Grouper insulin-like growth factor II gene, carrier and recombinant strain containing the gene and application thereof

Abstract

The present invention discloses one kind of rockfish insulin-like growth factor II gene. The gene is gene segment of rockfish insulin-like growth factor II gene whole length cDNA obtained with rockfish liver total RNA as template and through RT-PCR and RACE process. The present inventon also constitutes carrier and recombinant strain containing the gene. Utilizing the present invention can obtain recombinant rockfish insulin-like growth factor II protein with stable source and low cost and further prepares fry growth promoter or additive suitable for sea water fishes.

Description

Technical field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a grouper insulin-like growth factor II gene, a vector and a recombinant strain containing the gene, and the application of the gene in the preparation of a fry seedling growth promoter or feed additive. Background technique

[0002] Insulin-like growth factor II (Insulin-like growth factor-II) is a protein composed of 70 amino acids. Because its structure is similar to proinsulin (proinsulin), it is similar to insulin (INS) and insulin-like growth factor- I (IGF-I) and relaxin are collectively called INS / IGF / relaxin family proteins. IGFs are single-chain proteins with a molecular weight of about 7500D. The structure is similar to proinsulin. Its primary structure includes four regions: BCAD. The three regions of B, C, and A are connected to the B chain and C peptide (connecting peptide) of proinsulin respectively. It is homologous to the A chain, and differs fr...

Examples

Embodiment 1

[0031] Example 1: Synthesis of the middle fragment of the insulin-like growth factor II gene of Epinephelus nidus

[0032] According to the published homology comparison results of the cDNA sequence of insulin-like growth factor II between humans and various animals, a set of primers was designed and synthesized using the full cDNA sequence of the seabass, which is closest to the grouper, as a primer design template. Degenerate primers, a total of one upstream primer and one downstream primer. The upstream primer (Sense) is 20 bases from the 262nd base [5'-CCCTGACGCTCTACGT(A / T)GTG], and the downstream primer (Anti-sense) is 21 bases from the 564th base [ 5'-GCTTCCTTGGGACTTCCTGTT]. Touch-down PCR uses the cDNA obtained by reverse transcription of total RNA from the liver of Epinephelus obliquus as a template, and the original nucleotide sequence from position 262 to position 564 is amplified by PCR. The reaction conditions are: 95°C pre-denaturation for 5 minutes ; The following ar...

Embodiment 2

[0035] Example 2: PCR amplification of cDNA library of Epinephelus obliquus

[0036] The cDNA of Epinephelus obliquus from the reverse transcription of GeneRacer Kit was used as the template, and the primers were GeneRacer Kit 5'Primer and GeneRacer Kit 3’Primer respectively. The PCR method was used to amplify the cDNA library of Epinephelus obliquus. Reaction The conditions are: 94°C pre-denaturation for 5 minutes; the following is 40 cycles, divided into two stages: (1) 94°C denaturation for 30 seconds, drop 0.5°C from 68°C to 54°C in each cycle, annealing for 30 seconds, total 32 Cycle, 72°C extension for 2 minutes; (2) 94°C denaturation for 30 seconds, 50°C annealing for 30 seconds, a total of 8 cycles, 72°C extension for 2 minutes; finally 72°C extension for 7 minutes.

[0037] See the electrophoresis identification diagram of PCR amplification products figure 2 .

Embodiment 3

[0038] Example 3: Synthesis of the 5'-end fragment of the insulin-like growth factor II gene of Epinephelus sinensis

[0039] The operation is carried out according to the GeneRacer Kit Protocol. According to the requirements of the kit, two downstream primers, GSP2 and GSP3, were designed to perform nested PCR based on the sequence of the sequenced IGF-II intermediate fragment. The first downstream primer (GSP2) is 21 bases [5'-CTTTCGGACTTGGCGGGTTTG] from the 493th base of the sequenced IGF-II intermediate fragment sequence, and the second downstream primer (GSP3) is from the 437th base The base starts from 22 bases [5'-ACGGAAACAACACTCCTCTACG].

[0040] In the first PCR, the diluted PCR product (1∶100) obtained in the above example 3 was used as the template, and the original nucleotide sequence from position 493 to the 5'end was amplified by the landing PCR method. The reaction conditions were: 95°C pre-denaturation 5 The following is 40 cycles, divided into two stages: (1) Dena...

Technical field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a grouper insulin-like growth factor II gene, a vector and a recombinant strain containing the gene, and the application of the gene in the preparation of a fry seedling growth promoter or feed additive. Background technique

[0002] Insulin-like growth factor II (Insulin-like growth factor-II) is a protein composed of 70 amino acids. Because its structure is similar to proinsulin (proinsulin), it is similar to insulin (INS) and insulin-like growth factor- I (IGF-I) and relaxin are collectively called INS / IGF / relaxin family proteins. IGFs are single-chain proteins with a molecular weight of about 7500D. The structure is similar to proinsulin. Its primary structure includes four regions: BCAD. The three regions of B, C, and A are connected to the B chain and C peptide (connecting peptide) of proinsulin respectively. It is homologous to the A chain, and differs fr...

Examples

Embodiment 1

[0031] Example 1: Synthesis of the middle fragment of the insulin-like growth factor II gene of Epinephelus nidus

[0032] According to the published homology comparison results of the cDNA sequence of insulin-like growth factor II between humans and various animals, a set of primers was designed and synthesized using the full cDNA sequence of the seabass, which is closest to the grouper, as a primer design template. Degenerate primers, a total of one upstream primer and one downstream primer. The upstream primer (Sense) is 20 bases from the 262nd base [5'-CCCTGACGCTCTACGT(A / T)GTG], and the downstream primer (Anti-sense) is 21 bases from the 564th base [ 5'-GCTTCCTTGGGACTTCCTGTT]. Touch-down PCR uses the cDNA obtained by reverse transcription of total RNA from the liver of Epinephelus obliquus as a template, and the original nucleotide sequence from position 262 to position 564 is amplified by PCR. The reaction conditions are: 95°C pre-denaturation for 5 minutes ; The following ar...

Embodiment 2

[0035] Example 2: PCR amplification of cDNA library of Epinephelus obliquus

[0036] The cDNA of Epinephelus obliquus from the reverse transcription of GeneRacer Kit was used as the template, and the primers were GeneRacer Kit 5'Primer and GeneRacer Kit 3’Primer respectively. The PCR method was used to amplify the cDNA library of Epinephelus obliquus. Reaction The conditions are: 94°C pre-denaturation for 5 minutes; the following is 40 cycles, divided into two stages: (1) 94°C denaturation for 30 seconds, drop 0.5°C from 68°C to 54°C in each cycle, annealing for 30 seconds, total 32 Cycle, 72°C extension for 2 minutes; (2) 94°C denaturation for 30 seconds, 50°C annealing for 30 seconds, a total of 8 cycles, 72°C extension for 2 minutes; finally 72°C extension for 7 minutes.

[0037] See the electrophoresis identification diagram of PCR amplification products figure 2 .

Embodiment 3

[0038] Example 3: Synthesis of the 5'-end fragment of the insulin-like growth factor II gene of Epinephelus sinensis

[0039] The operation is carried out according to the GeneRacer Kit Protocol. According to the requirements of the kit, two downstream primers, GSP2 and GSP3, were designed to perform nested PCR based on the sequence of the sequenced IGF-II intermediate fragment. The first downstream primer (GSP2) is 21 bases [5'-CTTTCGGACTTGGCGGGTTTG] from the 493th base of the sequenced IGF-II intermediate fragment sequence, and the second downstream primer (GSP3) is from the 437th base The base starts from 22 bases [5'-ACGGAAACAACACTCCTCTACG].

[0040] In the first PCR, the diluted PCR product (1∶100) obtained in the above example 3 was used as the template, and the original nucleotide sequence from position 493 to the 5'end was amplified by the landing PCR method. The reaction conditions were: 95°C pre-denaturation 5 The following is 40 cycles, divided into two stages: (1) Dena...

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