Protein for specific targeted fish egg cell transportation and preparation method and application thereof

An egg cell-specific technology, which is applied in the field of proteins specifically targeting fish egg cell transport and its preparation, can solve the problems of high price, lack of precision, and long time-consuming, and achieve low price, high accuracy, and short time-consuming Effect

Active Publication Date: 2021-07-23
HUNAN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, methods for labeling fish eggs mainly use non-specific fluorescent staining, immunofluorescent antibody labeling, and preparation of Oct4-EGFP transgenic fish. Although they have certain advantages, there are still some shortcomings, such as: lack of precision, toxicity , expensive, time-consuming, etc.

Method used

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  • Protein for specific targeted fish egg cell transportation and preparation method and application thereof
  • Protein for specific targeted fish egg cell transportation and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] A protein specifically targeting fish egg cell transport of the present invention has an amino acid sequence as shown in SEQ ID NO: 2, and a nucleotide sequence of its encoding gene as shown in SEQ ID NO: 1.

[0026] The preparation method of the protein specifically targeting the transportation of fish egg cells comprises the following steps:

[0027] (1) Constructing a protein expression vector: link the gene encoding the protein specifically targeting fish egg cell transport into the protein expression vector. The specific steps are: analyze various fish vitellogenin sequences, and obtain its specificity through analysis. Identify the region, use high-fidelity RT-PCR to obtain the nucleic acid sequence containing the sequence from the RNA material of the female zebrafish liver, conduct codon analysis on the sequence, and perform synonymous mutations to ensure that the induction can proceed smoothly. The sequence (as shown in SEQ ID NO: 1) is connected to the prokaryo...

Embodiment 2

[0030] A protein carrying green fluorescent protein specifically targeting fish egg cell transport, its amino acid sequence is shown in SEQ ID NO: 8, and its coding gene nucleotide sequence is shown in SEQ ID NO: 7.

[0031] The preparation method of the protein specifically targeting the transportation of fish egg cells comprises the following steps:

[0032] (1) Construction of a fusion protein expression vector: the nucleotide sequence (as shown in SEQ ID NO: 1) and the green fluorescent protein nucleotide sequence (as shown in SEQ ID NO: 3) of the protein specifically targeted to fish egg cell transport; The above two sequences are connected to the same protein expression vector (pET30a or pET28a) through the Link sequence (nucleotide sequence shown in SEQIDNO: 5) by means of homologous recombination to ensure that the two can be fused, expressed and folded correctly (nucleoside acid sequence as shown in SEQID NO: 7);

[0033] (2) Fusion protein expression and purificatio...

Embodiment 3

[0034] Example 3: Targeting of specific targeting protein-green fluorescent fusion protein into goldfish egg cells

[0035] The application of the specific targeting protein-green fluorescent fusion protein obtained in Example 2 to target goldfish egg cells includes the following steps:

[0036] (1) Selection of female eyeball goldfish: select sexually mature and robust female eyeball goldfish;

[0037] (2) Multiple eye injections: After MS222 anesthesia, inject 100-200 μL of purified fusion protein (protein concentration: 1-5 mg / mL) into the eye socket with an insulin injection needle (specification: U40, 32G, 1 mL);

[0038] (3) Anatomical observation: 3 to 7 days after injection, after anesthesia, the gonads were taken and observed under a stereoscopic fluorescence microscope (see figure 1 ).

[0039] Depend on figure 1 It can be seen that the specific targeting protein-green fluorescent fusion protein of the present invention is used to inject female eyeball goldfish, w...

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Abstract

The invention discloses a protein for specific targeted fish egg cell transportation. The amino acid sequence of the protein is shown as SEQ ID NO: 2. The protein can carry other polypeptide (containing a fluorescent protein) substances to be transported to egg cells in a targeted manner, so that targeted marking of the egg cells is implemented; and the protein has the advantages of high precision, low toxicity, low price, short marking consumed time and the like, and is suitable for related research of fish ovary ova and the like. A preparation method of the protein comprises the following steps: cloning a coding gene of the protein for specific targeted fish egg cell transportation into a protein expression vector, and then transferring successfully constructed protein expression vector plasmids into escherichia coli to carry out induced expression so as to finally obtain the protein for specific targeted fish egg cell transportation. An application method of the protein comprises the following step: injecting the protein for specific targeted fish egg cell transportation into female fishes to enable the protein to enter ova. The preparation method and the application method which are disclosed by the invention are simple in operation and short in time consumption.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a protein specifically targeting fish egg cell transport, a preparation method and application thereof. Background technique [0002] At present, methods for labeling fish eggs mainly use non-specific fluorescent staining, immunofluorescent antibody labeling, and preparation of Oct4-EGFP transgenic fish. Although they have certain advantages, there are still some shortcomings, such as: lack of precision, toxicity , expensive, time-consuming, etc. Therefore, it is of great significance to this field to develop a labeling method with high precision, low toxicity, low cost, and short labeling time. Contents of the invention [0003] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology, and to provide a specific recognition sequence that can transport substances such as other polypeptides (in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46C07K19/00C12N15/12C12N15/62C12N15/70C12P21/02A01K67/027
CPCC07K14/461C12N15/70A01K67/027C07K2319/60A01K2207/10A01K2227/40Y02A40/81
Inventor 肖亚梅彭亮跃范云鹏刘文彬刘锦辉符文
Owner HUNAN NORMAL UNIVERSITY
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