A mylopharyngodon recombination parvalbumin and a preparation method thereof

Abstract

The present application relates to the technical field of genetic engineering, in particular to a kind of recombined parvalbumin of silver carp and its preparation method.To solve the technical problems such as lack of recombined parvalbumin of silver carp at present, the present application provides the recombined parvalbumin of silver carp as shown in SEQ ID NO:1, the amino acid sequence of the recombined parvalbumin of silver carp includes 6 histidine (His) tags;Based on the coding gene of the recombined parvalbumin of silver carp and pET-28a (+), recombination vector is constructed, and the recombination vector is transformed into escherichia coli, and gene engineering bacteria can be obtained, so as to induce expression of the recombined parvalbumin of silver carp with amino acid sequence consistent with silver carp parvalbumin sequence in large quantities.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a recombinant small albumin from silver carp and its preparation method. Background Technology

[0002] Fish allergies not only affect human health but also pose safety hazards to the seafood processing industry. Eliminating fish allergies is of great practical significance for ensuring human health and promoting industrial development. As a major fish allergen, the isolation, extraction, and sensitization mechanism of parvalbumin are key technical aspects in solving the allergy problem. However, the current difficulty in purifying high-quality, active natural allergens limits allergy research; therefore, recombinant proteins as alternatives to natural allergens are of great importance.

[0003] Traditional parvalbumin extraction techniques suffer from drawbacks such as limited raw material availability, complex extraction procedures, unstable product yield, low purity, severe impurity interference, poor stability, and uneven sensitizing activity. Compared to traditional parvalbumin extraction techniques, recombinant protein induced expression and purification offer advantages such as low raw material costs, simple purification procedures, high product yield, high purity, strong stability, controllable sensitizing activity, and excellent safety. Prokaryotic expression technology is widely used in recombinant protein preparation due to its ease of operation, low culture costs, and well-defined genetic background. Among these, the pET system based on the T7 promoter and... E. coli The BL21(DE3) host bacterial combination has become the preferred platform for exogenous protein expression due to its simple operation process, high expression stability, short culture cycle and large protein yield.

[0004] However, there are currently no reports on the expression of recombinant albumin in silver carp, while most recombinant albumin expression in other organisms adopts eukaryotic expression methods. This method is inefficient and costly in obtaining recombinant albumin, and there is an urgent need to develop more efficient and economical prokaryotic expression schemes. Summary of the Invention

[0005] Based on this, the present invention provides a recombinant small albumin from silver carp and its preparation method, which solves at least one problem in the prior art.

[0006] In a first aspect, the present invention provides a recombinant small albumin from silver carp, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0007] Secondly, the present invention provides a method for preparing the recombinant small albumin from silver carp, comprising the following steps: The coding gene of the recombinant small carp albumin was constructed based on the amino acid sequence of the recombinant small carp albumin, and the coding gene was linked to the backbone plasmid to obtain the recombinant vector. The recombinant vector was transformed into host bacteria to obtain genetically engineered bacteria; The genetically engineered bacteria were cultured, and recombinant silver carp albumin was isolated from the cultured genetically engineered bacteria.

[0008] In some optional embodiments, the isolation of recombinant silver carp albumin from cultured genetically engineered bacteria includes: disrupting the cell walls of the cultured genetically engineered bacteria and performing affinity chromatography based on the His tag of the recombinant silver carp albumin to obtain the recombinant silver carp albumin. The cell wall disruption can be performed by ultrasound to release the intracellular recombinant silver carp albumin from the genetically engineered bacteria, facilitating subsequent centrifugation and chromatographic purification. After affinity chromatography, dialysis can be performed to improve the purity of the recombinant silver carp albumin. After dialysis, the recombinant silver carp albumin can be further enriched and concentrated by freeze-drying to obtain a higher concentration of recombinant silver carp albumin.

[0009] Thirdly, the present invention provides a gene encoding the recombinant small albumin of silver carp, which encodes the recombinant small albumin of silver carp.

[0010] In some alternative embodiments, the nucleotide sequence of the encoding gene is shown in SEQ ID NO:2.

[0011] Fourthly, the present invention provides a recombinant vector for expressing the recombinant carp albumin, the recombinant vector comprising a backbone plasmid and a gene encoding the recombinant carp albumin.

[0012] In some optional embodiments, the backbone plasmid is pET-28a (+).

[0013] Fifthly, the present invention provides a genetically engineered bacterium expressing the recombinant carp albumin, the genetically engineered bacterium comprising the recombinant vector for expressing the recombinant carp albumin.

[0014] In some optional embodiments, the host bacterium of the genetically engineered bacteria is Escherichia coli.

[0015] In some optional embodiments, the host bacteria of the genetically engineered bacteria are E. coli BL21(DE3).

[0016] Due to the adoption of the above technical solutions, the embodiments of the present invention have at least the following beneficial effects: (1) For the first time, a recombinant silver carp albumin with an amino acid sequence identical to that of silver carp albumin was provided, which can be effectively applied to the study of albumin allergy; (2) The amino acid sequence of recombinant small albumin from silver carp contains 6 histidine (His) tags. His tags can be used to purify recombinant small albumin from silver carp, which can improve the purity of recombinant protein to meet higher application requirements. (3) A recombinant vector was constructed based on the coding gene of recombinant small albumin of silver carp and pET-28a (+), and the recombinant vector was transformed into Escherichia coli to obtain genetically engineered bacteria, which can induce the expression of recombinant small albumin of silver carp in large quantities; (4) The biologically active target protein can be obtained in large quantities and efficiently through the preparation method of recombinant small albumin from silver carp, which can be effectively applied to industrial production and has important commercial value. Attached Figure Description

[0017] Figure 1 This is an SDS-PAGE electrophoresis image of the supernatant in Example 4 of the present invention.

[0018] Figure 2 This is the mass spectrometry sequence of the recombinant small albumin from silver carp in Example 4 of the present invention. Detailed Implementation

[0019] The following will provide a clear and complete description of the concept and technical effects of the present invention, so as to fully explain the purpose, solution and effects of the present invention.

[0020] Example 1: Design of the gene encoding recombinant albumin in silver carp Using the original nucleotide sequence of silver carp carp parvalbumin registered in NCBI's GenBank (GenBank: FJ216937.1, https: / / www.ncbi.nlm.nih.gov / nuccore / FJ216937.1 / ) as a template, the DNA sequence of parvalbumin PV I was obtained (as shown in SEQ ID NO:3). The nucleotide sequence of the coding gene for recombinant silver carp parvalbumin was designed and constructed using SnapGene software (as shown in SEQ ID NO:2).

[0021] Among them, SEQ ID NO: 2 is ATGGGATCTTCACCACCACCACCACCACTCTTCTGGACTTGTCCCTAGGGGCTCTCACATGGCATTCGCTGGAATTCTGAATGAGGCTGACGTCACTGCAGCCCTGCAGGCCTGCCAAGCTGCTGACTCCTTCAAGTACAAGGACTTCTTCGCCAAGGTTGGCCTTTCCGCCAAGAGTCCTGATGACATCA AGAAGGCCTTTGCTGTCATTGACCAGGACAAGAGCGGCTTCATTGAGGAGGATGAGCTCAAACTGTTCCTGCAGGACTTCTCTGCTGGTGCCAGGGCACTCACTGATGCAGAGACAAAGGCCTTCTTGAAAGCTGGAGACTCTGATGGTGATGGCAAGATTGGAGTTGATGAGTTCGCCGTCCTGGTCAAGGCATAA.

[0022] SEQ ID NO: 3 is ATGGCATTCGCTGGAATTCTGAATGAGGCTGACGTCACTGCAGCCCTGCAGGCCTGCCAAGCTGCTGACTCCTTCAAGTACAAGGACTTCTTCGCCAAGGTTGGCCTTCCGCCAAGAGTCCTGATGACATCAAGAAGGCCTTTGCTGTCATTGACCAGGACA AGAGCGGCTTCATTGAGGAGGATGAGCTCAAACTGTTCCTGCAGGACTTCTCTGCTGGTGCCAGGGCACTCACTGATGCAGAGACAAAGGCCTTCTTGAAAGCTGGAGACTCTGATGGTGATGGCAAGATTGGAGTTGATGAGTTCGCCGTCCTGGTCAAGGCATAA.

[0023] Example 2: Synthesis of a recombinant vector for expressing recombinant small albumin from silver carp Shanghai Houji Biotechnology Co., Ltd. was commissioned to link the coding gene of recombinant small albumin from silver carp in Example 1 with pET-28a (+) to obtain a recombinant vector for expressing recombinant small albumin from silver carp.

[0024] Example 3: Preparation of genetically engineered bacteria expressing recombinant silver carp parvalbumin The recombinant vector for expressing recombinant carp carp albumin from Example 2 was transfected into competent Escherichia coli. E. coli BL21(DE3) was cultured overnight at 37°C on LB solid medium containing 25 mg / L kanamycin and single colonies were screened to obtain the prokaryotic expression engineered strain of recombinant Escherichia coli albumin, which is the genetically engineered strain expressing recombinant albumin from silver carp.

[0025] Example 4: Preparation of recombinant small albumin from silver carp A single colony of the genetically engineered bacteria expressing recombinant carp albumin from Example 3 was picked and cultured overnight at 37°C and 200 rpm in 5 ml of LB medium containing 25 mg / L kanamycin. Then, it was inoculated at a ratio of 1:50 into 200 ml of LB medium containing 25 mg / L kanamycin and cultured for 3 h at 37°C and 200 rpm to allow OD to develop. 600 The concentration was adjusted to 0.5-0.6; then the temperature was adjusted to 22℃ and cultured for 0.5 h. IPTG was added to make the final concentration 0.05 mM. After culturing for 24 h at 22℃ and 200 rpm, the cells were collected by centrifugation at 5000 rpm and 4℃ for 5 min. The cells were resuspended in 20 ml of loading buffer (PBS solution containing 20 mM imidazole, pH=7.4), and sonicated (sonication for 3 s, pause for 3 s, repeated for 20 min). The cells were centrifuged at 12000 rpm and 4℃ for 20 min and the supernatant was collected. Finally, insoluble microparticles in the supernatant were removed by filtering through a 0.22 μm filter membrane. His-tag affinity purification was performed using nickel ion packing material to obtain high-purity recombinant silver carp albumin. The obtained recombinant small albumin from silver carp was poured into a protein dialysis bag (molecular weight cutoff of 3.5 kDa), dialyzed into pure water, aliquoted, and frozen overnight at -80°C. Then it was freeze-dried in a freeze dryer for 48 hours and stored at -80°C.

[0026] The supernatant from Example 4 was subjected to SDS-PAGE and Coomassie Brilliant Blue staining to observe protein bands (e.g., Figure 1 (As shown in the figure). The results showed that recombinant small albumin from silver carp could be expressed in large quantities and existed in the supernatant in the form of a soluble protein.

[0027] Mass spectrometry identification of the recombinant small albumin from silver carp in Example 4 was performed, and the results are as follows: Figure 2As shown. The amino acid sequence of recombinant silver carp parvoprotein is SEQ ID NO: 1. SEQ ID NO: 1 is MGSSHHHHHHSSGLVPRGSHMAFAGILNEADVTAALQACQAADSFKYKDFFAKVGLSAKSPDDIKKAFAVIDQDKSGFIEEDELKLFLQDFSAGARALTDAETKAFLKAGDSDGDGKIGVDEFAVLVKA. HHHHHHSSGLVPRGSH represents the His tag and linker peptide added to the N-terminus of the recombinant protein; the 6×His tag (HHHHHH) is used for affinity chromatography with a nickel column to purify the recombinant silver carp parvoprotein, further improving its purity. The N-terminal His tag does not affect the activity of the recombinant parvoprotein and does not require tag removal.

[0028] The above description is merely a preferred embodiment of the present invention. The present invention is not limited to the above-described embodiments. Any embodiment that achieves the technical effects of the present invention by the same or equivalent means should fall within the protection scope of the present invention. Within the protection scope of the present invention, various modifications and variations can be made to the technical solutions and / or implementation methods.

Claims

1. A recombinant small albumin from silver carp, characterized in that, The amino acid sequence of the recombinant small albumin from silver carp is shown in SEQ ID NO:

1.

2. The method for preparing recombinant small albumin from silver carp according to claim 1, characterized in that, Includes the following steps: The coding gene of the recombinant small carp albumin described in claim 1 was constructed, and the coding gene was linked to a backbone plasmid to obtain a recombinant vector. The recombinant vector was transformed into host bacteria to obtain genetically engineered bacteria; The genetically engineered bacteria were cultured, and recombinant silver carp albumin was isolated from the cultured genetically engineered bacteria.

3. The preparation method according to claim 2, characterized in that, The process of isolating recombinant silver carp albumin from cultured genetically engineered bacteria includes: breaking the cell walls of the cultured genetically engineered bacteria and performing affinity chromatography based on the His tag of the recombinant silver carp albumin to obtain the recombinant silver carp albumin.

4. A gene encoding a gene, characterized in that, The coding gene encodes the recombinant small albumin from silver carp as described in claim 1.

5. The encoding gene according to claim 4, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO:

2.

6. A recombinant vector for expressing the recombinant albumin of silver carp, characterized in that, The recombinant vector includes a backbone plasmid and the encoding gene for the recombinant small albumin of silver carp as described in claim 5.

7. The recombinant vector according to claim 6, characterized in that, The backbone plasmid is pET-28a (+).

8. A genetically engineered bacterium expressing the recombinant albumin from silver carp, characterized in that, The genetically engineered bacteria include the recombinant vector for expressing the recombinant small albumin of silver carp as described in claim 6.

9. The genetically engineered bacterium according to claim 8, characterized in that, The host bacterium of the genetically engineered bacteria is Escherichia coli.

10. The genetically engineered bacterium according to claim 8, characterized in that, The host bacteria of the genetically engineered bacteria are E. coli BL21(DE3).

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