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Nile tilapia transforming growth factor TGF-beta 1 gene, related protein and application

A transforming growth factor, Nile tilapia technology, applied in the direction of transforming growth factor, anti-growth factor immunoglobulin, growth factor/inducing factor, etc., can solve the problem of tilapia disease, antibiotic deficiency, etc. Problems such as pertinence and poor effect of pathogenic bacteria variants

Inactive Publication Date: 2013-08-07
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Traditional treatment methods mainly use antibiotics, but antibiotics lack specificity, long-term use will cause pathogens to develop drug resistance, and will affect the water environment and involve food safety issues; there are also ways to add probiotics, Chinese herbal medicine additives, and vitamins to the feed. Wait to enhance the immunity of fish; some scholars have also developed a vaccine against disease, but found that the vaccine is not effective against the variants of pathogenic bacteria
None of these approaches have provided a breakthrough solution to tilapia disease

Method used

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  • Nile tilapia transforming growth factor TGF-beta 1 gene, related protein and application
  • Nile tilapia transforming growth factor TGF-beta 1 gene, related protein and application
  • Nile tilapia transforming growth factor TGF-beta 1 gene, related protein and application

Examples

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Effect test

Embodiment 1

[0040] Example 1 Synthesis of the Middle Fragment of Nile Tilapia Transforming Growth Factor β1 Gene

[0041] According to the homology comparison results of the cDNA sequence of the published fish transforming growth factor β1, a set of degenerate primers was designed and synthesized, and there was one upstream primer F1: 5'-GARGCBATTMGRRGHCAGAT-3' (shown in SEQ ID NO:7 ) and a downstream primer R1: 5'- GCMGAKGCWCCDGGRTTGTG-3' (shown in SEQ ID NO:8), wherein R=A / G; B=C / G / T; M=A / C; H=A / T / C; K=G / T; W=A / T; D=A / T / G. The cDNA obtained by reverse transcription of the total RNA of Nile tilapia spleen was used as a template and amplified by PCR method. The reaction conditions were: 94°C pre-denaturation for 3 minutes; the following was 40 cycles, divided into two stages: (1) Denaturation at 94°C for 30 seconds, 0.5°C per cycle from 65°C to 50°C, annealing for 30 seconds, a total of 30 cycles, extension at 72°C for 1 minute; (2) denaturation at 94°C for 30 seconds, annealing at 55°C...

Embodiment 2

[0044] Embodiment 2: the synthesis of Nile tilapia transforming growth factor β1 gene 3' end fragment

[0045] The upstream primer F2: 5'-TTTACCATGTCCATCCCTA-3' (shown in SEQ ID NO: 9) was designed according to the obtained intermediate fragment. According to the alignment of the existing fish transforming growth factor β1 cDNA, it was found that the stop codon was very Conservative, so a specific downstream primer R2 is designed here: 5'-TTAGCTACACTTGCAGGACTT-3' (shown in SEQ ID NO: 10). The cDNA obtained by reverse transcription of the total RNA of the Nile tilapia spleen was used as a template and amplified by PCR method. The reaction conditions were: 94°C pre-denaturation for 3 minutes; 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, and 72°C extension 30 seconds for a total of 30 cycles; 72°C extension for 10 minutes.

[0046] The electrophoresis identification chart of the PCR amplification product is shown in figure 2 . from figure 2 It can be see...

Embodiment 3

[0048] Embodiment 3: the synthesis of Nile tilapia transforming growth factor β1 gene 5' end fragment

[0049] Three downstream primers of 5'RACE, 5'-R1: 5'-GTTGAACCTGTTAAGCACCT-3' (shown in SEQ ID NO: 11), 5'-R2: 5, were designed based on the sequence of the cloned intermediate fragment of transforming growth factor β1 '-TGCTCCTCATCCTGCTTCT-3' (shown in SEQ ID NO: 12) and 5'-R3: 5'-CTGGTGCTGTTGTAGAGTGATAG-3' (shown in SEQ ID NO: 13) for nested PCR. For the first PCR, the total cDNA of Nile tilapia spleen was tailed by TdT as a template, 5'-R1 and universal primer 5'-CDS: 5'-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3' (shown in SEQ ID NO: 14 ) as primers, amplified by landing PCR method, the reaction conditions are: 94°C pre-denaturation for 3 minutes; the following is 40 cycles, divided into two stages: (1) 94°C denaturation for 20 seconds, from 60°C to 50°C every (2) Denaturation at 94°C for 20 seconds, annealing at 50°C for 20 seconds, 20 cycles in total, extension at 72°C for 1 min...

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Abstract

The invention discloses a Nile tilapia transforming growth factor TGF-beta 1 gene, a related protein and application, and belongs to the technical field of biological genetic engineering. The nucleotide sequence of the Nile tilapia transforming growth factor TGF-beta 1 gene is expressed by SEQIDNO:1; and the amino acid sequence of the related protein TGF-beta 1 is expressed by SEQIDNO: 2. Prokaryotic expression of the mature peptide gene of the Nile tilapia transforming growth factor TGF-beta 1 is performed so that recombinant mature peptide of the Nile tilapia transforming growth factor beta 1; and after the mice are immunized by the mature peptide, corresponding polyclone antibodies can be obtained. The recombinant mature peptide of the Nile tilapia transforming growth factor beta 1 and the prepared polyclone antibodies can be further used as immune stimulants and used for scientific research.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and specifically relates to a Nile tilapia transforming growth factor TGF-β1 gene, related proteins and applications. Background technique [0002] Transforming Growth Factor β (Transforming Growth Factor beta, TGF-β) superfamily is a kind of multi-effect cytokines, which play a role in various physiological functions such as inhibition of tumor, cell proliferation, cell differentiation, tissue formation, lineage determination, cell migration and cell apoptosis. play an important role in the process. TGF-β superfamily members include transforming growth factor β (TGF-β), activin (Activin) and nodal / bone morphogenic protein (Bone Morphogenetic Protein, BMP) three families. TGF-β in a narrow sense usually refers to the TGF-β family. TGF-β1 is encoded as a precursor protein, and each TGF-β1 is encoded by a separate gene. TGF-β1 precursor protein is a single-chain protein wi...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/495C12N15/11C12N15/63C12N15/70C12N1/21C12P21/02C07K16/22C12R1/19
Inventor 吴金英詹绪良李文笙
Owner SUN YAT SEN UNIV
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