, including CCK-58, CCK-39, CCK-33 and CCK-12. These macrom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A23K10/18C12N7/01
Inventor 曹永长覃健萍毕英佐舒鼎铭马静云谢青梅
Owner SOUTH CHINA AGRI UNIV
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