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Primer and method for preparing Beagle antimicrobial peptide beta-defensin recombinant protein CBD127 by using primer

A technology of recombinant protein and beagle dog, applied in biochemical equipment and methods, mammalian protein, cationic antibacterial peptide, etc., can solve the problems of cumbersome steps, high cost, affecting biological activity, etc. The effect of low and low operation difficulty

Inactive Publication Date: 2014-01-29
QINGDAO AGRI UNIV
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Problems solved by technology

[0007] The amount of naturally obtained canine β-defensins is extremely limited, and the extraction steps from the body are cumbersome and costly. β-defensins are mainly obtained through chemical synthesis and genetic engineering methods
The chemical synthesis method can easily change the primary structure of the polypeptide during the synthesis process, but the cost of the chemical synthesis method is expensive, and it is also difficult to ensure the correct pairing of the three pairs of disulfide bonds in the molecule, which affects its biological activity and seriously affects the defense. element research and application

Method used

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  • Primer and method for preparing Beagle antimicrobial peptide beta-defensin recombinant protein CBD127 by using primer
  • Primer and method for preparing Beagle antimicrobial peptide beta-defensin recombinant protein CBD127 by using primer
  • Primer and method for preparing Beagle antimicrobial peptide beta-defensin recombinant protein CBD127 by using primer

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Embodiment 1

[0046] (1) Cloning and sequencing of beagle β-defensin gene CBD127

[0047] Mammalian antimicrobial peptides play an important role in the innate immunity of animals. According to the nucleotide sequence of canine β-defensin gene published by Genbank (accession number: DQ011996.1), the present invention uses Primer5.0 software to design primers: SEQ ID NO: 15'-ATGAAGCTCATCCTGATCAT-3', SEQ ID NO: 25'-TTATGTAGAATTTGTCTTG -3', the CBD127 gene was amplified from the testicular tissue of a Beagle dog, and the fragment size was 303bp (SEQ ID NO: 3 Figure 5 ). (Testis tissue was taken out in liquid nitrogen, ground in a grinder, and then stored in 50mL bottles and stored at -80°C. The total RNA of each tissue sample was extracted according to the instructions of TRIzol reagent. The cDNA reverse transcription reagent was used to reverse Record enzyme M-MLV, reverse transcribe into cDNA according to the instructions, and then use the cDNA as a template to carry out PCR amplification...

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Abstract

The invention relates to the technical field of biology and specifically relates to a primer and a method for preparing Beagle antimicrobial peptide beta-defensin recombinant protein CBD127 by using the primer. The sequence of the primer is as follows: the upstream primer is 5'-ATGAAGCTCATCCTGATCAT-3', and the downstream primer is 5'-TTATGTAGAATTTGTCTTG-3'; and the preparation method comprises the following steps: (1) cloning and sequencing Beagle beta-defensin gene CBD127; (2) constructing and identifying an expression vector; (3) performing fusion expression of the recombinant protein; (4) identifying the recombinant protein; (5) performing solubility analysis on an expression product; (6) purifying a fusion protein; and (7) performing biological activity detection on the Beagle recombinant protein. The method provided by the invention has the advantages of small difficulty in operation, strong controllability and low cost, and can lay a foundation for immunological research and industrial mass production of defensins.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer and a method for preparing the beagle antibacterial peptide β-defensin recombinant protein CBD127 using the primer. Background technique [0002] Defensins are a class of cationic active peptides rich in cysteine, which are widely distributed in animals, plants and insects. Because of its broad-spectrum antimicrobial activity, it is named "defensins". Antibacterial tests in vitro show that defensins can have a strong killing effect on pathogenic microorganisms such as Gram-positive and negative bacteria, fungi, mycobacteria, spirochetes, and togaviruses, and constitute the first line of defense for the host against foreign pathogenic infection . [0003] Beagle is an internationally recognized general-purpose experimental dog. It is widely used in medicine and health because of its small size, docile temperament, good homogeneity, easy to hold, stable genetic performance, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C07K19/00C12N15/62
CPCC07K14/4723
Inventor 尹燕博冯培祥潘福星
Owner QINGDAO AGRI UNIV
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