CN110004153A - Chicken recombinant C EBP γ albumen, its DNA sequences encoding and application - Google Patents

Abstract

The invention discloses a DNA sequence encoding chicken recombinant CEBPγ protein, and also discloses a recombinant expression vector and transgenic recombinant Escherichia coli containing the DNA sequence. The invention also discloses a chicken recombinant CEBPγ protein and a preparation method and application thereof. Compared with the prior art, the DNA sequence of the chicken Cebpγ gene provided by the present invention can realize its soluble expression in Escherichia coli under the corresponding temperature and inducer concentration, and the preparation method of the recombinant protein has the advantages of easy method, simple operation and high expression. The amount is high and the cost is low, and the chicken CEBPγ recombinant protein with higher purity can be obtained by the method provided by the invention.

Description

Chicken recombinant CEBPγ protein, its coding DNA sequence and application

technical field

The invention relates to a method for prokaryotic expression and purification of recombinant genes in the field of genetic engineering, in particular to a method for expressing and purifying chicken CEBPγ recombinant protein in Escherichia coli.

technical background

Mcknight and colleagues first discovered the CEBPs family in rat liver in 1987, namely CCAAT enhancer binding protein (CEBPs) is a group of proteins belonging to the basic region leucine zipper (bZIP) family. Transcriptional regulators, six members have been found: α, β, γ, δ, ε and ζ. CEBPs have a variety of functions. They can either form heteromultimers through C/EBPs family members or combine with other transcription factors. It acts on the corresponding regulatory elements, so as to play its specific role in a specific tissue or cell, and form a complex and delicate regulatory network. , energy metabolism, blood production and other aspects play an important role. CEBPγ, a member of the CEBPs family, contains a DNA-binding domain near the C-terminus, but lacks the transactivation domain of CEBPs family proteins and cannot form homodimers, but can only form heterodimers with other CEBPs family members The formation of heterodimers between CEBPγ and CEBPβ can promote cell proliferation and inhibit senescence. CEBPβ plays an important role in the early growth and differentiation of adipocytes through PPARγ. In poultry production, fat deposition is mainly caused by the increase of adipocytes and cell enlargement caused by the accumulation of lipid droplets in adipocytes. Excessive fat deposition will affect animal health, reproduction, and lean meat yield. , affecting production efficiency. Therefore, to understand the structure of chicken CEBPγ and its function in the growth and differentiation of adipocytes will help to solve the many adverse effects caused by excessive fat accumulation in chickens at the genetic and breeding level. The present invention utilizes genetic engineering to express chicken recombinant protein CEBPγ in Escherichia coli system, can deeply analyze its molecular structure and prepare recombinant antigens for antibody preparation, and provides important material guarantee for further research on the role of CEBPγ in poultry.

SUMMARY OF THE INVENTION

Object of the invention: The first object of the present invention is to provide a DNA sequence of chicken CEBPγ recombinant protein.

Another object of the present invention is to provide a chicken CEBPγ recombinant protein.

Another object of the present invention is to provide a recombinant expression vector containing the DNA sequence of the chicken CEBPγ recombinant protein.

Another object of the present invention is to provide a transgenic recombinant Escherichia coli containing the DNA sequence of the chicken CEBPγ recombinant protein.

Another object of the present invention is to provide a method for preparing chicken CEBPγ recombinant protein and its application.

Technical solution: In order to solve the above problems, the present invention provides a DNA sequence of chicken CEBPγ recombinant protein, the nucleotide sequence of which is shown in SEQ ID NO: 1.

The amino acid sequence of the chicken CEBPγ recombinant protein of the present invention is shown in SEQ ID NO: 2.

The above DNA sequence was inserted into the E. coli expression vector to obtain a recombinant expression vector expressing the DNA of the chicken CEBPγ recombinant protein. Wherein, the E. coli expression vector is pET30a.

A transgenic recombinant Escherichia coli strain is obtained by transforming the recombinant expression vector into a BL21 (DE3) strain, and screening to obtain a transgenic recombinant Escherichia coli strain.

A preparation method of chicken CEBPγ recombinant protein, comprising the following steps:

(1) According to the protein sequence of the chicken Cebpγ gene published in GenBank, by optimizing the codons in the gene, redesign the nucleotide sequence of the gene (see seq1 for the specific sequence) to adapt it to the requirements of the translation system of Escherichia coli ;

(2) Insert the gene fragment of the synthesized chicken CEBPγ polypeptide into the E. coli expression vector pET30a, and add 6 His tags at the C-terminal to obtain the recombinant expression vector pET30a-gCebpγ-hc;

(3) The recombinant expression vector pET30a-gCebpγ-hc was transformed into Escherichia coli BL21(DE3), and the transgenic recombinant Escherichia coli strain BL21(DE3)-pET30a-gCebpγ-hc was obtained by kanamycin screening;

(4) The soluble expression of chicken CEBPγ recombinant protein was achieved by optimizing the temperature and IPTG induction concentration conditions; a nickel column affinity chromatography one-step purification method was established to obtain high-purity chicken CEBPγ recombinant protein.

The invention also discloses the application of the above chicken CEBPγ recombinant protein in research and production of chicken animal husbandry.

Beneficial effect: compared with the prior art, the present invention has the following advantages: the DNA sequence of the chicken Cebpγ provided by the present invention can realize its soluble expression in Escherichia coli under corresponding temperature and inducer concentration, and the preparation of recombinant protein The method has the advantages of easy method, simple operation, high expression amount and low cost, and the chicken CEBPγ recombinant protein with high purity can be obtained by the method provided by the present invention. Its main advantage is that the expression of the chicken Cebpγ gene fragment is optimized, and its expression in E. coli is improved, and the expression of soluble recombinant protein is realized. Through the one-step purification method of nickel ion affinity chromatography, high-purity can be obtained. Chicken C/EBPγ recombinant protein.

Description of drawings

Figure 1: Analysis results of the GC content of the chicken Cebpγ gene optimized fragment (the Y axis represents the GC content of the site (window size is 30bp), and the X axis represents the oligonucleotide residue site);

Figure 2: Codon adaptation index analysis results of chicken Cebpγ gene optimized fragments (Y-axis is the relative usage frequency of codons in E. coli, X-axis represents the corresponding position of each codon in the gene fragment);

Figure 3: Expression analysis results of recombinant protein after chicken Cebpγ gene optimization (M is Premixed ProteinMarker; 1-4 are 0.05mmol/L, 0.1mmol/L, 0.5mmol/L, 1.0mmol/L IPTG induced at 37℃, respectively The total bacteria lysate of 4h, A is the recombinant bacteria constructed by the natural fragment of chicken C gene, and B is the recombinant bacteria constructed by the optimized fragment of chicken C/EBPγ gene);

Figure 4: Soluble expression analysis results of recombinant protein after chicken Cebpγ gene optimization (M is PremixedProtein Marker; 1 is total bacterial lysis protein, 2 is bacterial lysis supernatant, 3 is bacterial lysis precipitate; A, B, C are respectively At 18°C, the induction results of different concentrations of IPTG (the final concentration was 0, 0.05, and 1.0 mmol/L in turn);

Figure 5: Purification results of chicken CEBPγ recombinant protein (M is Premixed Protein Marker; 1 is the total protein of BL21(DE3)-pET 30a blank strain induced lysis (control), 2 is the total protein of cell lysis that has not been induced by recombinant bacteria, 3 is The total protein of bacterial cell lysis induced by recombinant bacteria, 4 is the supernatant of bacterial cell lysis induced by recombinant bacteria, 5 is Ni column purification column liquid, 6 is washing solution, and 7-10 are imidazole eluates of different concentrations (imidazole eluates). Concentrations are 150, 200, 250, 300 mmol/L in sequence); induction conditions: 18°C, final IPTG concentration of 0.05 mmol/L, induction for 12 h);

Figure 6: Western blot identification results of recombinant protein CEBPγ.

Detailed ways

The present application will be described in detail below with reference to specific embodiments.

Embodiment 1: A kind of optimized expression of chicken CEBPγ recombinant protein and preparation method thereof

1. Optimization and synthesis of the expression sequence of the chicken Cebpγ gene

According to the cDNA and protein sequences of the chicken Cebpγ gene (NM_206859.1) provided by the GenBank database, the sequence analysis was carried out, and the dominant codon of the protein in E. In principle, the coding region sequence of the cDNA sequence of the chicken Cebpγ gene should be optimized, as shown in Figure 1-2. According to the diagram, it can be seen that the GC content of the optimized gene fragment is about 51%, and the selected codons are biased towards the commonly used E. coli. a. The sequence is shown in SEQ ID NO: 1, and the Nde I and Xho I restriction site sequences were added at the 5' and 3', respectively, and cloned into the pET30a expression plasmid to obtain the recombinant plasmid pET30a-gCebpγ-hc.

SEQ ID NO: 1

ATGAGCAAAACCAGCCCGCAAAATACCGCAACCGATGCAAACGGCGTTAGCGTTATCCATACCCAAGCACATAGTAGCGGTCTGCAACAAGTTCCGCAACTGGTTCCGGTTTCTCCGGGTGGTGGCGGTAAAGCAGTTCCGCCGAGTAAACAGGGCAAAAAGAACAGCTTCGTCGACCGCAACTCAGACGAATATCGTCAGCGTCGCGAACGTAACAACATGGCGGTCAAAAAATCCCGCCTGAAAAGCAAACAGAAAGCGCAGGATACCCTGCAACGCGTTACCCAACTGAAAGAAGAGAACGAGCGCCTGGAAGCGAAAATCAAACTGCTGACCAAAGAGCTGAGCGTCCTGAAAGACCTGTTTCTGGAACACGCACATAGCCTGGCAGATAACGTTCA ACCGGTTGGTACCGAAAGCACCACCACCTCTGCAGAAAATAGCGGTCAACTCGAGCACCACCACCACCACCACTGA

2. Construction of chicken Cebpγ gene recombinant expression strain BL21(DE3)-pET30a-gCebpγ-hc

The recombinant plasmid was transformed into BL21 (DE3) E. coli by the CaCl ₂ method, and 50 μg/mL kanamycin (kana) resistance was screened. The positive colonies were picked, and after culturing at 37°C for 4 hours, 50 μL of bacterial liquid was collected, heated at 100°C for 15 min, and centrifuged at 12,000 rpm for 3 min. The lysed bacterial liquid was used as a template for identification by PCR.

3. Inducible expression of chicken CEBPγ recombinant protein

The positive bacteria detected by PCR were picked and inoculated into 5 mL of LB medium containing 50 μg/mL kana, shaken at 37°C and 225 rpm for 12 h, and the cultured bacteria were inoculated into 3 mL of fresh LB medium (containing 50 μg/mL) at a ratio of 1:25. mL of kana), shake at 37°C and 225rpm until the OD ₆₀₀ is about 0.6-0.8, add IPTG of different final concentrations, continue to culture at 37°C and 225rpm for 4h, take 100μL of culture medium, centrifuge at 12,000rpm for 3min, discard the Collect the bacterial cells, add 100 μL of 1× SDS loading buffer, heat at 100 °C for 15 min, centrifuge at 12,000 rpm for 3 min, take the supernatant, and conduct electrophoresis on a 10% SDS-PAGE polyacrylamide gel for identification. The results are shown in Figure 3 As shown, after the Cebpγ gene was optimized, the expression of the recombinant protein was significantly increased, while the expression of the recombinant protein was basically not detected in the natural gene fragment in E. coli. Therefore, BL21(DE3)-pET30a-gCebpγ-hc was induced to express recombinant protein at low temperature (18°C) for 12 h with IPTG of 0.05mmol/L and 1.0mmol/L respectively, and the soluble expression of recombinant protein was detected. The results are shown in Figure 4 As shown, although most of the recombinant proteins formed inclusion bodies, the recombinant proteins were clearly seen in the lysis supernatant, and the soluble recombinant protein obtained under the induction condition of 0.05 mmol/L was significantly higher than that induced by 0.1 mmol/L.

4. Purification of Chicken CEBPγ Recombinant Protein

The recombinant bacteria were induced to express soluble CEBPγ protein by 0.05mmol/L IPTG at 18°C for 12h, centrifuged at 12,000g for 10min at 4°C, and the bacterial pellet was collected. Then use equilibration buffer (50 mmol/L phosphate buffer, 0.5 mol/L NaCl, 1 mmol/L MgCl ₂ , 80 mmol/L imidazole, 1 mmol/L PMSF, 1 mmol/L MgCl ₂ , 0.5% Tween-20, pH 7.2) After washing the cells once, resuspend the cells with equilibration buffer (ie lysate) containing 2 mg/mL lysozyme. After shaking and digesting on ice for 45 min, under the power of 550w, the ultrasonic working time was 2 sec and the working interval was 4 sec. 10min, and then repeat the above sonication once after standing in an ice-water bath for 10min. Centrifuge at 4°C and 12,000 rpm for 45 min; filter the supernatant with a 0.45 μm filter to collect the supernatant. Wash the equilibrated Ni-NTA affinity chromatography column with equilibration solution for 10 column volumes, flow the sample through the Ni-NTA affinity chromatography column to adsorb the recombinant protein, and wash and remove it with 10 column volumes of equilibration buffer containing 100 mmol/L imidazole. The non-specifically adsorbed impurity proteins were then eluted with an eluent containing 150, 200, 250, and 300 mmol/L imidazole (50 mmol/L phosphate buffer, 0.5 mol/L NaCl, pH 7.2), respectively, to collect the target protein. The purification results are shown in Figure 5. Ni-NTA can adsorb the soluble recombinant protein in the lysate. When the imidazole concentration reaches 200 mmol/L, the adsorbed recombinant protein is eluted out, and with the increase of the imidazole concentration, the recombinant protein is washed out. Removal increases, and the purity is above 99%.

The purified chicken CEBPγ recombinant protein was identified by Western blot, and the results are shown in Figure 6. Among them, 1 is the total protein of BL21(DE3)-pET 30a blank strain induced lysis (control), and 2 is the uninduced lysis of the recombinant bacteria. Total protein, 3 is the total protein of bacterial cell lysis induced by recombinant bacteria, 4 is the supernatant of bacterial cell lysis induced by recombinant bacteria, 5 is Ni column purification column liquid, 6 is washing solution, and 7-10 are imidazole of different concentrations respectively. Eluent (imidazole concentration is 150, 200, 250, 300 mmol/L in sequence); the primary antibody is rabbit anti-6×His polypeptide antibody, and the detection result shows that a detection band is obtained at the position of 20KDa, indicating that the CEBPγ recombinant protein of chicken induces expression and purification was successful.

sequence listing

<110> Changshu Institute of Technology

<120> Chicken recombinant CEBP gamma protein, its coding DNA sequence and application

<160> 2

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atgagcaaaa ccagcccgca aaataccgca accgatgcaa acggcgttag cgttatccat 60

acccaagcac atagtagcgg tctgcaacaa gttccgcaac tggttccggt ttctccgggt 120

ggtggcggta aagcagttcc gccgagtaaa cagggcaaaa agaacagctt cgtcgaccgc 180

aactcagacg aatatcgtca gcgtcgcgaa cgtaacaaca tggcggtcaa aaaatcccgc 240

ctgaaaagca aacagaaagc gcaggatacc ctgcaacgcg ttacccaact gaaagaagag 300

aacgagcgcc tggaagcgaa aatcaaactg ctgaccaaag agctgagcgt cctgaaagac 360

ctgtttctgg aacacgcaca tagcctggca gataacgttc aaccggttgg taccgaaagc 420

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Met Ser Lys Thr Ser Pro Gln Asn Thr Ala Thr Asp Ala Asn Gly Val

1 5 10 15

Ser Val Ile His Thr Gln Ala His Ser Ser Gly Leu Gln Gln Val Pro

20 25 30

Gln Leu Val Pro Val Ser Pro Gly Gly Gly Gly Lys Ala Val Pro Pro

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Ser Lys Gln Gly Lys Lys Asn Ser Phe Val Asp Arg Asn Ser Asp Glu

50 55 60

Tyr Arg Gln Arg Arg Glu Arg Asn Asn Met Ala Val Lys Lys Ser Arg

65 70 75 80

Leu Lys Ser Lys Gln Lys Ala Gln Asp Thr Leu Gln Arg Val Thr Gln

85 90 95

Leu Lys Glu Glu Asn Glu Arg Leu Glu Ala Lys Ile Lys Leu Leu Thr

100 105 110

Lys Glu Leu Ser Val Leu Lys Asp Leu Phe Leu Glu His Ala His Ser

115 120 125

Leu Ala Asp Asn Val Gln Pro Val Gly Thr Glu Ser Thr Thr Thr Ser

130 135 140

Ala Glu Asn Ser Gly Gln His His His His His His

145 150 155

Claims (7)

1. a kind of DNA sequence dna for encoding chicken recombinant C EBP γ albumen, which is characterized in that its nucleotide sequence such as SEQ ID NO:1 It is shown.

2. a kind of recombinant expression carrier containing chicken recombinant C EBP γ protein DNA sequence described in claim 1.

3. a kind of transgenosis recombinant escherichia coli strain containing chicken recombinant C EBP γ protein DNA sequence described in claim 1.

4. application of the chicken recombinant C EBP γ protein DNA sequence described in claim 1 in preparation chicken SIRT1 recombinant protein.

5. a breeder recombinant C EBP γ albumen, which is characterized in that amino acid sequence is as shown in SEQ ID NO:2.

6. the preparation method of chicken recombinant C EBP γ albumen described in claim 5, which comprises the following steps:

(1) according to chicken Cebp γ gene encode amino acid polypeptide sequence, preferences of combining cipher in Escherichia coli and The analysis of RNA secondary structure optimizes the cDNA segment of chicken Cebp γ gene；

(2) pET30a-gCebp γ-hc recombinant expression carrier is constructed with the segment synthesized in step (1)；

(3) BL21 (DE3)-pET30a-gCebp γ-hc recombinant bacterial strain is constructed；

(4) inducing expression, purifying and the identification of chicken CEBP γ recombinant protein.

7. application of the chicken CEBP γ recombinant protein in terms of production described in claim 5.