CN103614380A - Preparation and expression method of chicken beta-defensin 9 yeast engineered bacteria - Google Patents

Abstract

The invention discloses preparation and an expression method of a chicken beta-defensin 9 yeast engineered bacteria, and belongs to the fields of biological technology and gene engineering pharmaceutical technology. The preparation process comprises: utilizing a pichia yeast consistently-used codon to optimize chicken beta-defensin 9 gene, adding a Kex2 restriction enzyme cutting site coding sequence at the front terminal of the gene, and artificially synthesizing the gene; then inserting the synthetic beta-defensin 9 gene sequence into a yeast constitutive expression vector, integrating into a the chromosome of a methylotrophy type pichia yeast by homologous recombination; and screening for multiple times to obtain the yeast engineered bacteria for producing chicken beta-defensin 9. The yeast engineered bacteria is capable of continuously performing high-level protein expression after fermentation and culturing, the expressed product is high in yield, is relatively close to the natural structure, is secreted in the broth, and also has relatively strong anti-foodborne pathogenic activity. The broth is subjected to preliminary separation and can be directly used as a forage additive after concentrated or dried, and the forage additive is applied to animal breeding industry, so that the production cost of defensin is reduced and the economic benefit of a production enterprise is improved. The technical scheme provided by the invention helps to establish a technological base for industrial large-scale production and application of beta-defensin 9 and development of poultry forage antibiotic additive substitutes.

Description

Chicken β-defensin 9 Yeast engineering bacteria preparation and expression methods thereof

Technical field

The invention belongs to biotechnology and genetic engineering pharmaceutical technical field, be specifically related to a kind of chicken β-defensin 9 Yeast engineering bacteria preparation and expression methods thereof.

Background technology

From the 1950's, antibiotic sub-therapeutic dose is just added to the growth that promotes animal in feed.The abuse of a large amount of microbiotic in aquaculture, has caused drug tolerant bacteria constantly to occur, some infectious pathogens even serious threat to the development of human health and livestock industry.Along with the propagation with resistance cause of disease that improves constantly of people's attention rate, Europe has started to forbid using antibiosis usually to promote growth of animal in animal-feed.Therefore, find a kind of natural free of contamination novel fodder additive that can substitute antibiotics extremely urgent, it had both had antibacterial, can promote growth of animal again, to reduce feeding cost.

Alexin belongs to a subtribe of antibacterial peptide family, is the little peptide of positively charged ion that the class that is distributed widely in animals and plants circle is rich in halfcystine.Alexin has broad-spectrum anti-microbial activity, bacterium, fungi, spirochete, virus etc. are all had to resistance, and sex pheromone is difficult for producing resistance, it is the important defence line that host resists external invasive organism invasion and attack, simultaneously due to its persistent expression in the multiple different tissues organ of animal body, also a part that is considered to animal innate immune system, has important physiologic function.Its principal character is 3 pairs of disulfide linkage that 6 cysteine residues form, the nearly 38-42 of a mature peptide amino acid, and molecular mass is about 3-4kDa.According to the difference of space structure, alexin can be divided into 3 large classes: α-alexin, beta-alexin and θ-alexin.Fowl alexin belongs to beta-alexin class, and the 3 pairs of disulfide linkage are respectively Cys-1～Cys-5, Cys-2～Cys-4 and Cys-3～Cys-6 pairing is connected to form, and are rich in arginine, have wetting ability and lipotropy, characteristic β-laminated structure.Up to now, in chicken body, found 14 kinds of beta-alexins, these chicken phylaxin genes are by difference called after AvBD1～14.Chicken β-defensin 9(AvBD9 wherein) be distributed widely in all kinds of organ-tissues of chicken, especially in each organ-tissues of Digestive tract such as mucous bursa, esophagus, crop, liver and stomach, expression amount is higher.Digestive tube is animal body and food borne pathogens contact site, is also that it invades position.AvBD9 has good biological safety, and has stronger anti-microbial activity, can suppress or kill pathogenic bacteria, and this is healthy to chicken digestive tube, especially to preventing, in the body local defenses such as upper digestive tract infection and inflammation, plays an important role.In Production of Livestock and Poultry, can be used for disease preventing and treating, promote animal health cultivation, there is potential application and exploitation value, and molecular recombination technology is the effective way of protein or peptide series products Application and Development.

Pichia pastoris phaff (Pichia pastoris) expression system both had advantages of the expression amount of prokaryotic expression system high, can large scale culturing, there is again modification after the protein translation of eukaryotic expression system, processing and folding advantage, and the genetic stability of foreign gene is better, it is one of eukaryotic expression system of current widespread use.Pichia yeast expression system has very high biological safety, obtains the extensive approval that comprises U.S. FDA.Pichia spp self secretion is the albumen of amount seldom, is easy to the separation and purification of heterologous protein, and the modification of the albumen of its secretion existence translation post-treatment, makes it conformation and activity closer to native protein, is therefore more suitable for the expression of eukaryotic gene.Again because of advantages such as its expression level are stable, zymotechnique is ripe, and become one of at present most popular heterologous protein gene expression system.Alcohol oxidase AOX1 promotor (pAOX1) is to apply more promotor during P.pastoris expresses, and its methanol induction is very strong.But in fermenting process, need to use the methyl alcohol of toxic volatile as inductor, need the conversion of carbon source, thereby inconvenient operation, and the production cycle of foreign protein is longer.Glyceraldehyde-3-phosphate dehydrogenase promotor (pGAP) is the composing type strong promoter of P.pastoris, has been used for expressing many heterologous proteins, and has obtained the output similar to pAOX1.In addition, GAP promotor has many advantages: recombinant bacterium fermenting process is simple, safe and do not need storage and the transportation of a large amount of methyl alcohol, is more suitable for large scale fermentation and produces, and has the potential of cultured continuously, and has reduced the production cost of target protein.

Yeast single cell protein has obvious advantage as feed, 50%-60% as about in protein content in yeast dry matter; In yeast, contain abundant enzyme, contain beta-glucan (57.0%), mannosans (6.6%), glycoprotein (22.0%) and chitin isoreactivity composition; In protein, contain the Methionin, methionine(Met) and the tryptophane that in animal body necessary each seed amino acid, particularly plant feed, lack more, biological value is better than plant protein greatly, and the digestibility of single cell protein is up to 85%～90%; Both animal was had to growth promoting function, and can improve immunity of organism again, for animal, yeast feed is a kind of desirable biological activity protein feed.

Summary of the invention

AvBD9 gene of the present invention is a kind of gene after avian β-defensins 9 gene optimizations that derive from chicken are processed, its optimization process is to carry out according to pichia spp preferences codon, the codon agg that yeast is of little use, cgt, ggg, ggc, gca etc. are optimized for preference codon aga, ggt, gct etc., utilize and obtain after DNASTAR software analysis, and add Kex2 restriction enzyme site encoding sequence AAAAGA at its 5 ' end, through this processing, can make target gene better express in pichia spp, and secrete the active mature peptide closer to natural structure.

The present invention is intended to disclose a kind of method of expressing chicken AvBD9 in yeast expression system, realize first chicken AvBD9 gene persistence high level expression in yeast, and constitutive expression, without methanol induction, and chicken AvBD9 expression product is secreted in fermented liquid substratum, get supernatant liquor, carry out separation and purification treatment, and develop a kind of green, safety, efficient novel fodder additive.

The Yeast engineering bacteria that can secrete AvBD9 of the present invention is achieved by the following technical solution.

Adopt recipient bacterium GS115, constitutive expression carrier plasmid pGHK α; 41 amino acid whose gene fragments of target protein; During construction of recombinant plasmid, between the EcoR I of plasmid pGHK α and Not I site, insert chicken AvBD9 goal gene fragment; Recombinant plasmid, through Sac I and Bgl II linearization for enzyme restriction, is removed Amp resistant gene, and electricity is converted into efficient Pichia pastoris GS115 competent cell; Utilize the dull and stereotyped primary dcreening operation of MD and PCR evaluation and screening to go out positive recombinant, and through the high copy of G418 resistance screening recon; Under identical condition, the bacterial strain of different copy numbers is carried out respectively to culture expression foreign protein, measure protein concn in nutrient solution, select high expression level bacterial strain; Recycling high expression level bacterial strain carries out fermentation culture, and expression product direct secretion, outside born of the same parents, contains protein product---the AvBD9 of expression in centrifugal resulting supernatant liquor.

The expression method of AvBD9 in yeast expression system, comprises the following steps:

(1) optimization design of goal gene AvBD9 is with synthetic;

(2) double digestion AvBD9 gene and carrier pGHK α, connect construction recombination plasmid, transforms Host Strains Pichia pastoris GS115;

(3) recombination microzyme screening, through cultivating, is AvBD9 in centrifugal gained supernatant liquor.

AvBD9 recombinant bacterial strain, its preserving number: CGMCC No7832; Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center; Depositary institution address: No. 3, No. 1, Beichen Lu, Chaoyang District, Beijing City institute; Preservation date: 2013-06-27; Classification And Nomenclature: pichia pastoris phaff; Latin name: Pichia pastoris.

Described fermentation culture be picking recombination microzyme list colony inoculation in 5mL YPD liquid nutrient medium, 30 ℃, 200rpm incubated overnight, then transfer and cultivate 2 days in 28 ℃ of 50mL improvement MD liquid nutrient mediums, 200rpm.

Before described conversion Host Strains, first recombinant plasmid is carried out to linearization for enzyme restriction by Sac I and Bgl II, to remove Amp resistant gene, increase security.

5 of described goal gene AvBD9 ' end adds Kex2 restriction enzyme site encoding sequence AAAAGA.

Described double digestion restriction enzyme used is EcoR I and Not I.

The advantage that the present invention has with respect to prior art and useful achievement.

AvBD9 recombinant bacterial strain of the present invention, the prior art comparing, it is easy and simple to handle, quick that recipient bacterium is selected, good stability and the pichia spp that is easy to high density fermentation are as genetic expression host, not only avoided the host expresses potential waste that in prokaryotic expression, fusion rotein causes and formed inclusion body, and remove the complicated procedures that removes carrier proteins from, and can carry out the modification of protein translation post-treatment.

The present invention's expression vector self used with signal Toplink by expression product direct secretion in culture supernatant, be easy to separating-purifying, also be easy to detect, and the Kex2 restriction enzyme site of introducing by AvBD9 gene can further improve the cutting efficiency of signal peptide, produce more mature peptides close to natural structure.

The present invention is also according to the codon preference of pichia spp, to the encoding gene of AvBD9, carried out codon optimized, eliminated the possibility that AvBD9 encoding gene is affected by codon utilization in heterologous expression system, the gene of the best codon design obtaining thus can increase protein output, makes protein production more effective and economical.

Expression vector used in the present invention is composing type carrier, sustainability secreting, expressing AvBD9, without methanol induction, also without storage and the transportation of methyl alcohol in producing, security is higher, for analysis and research and large-scale industrialization are from now on produced and applied and lay a good foundation.

Accompanying drawing explanation

Fig. 1 is gene complete sequence after AvBD9 optimizes;

Fig. 2 is that the PCR of recombinant plasmid identifies (M:DNA marker DL2000; 1: the PCR product of plasmid pGHK α; 2: the PCR product of recombinant plasmid pGHK α-AvBD9);

Fig. 3 is that the PCR of restructuring yeast strains identifies (M:DNA marker DL2000; The pcr amplification product of 1:GS115/pGHK α; The pcr amplification product of 2-9:GS115/pGHK α-AvBD9 bacterial strain);

Fig. 4 is standard protein BSA concentration and absorbancy dependency curve;

Tricine-SDS-PAGE analysis (the M:Protein marker that Fig. 5 is restructuring AvBD9; The expression supernatant of 1:GS115/pGHK α; 2,3: the expression supernatant of restructuring GS115/pGHK α-AvBD9);

Fig. 6 is the antimicrobial spectrum of restructuring AvBD9;

Fig. 7 is expression carrier used thereof plasmid pGHK α.

Embodiment

According to drawings and embodiments the present invention is described in further detail below.

Bacterial strain uses therefor in literary composition:

ATCC U.S. representative microbial DSMZ

Candida albicans (ATCC10231) Candida albican(ATCC10231)

Streptococcus aureus (ATCC25923) Staphylococcus aureu (ATCC25923)

Excrement intestines suis (ATCC29212) E.faecalis(ATCC29212)

Pseudomonas aeruginosa Pseudomonas aeruginosa ATCC27853

Salmonella paratyphi A Salmonella paratyphi A ATCC9150

White dysentery Salmonellas Salmonella pullorum S2

CMCC Chinese medicine bacterium preservation administrative center

Intestinal bacteria (CMCC44102) Escherichia coli(CMCC44102)

Enterobacter cloacae Enterobacter cloacae CMCC45301

Citrobacter Citrobacter CMCC48017

Moscow' paratyphi B Salmonella paratyphi B CMCC50094

Pichia pastoris GS115 is purchased from Life Technologies Corporation

The present invention utilizes pichia yeast expression system to build engineering bacteria, composing type secreting, expressing goes out AvBD9 albumen, molecular weight is about 5kDa left and right, and the ratio in expressing supernatant classifies 11.38% as, and the food borne pathogenses such as Salmonellas, intestinal bacteria are had to stronger bacteriostatic activity.

Embodiment 1:

In the present invention, adopting recipient bacterium is that Pichia pastoris GS115, vector plasmid are pGHK α; Goal gene is through 41 codon optimized nucleotide fragments that amino acid is corresponding; During construction of recombinant plasmid, between the EcoR I of plasmid pGHK α and Not I site, insert chicken AvBD9 goal gene fragment; Recombinant plasmid is through Sac I and Bgl II successively linearization for enzyme restriction, and electricity is converted into efficient Pichia pastoris GS115 competent cell; Utilize the dull and stereotyped primary dcreening operation of MD and PCR evaluation and screening to go out positive recombinant, and through the high copy of G418 resistance screening recon, then by protein expression level, select high expression level bacterial strain, carry out again fermentation culture, expression product direct secretion, outside born of the same parents, contains target protein product---the AvBD9 of expression in centrifugal resulting supernatant liquor.

Embodiment 2:

The concrete operation step of chicken β-defensin 9 Yeast engineering bacteria preparations and expression method is as follows:

(1) AvBD9 gene order is codon optimized and synthetic, and the gene order after optimization as shown in Figure 1, up: original series.Descending: codon optimized rear sequence.Add boldface type: the base of change.Underscore: restriction endonuclease cleavage site.Italic: Proteinase K ex2 restriction enzyme site encoding sequence.In square frame: terminator codon;

AvBD9 gene order (the accession number: AY534894.1) of announcing according to GenBank, with reference to pichia spp preference codon, under the prerequisite that does not change aminoacid sequence, utilize DNASTAR software optimization gene order, and add EcoR I restriction enzyme site and Kex2 restriction enzyme site encoding sequence AAAAGA at 5 of gene ' end, at 3 of gene ' end, add TAA site and Not I restriction enzyme site, full length gene after optimization is 146bp, and by life work biotechnology (Shanghai), limited-liability company is synthetic.

(2) Construction and identification of recombinant expression plasmid;

As shown in Figure 2, in related experiment of the present invention, with restriction enzyme EcoR I, Not I double digestion plasmid pUC57-AvBD9 and carrier pGHK α (as shown in Figure 7).Glue reclaims object product and connects by T4DNA Ligase, and Transformed E .coli DH5 α, screening positive clone, take pG1 and pG2 as primer (pG1:5 '-GTCCCTATTTCAATCA ATTGAA-3 ' and pG2:5 '-GCAAATGGCATTCTGACATCC-3 ', by life work biotechnology (Shanghai), limited-liability company is synthetic), after PCR checking, send the order-checking of raw work biotechnology (Shanghai) limited-liability company, the recombinant plasmid called after pGHK α-AvBD9 that checks order correct.Result shows, pcr amplification product is through agarose gel electrophoresis analysis, and empty plasmid carrier expands band and is about 462bp, and recombinant expression plasmid expands band and is about 572bp, conforms to theoretical value.By the transformed bacteria sample presentation order-checking that contains recombinant expression plasmid, sequencing result shows really to contain goal gene fragment in the plasmid of this transformed bacteria, and reading frame is correct, and expression plasmid pGHK α-AvBD9 successfully constructs.

(3) recombinant plasmid electricity is converted into Pichia pastoris GS115;

The E.coli DH5 α recombinant bacterium that contains recombinant plasmid pGHK α-AvBD9 is cultivated in a large number, extracting plasmid, and use Sac I and Bgl II successively enzyme excision remove Amp resistant gene, reclaim the object fragment after linearizing.The parameter that electric conversion instrument is set is 1.5kV, 25 μ F and 186 Ω, and object fragment electric shock is converted into Pichia pastoris GS115 competent cell.

(4) screening of recombination yeast engineering bacteria;

As shown in Figure 3, through the dull and stereotyped primary dcreening operation of MD, picking list bacterium colony, with yeast cell lysate lysing cell, obtain pastoris genomic dna, take it as template, with primer pG1 and pG2, carry out PCR and identify positive transformant, and point is connected to containing the high copy of screening recombinant bacterium on the YPD flat board of 0.5mg/mL G418, then by protein expression level, select high expression level bacterial strain.Pcr amplification product is through agarose gel electrophoresis analysis, and the band of 572bp, consistent with expection as seen; And the band of the same visible 462bp of empty carrier recombinant bacterial strain GS115/pGHK α processing of warp.

(5) expression of restructuring AvBD9;

Picking restructuring yeast strains, is inoculated in the triangular flask that contains 5mL YPD substratum, in 30 ℃ of 200rpm overnight incubation.Get overnight culture and be inoculated in the 500mL triangular flask containing 50mL fermention medium with 1% ratio row, in 28 ℃ of 200rpm, cultivate 48h.And in 4 ℃ of centrifugal 10min of 10000 * g, get supernatant liquor, be AvBD9.

(6) express the mensuration of supernatant protein concentration

As shown in Figure 4, adopt BCA determination of protein concentration test kit, the bovine serum albumin (BSA) of take is standard substance, measures the OD under different protein concentrations _570nmvalue, take protein concentration as X-axis, OD _570nmvalue is Y-axis, drawing standard albumen curve.By the culture supernatant dilution certain multiple of each recombinant bacterial strain, measure respectively its protein concentration, the relation of icp gene copy number and protein concentration, and select high expression level bacterial strain, as shown in the table.

Table1The?concentration?of?proteins

(7) Tricine-SDS-PAGE identifies expression product

According to the gel formula of following table and concentration, prepare protein electrophoresis gel.Get the appropriate supernatant liquor of high expression level strain, after concentrated, carry out Tricine-SDS-PAGE analysis, as shown in Figure 5.At relative molecular mass, be visible specific proteins band within the scope of 4.1～6.5kDa, proved that target protein has obtained secreting, expressing.Through gel imaging system scanning analysis, the expression amount of target protein accounts for 11.38% of supernatant total protein.And express the total protein concentration in supernatant according to it, calculating restructuring AvBD9 expression amount is 66.18mg/L.

(8) restructuring AvBD9 Analysis of Antimicrobial Activity

Adopt agar diffusion method, with intestinal bacteria CMCC44102, Salmonella paratyphi A ATCC9150, Pseudomonas aeruginosa ATCC27853, white dysentery Salmonellas S2, citrobacter CMCC48017, excrement intestines hammer ATCC29212, enterobacter cloacae CMCC45301 is test organisms, get respectively above-mentioned each bacterium liquid 20 μ L in logarithmic phase mid-term and be added to the LB solid medium that 20mL is chilled to 50 ℃, mix, pour 9cm flat board into, after it solidifies.With the punching of 7mm sterilizing punch tool, in hole, drip 40 μ L recombinant bacterium supernatant concentrated solutions, cultivate 6～12h, observe and record antibacterial circle diameter for 37 ℃.To add 40 μ L penbritins (Amp, 100 μ g/mL) or the positive contrast of kantlex (Kan, 250 μ g/mL), with expression supernatant concentrated solution and the negative contrast of PBS of the GS115/pGHK α that processes equally.Result shows: restructuring AvBD9 as intestinal bacteria, Salmonellas, Pseudomonas aeruginosa, enterococcus faecalis etc. all have significant fungistatic effect, can form inhibition zone clearly, as Fig. 6 to pathogenic bacteria.AvBD9 is as shown in the table to the antibacterial circle diameter size of each pathogenic bacteria in restructuring.

Table2The?antimicrobial?diameter?of?AvBD9to?pathogenic?microbes?in?this?study

Note: on flat board, sample bore dia is 7mm; * represent that microbiotic used is Kan; Obvious inhibition zone is not observed in-expression.

Above-described embodiment is only explanation technical conceive of the present invention and feature, and its object is to allow the personage who is familiar with this art can understand content of the present invention and be implemented, and can not limit the scope of the invention with this.All equivalences that spirit is done according to the present invention change or modify, and all should be encompassed in protection scope of the present invention.

Claims (10)

1. optimize an AvBD9 gene, it is characterized in that, its gene order is as shown in sequence table.

2. optimization AvBD9 gene according to claim 1, it is characterized in that, described optimization AvBD9 gene is that the codon that is of little use of the yeast in AvBD9 gene is replaced to its habitual codon design gene order, and AvBD9 gene is added to EcoR I restriction enzyme site and Kex2 restriction enzyme site encoding sequence AAAAGA at 5 of gene ' end, at 3 of gene ' end, add that TAA site and Not I restriction enzyme site obtain, the full length gene of optimizing AvBD9 gene is 157bp.

3. chicken β-defensin 9 Pichia yeast engineering preparation methods, is characterized in that, comprise step:

(1) optimizing AvBD9 gene obtains;

(2) double digestion AvBD9 gene and carrier pGHK α, connect and be built into recombinant plasmid, and be converted into conversion Host Strains Pichia pastoris GS115 acquisition restructuring yeast strains.

4. chicken β-defensin 9 Pichia yeast engineering preparation methods according to claim 3, is characterized in that, the restriction enzyme that double digestion AvBD9 gene and carrier pGHK α are used is EcoR I and Not I.

5. chicken β-defensin 9 Pichia yeast engineering preparation methods according to claim 3, is characterized in that, the recombinant plasmid use Sac I that step (2) is obtained and Bgl II respectively enzyme cut out Amp resistant gene.

6. chicken β-defensin 9 Pichia yeast engineering preparation methods according to claim 3, is characterized in that, the transform mode of described recombinant plasmid is electric shock conversion.

7. chicken β-defensin 9 Pichia yeast engineering expression methods, is characterized in that, by the restructuring yeast strains of claim 3 gained, through screening, fermentation culture, is chicken β-defensin 9 in centrifugal gained supernatant liquor.

8. chicken β-defensin 9 Pichia yeast engineering expression methods according to claim 7, it is characterized in that, described screening is through the dull and stereotyped primary dcreening operation of MD, single bacterium colony of restructuring yeast strains described in picking, with yeast cell lysate lysing cell, obtain pastoris genomic dna, take it as template, with primer pG1:5 '-GTCCCTATTTCAATCAATTGAA-3 ' and pG2:5 '-GCAAATGGCATTCTGACATCC-3 ', carry out PCR and identify positive transformant, and point is connected to containing the high copy of screening recombinant bacterium on the YPD flat board of 0.5mg/mL G418.

9. chicken β-defensin 9 Pichia yeast engineering expression methods according to claim 7, it is characterized in that, described fermentation culture is that single colony inoculation of picking restructuring yeast strains is in 5mL YPD liquid nutrient medium, 30 ℃, 200rpm incubated overnight, then transfer in 28 ℃ of 50mL MD liquid nutrient mediums, 200rpm shaking culture 2 days.

10. chicken β-defensin 9 Pichia yeast engineering expression methods according to claim 9, it is characterized in that, described MD substratum includes 1～3%wtD-glucose, 1.3～1.4%wtYNB, 0.0003～0.0005 ‰ wt vitamin H, 0.1～0.3%wt calcium carbonate.

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