CN1294261C - High efficiency production method for recombinant silkworm antibacterial peptide CM4 - Google Patents
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Abstract
The present invention relates to a method for expressing and purifying recombinant protein in the field of gene engineering, particularly to a high-efficiency production method for recombinant silkworm antibacterial peptide CM4, which comprises the following processes: (1) using a recombinant DNA technology to build an ABP-CM4 eukaryotic expression vector (shown in drawing 1); (2) transforming host cell pichia yeast by the ABP-CM4 eukaryotic expression vector to build expression engineering bacteria; (3) culturing the engineering bacteria by fermentation, and performing methanol inducing expression; (4) purifying the expression products through the chromatography of a molecular sieve column to obtain the products of recombinant silkworm antibacterial peptide CM4. In the present invention, the gene engineering technology is used to efficiently express the recombinant antibacterial peptide CM4 in the eukaryotic host cells; thus, the present invention has the advantages of high expression efficiency, simple separation and purification, easy operation, easy magnification and favorable stability, and is suitable for large-scale industrial production. The present invention has important value for developing novel high-efficiency anti-infection medicines, and has wide application prospects in pharmaceutical industry.
Description
Technical field
The present invention relates to the expression and the purifying of genetically engineered field recombinant protein, especially relate to the high-efficiency method for producing of recombinant silkworm antibacterial peptide CM 4.
Background technology
Antibacterial peptide is the molecule effector of innate immunity, and they contain 15-45 amino-acid residue usually, and net charge is for just.The halfcystine linear peptides that do not contain of Cecropins type is found the earliest, derives from insect.Have now found that antibacterial peptide, albumen have more than 800, derive from animal, plant field.The main action target spot of antibacterial peptide is the film of bacterium, and the process of killing and wounding must be faster than the speed of bacterial growth.It is found that many traditional microbiotic, particularly penicillin, generation that can the inducible resistance bacterial strain.In order to solve the problem of antibiotic resistance, develop new microbiotic or the antibacterial peptide particularly important that just seems.Antibacterial peptide has good antibiotic selectivity and unique antibiotic pattern, and can not induce the generation Resistant strain, is degraded easily, is difficult for enrichment in microbe, therefore, is believed to become alternative antibiotic choice drug.
Silkworm antibacterial peptide CM 4 is extracted from the fortunatus hemolymph by people such as Zhang Shuanquan, similar to the member of Cecropins family, it also has amphipathic α-Luo Xuanjiegou zone, belong to Cecropins family, contain 35 amino-acid residues, molecular weight is about 3.5KD, and very strong sterilizing ability is arranged, and bacterium, pathogenic bacteria, fungi and virus are all had effect.
At present, many antibacterial peptides are being developed patent medicine, but the natural output of antibacterial peptide is very low, and prices are rather stiff for synthetic peptide, and this becomes a bottleneck of exploitation patent medicine, produce antibacterial peptide by genetic engineering technique and have broad application prospects.
Have report once to adopt the formal representation silkworm antibacterial peptide CM 4 of escherichia expression system with fusion rotein, the result is not very good.In recent years, be that the research of genetically engineered recipient bacterium is subjected to people day by day and payes attention to the yeast.Especially methyl alcohol nutritious yeast expression system, this system has become one of very successful exogenous protein expression system, have many advantages: (1) utilizes alcohol oxidase (the alochol oxidaseI AOXI) promotor that is subjected to methanol induction, can strictly control expression of exogenous gene, the high expression level no matter born of the same parents are interior, born of the same parents all can realize foreign gene outward; (2) the pichia spp growth is quick, and culture condition is simple, is fit to high-density culture, and wet cell weight can reach 450g in the every liter of nutrient solution in fermentation back, helps improving target protein output; (3) pichia spp is as a kind of eucaryotic organism, and the albumen processing after can translating is so that foreign protein obtains correct advantages such as folding and modification.Also there is report to express antibacterial peptide (transformation of yellow Yadong antibacterial peptide AD gene and the expression South China Science ﹠ Engineering University journal in pichia spp both at home and abroad with Bichi yeast system, 2002,30 (2), 13-16), but pichia yeast expression system secreting, expressing silkworm antibacterial peptide CM 4 does not still have report at present.
Summary of the invention
The invention provides a kind of production method that adopts eukaryotic expression system express recombinant silkworm antibacterial peptide CM 4.
Technical scheme of the present invention is: a kind of production method of recombinant silkworm antibacterial peptide CM 4 may further comprise the steps;
(1) uses recombinant DNA technology, make up ABP-CM4 carrier for expression of eukaryon pPICZaA-CM4;
(2) ABP-CM4 carrier for expression of eukaryon transformed host cell Pichia yeast, the construction expression engineering bacteria;
(3) fermentation culture engineering bacteria carries out methanol induction and expresses;
(4) expression product gets the recombinant silkworm antibacterial peptide CM 4 product through sephadex G50 molecular sieve column chromatography purifying.
Optimized technical scheme recommendation step of the present invention (3) is specially: express the about 20h to OD600 of engineering bacteria and reach 2-6 with BMGY, 28 ℃～30 ℃ fermentation culture; Centrifugal, be about 1.0,20 ℃～30 ℃ to OD600 and cultivate about 72h with BMMY is resuspended, adding 100% methyl alcohol to methyl alcohol concentration expressed in percentage by volume every 24h is 0.5-3%, preferably 0.5%.
Step (4) is specially: express the supernatant freeze-drying, be dissolved in aqua sterilisa, boil, low-temperature centrifugation is removed precipitation,
Molecular sieve column chromatography: separating medium is sephadex G50, uses 0.05M, 5 column volumes of PH5.1 Ammonium Acetate damping fluid balance, last sample; 0.05M PH5.1 Ammonium Acetate buffer solution elution is collected the albumen elution peak, gets product.
The further optimized technical scheme of the present invention is to add the acid hydrolysis casein in step (3) culture expression process.
Using gene engineering technique of the present invention has efficiently expressed recombinant antibacterial peptide CM4 in eukaryotic host cell, CM4 has germicidal action to multiple fungi through this recombinant antibacterial peptide of experimental verification, and expression efficiency height of the present invention, separation and purification is simple, easy to operate, easily amplify, good stability is suitable for large-scale industrial production.Therefore, the present invention has important value to developing new and effective anti-infectives, has broad application prospects at pharmaceutical industry.
Description of drawings
Fig. 1 is the expression vector establishment synoptic diagram;
Fig. 2 is the synoptic diagram of the relation of acid hydrolysis casein concentration and product expression amount;
Fig. 3 is the synoptic diagram of the relation of methanol concentration and product expression amount;
Fig. 4 is the synoptic diagram of the relation of induction time and product expression amount;
Fig. 5 is chromatography and anti-microbial activity analysis chart;
Fig. 6 is the Tricine-SDS-PAGE electrophoretic analysis of purified product; Wherein swimming lane N is a natural antibacterial peptide; Swimming lane M is small molecules Marker; Swimming lane R is a purified product of the present invention;
Fig. 7 is that the acidity of purified product is surveyed the electrophoretic analysis of living; Wherein 4 is inhibition zone;
Fig. 8 is the bacteriostatic action of purified product to aspergillus niger; The wherein negative contrast of C, 1,2,3 is inhibition zone;
Fig. 9 is the bacteriostatic action of purified product to viride; The wherein negative contrast of C, 1,2,3 is inhibition zone;
Embodiment
The present invention is further described below in conjunction with drawings and Examples.
Embodiment 1:
(1) construction of expression vector: according to the preference codon and the antibacterial peptide CM 4 aminoacid sequence of pichia spp gene translation, the multiple clone site of pPIC9, synthetic two primers of design are as follows:
ABP-F?5′agatggaagattttcaagaagatcgagaaggtcggtcaaaacatcagagacggtatcgt3′
ABP-R?5′aatagtagcagcttgaccgacgacagcgacagctggaccagccttgacgataccgtctc3′
Restriction enzyme site is Xho I and Xba I, and expression plasmid is pPICZaA, and vector construction is seen Fig. 1;
(2) make up genetic engineering bacterium: transform the host bacterium with above-mentioned expression vector, host strain is yeast GS115;
(3) abduction delivering: express the about 20h to OD600 of engineering bacteria and reach 2-6 with BMGY, 28 ℃～30 ℃ fermentation culture; Centrifugal, be about 1.0,20 ℃～30 ℃ cultivations with BMMY is resuspended to OD600, adding 100% methyl alcohol to methyl alcohol concentration expressed in percentage by volume every 24h is 0.5-3%; Analyze the different final concentrations of methyl alcohol as shown in Figure 3 to the influence expressed.
(4) purifying: express the supernatant freeze-drying, be dissolved in the aqua sterilisa; Boil 10～30min, 4 ℃, the centrifugal 5min of 10000rpm;
Adopt sieve chromatography (separating medium is sephadex G50), use 0.05M, (1.5 * 40cm), flow velocity is 1ml/min to 5 column volumes of PH5.1 Ammonium Acetate damping fluid balance; Sample on the above-mentioned sample; 0.05M, PH5.1 Ammonium Acetate buffer solution elution (0.5ml/min), collecting has bacteriostatic activity albumen elutriant.
Embodiment 2: substantially the same manner as Example 1, difference is, in the abduction delivering process, adds 1-3% (W/V) acid hydrolysis casein and suppress degraded in BMMY.The relation of analysis acid hydrolysis casein addition and expression product as shown in Figure 2.
Adopt sieve chromatography (separating medium is sephadex G50), use 0.05M, (1.5 * 40cm), flow velocity is 0.5ml/min to 5 column volumes of PH5.1 Ammonium Acetate damping fluid balance; Sample on the above-mentioned sample; 0.05M, PH5.1 Ammonium Acetate buffer solution elution (0.15ml/min), chromatography and anti-microbial activity are analyzed shown in Figure 5, and collecting has bacteriostatic activity albumen elutriant.
Embodiment 3: the method identical with embodiment 2, the relation of analyzing methanol induction incubation time and expression product as shown in Figure 4.
Embodiment 4:Tricine-SDS-PAGE detects
The separation gel (urea) for preparing 15% concentration, the middle glue of 10% concentration and the concentrated glue of 4% concentration; Final purification of samples concentrates 10 times, with 2 * sample-loading buffer (4%SDS, 12% glycerine, 50mMTris, 2% mercaptoethanol, 0.01% tetrabromophenol sulfonphthalein, 6N HCl transfers PH to 6.8) mix, go up the sample electrophoresis behind the boiling water bath 5min, stationary liquid (50% methyl alcohol, 10% acetic acid) fixing 30min, the Kao Masi light blue G-250 1h that dyes decolours with destainer again.As shown in Figure 6, the sample after the separation and purification is through the Tricine-SDS-PAGE check and analysis, and every liter of fermented liquid can obtain purity greater than 96% sample 15mg.
Embodiment 5: acid electrophoresis is identified
Preparation 15% continuous glue (activity), sample mix with sample-loading buffer directly goes up sample, and 100V runs 5h; Glue is used among the sterilising liq LB and 30min, and one deck contains 1%E.coliK on the glue upper berth
12D
31Solid LB, put 37 ℃ and spend the night, as shown in Figure 7, inhibition zone 4 appears.
Embodiment 6: the responsive fungi screening of purified product
Aspergillus niger (Aspergillus niger), flavus (Aspergillus flavus), Aspergillus parasiticus (Aspergillus parasiticus), viride (Trichodermaviride), beading sickle spore (Fusarium moniliforme), cotton rot (Fusariumoxyporium), pink sickle spore (Fusarium roseum), Candida albicans (Candidaalbicans) etc. are inoculated on the potato culture (PDA), 28 ℃, 5 days.Use aseptic water washing, 100ul spore suspension (10
4/ ml) coat potato culture, and in the last hole of beating 5mm, hole c adds not inductive supernatant for contrast, all the other each holes add purified product, cultivate 3 days, and with Fig. 8, shown in Figure 9 basic identical, inhibition zone 1,2,3 occurs for 28 ℃.Show that above-mentioned bacterial strains is all to the recombinant silkworm antibacterial peptide CM 4 sensitivity.
Claims (4)
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| CN102191251A (en) * | 2011-03-25 | 2011-09-21 | 盐城星海饲料有限公司 | Method for expressing Cecropin CM4 gene in sf9 insect cells |
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