A kind of recombinant retinol binding protein 4 and its preparation method and application

Abstract

The invention discloses a recombinant retinol binding protein 4 and its preparation method and application. In view of the uncontrollable regeneration of the existing recombinantly expressed RBP4 protein, poor immunogenicity, unstable performance, and poor performance of antibodies obtained from immunized animals, the present invention obtains a mutant recombinant RBP4 protein by replacing amino acids of the RBP4 protein, and utilizes PCR technology The recombinant RBP4 gene was obtained, and the recombinant RBP4 gene was double-digested and connected to the double-digested carrier to obtain an expression vector, and then the expression vector was transformed into Escherichia coli Origamin B competent cells to obtain an RBP4 expression strain, and the recombinant RBP4 was obtained after purification. The recombinant RBP4 prepared by the invention has the biological activity of the natural protein, its stability and immunogenicity are better than that of the non-mutated recombinant protein, and it can be applied to the preparation of RBP4 antigen and antibody.

Description

technical field

[0001] The invention relates to the field of biotechnology, in particular to a prokaryotic expression recombinant retinol binding protein 4 and its preparation method and application. Background technique

[0002] Retinol-binding protein (RBP) is a class of proteins responsible for binding and transporting vitamin A retinol-like active metabolites in vivo. The retinol-binding protein 4 (RBP4) gene is located at 10q, the full length of the mRNA is 914bp, it is composed of 181 amino acids, and the relative molecular mass is 21kD. It is a secreted RBP in the retinol-binding protein family and is a single peptide chain Carrier protein, which has a site that specifically binds a molecule of vitamin A, can transport vitamin A from the liver to the target organ, and can realize the intracellular transport and metabolism of vitamin A. RBP4 plays an important role in assisting vitamin A to exert its physiological functions .

[0003] At present, the methods used for...

Examples

Embodiment 1

[0030] [Example 1] Preparation of recombinant RBP4(20)

[0031] (1) Recombinant RBP4(20) protein design:

[0032] According to the amino acid sequence of the RBP4 protein published on the NCBI website and the characteristics of the sequence, amino acids 1-18 are signal peptides, and the mature protein sequence is amino acids 19-201, with a total of 183 amino acids. The amino acid sequence is shown in the sequence table SEQID NO. As shown in 4, replace the F at position 20 with Y to obtain the amino acid sequence shown in the sequence listing SEQ ID NO.1.

[0033] (2) Optimization and acquisition of recombinant RBP4(20) gene sequence:

[0034] The mutated amino acid sequence SEQ ID NO.1 was reverse-translated into the nucleotide sequence SEQ ID NO.5 with Escherichia coli dominant codons, and the recombinant RBP4 composed of Escherichia coli dominant codons ( 20) Genes.

[0035] The RBP4(20) gene is designed into two segments A and B. The first round of PCR reaction system is...

Embodiment 2

[0053] [Example 2] Preparation of recombinant RBP4(35)

[0054] (1) Recombinant RBP4(35) protein design:

[0055] In the amino acid sequence of retinol binding protein 4, L at position 35 was replaced with G to obtain the amino acid sequence shown in SEQ ID NO.2 of the sequence table.

[0056] (2) Optimization and acquisition of recombinant RBP4(35) gene sequence:

[0057] The mutated amino acid sequence SEQ ID NO.2 was reverse-translated into the nucleotide sequence SEQ ID NO.6 with the dominant codons of Escherichia coli, and the recombinant RBP4(35) composed of the dominant codons of Escherichia coli was obtained by annealing extension PCR technique Gene.

[0058] The RBP4(35) gene was designed into two segments A and B. The first round of PCR reaction system was 2×PCR reaction solution, double distilled water and primers from Biotec Company. The PCR reaction conditions were 94°C for 1 minute, followed by 94°C for 1 minute , 55°C for 1 minute, 72°C for 1 minute, 5 cycles...

Embodiment 3

[0076] [Example 3] Preparation of recombinant RBP4 (20-35)

[0077] (1) Recombinant RBP4(20-35) protein design:

[0078] In the amino acid sequence of retinol binding protein 4, F at position 20 is replaced by Y while L at position 35 is replaced with G to obtain the amino acid sequence shown in the sequence table SEQ ID NO.3.

[0079] (2) Optimization and acquisition of recombinant RBP4(20-35) gene sequence:

[0080] The mutated amino acid sequence SEQ ID NO.3 was reverse-translated into the nucleotide sequence SEQ ID NO.7 with the dominant codons of Escherichia coli, and the recombinant RBP4 (20- 35) Genes.

[0081] Design the RBP4(20-35) gene into two segments, A and B. The first round of PCR reaction system is 2×PCR reaction solution, double distilled water, and primers from Biotech Company. The PCR reaction conditions are 94°C for 1 minute, and then 1 minute, 55°C for 1 minute, 72°C for 1 minute, 5 cycles; the PCR reaction system for the next two rounds is 2×PCR reacti...

technical field

[0001] The invention relates to the field of biotechnology, in particular to a prokaryotic expression recombinant retinol binding protein 4 and its preparation method and application. Background technique

[0002] Retinol-binding protein (RBP) is a class of proteins responsible for binding and transporting vitamin A retinol-like active metabolites in vivo. The retinol-binding protein 4 (RBP4) gene is located at 10q, the full length of the mRNA is 914bp, it is composed of 181 amino acids, and the relative molecular mass is 21kD. It is a secreted RBP in the retinol-binding protein family and is a single peptide chain Carrier protein, which has a site that specifically binds a molecule of vitamin A, can transport vitamin A from the liver to the target organ, and can realize the intracellular transport and metabolism of vitamin A. RBP4 plays an important role in assisting vitamin A to exert its physiological functions .

[0003] At present, the methods used for...

Examples

Embodiment 1

[0030] [Example 1] Preparation of recombinant RBP4(20)

[0031] (1) Recombinant RBP4(20) protein design:

[0032] According to the amino acid sequence of the RBP4 protein published on the NCBI website and the characteristics of the sequence, amino acids 1-18 are signal peptides, and the mature protein sequence is amino acids 19-201, with a total of 183 amino acids. The amino acid sequence is shown in the sequence table SEQID NO. As shown in 4, replace the F at position 20 with Y to obtain the amino acid sequence shown in the sequence listing SEQ ID NO.1.

[0033] (2) Optimization and acquisition of recombinant RBP4(20) gene sequence:

[0034] The mutated amino acid sequence SEQ ID NO.1 was reverse-translated into the nucleotide sequence SEQ ID NO.5 with Escherichia coli dominant codons, and the recombinant RBP4 composed of Escherichia coli dominant codons ( 20) Genes.

[0035] The RBP4(20) gene is designed into two segments A and B. The first round of PCR reaction system is...

Embodiment 2

[0053] [Example 2] Preparation of recombinant RBP4(35)

[0054] (1) Recombinant RBP4(35) protein design:

[0055] In the amino acid sequence of retinol binding protein 4, L at position 35 was replaced with G to obtain the amino acid sequence shown in SEQ ID NO.2 of the sequence table.

[0056] (2) Optimization and acquisition of recombinant RBP4(35) gene sequence:

[0057] The mutated amino acid sequence SEQ ID NO.2 was reverse-translated into the nucleotide sequence SEQ ID NO.6 with the dominant codons of Escherichia coli, and the recombinant RBP4(35) composed of the dominant codons of Escherichia coli was obtained by annealing extension PCR technique Gene.

[0058] The RBP4(35) gene was designed into two segments A and B. The first round of PCR reaction system was 2×PCR reaction solution, double distilled water and primers from Biotec Company. The PCR reaction conditions were 94°C for 1 minute, followed by 94°C for 1 minute , 55°C for 1 minute, 72°C for 1 minute, 5 cycles...

Embodiment 3

[0076] [Example 3] Preparation of recombinant RBP4 (20-35)

[0077] (1) Recombinant RBP4(20-35) protein design:

[0078] In the amino acid sequence of retinol binding protein 4, F at position 20 is replaced by Y while L at position 35 is replaced with G to obtain the amino acid sequence shown in the sequence table SEQ ID NO.3.

[0079] (2) Optimization and acquisition of recombinant RBP4(20-35) gene sequence:

[0080] The mutated amino acid sequence SEQ ID NO.3 was reverse-translated into the nucleotide sequence SEQ ID NO.7 with the dominant codons of Escherichia coli, and the recombinant RBP4 (20- 35) Genes.

[0081] Design the RBP4(20-35) gene into two segments, A and B. The first round of PCR reaction system is 2×PCR reaction solution, double distilled water, and primers from Biotech Company. The PCR reaction conditions are 94°C for 1 minute, and then 1 minute, 55°C for 1 minute, 72°C for 1 minute, 5 cycles; the PCR reaction system for the next two rounds is 2×PCR reacti...

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