CN102703457A - Method for preparing and expressing antibacterial peptide gene - Google Patents

Abstract

The invention relates to an antibacterial peptide gene and a preparation method thereof. The antimicrobial peptide gene of the present invention is the Ascaris suum antimicrobial peptide Cecropin P2, and the coded Ascaris suum antimicrobial peptide Cecropin P2 is first isolated from mammals. The antimicrobial peptide gene of the present invention is a gene Cecropin P2' after optimizing the antimicrobial peptide gene Cecropin P2 of Ascaris suum, and its sequence is SEQ №1, in its sequence; the sixth codon is optimized from the original ctc to ctg ; The ninth, twelfth and eighteenth codons were optimized from aaa to aag. After this optimization process, the target gene can be better expressed in the Pichia pastoris system.

Description

Preparation and expression method of an antimicrobial peptide gene

technical field

The invention relates to an antibacterial peptide gene and a preparation method thereof.

Background technique

In recent years, the extensive abuse of antibiotics and chemical drugs in the breeding industry has led to the continuous emergence of bacterial resistance, and some infectious diseases have seriously threatened human health and the development of animal husbandry. Residues of chemical drugs in meat products have laid a huge hidden danger for human health. Therefore, it is imminent to find new biological agents that can replace antibiotics and chemical drugs. Antimicrobial peptides (antimicrobial peptides or antibacterial peptides) are a class of active peptide substances with antibacterial activity, and are an important part of the natural defense system in organisms. Good, good water solubility, not easy to produce drug resistance and so on. At the same time, antimicrobial peptides have broad-spectrum antibacterial activity, showing significant activity against fungi, Gram-positive bacteria, Gram-negative bacteria, and some enveloped viruses, and also have selective killing effects on protozoa and tumor cells, but It has no obvious toxic and side effects on normal mammalian and insect cells.

The acquisition of antimicrobial peptides in the prior art mainly includes natural extraction, chemical synthesis or recombinant expression. Due to the high cost and low yield of natural extraction and chemical synthesis, antimicrobial peptides are mostly obtained by recombinant expression. The existing systems for the expression of antimicrobial peptides mainly include Escherichia coli expression system, yeast expression system, insect cell/baculovirus group expression system and mammalian cell expression system. The initial research on the expression of antimicrobial peptides was carried out in the Escherichia coli expression system. The prokaryotic expression system has the characteristics of low cost, easy operation, clear genetic background, etc., and also has a large number of expression vectors to choose from. However, since the prokaryotic expression system does not have the expression regulation mechanism similar to eukaryotes and the function of post-translational processing and modification of proteins, its products often form inclusion bodies, which must undergo a series of treatments such as denaturation and renaturation to restore activity. relatively difficult. The insect cell/baculovirus group expression system has the advantages of simplicity and high efficiency, and has the ability to post-translationally modify, process and transfer foreign proteins. When the source protein is expressed, the virus has already started apoptosis. Although the mammalian cell expression system has certain advantages in gene post-transcriptional modification, protein processing, secretion, glycosylation, etc., its expression cost is too high and it is not suitable for large-scale production.

Contents of the invention

The invention provides an antimicrobial peptide gene different from the prior art and a preparation method thereof.

The antimicrobial peptide gene of the present invention is Ascaris suum antimicrobial peptide Cecropin P2, and the coded Ascaris suum antimicrobial peptide Cecropin P2 is first isolated from mammals.

The antimicrobial peptide gene of the present invention is a gene Cecropin P2' after optimizing the antimicrobial peptide gene Cecropin P2 of Ascaris suum, its sequence is SEQ№1, in its sequence; the sixth codon is optimized from the original ctc to ctg ; The ninth, twelfth and eighteenth codons were optimized from aaa to aag. After this optimization process, the target gene can be better expressed in the Pichia pastoris system.

The method for preparing the Ascaris suum antimicrobial peptide Cecropin P2' gene after optimized treatment of the present invention is as follows: first, use the oligonucleotide sequences SEQ №2 and SEQ №3 as primer templates to carry out PCR reaction to obtain product 1, and then The product 1 and SEQ№4 are mutually used as primer templates for PCR reaction to obtain the target gene SEQ№1.

The method for preparing the yeast vector with the Ascaris suum antimicrobial peptide Cecropin P2' gene after optimized treatment according to the present invention is as follows: clone SEQ No. 1 on the pMD18-T simple vector to obtain the recombinant plasmid pMD18-T -Cecropin P2'-His6, pMD 18-T-Cecropin P2'-His6 recombinant plasmid and Pichia pastoris expression vector pPIC9K were digested with restriction endonucleases EcoR I and Not I, and the digested products were recovered by T4 DNA Ligase After ligation, the competent cell DH5α was transformed to obtain the recombinant expression plasmid pPIC9K-Cecropin P2'-His6. The recombinant expression plasmid was linearized by Sal I single-enzyme digestion, purified by agarose gel electrophoresis, and recovered from the gel, and frozen for later use. Inoculate Pichia pastoris GS115 on the YPD medium plate, and culture the GS115 competent cells to obtain the precipitate. 80 μl and 10 μl (20 μl) of the linearized recombinant expression plasmid pPIC9K-CecropinP2 of the GS115 competent cells treated in the previous step After '-His6 was mixed, electroporation treatment was carried out. The parameters of electroporation treatment were 1.5kV, 25μF, 200Ω, 5ms, and the transformant Cecropin P2'-His6-P was obtained through screening and identification by MM, MD and antibiotic G418.

The method of inducing expression with the yeast vector prepared by the above method is to inoculate the obtained transformant Cecropin P2'-His6-P in 25ml BMGY, and culture at 28°C with shaking at 200rpm until the OD ₆₀₀ reaches 2-6, and at 4°C Collect bacteria by centrifugation at 3000rpm for 5min, resuspend in 100ml of BMMY (buffered compound methanol medium), shake and culture at 30°C and 200rpm for 96h, sample 1ml every 24h, and add methanol to a final concentration of 1%, take out The sample product was centrifuged at 4° C. and 3000 rpm for 5 minutes, and the supernatant was collected, and then separated and purified.

The antibacterial peptide gene of the present invention can be used in the preparation of antibacterial drugs.

The present invention has the following advantages:

1. The antimicrobial peptides of the present invention are taken from mammals, and it is expected that they will have better effects and effects when used in mammals. Cecropin P2 was isolated from nematodes by Pillai A et al. in 2004 through bacterial induction. Its mature peptide has a molecular weight of about 4.2 kDa, does not contain cysteine (cys), does not form intramolecular disulfide bonds, and has The structure is similar to the insect cecropin antibacterial peptide Cecropins, which is a typical amphipathic structure and has potential antibacterial effects, but so far, no research on its expression, purification and function has been reported.

2. The present invention expresses, optimizes and purifies the Ascaris suum antimicrobial peptide Cecropin P2 for the first time in the world, and the expressed and purified Cecropin P2 has antibacterial activity.

3. According to the codon preference of Pichia pastoris, the present invention optimizes the codons of the coding gene of Ascaris suum antimicrobial peptide Cecropin P2'. In the heterologous expression system of the coding gene of Cecropin P2' of the present invention, the expression of the recombinant protein may be affected by codon utilization. Therefore, the redesign of the gene utilizing optimal codons can increase protein production and make protein production more efficient and economy.

4. In the fusion gene constructed by the present invention, a coding sequence of 6 histidines is introduced at the 3' end, and the coding sequence of 6 histidines introduced at the 3' end can facilitate subsequent protein expression and purification. Nickel column binding tag, because histidine can specifically bind nickel ions, the recombinant protein with histidine tag can be adsorbed by nickel column, which is beneficial to the separation and purification of target protein by nickel column in the later stage, so as to achieve the purpose of purification , in the process of protein expression and purification in other fields, this technology has been widely used.

5. The Ascaris suum antimicrobial peptide Cecropin P2' expressed in the present invention comes from the organism itself, and has little or no toxicity to the organism. For this part, see item 9 in the specific embodiment. Therefore, the antibacterial drug prepared by using the gene of the present invention will be a safer antibacterial drug compared with traditional antibiotic drugs.

6. The present invention uses the Pichia pastoris expression system for expression, which is easy to operate, can use fermenters to achieve large-scale production, strictly controls the quality, obtains a large amount of target protein, and better exerts its role.

7. The present invention uses the Pichia pastoris expression system for expression, and selects the pPIC9K vector for secretory expression. Compared with prokaryotic expression, the expression level is higher, and the protein can be better folded into a natural conformation in the eukaryotic system, and can be It exists in soluble form, and its activity is much higher than that of inclusion body protein. Yeast grows extremely fast, and the alcohol oxidase gene (AOX1) promoter used in the pPIC9K vector used in the present invention is a strong inducible promoter, which can effectively regulate the high-efficiency expression of foreign genes, and its expression yield is about the same as that of bacteria, insects or 10-100 times the output of expression systems such as mammalian cells. See the following references:

[1] Kong Fanhong, Zhong Weixia, Wang Hongfa, etc. Research progress on the expression system of biological antimicrobial peptide Pichia pastoris [J]. Chinese Journal of Pathogenic Biology, 2009, 4(9): 700-702.

[2] Wang Qinglu, Li Qiaoqiao, Zhang Jie, etc. Characteristics and application of Pichia pastoris expression system [J]. Biotechnology Bulletin, 2006, 17(4): 640-643.

[3] Cregg J M, Cereghino J L, Shi J, et al. Recombinant protein expression in Pichia pastoris [J]. Mol Biotechnol, 2000, 16(1): 23-52.

8. The present invention uses the Tristricine-SDS-PAGE system suitable for the separation of small molecular proteins and peptides for detection, and successfully obtains the expressed protein with a molecular weight of about 5kDa.

9. In the process of Tristricine-SDS-PAGE, the present invention utilizes three-layer gel (5% stacking gel, 10% interlayer gel, 18% stacking gel) to separate and detect the target protein. Among them, the use of three-layer glue to separate and detect proteins has been applied in related fields. The use of three-layer glue is to enable better separation of small molecular proteins. The interlayer glue plays a role between the separation gel and the stacking gel. The role of the buffer. In order to separate proteins with a molecular weight of about 5kDa, the present invention explored the formula of the gel and the concentration of each layer of gel, and finally obtained a better separation effect, see item 8 of the specific implementation part and accompanying drawing 6.

Description of drawings

Fig. 1 is the electrophoresis figure of the Cecropin P2 ' target gene that the present invention obtains through overlapping polymerase chain reaction; Fig. 2 is the PCR identification nucleic acid electrophoresis figure of recombinant expression plasmid pPIC9K-Cecropin P2 '-His6 of the present invention; Fig. 3 is this Recombinant expression plasmid pPIC9K-Cecropin P2'-His6 of the invention is identified by restriction nucleic acid electrophoresis; Figure 4 is the nucleic acid linearized by the restriction endonuclease Sal I of the recombinant expression plasmid pPIC9K-Cecropin P2'-His6 of the present invention Electrophoresis figure; Fig. 5 is the PCR identification electrophoresis figure of Cecropin P2'-His6-P recombinant yeast transformant of the present invention; Fig. 6 is the Tris tricine-SDS-PAGE electrophoresis figure of Cecropin P2'-His6-P yeast expression product of the present invention Fig. 7 shows Pichia pastoris codon preference; Fig. 8 is the bacteriostatic test result of Cecropin P2 of the present invention to Escherichia coli and Staphylococcus aureus.

Detailed ways

The specific embodiment of the present invention is as follows:

Main material used in the present invention

Strains and vectors: Escherichia coli competent cells JM109, DH5α, DNA Marker and pMD18-Tsimple vectors were purchased from TaKaRa Biotech; Pichia pastoris GS115 strain (his ^- , mut ⁺ ), the expression vector pPIC9K was preserved for this experiment; Escherichia coli CMCC44102 strain and Staphylococcus aureus CMCC26003 strain were purchased from China Veterinary Microorganism Culture Collection Management Center.

Main reagents and kits: protein marker, ethylene glycol, Tricine, Acryl/Bis Solution, Yeast Nitrogen Base (YNB), etc. were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; antibiotic G418 was a product of Invitrogen; yeast extract (Yeast extract) and peptone (Trptone) are products of OXOID company; agar powder (Agar), agarose, Coomassie brilliant blue R-250, sorbitol, etc. were purchased from Sigma company; methanol, ethanol, ethylene glycol, isopropanol, Phosphoric acid and glycerin were purchased from Tianjin Chemical Reagent Co., Ltd.; glucose, sodium chloride, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate were purchased from Beijing Chemical Plant; glycine was a product of Genearay Biotech; ammonium persulfate was purchased from Baoxin Biotechnology Co., Ltd.; T4 DNA Ligase, Ex Taq, restriction endonuclease Not I, EcoR I, Sal I, etc. were purchased from TaKaRaBiotech.

Plasmid extraction kits, PCR purification and recovery kits, gel recovery kits, and pMD18-T vector ligation kits were all purchased from TaKaRa Biotech.

Main instruments: high-speed refrigerated centrifuges, pipettes, and PCR instruments were purchased from Eppendorf, Germany; electronic balances were purchased from Beijing Saiduosi Instrument System Co., Ltd.; electric thermostatic incubators were purchased from Shanghai Precision Experimental Equipment Company; DYY-6C electrophoresis The instrument and horizontal shaker were purchased from Beijing Liuyi Instrument Factory; the electrothermal constant temperature water tank was purchased from Shanghai Jinghong Experimental Equipment Co., Ltd.; the nucleic acid electrophoresis tank and protein electrophoresis tank were purchased from Beijing Junyi Dongfang Electrophoresis Equipment Co., Ltd.; - Rad company, 3KDa, 10KDa, 30KDa ultrafiltration tubes Amicon-Ultra-15 were purchased from Millipore company.

The specific operation is as follows:

1. Primer Synthesis

The present invention designs three oligonucleotide sequences based on the antibacterial peptide Cecropin P2' coding gene (Genbank retrieval sequence number is AB186037) and the multiple cloning site of Pichia pastoris expression vector pPIC9K.

C1-F (48bp): 5'-GCC GAATTC AGTTGGCTCAGCAAAACCTACAAGAAACTCGAGAACTCA-3' (EcoR I restriction site introduced at the 5' end), that is, SEQ No. 2.

C2-R (58bp): 5'-GTATGGCGATAGCAATGCCTTCTGAGATGCGCTTTTTTGCTGAGTTCTCGAGTTTCTT-3', namely SEQ No.3.

C3-F (60bp): 5'-ATTGCTATCGCCATACAGGGTGGTCCGCGTCATCATCATCATCATTAA GCGGCCGC C (the 3' end introduces a Not I restriction site, with a 6×his tag in the box), that is, SEQ №4.

Wherein C2-R is a reverse complementary sequence, there is 18 bp nucleotide sequence complementary pairing between C1-F and C2-R sequences, and there is 16 bp nucleotide sequence complementary pairing between C2-R and C3-F sequences. The C1-F and C3-F sequences have EcoR I and Not I restriction sites respectively, and the C3-F sequence also contains the coding sequence of his tag.

2. Polymerase Chain Reaction Amplification of the Target Gene

(1) Amplification of the target gene:

C1-F and C2-R are primers and templates for each other. The PCR reaction conditions are: 94°C for 5 minutes, (94°C for 30s, 52°C for 30s, 72°C for 40s) for 30 cycles, and 72°C for 10 minutes to obtain PCR product 1. PCR product 1 and C3-F serve as primers and templates for each other. The PCR reaction conditions are: 94°C for 5 minutes, (94°C for 30s, 51°C for 30s, 72°C for 40s) for 30 cycles, and 72°C for 10 minutes to obtain PCR product 2, which is the target Gene sequence (this sequence is SEQ№1), as follows:

gcc gaa ttc agt tgg ctg agt aat acc tac aag aat ctc gag aac 45

Ala Glu Phe Ger Trp Leu Ger Lys Thr Tyr Lys Lys Leu Glu Asn

1 5 10 15

tca gca aat aag cgc atc tca gaa ggc att gct atc gcc ata cag 90

Ger Gla Lys Lys Arg Ile Ger Glu Gly Ile Gla Ile Gla Ile Gln

16 20 25 30

ggt ggt ccg cgt cat cat cat cat cat cat taa gcg gcc gcc 132

Gly Gly Pro Arg His His His His His His His Terminated *** *** ***

31 35 40

(2) Amplification result:

The three oligonucleotide fragments were mutually used as primers for overlapping PCR, and the obtained products were subjected to 2% agarose gel electrophoresis to obtain a band of the desired size, as shown in Figure 1, with a size of 132 bp.

3. Construction and identification of recombinant expression plasmids

(1) Construction of recombinant expression plasmids:

Through T-A cloning, the PCR product was cloned on the pMD18-T simple vector, and the cloned vector was named pMD18-T-Cecropin P2'-His6. In the relevant experiments of the present invention, the cloning product was double-digested with restriction endonuclease EcoR I and Not I to pMD18-T-Cecropin P2'-His6 recombinant plasmid and Pichia expression vector pPIC9K, after the digestion product was recovered, it was After T4 DNA Ligase ligation, the competent cells DH5α were transformed, and the positive clones screened were identified by PCR and sequenced. The correct recombinant expression plasmid was identified as pPIC9K-Cecropin P2'-His6.

(2) Identification results:

Recombinant cloning plasmid pPIC9K-Cecropin P2'-His6 and Pichia pastoris expression vector pPIC9K were digested with restriction endonucleases EcoR I and Not I, ligated and transformed into E.coli DH5α competent cells to obtain recombinant expression plasmid pPIC9K -Cecropin P2'-His6. Using the pPIC9K universal primer as the amplification primer, the obtained recombinant plasmid was identified by PCR and enzyme digestion, and the product was detected by 1% agarose gel electrophoresis, and a band of the target size was detected at the correct position (Figure 2, Figure 3) . The positive clones were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing, and the sequencing results were analyzed by bioinformatics software. The results proved that the target gene was not mutated and the insertion position on the vector was correct.

4. Preparation of yeast-related reagents

10×D (20% D-glucose): D-glucose 200g, fully dissolved in 1000ml deionized water, autoclaved for 15min, and stored at 4°C for later use.

10×YNB: YNB 134g, fully dissolved in deionized water, dilute to 1000ml, filter and sterilize, store at 4°C for later use.

10×M (5% methanol): add 50ml of methanol into 950ml of deionized water, mix well, filter and sterilize, store at 4°C for later use.

10×GY (10% glycerol): add 100ml glycerin to 900ml deionized water, mix well, sterilize by autoclaving for 15min, store at 4°C for later use.

500×B (0.002% biotin): 200 mg of biotin was added to 1000 ml of deionized water, mixed evenly, sterilized by filtration, and stored at 4°C for later use.

pH 6.0 phosphate buffer: 30.125g dipotassium hydrogen phosphate, 118.125g potassium dihydrogen phosphate, fully dissolved in deionized water, set the volume to 1000ml, adjust the pH to 6.0 with KOH or phosphoric acid, autoclave, store at 4°C spare.

YPD medium: 20g of peptone, 10g of yeast extract, fully dissolved in 900ml of deionized water, autoclaved for 20min, after cooling, add 100ml of 10×D, and store at 4°C for later use. When preparing YPD plates, add 15g/L Agar.

MD medium: Dissolve 15g of Agar in 800ml of deionized water, autoclave for 20min, cool to about 60°C, add 100ml of 10×YNB, 100ml of 10×D, 2ml of 500×B, mix well, pour plate, store at 4°C for later use.

MM medium: Dissolve 15g of Agar in 800ml of deionized water, autoclave for 20min, cool to about 60°C, add 100ml of 10×YNB, 100ml of 10×M, 2ml of 500×B, mix well, pour plate, store at 4°C for later use.

BMGY medium: 20g of peptone, 10g of yeast extract, fully dissolved in 700ml of deionized water, autoclaved for 20min, after cooling, add 100ml of 10×YNB, 100ml of 10×GY, 100ml of pH 6.0 phosphate buffer, 2ml of 500×B , stored at 4°C for later use.

BMMY medium: 20g of peptone, 10g of yeast extract, fully dissolved in 700ml of deionized water, autoclaved for 20min, after cooling, add 100ml of 10×YNB, 100ml of 10×M, 100ml of pH 6.0 phosphate buffer, 2ml of 500×B , stored at 4°C for later use.

5. Cecropin P2'-pPIC9K-His6 electrotransformed Pichia pastoris GS115

5.1 Linearization

The recombinant expression plasmid was linearized by single enzyme digestion with Sal I, and the linearized product was detected by 0.8% agarose gel electrophoresis. The company's DNA gel recovery kit operation method performs gel recovery of the target fragments and freezes them at -20°C for later use.

5.2 Preparation of GS115 Competent Cells

(1) Inoculate Pichia pastoris GS115 strain frozen at -70°C on a YPD solid culture plate by streaking method, and culture at a constant temperature of 28°C for 2 days;

(2) Pick a single colony from the YPD culture plate, inoculate it in 5ml YPD liquid medium, and culture it with shaking at 28°C and 200rpm for 16-18h;

(3) Inoculate 1ml of the overnight culture into 100ml of fresh YPD liquid medium, culture at 28°C with shaking at 200rpm until the OD value (OD ₆₀₀ ) reaches 1.0-1.3;

(4) Centrifuge the culture in step 3 at 3000rpm at 4°C for 5min, and discard the supernatant;

(5) Suspend and wash the precipitate in step 4 with 100ml pre-cooled sterilized water, centrifuge at 3000rpm at 4°C for 5min, and discard the supernatant;

(6) Suspend and wash the precipitate in step 5 with 50 ml pre-cooled sterilized water, centrifuge at 3000 rpm at 4°C for 5 min, and discard the supernatant;

(7) Suspend the precipitate in step 6 with 4.5ml ice-precooled 1M sorbitol, divide into 1.5ml centrifuge tubes, centrifuge at 3000rpm at 4°C for 5min, and discard the supernatant;

(8) Suspend the precipitate in step 7 with 1 ml of ice-cold 1M sorbitol to a final volume of 1.5 ml, and mark it as "competent".

5.3 Electroconversion:

(1) After fully mixing 80 μl of GS115 competent cells marked as competent and 10 μl of linearized DNA, transfer the mixture into a pre-ice-bathed electroporation cuvette;

(2) Place the electric transfer cup on ice for 5 minutes;

(3) Electric shock, electric shock parameters: Cuvette Gap: 2mm; Voltage: 1.5kV; Capacitance: 25μF; Resistance: 400Ω; Electric shock time: 5ms.

(4) Immediately after electric shock, add 1ml of ice-cooled 1M sorbitol, and transfer the solution to a new sterilized centrifuge tube;

(5) Divide the solution in the centrifuge tube into 200-300 μl aliquots, and spread them on the pre-prepared MD plates;

(6) Cultivate the MD plate at a constant temperature of 28°C until a single clone is produced.

6. Screening and identification of Mut ⁺ transformants and high copy transformants

(1) Screening and identification of transformants

The single clones grown on the MD plate were transferred to MD and MM (Minimal Methanolmedium, minimal methanol medium) agar plates respectively, incubated at 28° C. for 2 days, and the growth of the transformants was observed. Transformants that grow well on both MD and MM plates are methanol utilization fast (Mut ⁺ ) transformants, grow well on MD plates, and transformants that do not grow or grow slowly on MM plates are methanol utilization slow types ( Mut ^s ) transformant. Inoculate the Mut ⁺ transformants obtained by screening on single clone colonies to YPD plates containing 0.5, 1.0, 2.0, 3.0 mg/ml G418 respectively, and culture them statically at 28°C to screen for high-copy yeast transformants with Mut ⁺ phenotype. The PCR template of the Mut ⁺ transformant was prepared by boiling method, and the pPIC9K universal primer was used to carry out PCR identification on the screened transformant.

(2) Identification results:

The obtained Cecropin P2'-His6-P transformants were subjected to colony PCR by boiling method, and the PCR results were detected by 1% gel electrophoresis, and 12 Cecropin P2'-His6-P positive clones were identified (Figure 5) .

7. Induced Expression

The transformants identified as positive by PCR were inoculated in 25ml BMGY (buffered compound glycerol medium), cultured with shaking at 200rpm at 28°C until the _OD600 reached 2-6, centrifuged at 3000rpm at 4°C for 5min to collect the bacteria, and resuspended in In 100ml of BMMY (buffered complex methanol medium), shake culture at 30°C and 200rpm for 96h, sample 1ml every 24h, and add methanol to a final concentration of 1%. The sample product taken out was centrifuged at 4°C and 3000rpm for 5min, and the supernatant was collected and frozen at -20°C until testing.

8. Tris tricine-SDS-PAGE detection of recombinantly expressed Cecropin P2'

(1) Preparation of relevant solutions

5×Tris-glycine buffer: Weigh 15.1g Tris, 94g glycine, 5.0g SDS, and make up to 1L.

Coomassie Brilliant Blue R-250 Staining Solution: Dissolve 0.25g Coomassie Brilliant Blue R-250 in 45ml of methanol, 45ml of water, and 10ml of glacial acetic acid, and store at room temperature.

Coomassie Brilliant Blue staining solution: 100ml glacial acetic acid, 50ml ethanol, 850ml water, mix thoroughly and store at room temperature.

10% ammonium persulfate: Weigh 10 g of ammonium persulfate, add 10 ml of deionized water, stir to dissolve, and store at 4°C.

10×Anode buffer solution: Weigh 48.44g Tris, add 160ml deionized water to fully dissolve, adjust the pH to 8.9, and set the volume to 200ml.

10×Tris-Tricine-SDS buffer (0.1M Tris, 0.1M Tricine, 0.1% SDS).

(2) Gel formula and concentration

(3) Tris tricine-SDS-PAGE detection of recombinantly expressed Cecropin P2'

Concentrate the supernatant stored at -20°C and mix it with an equal volume of 2× loading buffer, bathe in boiling water for 7-10 minutes, then centrifuge at 10,000 rpm for 5 minutes, take 15 μl of the supernatant for Tris tricine-SDS-PAGE electrophoresis ^{[ 7]} . The separation gel concentration of Tris tricine-SDS-PAGE electrophoresis was 18%, the interlayer gel concentration was 10%, and the stacking gel concentration was 5%.

① Glue making: After cleaning and drying the electrophoretic glass plate, fix it on the glue distribution rack according to the operating instructions. Prepare 18% Tristricine-SDS-PAGE electrophoresis gel according to the instructions, in which the concentration of separating gel is 18%, the concentration of interlayer gel is 10%, and the concentration of stacking gel is 5%. Pour it between two glass plates in turn, and wait for it to solidify.

②Installation: Carefully remove the comb, rinse the sample tank several times with deionized water, put it into the electrophoresis tank, then add 1×Tris-Tricine-SDS buffer to the cathode of the electrophoresis tank, and add 1×anode buffer to the anode.

③Preparation, loading and electrophoresis of samples: Concentrate the expression supernatant 50 times with an ultrafiltration tube, add an equal amount of 2×SDS-PAGE loading buffer, mix the two, and boil in a water bath for 5-10 minutes , after cooling, load 5-10ul/well. After adding the sample, set the voltage to 60V. After the bromophenol blue enters the interlayer gel, adjust the voltage to 80V. After the bromophenol blue enters the separation gel, adjust the voltage to 120V. Continue constant voltage electrophoresis until the bromophenol blue reaches Gel bottom layer.

④ Staining: After the electrophoresis is completed, carefully take out the gel plate from the electrophoresis tank, rinse it with distilled water, take out the film with a gel pad and rinse it with distilled water, then fix it on a horizontal shaker, pour the staining solution, and stain at room temperature 3 hours.

⑤ Decolorization: After the staining, pour out the staining solution, rinse the film with distilled water, add fresh decolorization solution, place on a horizontal shaker for 5-8 hours, and replace the decolorization solution 3-4 times during the period. After the blue background is removed, place the gel in distilled water to stop the decolorization and observe the results.

(4) Test results of recombinant protein Tris tricine-SDS-PAGE

The expression product of Cecropin P2'-pPIC9K-His6 was detected by 18% Tris tricine-SDS-PAGE electrophoresis, and a single band of target size was detected (Figure 6).

9. Cytotoxicity test of recombinant expressed protein Cecropin P2'

After the purified recombinant protein Cecropin P2 was filtered with a 0.22μl filter, it was co-cultured with MDBK cells (bovine kidney cells), CHO cells (hamster ovary cells) and DC cells (dendritic cells) in different volumes from 10ul to 120ul for two days. observed under a microscope. The results showed that the recombinant protein had no obvious effect on the growth and proliferation of the above three types of cells, which preliminarily indicated that the fusion protein had no attack on these three types of cells.

10. Antibacterial activity test of recombinant protein

(1) Antibacterial activity test of recombinant protein

In this experiment, the agarose diffusion method was used to detect the antibacterial activity of the recombinantly expressed antimicrobial peptide Cecropin P2 (Han Jinhui, et al., 2008). Take 300 μl of Escherichia coli and Staphylococcus aureus cultures in the logarithmic growth phase and mix them with 25ml of the corresponding solid medium at about 45°C. After solidification, paste a sterile filter paper with a diameter of about 5mm on the surface of the medium Add 5 μl of the concentrated expression supernatant of the sample to be tested dropwise on the filter paper sheet, use the yeast induction supernatant containing pPIC9K empty vector as a negative control, and observe after culturing at 37°C for 12 hours.

(2) Antibacterial test results

The antibacterial activity of the recombinant antimicrobial peptide was detected by the agarose diffusion method. After 12 hours of constant temperature cultivation at 37°C, the results showed that on the agarose plates of Escherichia coli and Staphylococcus aureus, the filter paper with the recombinant antibacterial peptide dropped was In the center, there are antibacterial zones with diameters of about 1.5cm and 3cm, respectively. This result shows that the recombinant antimicrobial peptide has a certain antibacterial activity against both Escherichia coli and Staphylococcus aureus, see Figure 8.

Claims (7)

1. An antimicrobial peptide gene, characterized in that the antimicrobial peptide gene is the Ascaris suum antimicrobial peptide CecropinP2. 2. The antimicrobial peptide gene according to claim 1, characterized in that the optimized gene Cecropin P2' includes SEQ No. 1 sequence, wherein; the sixth codon is optimized to ctg from the original ctc; the ninth, The twelfth and eighteenth codons were optimized from aaa to aat. 3. the antimicrobial peptide gene preparation method described in claim 2 is characterized in that at first carry out PCR reaction with oligonucleotide sequence SEQ No. 2 and SEQ No. 3 as primer template mutually, obtain product 1, then product 1 and SEQ No. 4 are used as primer templates to carry out PCR reaction to obtain the target gene SEQ№1. 4. the method for preparing yeast carrier with the gene described in claim 3 is characterized in that SEQ No. 1 is cloned on pMD18-T simple carrier, obtains recombinant plasmid pMD18-T-Cecropin P2'-His6, uses restriction endonuclease Enzymes EcoR I and Not I double-enzyme digest pMD18-T-Cecropin P2'-His6 recombinant plasmid and Pichia pastoris expression vector pPIC9K, after the digested products are recovered, they are ligated with T4 DNA Ligase and then transformed into competent cells DH5α to obtain the recombinant expression plasmid pPIC9K -Cecropin P2'-His6, the recombinant expression plasmid was linearized by Sal I single enzyme digestion, purified by agarose gel electrophoresis, recovered from the gel, and frozen for later use, and then inoculated Pichia pastoris GS115 on the YPD medium plate, After culturing the GS115 competent cells obtained from the precipitate, mix 80 μl of the GS115 competent cells treated in the previous step with 10 μl (20 μl) of the linearized recombinant expression plasmid pPIC9K-Cecropin P2'-His6, and perform electroporation treatment. The parameters of the transformation treatment were 1.5kV, 25μF, 200Ω, 5ms, and the Cecropin P2'-His6-P transformant was obtained through screening and identification by MM, MD and antibiotic G418. 5. The yeast vector induced expression method prepared according to claim 4, characterized in that the obtained transformant Cecropin P2'-His6-P is inoculated in 25ml BMGY, and shaken at 200rpm at 28°C until the _OD600 reaches 2-6 Centrifuge at 3000rpm for 5min at 4°C to collect the bacteria, resuspend in 100ml of BMMY (buffered compound methanol medium), shake and culture at 30°C and 200rpm for 96h, sample 1ml every 24h, and add methanol to a final concentration of 1%, the sample product taken out was centrifuged at 4°C and 3000rpm for 5min, the supernatant was collected, and then separated and purified. 6. The yeast vector-induced expression method according to claim 5, characterized in that the target protein is separated and detected by utilizing 5% stacking gel, 10% interlayer gel and 18% stacking gel. 7. The application of the antimicrobial peptide gene described in claim 2 in the preparation of antibacterial drugs.

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