CN101509007B - Synthesis of LfcinB15-Mag12 encoding gene and expression method in colon bacillus - Google Patents

Abstract

The invention provides a method for synthesizing and expressing the LfcinB15-Mag12 coding gene in Escherichia coli, and designing the active gene fragment encoding Lfcin B1-15 amino acids and Magainin (1-12) according to the codon preference of E.coli Active gene fragment, use the expression vector pET-32a to construct the recombinant expression vector pET-32a-Lfcin B15-Mag12, there are codons encoding 6 histidine tags before the multiple cloning site of pET-32a, and a stop codon is added at the same time TAG, TAA. The present invention successfully expresses LfcinB15-Mag12 in Escherichia coli using genetic engineering methods, laying a theoretical and practical foundation for the large-scale production of hybrid peptides and other valuable peptides by genetic engineering methods, and is useful for the establishment of peptide genetic engineering bacteria and antibacterial peptide research The common technology is of great significance.

Description

Synthesis of LfcinB15-Mag12 coding gene and method for its expression in Escherichia coli

(1) Technical field

The invention belongs to the application field of agriculture, animal husbandry and veterinary medicine, and specifically relates to an LfcinB15-Mag12 coding gene technology. the

(2) Background technology

The development of antibiotics has a history of more than 50 years. Because it can prevent the occurrence of livestock and poultry diseases, improve animal production capacity, and improve the efficiency of breeding industry, it has always been an important part of the additive premix. However, the long-term use of antibiotic additives can cause many pathogenic bacteria to develop drug resistance, and at the same time, serious drug residues will be produced in animal products, which will have adverse effects on the further prevention and treatment of livestock and poultry diseases and human health. Therefore, the use of antibiotic additives is increasingly strictly restricted in countries all over the world. Since January 1, 2006, the European Union has completely banned the addition of antibiotic feed additives for the purpose of health care and growth promotion in feed. In order to find alternatives to antibiotics and produce green feed additives that can effectively prevent the occurrence of livestock and poultry diseases, promote animal growth, and have little toxic and side effects, no residue, and no drug resistance, many animal husbandry and veterinary workers at home and abroad have conducted a large number of research has made some progress. The antibiotic substitutes they studied mainly include: enzyme preparations, acidifiers, probiotics, probiotics, plant extracts, Chinese herbal medicines, saccharoterpenoids, etc. However, these antibiotic substitutes all have shortcomings such as high production costs, ineffective effects, and immature extraction and production techniques. the

Antimicrobial peptides are a class of small molecule polypeptides that resist external microbial invasion and eliminate mutant cells in animals. They have the advantages of broad-spectrum antibacterial, non-toxic, non-drug resistance, no residue and pollution, etc., and they have good thermal stability. It is small, suitable for use in the feed production process, has the same performance as antibiotics and fully meets the needs of safe production of livestock products, and has great potential as a new generation of feed additives. In recent years, the research and development of antimicrobial peptides has become a research hotspot. On the one hand, people continue to search for new cationic antimicrobial peptides, and on the other hand, they modify known natural antimicrobial peptides in order to obtain efficient and safe antibacterial drugs. However, the sources of natural antimicrobial peptides are limited, the extraction is difficult, and most of them have the defects of unsatisfactory activity or hemolytic toxicity. Therefore, using naturally occurring antimicrobial peptides as templates to design synthetic derivatives is more important for the study of antimicrobial peptide mechanism of action and synthetic activity. Antimicrobial peptides with lower toxicity, more stability and broader spectrum are of great significance, and the method of obtaining antimicrobial peptides by constructing bioreactors through genetic engineering technology shows broad prospects and great potential. the

(3) Contents of the invention

The object of the present invention is to provide a coding gene for the hybrid peptide LfcinB15-Mag12 designed according to the codon preference of Escherichia coli, and divide the forward and reverse strands of the coding gene into four polynucleotide chains to synthesize them, and use genetic engineering methods to generate them in Escherichia coli LfcinB15-Mag12 was successfully expressed. the

The object of the present invention is achieved like this: according to E.coli codon bias, design the active gene fragment of encoding Lfcin B 1-15 amino acids and the active gene fragment of Magainin (1-12), utilize expression vector pET-32a to construct recombination In the expression vector pET-32a-Lfcin B15-Mag12, there are codons encoding 6 histidine tags before the multiple cloning site of pET-32a. In pET-32a-Lfcin B15-Mag12, Lfcin B15-Mag12 will be encoded The 27-amino acid gene fragment was cloned after the codon encoding the histidine tag, between the codons of the restriction endonuclease Sal I and Nco I recognition sites, and the stop codons TAG and TAA were added at the same time.

The four-segment synthetic sequence is as follows:

Fragment 1: 5'-CATGGCTTTCAAATGCCGCCGTTGGCAGTGGCGTTGGAAAAAACTGGGTGCGGGTATCG-3'

Fragment 2: 5'-GTAAATTCCTGCACTCTGCTAAAAAATTC TAGTAA G-3'

Fragment 3: 5'- TCGACTTACTAGAATTTTTTAGCAGAGGTGCAGGAATTTACCGATACCCGCACCCAGTTT -3'

Fragment 4: 5'-TTTCCAACGCCACTGCCAACGGCGGCATTTGAAAGC-3'

The underlined part is the added stop codon TAG, TAA, the italic part is the recognition sequence of the restriction endonuclease Sal I, Nco I, and the rest is the bovine lactoferrin-Magainin hybrid peptide gene. the

The present invention also has some technical features: in order to facilitate direct cloning into the pET-32a treated with double enzyme digestion, the sticky end of the restriction site is directly designed into the Lfcin B15-Mag12 gene, that is, in the Lfcin B15-Mag12 gene The upstream and downstream of the restriction endonucleases Sal I and Nco I recognition sites were added to the fragments digested. the

The technical characteristics of the present invention have:

1. According to the codon preference of Escherichia coli, the coding gene of the hybrid peptide LfcinB15-Mag12 was designed, and the positive and negative strands of the coding gene were synthesized into four polynucleotide chains. After annealing, the hybrid peptide LfcinB15-Mag12 was successfully obtained. coding genes. the

2. Directly introduce cohesive ends at both ends of the synthesized LfcinB15-Mag12 coding gene fragment, connect it into the expression vector pET-32a, and construct the expression vector pET-32a-LfcinB15-Mag12, which is completely consistent with the design by sequencing. the

3. The expression vector pET-32a-LfcinB15-Mag12 was transformed into the expression host E.coli DH5α, induced with 0.1mmol/L IPTG at 30°C for 3h, and a specific induced expression band with the expected molecular weight of 23.9kD was produced, which was analyzed by Western blot , the induced band with a molecular weight of 24kDa is a TRX fusion protein, which proves that the fusion protein Trx-S-His-LfcinB15-Mag12 is successfully expressed. the

4. Optimize the optimal induction conditions. Under the determined optimal induction conditions, that is, when the cell density reaches OD600 = 0.6, induction begins, 0.3mmol/L IPTG, 30°C induction for 4h, the expression of fusion protein accounted for 22% of total body protein. the

5. Purify and recover the fusion protein Trx-S-His-LfcinB15-Mag12 through NTA-affinity chromatography column, the purity is higher, reaching more than 95%, but the yield is low, and on average, only 19mg fusion protein is obtained per liter of induced bacterial culture . the

6. Use enterokinase to cleavage the fusion protein Trx-S-His-LfcinB15-Mag12 according to the ratio of enzyme: protein ratio of 1:100, complete cleavage can be achieved at 23°C for 6 hours, and ultrafiltration through an ultrafiltration tube with a molecular weight cut-off of 5kDa Afterwards, a relatively pure hybrid peptide LfcinB15-Mag12 was obtained, and an average of 200 μg of LfcinB15-Mag12 was obtained per liter of induced bacterial culture. the

7. The antibacterial activity of the recombinant hybrid peptide LfcinB15-Mag12 was detected by the thin-layer plate agarose disc diffusion method, and it had obvious inhibitory effect on Staphylococcus aureus ATCC25923, indicating that this experiment obtained recombinant LfcinB15-Mag12 with biological activity , the heterologous expression of the antibacterial peptide LfcinB15-Mag12 was successfully realized for the first time by using genetic engineering methods. the

According to the codon preference of Escherichia coli, the present invention designs the coding gene of the hybrid peptide LfcinB15-Mag12, divides the positive and negative strands of the coding gene into four polynucleotide chains and synthesizes them. coding genes. In this study, LfcinB15-Mag12 was successfully expressed in Escherichia coli using genetic engineering methods, which laid a theoretical and practical foundation for the large-scale production of hybrid peptides and other valuable peptides by genetic engineering methods. The common technology is of great significance. the

(4) Description of drawings

Fig. 1 is the antibacterial figure of the present invention;

Fig. 2 is a process flow diagram of the present invention;

Fig. 3 is the schematic diagram of the synthetic LfcinB15-Mag12 coding gene of purification, and wherein 1 is DNA molecular weight standard, and 2 is the Lfcin B15-Mag12 gene that is used for constructing pET-32a-Lfcin B15-Mag12 of purification;

Fig. 4 is the schematic diagram of positive recombinant plasmid pET-32a and pET-32a-Lfcin B15-Mag12 screened by PCR method, 1 is the DNA molecular weight standard, 2 is the PCR amplification product of pET-32a blank control as template, and 3 is the PCR amplification product containing pET- 32a-LfcinB15-Mag12 colony is the PCR amplification product of the template;

Figure 5 is a schematic diagram of SDS-PAGE analysis of the expression products of pET-32a and pET-32a-Lfcin B15-Mag12 transformants, 1 is the protein molecular weight standard, 2 is the expression products of pET-32a transformants, and 3 is the uninduced pET-32a- The Lfcin B15-Mag12 transformant induces the expression product, and 4 is the pET-32a-Lfcin B15-Mag12 transformant induces the expression product. the

(5) Specific implementation methods

Below in conjunction with accompanying drawing and specific embodiment the present invention will be further described:

In conjunction with Fig. 2, the technical route taken in this embodiment is:

1. Directly introduce cohesive ends at both ends of the synthetic LfcinB15-Mag12 coding gene fragment, connect it into the expression vector pET-32a, and construct the expression vector pET-32a-LfcinB15-Mag12;

2. Transform the expression vector pET-32a-LfcinB15-Mag12 into the expression host E.coli BL21(DE3), induce it with 0.1mmol/LIPTG at 30°C for 3h, and produce a specifically induced expression band with the expected molecular weight of 23.9kD;

3. Optimize the optimal induction conditions. Under the determined optimal induction conditions, the induction starts when the cell density reaches OD600=0.6, 0.3mmol/L IPTG, 30°C induction for 4h, the expression of the fusion protein accounted for 22% of total body protein;

4. The fusion protein Trx-S-His-LfcinB15-Mag12 was purified and recovered by NTA-affinity chromatography column, with a purity of more than 95%, and an average of only 19 mg of fusion protein was obtained per liter of induced bacterial culture;

5. Cleavage the fusion protein Trx-S-His-LfcinB15-Mag12 with enterokinase according to the ratio of enzyme: protein mass ratio of 1:100, complete cleavage can be achieved at 23°C for 6 hours, and ultrafiltration through ultrafiltration tube with a molecular weight cut-off of 5kDa Obtain relatively pure hybrid peptide LfcinB15-Mag12 afterwards, obtain LfcinB15-Mag12200 μ g per liter of induced bacterial culture on average;

6. The antibacterial activity of the recombinant hybrid peptide LfcinB15-Mag12 was detected by thin-layer plate agarose disk diffusion method. the

The specific implementation process of this embodiment:

1. Synthesis of LfcinB15-Mag12 gene code

According to E.coli codon bias (as shown in Table 1, Grojean et al., 1982; Hale et al., 1998) design the active gene fragment encoding LfcinB 1-15 amino acids and the active gene fragment of Magainin (1-12), as To express recombinant LfcinB15-Mag12, use the expression vector pET-32a to construct the recombinant expression vector pET-32a-Lfcin B15-Mag12, and there are codons encoding 6 histidine tags before the multiple cloning site of pET-32a, so in pET In -32a-Lfcin B15-Mag12, the gene fragment encoding Lfcin B15-Mag12 27 amino acids was cloned after the codon encoding the histidine tag and between the codons of the restriction endonuclease SalI and NcoI recognition sites. At the same time, stop codons TAG and TAA were added. the

In order to facilitate direct cloning into pET-32a treated with double digestion, the sticky end of the restriction site was directly designed into the LfcinB15-Mag12 gene, that is, restriction endonucleases were added to the upstream and downstream of the Lfcin B15-Mag12 gene Fragments of enzymes Sal I and Nco I recognition sites after digestion. the

The four-segment synthetic sequence is as follows:

Fragment 1: 5'-CATGGCTTTCAAATGCCGCCGTTGGCAGTGGCGTTGGAAAAAACTGGGTGCGGGTATCG-3'

Fragment 2: 5'-GTAAATTCCTGCACTCTGCTAAAAAATTC TAGTAA G-3'

Fragment 3: 5'- TCGACTTACTAGAATTTTTTAGCAGAGGTGCAGGAATTTACCGATACCCGCACCCAGTTT -3'

Fragment 4: 5'-TTTCCAACGCCACTGCCAACGGCGGCATTTGAAAGC-3'

The underlined part is the added stop codon TAG, TAA, the italic part is the restriction endonuclease SalI, NcoI recognition sequence, and the rest is the bovine lactoferrin-Magainin hybrid peptide gene. the

Table 1 The frequency of codon usage in E.coli

Continued Table 1

a usage frequency a usage frequency a usage frequency Arg AGC 0.00 Tyr TAT 0.18 Pro CCG 0.87 Arg AGA 0.00 Tyr TAC 0.82 Pro CCA 0.13 Ser AGT 0.00 Leu TTG 0.00 Pro CCT 0.00 Ser AGC 0.17 Leu TTA 0.00 Pro CCC 0.00 Lys AAG 0.18 Phe TTT 0.17 Lys AAA 0.82 Phe TTC 0.83

1.1. Phosphorylation of the 5' end of polynucleotide fragments 2 and 4

Chemically synthesized polynucleotide fragments, whose 5' end is in the form of -OH, need to be phosphorylated before the ligation reaction can be performed. In the presence of ATP, T4 polynucleotide kinase can transfer the γ-phosphate group of ATP To the 5' end of the nucleotide fragment, replace -OH. the

reaction system:

Gene Fragment 2 or 4 10μL

10×T4 polynucleotide kinase buffer 5μL

ATP 10μL

T4 polynucleotide kinase 2 μL (20 units)

Water 23μL

Total volume 50μL

Inactivate T4 polynucleotide kinase activity in a water bath at 37°C for 1 hour and at 65°C for 20 minutes. the

1.2. Annealing and ligation of polynucleotide fragments

reaction system

Gene fragment 2 phosphorylation reaction system solution 10 μL

Gene fragment 4 phosphorylation reaction system solution 10 μL

Gene Fragment 1 5 μL

Gene Fragment 3 5 μL

3mol/L NaCl solution 4μL

Water 6μL

Total volume 40μL

The above reaction system was denatured at 95°C for 5 minutes, then naturally lowered to 40°C, maintained for 2 hours, added 5 μL of 10× ligase buffer, 0.2 μL (2 units) of T4 DNA ligase, supplemented with 50 μL of H ₂ O, and ligated overnight at 16°C

1.3. Recovery of the gene encoding Lfcin B15-Mag12

1) Perform 2.5% gel electrophoresis on the annealed and ligated polynucleotide fragments, recover using the Qiagen gel recovery kit, and perform according to the kit operation manual;

2) Cut off the gel piece containing the target DNA fragment under the ultraviolet light, make it as small as possible, and put it into a pre-weighed 1.5mL centrifuge tube;

3) Weigh the centrifuge tube containing the gel block again, calculate the mass of the gel block, add the sol solution at a ratio of 600 μL per 100 mg of the gel block, and bathe in water at 50°C for 10 min to completely dissolve the agarose gel block, every 2 min Mix once;

4) Add isopropanol at a ratio of 100 μL per 100 mg of gel block, and mix well;

5) Transfer the gel solution into the adsorption column, centrifuge for 1 min, pour off the liquid in the collection tube, and then put the adsorption column into the same collection tube;

6) Add 750 μL of washing solution to the adsorption column, let it stand for 2 minutes, centrifuge for 1 minute, pour off the liquid in the collection tube, and put the adsorption column into the same collection tube;

7) centrifuge for 1min, and remove the washing solution as much as possible;

8) Put the adsorption column into a clean 1.5mL centrifuge tube, add 30-50μL eluent to the center of the adsorption membrane, let it stand for 1min, centrifuge for 1min, and store the 1.5mL centrifuge tube (containing DNA) at -20°C spare. the

2. Construction of recombinant vector

2.1. Enzyme digestion treatment of plasmid pET-32a

Sal I, and Nco I digestion plasmid pET-32a reaction system:

Plasmid pET-32a 2μg

10×Sal Ibuffer 5μL

Nco I 1μL (20 units)

Sal I 1μL (20 units)

Water 37μL

Total volume 50μL

37°C water bath for 3h, 65°C for 20min to inactivate the enzyme activity. the

2.2. Purification and recovery of linearized plasmid pET-32a

The enzyme digestion treatment reaction system of plasmid pET-32a was subjected to 0.8% gel electrophoresis, purified and recovered linear plasmid pET-32a according to the method in 2.2.1.4, and stored at -20°C for future use. the

2.3. Preparation of Competent E.coli

1) Take the frozen-preserved host bacteria, inoculate the slant with an inoculation loop, revive and culture for 2 generations, then pick a single colony, transfer it to a 250mL Erlenmeyer flask containing 50mL of LB medium, and culture it overnight at 37°C with shaking. the

2) Take 500 μL of bacterial liquid and transfer it into fresh 50 mL LB medium. When the bacterial cell density grows to OD600=0.4, transfer the bacterial liquid to a sterile, single-use, 50 mL polypropylene pre-cooled with ice. Tubes were placed on ice for 10 min. the

3) Centrifuge at 4100 rpm for 10 min at 4°C to recover the cells. the

4) Pour out the culture solution and invert the tube for 1 min to make the last trace of the culture solution flow out. the

5) Resuspend each cell pellet with 30 mL of pre-cooled 0.1 mol/L MgCl ₂ -CaCl ₂ solution (80 mmol/L MgCl ₂ , 20 mmol/L CaCl ₂ ) for every 50 mL of initial culture medium.

6) Centrifuge at 4100 rpm for 10 min at 4°C to recover the cells. the

7) Pour out the culture solution and invert the tube for 1 min to drain the last trace of the culture solution. the

8) Resuspend each 50 mL of initial culture with 2 mL of ice-cold 0.1 mol/L CaCl ₂ and set aside.

2.4. Ligation and transformation of Lfcin B15-Mag12 coding gene and plasmid pET-32a

Ligation system between gene encoding LfcinB and plasmid pET-32a

10×ligase buffer 1μl

                                                                        

Plasmid pET-32a 2μl (100ng)

T4DNA ligase 0.1μL

H ₂ O 5.9 μL

Total volume 10μL

Ligate overnight at 16°C and transform E.coli BL21(DE3). the

1) Use a pre-cooled sterile tip to draw 200 μL from each portion of competent cell suspension prepared with CaCl ₂ solution and transfer it to a sterile centrifuge tube, and add DNA to each tube (the amount used shall not exceed 10 μl, and the DNA in it shall be less than 50ng), swirl gently to mix the contents, and place on ice for 30min;

2) Put the tube into a circulating water bath preheated to 42°C, and leave it for exactly 90 seconds without shaking the tube;

3) Quickly transfer the tube to an ice bath to cool the cells for 2 minutes;

4) Add 800 μl of SOC medium to each tube, warm the medium to 37°C with a water bath, then transfer the tube to a shaker, and incubate at 37°C for 45 minutes to recover the bacteria and express the antibiotic marker gene encoded by the plasmid;

5) An appropriate volume (up to 200 μl per 90 mm plate) is transferred to the LB agar medium containing 20 mmol/L MgSO and ₁₀₀ μg/mL ampicillin with transformed competent cells;

6) Place the plate at room temperature until the liquid is absorbed, invert the plate, and incubate at 37°C for 12-16 hours. the

2.5. PCR method to screen positive clones

Primers used:

Primer 1: 5′-GGG CTG GCA AGC CAC GTT TGG TG-3′,

Primer 2: 5′-CCG GGA GCT GCA TGT GTC AGA GG-3′,

reaction system:

10×buffer(Mg ²⁺ ) 2μL

Primer1 0.4μL

Primer2 0.4μL

dNTP(2.5mmol/L) 0.4μL

Taq DNA polymerase 0.4μL

H ₂ O 16.4 μL

Total volume 20μL

Pick up positive colonies with a sterile toothpick, turn them slightly in the above reaction system solution for several times, and use the colonies without adding colonies and containing pGEX-4T-2 as templates respectively as negative and positive controls for PCR amplification.

Reaction conditions: 94°C pre-denaturation for 5 minutes

72℃ Extend 1min

72℃ Extend 5min

4°C 5min

PCR amplification products were detected by 2.5% agarose gel electrophoresis, and positive clones were further sequenced for verification. the

2.6. Small amount of plasmid extraction by SDS alkaline lysis method

Principle: Alkaline denaturation extraction of plasmid DNA is based on the difference between denaturation and renaturation of chromosomal DNA and plasmid DNA to achieve the purpose of separation. Under alkaline conditions with a pH as high as 12.6, the hydrogen bonds of chromosomal DNA are broken, and the double-helix DNA structure is untied and denatured. Most of the hydrogen bonds of the plasmid DNA are also broken, but the supercoiled covalently closed and circular two complementary strands are not completely separated. When the pH is adjusted to neutral with NaAc high-salt buffer of pH 4.8, the The plasmid DNA restores its original configuration and is stored in the solution, while the chromosomal DNA cannot be refolded and forms a tangled network structure. Through centrifugation, the chromosomal DNA, unstable macromolecular RNA, and protein-SDS complexes are precipitated together. down and removed. the

experiment procedure:

1) Pick a single colony containing the positive clone of interest, inoculate it into 2 mL of LB culture medium containing 100 μg/mL Amp, and incubate overnight at 37°C with shaking;

2) Take 1mL of the culture in a 1.5mL centrifuge tube, centrifuge at the maximum speed of 4°C for 30s with a microcentrifuge, store the rest of the culture at 4°C, and blot the culture medium as dry as possible after centrifugation;

3) Resuspend the bacterial pellet in 100 μL of ice-cold alkaline lysate I and shake vigorously;

4) Add 200 μL of newly prepared alkaline lysate II to each tube of bacterial suspension, cap the tube tightly, quickly invert the centrifuge tube 5 times to mix the contents, do not shake, and place the centrifuge tube on ice;

5) Centrifuge at 4°C for 5 minutes, and take the supernatant;

6) Use 2 times the volume of absolute ethanol to precipitate nucleic acid at room temperature, shake and mix, and place at room temperature for 2 minutes;

7) Centrifuge at 4°C for 5 minutes to collect the precipitated nucleic acid;

8) Carefully suck out the supernatant, put the centrifuge tube upside down on a paper towel, so that all the liquid can flow out and drain, and use a disposable tip to remove the liquid droplets on the tube wall;

9) Add 1mL of 70% ethanol to the precipitate and invert the tightly capped test tube several times, centrifuge at 4°C for 2min, recover DNA and recover the precipitate;

10) Remove the alcohol droplets on the tube wall, place the open test tube at room temperature to evaporate the alcohol until there is no visible liquid in the test tube;

11) Redissolve the nucleic acid with 50 μL of TE containing DNase-removing RNase A (trypsinase) (20 μg/mL), shake gently for a few seconds, and store at -20°C. the

3. Expression of fusion protein and optimization of induction conditions

3.1. IPTG-induced expression of fusion protein

1) Transform the recombinant plasmid into E.coli BL21(DE3) according to the method in 2.4;

2) Inoculate the transformed colony in 5 mL of LB medium containing 100 μg/mL ampicillin, draw a line on the LB/ampicillin slant, and inoculate transformed bacteria containing the parental pET-32a vector as a control. The slant culture was cultured at 37°C for 12 hours and then stored at 4°C, and the liquid culture was cultured overnight in a shaker at 37°C;

3) Transfer 500 μL of the bacterial solution into 50 mL of LB medium containing 100 μg/mL ampicillin, culture on a shaker at 37°C until OD600=0.6, add 100mmol/L IPTG to a final concentration of 0.1mmol/L, and induce at 30°C for 3 Hour;

4) Take 1mL of bacterial liquid, centrifuge at 12000 rpm for 15s at high speed, and discard the supernatant;

5) Add 100 μL of ice-cold PBS, pipette evenly, pipette 5 μL into another centrifuge tube, and wait for SDS-PAGE analysis. the

3.2. SDS-PAGE analysis

Principle: Biomacromolecules, especially proteins, have different charges and molecular weights. After being treated with the anionic detergent SDS, the charges on the protein molecules are neutralized. During polyacrylamide gel electrophoresis, different proteins are distributed according to their molecular weight, and the electrophoretic mobility depends only on the molecular weight of the protein. Polyacrylamide gel electrophoresis consists of acrylamide monomer and methylene bisacrylamide, under the action of a catalyst, to form a three-dimensional network structure. Gel electrophoresis not only has a molecular sieve effect, but also a concentration effect. Due to the discontinuous pH gradient, the sample is compressed into a narrow zone, which improves the separation effect. Rapid staining with Coomassie Brilliant Blue allows timely observation of the electrophoretic separation effect. the

Experimental steps:

1) Wash the glass plate and fix it on the electrophoresis tank;

2) Prepare 15% separating gel according to the formula in Table 2, inject the separating gel into the glass interlayer, cover the upper part with a small amount of water, keep the surface of the gel flat, and prepare the concentrated gel after the separating gel is polymerized;

Table 2 Different concentrations of separation gel formulations

3) Prepare 5% concentrated gel according to Table 3, pour off a small amount of water on the surface of the separating gel, then pour into the gel, and insert the spotting comb;

Table 35% Stacking Gel Formula

4) Sample treatment: Add an equal volume of 2×SDS-PAGE sample treatment solution to the protein sample to be analyzed and identify, put it in a water bath at 100°C for 3-5 minutes, and cool it in an ice bath;

5) Adding samples: add the electrophoresis buffer to the electrophoresis tank, carefully pull out the spotting comb, and blow the sample hole with a syringe. After the sample is centrifuged at high speed, use a micro-sampler to draw and analyze the samples respectively, and determine the sample volume according to the protein concentration and the volume of the sample hole;

6) Initially use low voltage 40V constant voltage electrophoresis, after the bromophenol blue indicator is concentrated into a line in the stacking gel, perform 120V electrophoresis, and stop electrophoresis when the bromophenol blue indicator reaches the bottom edge;

7) Staining: Gently pry open the two layers of glass, take out the gel, cut the corners to mark, place in Coomassie Brilliant Blue R-250 staining solution, and stain overnight;

8) Decolorize the decolorization solution until the background is clean and the bands are clear. the

3.3. Optimization of induction conditions

3.3.1. Determination of the optimal concentration of inducer IPTG

Inoculate the transformed colonies in 5 mL of LB medium containing 100 μg/mL ampicillin, culture overnight in a shaker at 37°C, and add 500 μL of bacterial solution was cultured on a shaker at 37°C until OD ₆₀₀ =0.6, and 100 mmol/L IPTG was added to different Erlenmeyer flasks to final concentrations of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0 mmol/L, respectively. L, induced at 30°C for 3 hours; 5 μL was sampled for SDS-PAGE analysis.

3.3.2. Determination of the best induction time

Inoculate the transformed colonies in 5 mL of LB medium containing 100 μg/mL ampicillin, culture overnight in a shaker at 37°C, and add 500 μL of bacterial solution, after the bacterial concentration was grown to OD600 = 0.6, 100 mmol/L IPTG was added to a final concentration of 0.3 mmol/L, induced at 30°C for 0.5, 1, 3, 5, and 7 hours, and 5 μL was sampled for SDS-PAGE analysis. the

3.4. Western blot analysis

Principle: The protein samples separated by SDS-PAGE are transferred to a solid phase support (such as nitrocellulose membrane). The solid phase support adsorbs proteins in the form of non-covalent bonds, and can maintain the type of polypeptide separated by electrophoresis and its biological activity. constant. Use the protein or polypeptide on the solid phase carrier as the antigen, react with the corresponding antibody, and then react with the enzyme or isotope-labeled secondary antibody, and then check the specific target gene separated by electrophoresis through substrate color development or autoradiography Expressed protein components. The His antibody used in this test is directly coupled with horseradish peroxidase, and after a one-step antigen-antibody reaction, 3,3'-diaminoaniline can be directly used for color reaction. the

3.4.1 Electrotransfer of proteins

1) SDS-PAGE electrophoresis is performed on the induced expression protein sample;

2) Open the transfer box and place it in a shallow dish. After fully soaking the sponge pad with transfer buffer, place it on the wall of the transfer box. Put a piece of 3MM Whatman filter paper on the sponge, place the gel, and use a test tube or The glass rod moves slowly on the surface of the gel to remove the air bubbles between the gel and the filter paper;

3) Prepare the transfer membrane, cut the PVDF membrane according to the size of the gel but each side is about 1mm larger than the gel, slowly put the membrane into distilled water at 45 degrees, the water will penetrate into the membrane and wet the entire surface, and then transfer the buffer Equilibrate in the liquid for 15 minutes;

4) Rinse the surface of the gel with transfer buffer, and place the wetted membrane directly on top of the gel to remove air bubbles;

5) Wet another piece of 3MM Whatman filter paper and place it on the anode side of the membrane to remove all air bubbles, and place another piece of sponge that has been finely moistened with transfer buffer on top of the filter paper;

6) Assemble the transfer box, put the cellulose membrane side against the positive electrode, and the glue side against the negative electrode, add buffer, connect the power supply, and transfer for 1 hour at a horizontal pressure of 100V;

3.4.2. Western blot detection of expressed protein-Western blot detection

1) Put the PVDF membrane into a plate and add the blocking solution (the amount is enough to soak the membrane), seal with 4% skim milk blocking solution, 37°C for 2 hours, discard the reaction solution;

2) Wash the membrane twice with TBST;

3) Dilute the antibody with TBST 1:5000, put the membrane into a plastic bag, add 0.1mL antibody diluent per square centimeter, and block for 1h;

4) Wash the membrane twice with TBST, 5 minutes each time;

5) Wash the membrane twice with TBS, 5 minutes each time;

6) Put the membrane into the chromogenic substrate developing solution, the bands appear within 10-30min, wash the membrane with distilled water, terminate the reaction, and dry in air. the

4. Purification of fusion protein

4.1. Cracking of E.coli

1) Inoculate E.coli BL21(DE3) containing the positive recombinant pET-32a-Lfcin B15-Ma12 in LB liquid medium containing 100 μg/mL Amp, and culture overnight at 37°C on a shaker;

2) Inoculate 0.5 mL of the above overnight culture into fresh LB (containing 100 μg/mL Amp) liquid medium;

3) Shake culture at 37°C for about 3 hours until OD600=0.6, add 100mmol/L IPTG to a final concentration of 0.3mmol/L, and continue to culture at 30°C for 3h;

4) Add GuNTA-0Buffer (20mM Tris-HCl pH7.9, 0.5M NaCl, 10% Glycerol, 6M Guanidium HCl) and PMSF at 1/20 of the cell growth volume. PMSF is made into a 200mM stock solution with absolute ethanol and stored at -20°C or 4°C; the working concentration of PMSF is 1mM; Note: PMSF sees water decomposition and needs to be added before use. the

5) Suspend the cells and ultrasonically break the cells on ice to reduce the viscosity. the

6) Leave it at room temperature for 30 minutes, mix it occasionally or stir it with a magnetic force. the

7) Centrifuge at 15000 rpm (above 20,000xg) at 4 degrees for more than 15 minutes. Take the supernatant and store it on ice or store at -20°C. the

4.2. Purification and elution of fusion protein by affinity chromatography

1) Purification of fusion protein by affinity chromatography

(1) The NTA resin was loaded into a suitable chromatography column (QIAgene), and the chromatography was washed with GuNTA-0Buffer 10 times the volume of NTA. the

(2) Add the sample to the NTA chromatography column, the flow rate is controlled at about 15mL/hour, and the breakthrough fraction is collected for SDS/PAGE analysis of protein binding. the

(3) The chromatography was washed with GuNTA-0 Buffer 5 times the volume of NTA, and the flow rate was controlled at about 30 mL/hour. the

(4) Elute with 5 times the NTA volume of GuNTA-20, GuNTA-40, GuNTA-60, GuNTA-100, GuNTA-500 respectively, and control the flow rate at about 15mL/hour, collect the eluate, and collect one NTA volume in each tube . the

(5) 15% polyacrylamide gel for SDS-PAGE detection. the

2) Regeneration of NTA resin

After the NTA resin has been used for several times (3-5 times), the binding efficiency has decreased, and the following method can be used to regenerate the resin to improve the service life of the resin and the binding efficiency of the protein. Before the regeneration of the NTA resin, it is necessary to drain all the solution from the lower end of the chromatography column. Estimate the volume of the NTA resin, and add the regeneration reagent to the chromatography column in the following order. After the previous regeneration solution is drained, add the next regeneration. dissolve. You need to prepare 25%, 50%, 75%, 100% (v/v) ethanol and deionized water by yourself. the

NTA regeneration steps:

(1) Drain all the solution from the lower end of the chromatography column and wash with Stripping Solution I 2 times the volume of NTA resin. the

(2) Wash with 2 times the volume of deionized water. the

(3) Wash with 3 times the volume of Stripping Solution II. the

(4) Wash with 1 volume of 25% ethanol. the

(5) Wash with 1 volume of 50% ethanol. the

(6) Wash with 1 volume of 75% ethanol. the

(7) Wash with 5 times the volume of 100% ethanol. the

(8) Wash with 1 volume of 75% ethanol. the

(9) Wash with 1 volume of 50% ethanol. the

(10) Wash with 1 volume of 25% ethanol. the

(11) Wash with 1 volume of deionized water. the

(12) Wash with 5 times the volume of Stripping Solution III. the

(13) Wash with 3 times the volume of deionized water. the

(14) If used immediately, wash with 5 times the volume of Ni Charging Solution, and then wash with 10 times the volume of the equilibrium solution (NTA-0Buffer or GuNTA-0Buffer). the

(15) If you want to store for a long time, add 1 volume of 20% ethanol, store at 4 degrees, and perform step 14 before use. the

3) Treatment of dialysis membrane

(1) Cut the dialysis membrane to an appropriate length wearing gloves and soak in distilled water for 15 minutes;

(2) Immerse in 10mmol/L sodium bicarbonate solution, heat to 80°C, and stir for at least 30min;

(3) Change to 10mmol/L Na2 EDTA and soak for 30min, and treat it three times with fresh EDTA in the same way;

(4) Wash with distilled water at 80°C for 30 minutes, then change to 20% ethanol, and store at 4°C for later use. the

4.3. Dialysis and concentration of eluted protein

(1) Rinse the dialysis bag with distilled water, clamp one end of the dialysis bag clamp, fill in 5mL of the eluted protein sample, exhaust the air, and clamp the other end of the dialysis bag clamp;

(2) Put it into 500mL distilled water, stir with a magnetic stirrer and dialyze at 4°C for 1h;

(3) Put the eluted His-Lfcin B15-Mag12 into 500mL PBS, stir with a magnetic stirrer and dialyze at 4°C for 4h;

(4) Replace with fresh buffer solution and continue dialysis for 4h;

(5) Put the dialysis bag into a small beaker, embed it in Sephadex G-200, and concentrate it to an appropriate volume at 4°C;

(6) Sampling 5 μL for SDS-PAGE, analysis and determination of protein purity and affinity chromatography. the

4.3.1. Determination of protein concentration by Bradford method

Principle: Coomassie Brilliant Blue G-250 is a protein dye, which is brown-red under acidic conditions. When the hydrophobic group it contains is combined with the protein through hydrophobic interaction, it turns blue. The blue color is related to the protein concentration. Proportional. After the protein is combined with Coomassie Brilliant Blue G-250, the light absorption peak shifts from 465nm to 595nm. The method for determining protein concentration not only has high sensitivity, but also has a wide concentration range, which is practical and convenient. The range of protein that can be determined by this method is 10μg/mL-1.0mg/mL. the

(1) Preparation of standard curve:

①Take 4 μL of BSA protein standard and dilute to 100 μL with PBS to make the final concentration 200 μg/mL;

② Add 1, 2, 4, 6, 8, 10, and 15 μL of the diluted standard to the sample wells of the 96-well ELISA plate, add PBS to make up to 20 μL, and the protein content in each well is 0, 0.2, 0.4, 0.8, 1.2, 1.6, 2.0, 3.0μg,

③ Add 200 μL Bradford reagent to each well, mix well, and place at room temperature for 5 minutes.

④ Measure the absorbance of A595 with a preheated microplate reader, and draw a standard curve. the

(2) Determination of eluted protein concentration:

① While configuring the sample for making the standard curve, take an appropriate volume of the eluted protein His-Lfcin B15-Mag1296-well microplate sample well, add PBS plus PBS to make up to 20 μL,

②Add 200 μL Bradford reagent to each well, mix well, and place at room temperature for 5 minutes.

③Use a preheated microplate reader to measure the absorbance of A595, and calculate the protein concentration in the sample according to the standard curve. the

5. Cleavage of fusion protein and purification of Lfcin B15-Mag12

5.1. Enterokinase cleaves the fusion protein Trx-S-His-Lfcin B15-Mag12

Add 1.5 μg Enterokinase to 100 μL of 1 mg/mL fusion protein Lfcin B15-Mag12, add 10 μL of 10× buffer solution, incubate at 23°C for reaction, take 5 μL of sample at 6 hours, mix with 5 μL 2×Tricine-SDS sample buffer, freeze at -20°C , to be analyzed by urea-Tricine-SDS-PAGE. the

5.2 Purification of Lfcin B15-Mag12 by ultrafiltration

1) Use Enterokinase to cleave the fusion protein Trx-S-His-Lfcin B15-Mag12 according to the instructions, and incubate at 22°C for 6 hours;

2) Put the above reaction solution into an ultrafiltration tube with a molecular weight cut-off of 5kDa, and centrifuge at 12000 rpm at 4°C for 30min;

3) Take samples to determine the protein concentration according to 4.3.1;

4) Collect the filtrate and store it at -20°C for future use. the

5.3. Urea-Tricine-SDS-PAGE analysis

Table 4 Urea-Tricine-SDS-PAGE gel formula

Separating gel Spacer glue stacking gel 49.5% (C=6%) gel stock solution (mL) 3.3 - - 49.5% (C=3%) gel stock solution (mL) - 0.60 0.45 Gel buffer (pH8.5) (mL) 3.3 1.0 1.4 Urea (g) 3.6 - - H ₂ O 1.0 1.40 1.9 10% APS (μL) 45 20 37.5 TEMED (μL) 4.5 20 3.75

1) Configure the gel according to the formula in Table 4, the configuration method and steps are the same as 3.2;

2) Add an equal volume of 2×Trincine sample buffer solution to the sample, cook in boiling water for 5 minutes, centrifuge at high speed after cooling, and perform electrophoresis according to 3.2;

3) Coomassie Brilliant Blue R-250 staining, 42°C for 1h, destaining solution until the background is clear (Schagger and von Jagow, 1987; Strom et al., 1992, 1993)

6. Activity identification of LfcinB15-Mag12

1) Inoculate the Staphylococcus aureus S.aureas ATCC25923 preserved on the slant into 5mL LB medium, and cultivate overnight on a shaker at 37°C;

2) Transfer 0.5 mL of the culture into fresh 50 mL LB, and continue culturing at 37°C for 2.5 h to the mid-logarithmic growth phase;

3) Take 5mL of bacterial liquid, centrifuge at 4100 rpm for 10min at 4°C to collect the bacterial cells;

4) Resuspend the bacteria in PBS, centrifuge at 4100 rpm for 10 minutes at 4°C to collect the bacteria;

5) Add PBS to resuspend the bacteria, adjust the OD620 to about 0.2, and the bacterial concentration is about 5×107 CFU/mL;

6) Prepare sterilized 42°C low-melting point agarose LB medium in advance, add 50 μL of the above bacterial solution (containing 2.5×106 CFU) to 10 mL of the medium, mix gently immediately, and place on a 90 mm×15 mm plate At this time, the thickness of the plate is about 1mm;

7) After the culture medium is solidified, use 2 pieces of sterile filter paper with a diameter of 3 mm to infiltrate the Lfcin B15-Mag12 solution and paste it on the plate, and 1 piece to infiltrate with PBS as a control;

8) After incubating the plate with its face up at 37°C for 1 hour, it was inverted and continued to incubate for 18 hours to observe the antibacterial effect. the

7. Phosphorylation, annealing, ligation of polynucleotides and purification of coding genes

The polynucleotide synthesized by solid-phase synthesis method has a hydroxyl form at its 3′ end, which is phosphorylated by T4 polynucleotide kinase before annealing and ligation. The gap was ligated by T4DNA ligase to form a complete gene fragment encoding Lfcin B15-Mag12, which was 95bp. After being recovered by 2.5% gel electrophoresis and a purification kit, purified gene fragments were obtained respectively (as shown in 2 in Figure 3) . the

8. PCR screening of recombinant expression vectors

The 5' and 3' universal sequencing primers of pET-32a are complementary to the bases of 57U20 and 327L20 of pET-32a respectively, and the recombinant plasmids are screened with this pair of sequencing primers. After 2.5% gel electrophoresis, as shown in Figure 4, the positive control , that is, the PCR product using the colony containing plasmid pET-32a as a template is a 270bp DNA band, while the amplified products of the positive recombinant plasmid pET-32a-Lfcin B15-Mag12 are respectively about 370bp DNA bands, indicating that in the recombinant plasmid The target segment has been connected in . the

9. Expression of fusion protein and optimization of induction conditions

9.1. SDS-PAGE analysis of primary induced expression of pET-32a-Lfcin B15-Mag12

It was identified by SDS-PAGE. As a result, pET-32a-Lfcin B15-Mag12 transformants produced a specific protein band of about 24kDa, which was consistent with the size of the expected protein (20.4+3.57KD). The negative control non-recombinant pET- The 32a transformant produced a 20.4KD band, while the culture sample of the plasmid bacteria that had not been induced by IPTG had no specific induction band (shown in Figure 5). the

10. Conclusion

1. According to the codon preference of Escherichia coli, the present invention designed and synthesized the coding gene of antimicrobial peptide LfcinB15-mag12 for the first time by artificial synthesis method.

2. Ligate the coding gene of synthetic LfcinB15-mag12 into the expression vector pET-32a, construct the expression vector pET-32a-LfcinB15-mag12, use E.coli BL (DE3) as the expression host, induce it through IPTG, and show through Western blot analysis , the induction band with a molecular weight of 24kDa is a TRX fusion protein, which proves that the fusion protein Trx-S-His-LfcinB15-Mag12 is successfully expressed. twenty two%. the

3. The fusion protein was purified and recovered by affinity chromatography, and the purity was higher, reaching more than 95%, but the yield was low. On average, only 19 mg of the fusion protein was obtained per liter of induced bacterial culture. the

4. After the fusion protein Trx-S-His-LfcinB15-Mag12 was treated with Enterokinase, the recombinant antibacterial peptide LfcinB15-Mag12 with antibacterial activity was obtained, and the heterologous expression of the hybrid peptide LfcinB15-Mag12 was successfully realized for the first time, and the average induction per liter Bacterial culture obtained 200 μg of LfcinB15-Mag12, which laid a theoretical and practical foundation for the production of hybrid peptide LfcinB15-Mag12 and other valuable peptides by genetic engineering methods, and is of great significance for the establishment of common technologies for peptide genetic engineering bacteria and antimicrobial peptide research. the

Claims (1)

1. the synthetic method of a LfcinB15-Mag12 encoding gene, it is characterized in that: according to E.coli codon-bias design coding 1-15 amino acid whose active gene fragment of Lfcin B and Magainin 1-12 amino acid whose active gene fragment, utilize expression vector pET-32a to make up recombinant expression vector pET-32a-Lfcin B15-Mag12, before the multiple clone site of pET-32a, there are 6 histidine-tagged codons of coding, in pET-32a-LfcinB15-Mag12, with 27 amino acid whose gene fragment clones of coding Lfcin B15-Mag12 behind the codon of encoding histidine label, restriction enzyme SalI, between the Nco I recognition site codon, add terminator codon TAG simultaneously, TAA

Divide four sections composition sequences as follows:

Fragment 1:

5’-CATGGCTTTCAAATGCCGCCGTTGGCAGTGGCGTTGGAAAAAACTGGGT

GCGGGTATCG-3’

Fragment 2:

5’-GTAAATTCCTGCACTCTGCTAAAAAATTCTAGTAAG-3’

Fragment 3:

5’-TCGACTTACTAGAATTTTTTAGCAGAGTGCAGGAATTTACCGATACCCGC

ACCCAGTTT-3’

Fragment 4:

5’-TTTCCAACGCCACTGCCAACGGCGGCATTTGAAAGC-3’。

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