Preparation method of fish skin mucous gland bioreactor and application of preparation method

A bioreactor and mucous gland technology, applied in the field of animal bioreactor preparation, can solve the problems of high cost, harsh culture conditions of isolated animal cells, low success rate and the like

Active Publication Date: 2019-12-31
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently commonly used bioreactors mainly include bacteria, yeast, plant cells or tissues, cultured animal and plant cells, living animals and plants or tissues (such as animal mammary glands), etc. They have their own advantages, but there are one or other defects: Proteins expressed in bacteria cannot undergo correct post-translational modification, proteins expressed in fungi are immunogenic, isolated animal cell culture conditions are harsh and costly, animal mammary gland bioreactors have a long development cycle and low success rate
[0006] However, the current existing technology has only progressed to using the reproductive system of fish as a bioreactor. Whether other systems can still be used as bioreactors and what kind of beneficial effects are still waiting for researchers to continue to study

Method used

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  • Preparation method of fish skin mucous gland bioreactor and application of preparation method
  • Preparation method of fish skin mucous gland bioreactor and application of preparation method
  • Preparation method of fish skin mucous gland bioreactor and application of preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Identification of skin-specific expression genes by genomics technology, and acquisition of fish skin and mucus gland cell-specific promoters

[0050] Total RNA was extracted from loach skin, a sequencing library was constructed, and transcriptome sequencing was performed on a high-throughput sequencing platform. The sequencing results were de novo assembled using Velvet software, and a total of 40,364 cDNA sequences were obtained. Through comparison and analysis of the Blast genome database, the genes and cDNA sequences highly expressed in the skin were obtained, and then PCR primers and RNA probes were designed according to the protein cDNA sequences, and RT-PCR and overall in situ hybridization analysis were performed. Among the highly expressed genes, lgals2b, rblec3 and glant8 were screened out, three genes that can be specifically expressed in loach skin and intestine, the results are shown in figure 2 , wherein (A) RT-PCR detects tissue distribution o...

Embodiment 2

[0051] Example 2: Proteomics technology to identify mucus-specific proteins, and obtain fish skin and mucus gland cell-specific promoters

[0052] Extract different tissue samples and mucus from loach gills, muscles, intestines, liver, etc., and perform sample pretreatment on proteins according to the requirements of proteomics mass spectrometry. Due to the lack of genomics data in loach, the present invention compares the mass spectrometry data with the NCBI bony fish database, analyzes samples between different tissues, and finds 160 specific proteins in loach mucus samples, the results are shown in image 3 , wherein (A) pre-processing steps for mass spectrometry loading of different histone samples. (B) Venn diagram of protein species identified in each tissue; muscle (Muscle), liver (Liver), intestine (Intestine), gill (Gill), mucus (Mucus). The GI number of the loach mucus-specific protein was converted into the UniProt KB format through the UniProt database, and the da...

Embodiment 3

[0053] Example 3: Search published literature and find genes or promoters specifically expressed in fish skin and mucous gland cells

[0054] To detect whether the existing krt8 promoter can drive the specific expression of the target gene in loach skin.

[0055] Construction of pT2-krt8-EGFP:

[0056] 1. Total RNA was extracted from 24-48hpf zebrafish embryos, and after grinding, follow the Total RNA was extracted according to the operating instructions of Reagent. Take 1 μL of RNA electrophoresis to check the quality of RNA and determine the concentration of RNA. Take 4 μg of newly prepared RNA for reverse transcription, and store the remaining RNA at -80°C after aliquoting.

[0057] 2. Synthesis of first-strand cDNA by reverse transcription

[0058] (1) Add the following reagents to a nuclease-free PCR tube in sequence: Total RNA, 5 μL (4 μg); Oligo-dTprimer, 1 μL Nuclease-free, 6 μL; total volume, 12 μL. (2) After mixing and centrifuging, put it into a PCR instrument...

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Abstract

The invention belongs to the technical field of preparation of animal bioreactors, and particularly relates to a preparation method of a fish skin mucous gland bioreactor and an application of the preparation method. The preparation method comprises the steps of identifying specific expression genes, promoters and protein secreting signal peptide of fish skin mucous gland cells, constructing transgene expression vectors which can specifically express endogenous or heterogenesis bioactive substances in fish skin and mucous gland cells, and developing transgenic fish which can stably inherit andsecrete bioactive substances to fish mucosa, wherein the bioactive substances secreted by mucous glands can be used for growth, stress resistance and disease resistance of animals and plants, healthcare and disease prevention and treatment of human, and enzymes for business. The developed fish skin mucous gland bioreactor disclosed by the invention has the characteristics that breeding and groupextending are easy, much skin mucus secretes, mucus collection is convenient, and the bioactive substances are easy to purify, and large-scale production and high-efficiency application of the fish skin mucous gland bioreactor can be realized.

Description

technical field [0001] The invention belongs to the technical field of animal bioreactor preparation, and in particular relates to a preparation method and application of a fish skin mucus gland bioreactor. This type of bioreactor can be used in the preparation of polypeptides, proteins, enzymes, vaccines and antibodies . Background technique [0002] Bioreactor refers to the transfer of DNA sequences encoding biologically active substances into the genomes of microorganisms, yeast, animal and plant cells or tissues, and living animals and plants, making them containers for the efficient expression of corresponding biologically active polypeptides or protein molecules. The use of bioreactors to produce biologically active polypeptides, proteins, enzymes, vaccines, antibodies, etc. is one of the frontiers of genetic engineering technology application research. [0003] Currently commonly used bioreactors mainly include bacteria, yeast, plant cells or tissues, cultured animal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85
CPCC12N15/8509C12N2800/106C12N2830/008A01K2207/05A01K2227/40A01K2267/01C12N2015/8518A01K67/0275A01K2217/052A01K2267/02Y02A40/81
Inventor 崔宗斌周同周波兰龙勇宋桂丽李青朱作言
Owner INST OF AQUATIC LIFE ACAD SINICA
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