A shrimp gene PIAV and polypeptides coded thereby

A coding, protein and polypeptide technology, applied in genetic engineering, plant gene improvement, recombinant DNA technology, etc., can solve the problems of lack of scientific support for breeding of improved varieties, backward level of immunomolecular biology research, etc.

Inactive Publication Date: 2005-06-22
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research level of immunology, especially immunomolecular biology, of invertebrates, including prawns, is far behind that of humans and higher animals, resulting in a lack of scientific support in solving problems such as diseases and breeding of improved species in invertebrates

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Cloning and determination of embodiment 1 PIAV cDNA sequence

[0031] 1. Extraction of mRNA

[0032] The mRNA extraction kit (Quiagen, Valencia, CA) was used to extract blood cells from normal prawns and disease-resistant prawns, respectively.

[0033] 2. Synthesis of Smart cDNA

[0034] The extracted mRNA was quickly reverse-transcribed into cDNA using a kit (Clontech, Palo Alto, CA).

[0035] 3. SSH

[0036] The specific operation was carried out according to the instructions of the kit (Clontech, Palo Alto, CA). A brief description is as follows: the reverse-transcribed cDNA is amplified by PCR with an appropriate number of cycles, and the amplified product is purified and recovered. Then digested with RsaI. Divide the digested cDNA of anti-virus shrimp into two parts, add an adapter to each, and then hybridize with excess cDNA of normal shrimp digested by enzyme. The two hybridization products were then crossed again. The results after hybridization were subj...

Embodiment 2

[0039] Example 2 Expression of PIAV in Escherichia coli

[0040] 1. Amplification of cDNA

[0041] The obtained cDNA is full-length, and the 24 nucleotides in front of it encode the signal peptide, so it is expressed as the nucleotide after the signal peptide. The cDNA sequence of PIAV was amplified with PCR oligonucleotide primers corresponding to the 5' and 3' ends of the open reading frame of the DNA to obtain the PIAV insert.

[0042] The 5' oligonucleotide primer is: 5'-CGCGGATCCGACATGGTTTGCCACCAAC-3' (SEQ ID No. 3), which contains the recognition site of the BamH I restriction endonuclease. The 3' oligonucleotide primer is: 5'-CGCGGAATTCTCAGCGCTCGCAAAGC-3' (SEQ ID No.4), which contains the recognition site of EcoR I restriction endonuclease, the stop codon PIAV partial coding sequence.

[0043] PCR program: ①95℃ for 2min

[0044] ②30 cycles: 94°C 15sec; 56°C 30sec; 72°C 1min

[0045] ③68℃10min

[0046] 2. Recombination of PIAV DNA

[0047] The recognition sites of ...

Embodiment 3

[0051] Example 3 Antibody Preparation

[0052] The purified recombinant protein obtained in Example 2 was emulsified with an equal volume of complete Freund's adjuvant, and the mice were subcutaneously injected with 50-100 μg / 0.2 ml of the emulsified protein. Ten days later, the same antigen emulsified with incomplete Freund's adjuvant was re-injected for booster immunization to immunize animals to produce antibodies. Booster immunization was carried out at least 3 times every 10 days. The titer and specificity of the obtained antisera were analyzed.

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Abstract

The invention relates to a prawn gene nucleotide sequence, specifically, the invention relates to the cDNA sequence of prawn PLAV polypeptide, wherein the polypeptide protein is an antiviral protein. The invention also relates to polypeptides encoded by the nucleotide sequence, the use of these polynucleotides and polypeptides, and the process for preparing these polynucleotides and polypeptides.

Description

technical field [0001] The invention relates to a nucleotide sequence of a prawn gene. More specifically, the present invention relates to the cDNA sequence of the prawn PIAV polypeptide, which is an antiviral protein. The present invention also relates to the polypeptide encoded by the nucleotide sequence, the application of these polynucleotides and polypeptides, and the production method of said polynucleotides and polypeptides. Background technique [0002] Shrimp is a very important economic animal in aquaculture, but shrimp diseases have brought huge losses to the global aquaculture industry since the early 1990s, when the disease broke out in the world. The root cause of this result is viral disease, which is still a major problem in shrimp farming until now. Since the shrimp virus disease is still untreatable, we can only optimize the breeding environment, block the spread of the pathogen, improve the germplasm of the shrimp, and enhance the basic immunity of the s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/00C07K16/18C12N15/12C12N15/63C12P21/00C12Q1/68
Inventor 徐洵何南海
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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