CN100460502C - Genetically engineered bacteria for efficiently expressing recombinant human sweet gland antibacterial peptide, and its constructing method and use - Google Patents
Genetically engineered bacteria for efficiently expressing recombinant human sweet gland antibacterial peptide, and its constructing method and use Download PDFInfo
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- CN100460502C CN100460502C CNB2005100236744A CN200510023674A CN100460502C CN 100460502 C CN100460502 C CN 100460502C CN B2005100236744 A CNB2005100236744 A CN B2005100236744A CN 200510023674 A CN200510023674 A CN 200510023674A CN 100460502 C CN100460502 C CN 100460502C
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Abstract
This invention is genetic engineer bacterial of high-efficient expressive sweat gland recomposing antibacterial peptide and its composing facture and utilization. The genetic bacterial carries DH5 alpha, BL21(DE3) with recomposing plasmids that contain sweat gland antibacterial peptide-plasmid pET32a(+) of DCD-1L gene. The structure of this gene is: according the amino acid of DCD-1L, compose two DNA chains in leading medium form, and 26bp is between the two chains, obtain double-chain DNA of DCD-1L through PCR expansion, insert plasmid pET32a(+) through enzyme cutting and compose Pet-32a-DCD-1L and obtain the high-efficient expressive sweat gland recomposing antibacterial peptide. The relating method includes three processes: liquid culture; purification to get sweat gland peptide syncretic albumen and preparation of sweat gland antibacterial peptide. The strong points of this invention: low cost, high expressive amount, simple purification procedure, easy operation and high yield.
Description
Technical field
The present invention relates to a kind of genetic engineering bacterium and construction process and application of efficiently expressing recombinant human sweet gland antibacterial peptide, belong to technical field of bioengineering.
Background technology
Microbiotic has been the jinx of human infectious disease and inflammation since it produces always.But because antibiotic long-term and high dosage use, many bacterial strains produce resistance to it, even nearly all antibiotic " superbacteria " occurred tolerating.Therefore, seek that a kind of can to substitute antibiotic medicine extremely urgent.Antibacterial peptide claims peptide antibiotic again, because of its anti-microbial activity height, and has a broad antifungal spectrum, kind is many, and alternative scope is wide, and the target bacterial strain is difficult for producing reasons such as resistant mutation, is considered to substitute antibiotic ideal medicament.Simultaneously, antibacterial peptide not only has lethal effect to bacterium, fungi and protozoon, can also play regulating effect as the division growth of mitogen, signaling molecule pair cell, and along with the increase of antibacterial peptide concentration, and it can be antiviral and tumour cell etc.Antibacterial peptide has broad application prospects in disease treatment as polyfunctional molecule.
Dermcidin-1L Dermcidin is called for short DCD, is that scientist such as Schittek separates the small molecules antibacterial peptide that obtains, be made up of 110 amino acid calendar year 2001 from the human sweat.Dermcidin-1 is called for short DCD-1, is to be processed into 47 amino acid whose little peptides through proteolytic enzyme by 110 amino acid whose DCD of coding in sweat, promptly is made up of the amino acid of the 63-109 position of product before the DCD processing, has broad-spectrum antibacterial activity.Dermcidin-1L, i.e. DCD-1L is that 3 ' end at DCD-1 has added a leucine, has stronger anti-microbial activity than DCD-1.Recently, discovery DCD-1 can kill and reside in the chronic pathogenic bacterium-staphylococcus epidermidis that causes hospital infection on the medicine equipment, on concurrent present various skin cancer and the mammary cancer this expression of gene is arranged also.In sum, DCD-1L is expected in the future to become and has broad spectrum antibiotic activity and anti-tumor drug.External at present preparation DCD-1L can only be by separating or chemosynthesis from people's sweat.The shortcoming that background technology prepares DCD-1L is, and is from people's sweat separation DCD-1L, not only time-consuming but also require great effort; Chemosynthesis DCD-1L technical difficulty is big, synthetic cost height, purification difficult.Product with above-mentioned these method preparations costs an arm and a leg, and does not conform to China's actual conditions, therefore presses for the method for preparing DCD-1L of a kind of efficient and cheap of development.
Summary of the invention
Be the Dermcidin-1L of the low reorganization of production cost, at first must find and make up a kind of Dermcidin-1L that efficiently expresses, can produce the genetic engineering bacterium of Dermcidin-1L.
The technical problem that the present invention will solve is the genetic engineering bacterium that proposes a kind of efficiently expressing recombinant human sweet gland antibacterial peptide.This genetic engineering bacterium is the e. coli bl21 (DE3) that carries recombinant plasmid, and described recombinant plasmid is to contain the plasmid pET32a (+) that Dermcidin-1L is the DCD-1L gene.
Another technical problem that the present invention will solve provides a kind of construction process of genetic engineering bacterium of efficiently expressing recombinant human sweet gland antibacterial peptide.The present invention is by solving above technical problem by the following technical solutions.Amino acid according to DCD-1L is formed, two DNA chains that synthesize DCD-1L with the form of primer, article two, the 26bp complementation is arranged between the chain, obtain the DCD-1L double-stranded DNA by pcr amplification, cut through enzyme, insert plasmid pET-32a (+), be built into recombinant plasmid pET-32a-DCD-1L, transformed into escherichia coli, the genetic engineering bacterium of acquisition efficiently expressing recombinant human sweet gland antibacterial peptide.
Now be described with reference to the accompanying drawings technical scheme of the present invention.
A kind of construction process of genetic engineering bacterium of efficiently expressing recombinant human sweet gland antibacterial peptide is characterized in that, the concrete operations step:
The amplification of the first step DCD-1L dna sequence dna
Entrust the following base sequence of biotech company's chemosynthesis of specialty: DCD-1La-Dermcidin-1L 5 ' end 85bp single stranded DNA sequence, DCD-1L b--Dermcidin-1L 3 ' end 85bp single stranded DNA sequence, P5--5 ' holds primer, introduce BamHI restriction enzyme site and FactorXa site, P3--3 ' holds primer, introduce EcoRI restriction enzyme site and terminator codon TCA, DCD-1L a (85bp):
5’-TCTTCTTTGTTGGAGAAGGGTTTGGACGGTGCTAAGAAGGT GTTGGTGG
TTTGGGTAAG TTGGGTAAGG ACGCTGTTGA GGACT-3’
DCD-1L?b(85bp)：
5’-CAAAACAGAGTCCAAAACGTCCTTAACGTCGTGAACAGCACCCTTACCAA
CAGACTCCAA GTCCTCAACA GCGTCCTTAC CCAAC-3’
BamHI
P5：5’-GGCGGATCC
ATCGAAGGTCGTTCTTCTTTGTTGGAGAAGG-3’
Factor?Xa
EcoRI
P3：5’-CCGGAATTC
TCACAAAACAGAGTCC-3’
End
DCD-1L a and DCD-1L b be template each other, add Ex Taq archaeal dna polymerase, carry out pcr amplification, obtain double-stranded DNA, being complete DCD-1L gene fragment, is template with the double-stranded DNA, adds primer P5 and P3 and carries out pcr amplification again, reaction product reclaims with DNA GelExtraction Kit, obtains the DCD-1L dna fragmentation;
The second step structure contains the DCD-1L dna fragmentation of the genetic engineering bacterium of Dermcidin-1L gene order with two kinds of restriction enzymes double zyme cutting the first steps acquisitions, described two kinds of restriction enzymes are BamHI and EcoRI, purifying reclaims small segment, with same two kinds of restriction enzymes double zyme cutting plasmid pET-32a (+), thereby purifying reclaims big fragment, the small segment and the big fragment that connect above-mentioned recovery, the recombinant plasmid pET-32a-DCD-1L that must contain the DCD-1L dna fragmentation, pET-32a-DCD-1L is transformed into bacillus coli DH 5 alpha with recombinant plasmid, make and contain the Dermcidin-1L gene order, it is the genetic engineering bacterium of DCD-1L dna sequence dna, entrust the biotech company of specialty to check order this genetic engineering bacterium, confirm that this genetic engineering bacterium contains complete reorganization DCD-1L gene fragment;
The 3rd step made up the genetic engineering bacterium that efficiently expresses Dermcidin-1L
Genetic engineering bacterium extracting recombinant plasmid pET-32a-DCD-1L from second step built is transformed into e. coli bl21 (DE3), the genetic engineering bacterium of the Dermcidin-1L that obtains efficiently expressing.
The plasmid pET-32a (+) that above-mentioned structure relates to, bacillus coli DH 5 alpha, BL21 (DE3), restriction enzyme BamHI, EcoRI and Ex Taq archaeal dna polymerase all can be buied from market.Entrusting the synthetic described base sequence that gets and the biotech company of order-checking is the learned Bioisystech Co., Ltd in Shanghai.
Another technical problem that the present invention will solve is to propose a kind of application that efficiently expresses the genetic engineering bacterium of Dermcidin-1L, promptly proposes the method that the described genetic engineering bacterium of a kind of usefulness is produced Dermcidin-1L.
The present invention solves the problems of the technologies described above by following technical scheme.
The method that the described genetic engineering bacterium of a kind of usefulness is produced Dermcidin-1L is characterized in that the concrete operations step:
The first step liquid culture
The above-mentioned genetic engineering bacterium that efficiently expresses Dermcidin-1L that builds is cultured to OD in the LB liquid nutrient medium
600nm=0.4～1.0 o'clock, the adding final concentration was that the IPTG of 0.4～1.0mM carried out abduction delivering 3～5 hours, produces and accumulate the Dermcidin-1L fusion rotein of solubility expression;
The Dermcidin-1L fusion rotein that the second step purifying makes
Centrifugal collection thalline is with the resuspended thalline of damping fluid, after the carrying out ultrasonic bacteria breaking, centrifugal collection supernatant, carry out affinity chromatography, the fusion rotein component of collector's sweet gland antibacterial peptide is that the Millipore Amicon Ultra-15 ultrafiltration pipe of 5KD is concentrated into 2～8mg/mL with albumen with molecular weight cut-off;
The 3rd step preparation recombinant human sweet gland antibacterial peptide
With Factor Xa enzymolysis Dermcidin-1L fusion rotein, 23 ℃ of following cracking 2～5 hours, termination reaction is carried out affinity chromatography once more, collects and penetrates the peak, and lyophilize obtains recombinant human sweet gland antibacterial peptide.
The invention has the advantages that:
(1) produce recombinant human sweet gland antibacterial peptide by the method for genetic engineering technique, it is low to produce the recombinant human sweet gland antibacterial peptide cost than chemical synthesis process;
(2) method by genetic engineering technique makes up the genetic engineering bacterium that contains the Dermcidin-1L gene, has improved the expression amount of Dermcidin-1L greatly;
(3) will carry out affinity chromatography one time, just can obtain purer Dermcidin-1L fusion rotein from fermented liquid, purification step is easy, is easier to operation than the repeatedly chromatography of conventional art;
(4) just can obtain recombinant human sweet gland antibacterial peptide as long as carry out primary enzymolysis, purifying is very easy, the yield height.
Thereby produce recombinant human sweet gland antibacterial peptide with genetic engineering bacterium provided by the invention, and the output height, purifying process is easy, and production cost is lower.
Description of drawings
Fig. 1 is the structure synoptic diagram of recombinant plasmid pET-32a-DCD-1L.
Fig. 2 is the sequencing result figure of recombinant plasmid pET-32a-DCD-1L.
Fig. 3 is that the separation and purification and the enzyme of recombinant human sweet gland antibacterial peptide cut figure as a result, and wherein 1 is the standard protein molecular mass; 2 for carrying the fusion rotein of recombinant human sweet gland antibacterial peptide; 3 be the fusion rotein of recombinant human sweet gland antibacterial peptide through Factor Xa enzymolysis, 4 is the recombinant human sweet gland antibacterial peptide behind the purifying.
Fig. 4 is the mass spectrum of recombinant human sweet gland antibacterial peptide.
The result shows with the size of the prepared recombinant human sweet gland antibacterial peptide molecular weight of the technology of the present invention route consistent with its theoretical value.
Fig. 5 is the active figure of the anti-streptococcus aureus of recombinant human sweet gland antibacterial peptide.
Fig. 6 is the active figure of recombinant human sweet gland antibacterial peptide Chinese People's Anti-Japanese Military and Political College enterobacteria.
Fig. 7 is the active figure of the anti-Candida albicans of recombinant human sweet gland antibacterial peptide.
Embodiment
Below in conjunction with accompanying drawing,, further specify the present invention by embodiment.The experimental technique of all unreceipted actual conditionses among specification sheets and the embodiment, condition is carried out routinely.
The amplification of the first step DCD-1L dna sequence dna
Entrust the following base sequence of biotech company's chemosynthesis of specialty: DCD-1L a-Dermcidin-1L 5 ' end 85bp single stranded DNA sequence, DCD-1L b--Dermcidin-1L 3 ' end 85bp single stranded DNA sequence; P5--5 ' holds primer, introduces BamHI restriction enzyme site and FactorXa site, and P3--3 ' holds primer, introduces EcoRI restriction enzyme site and terminator codon TCA, DCD-1La (85b p):
5’-TCTTCTTTGTTGGAGAAGGGTTTGGACGGTGCTAAGAAGG?TGTTGGTGG
TTTGGGTAAG TTGGGTAAGG ACGCTGTTGA GGACT-3’
DCD-1L?b(85bp)：
5’-CAAAACAGAGTCCAAAACGTCCTTAACGTCGTGAACAGCACCCTTACCAA
CAGACTCCAA GTCCTCAACA GCGTCCTTAC CCAAC-3’
BamHI
P5：5’-GGCGGATCC
ATCGAAGGTCGTTCTTCTTTGTTGGAGAAGG-3’
EcoRI Factor?Xa
P3：5’-CCGGAATTC
TCACAAAACAGAGTCC-3’
End
DCD-1L a and DCD-1L b be template each other, adds Ex Taq archaeal dna polymerase, carries out pcr amplification by following condition: 94 ℃, and 3min; 45 ℃, 2min; 72 ℃, 10min obtains double-stranded DNA, promptly complete DCD-1L gene fragment.With the double-stranded DNA is template, adds primer P5 and P3 and carries out pcr amplification.Comprise in the PCR reaction system: 2 μ l templates, promptly double-stranded DCD-1L gene, 1.5 μ l 10X Taq buffer, 3 μ l Ex Taq archaeal dna polymerases, 4 μ l dNTP, each 2 μ l of primer P5 and P3,3.5 μ l sterilized waters.The pcr amplification condition is: 94 ℃ of 30s, and 58 ℃ of 30s, 72 ℃ of 45s circulate after 28 times 72 ℃ and extend 5min.Reaction product reclaims with DNA Gel Extraction Kit.
Second step made up the genetic engineering bacterium that contains the Dermcidin-1L gene order
DCD-1L dna fragmentation with two kinds of restriction enzymes double zyme cutting the first steps acquisitions, described two kinds of restriction enzymes are BamHI and EcoRI, purifying reclaims small segment, with same two kinds of restriction enzymes double zyme cutting plasmid pET-32a (+), purifying reclaims big fragment, the small segment and the big fragment that connect above-mentioned recovery obtain containing the recombinant plasmid pET-32a-DCD-1L of DCD-1L dna fragmentation.PET-32a-DCD-1L is transformed into bacillus coli DH 5 alpha with recombinant plasmid, makes to contain the Dermcidin-1L gene order, i.e. the genetic engineering bacterium of DCD-1L dna sequence dna.
The 3rd step made up the genetic engineering bacterium that efficiently expresses Dermcidin-1L
Genetic engineering bacterium extracting recombinant plasmid pET-32a-DCD-1L from second step built is transformed into e. coli bl21 (DE3), the genetic engineering bacterium of the Dermcidin-1L that obtains efficiently expressing.
2 one kinds of application that efficiently express the genetic engineering bacterium of Dermcidin-1L of embodiment are promptly produced Dermcidin-1L with described genetic engineering bacterium.Following affinity chromatography medium is the NTA-0 resin, and available from Novagen company, following Factor Xa is available from NEB company.
The first step liquid culture
The genetic engineering bacterium that builds among the embodiment 1 is inoculated in 20mL contains in the LB substratum of 100mg/mL Amp, 37 ℃, 210rpm is cultured to OD600=0.6-1.0.5000rpm, 4 ℃, centrifugal 5min, the thalline of collecting precipitation, 4 ℃ of preservations are spent the night.Contained the resuspended thalline of LB substratum of 100mg/mL Amp with 20mL in second day, 4% volume is inoculated in the LB substratum that 400mL contains 100mg/mL Amp.Be cultured to OD600nm=0.4～1.0.Adding final concentration is 0.4～1.0mM IPTG abduction delivering, 3～5h.After will shaking bottle and placing on ice ice bath 5min, 6000rpm, 4 ℃, centrifugal 5-10min collects thalline.Add the resuspended thalline of 100mL NTA-0 Buffer, add the PMSF that final concentration is 1mM simultaneously.The ultrasonic disruption thalline, centrifugal collection supernatant.See 1 of accompanying drawing 3.
The second step purifying makes into the sweet gland antibacterial peptide fusion rotein
Supernatant gets fusion rotein TrxA-DCD-1L with affinity column SNBC 3S NTA Resin separation and purification.NTA-0Bu ffer balance NTA-Resin with 5 times of NTA volumes.Sample is added among the NTA-Resin, use the NTA-0 of 2 times of NTA volumes respectively, NTA-20, NTA-40, NTA-60, NTA-80, NTA-100, NTA-200, the NTA-1000 Buffer wash-out of 5 times of NTA volumes, collect the elutriant of penetrating component and each component respectively, be used for SDS-PAGE analysing protein wash-out situation.Analyze the elutriant that contains fusion rotein TrxA-DCD-1L component molecular sieve Sephadex G-25 Coarse desalination through SDS-PAGE, concentrate with 5K ultrafiltration pipe then.See 2 of accompanying drawing 3.Protein concentration is measured with the Lowry method.
The 3rd step preparation Dermcidin-1L
Fusion rotein TrxA-DCD-1L cuts with Factor Xa proteolytic enzyme enzyme. and every milligram of fusion rotein adds the proteolytic enzyme of 1.5 units, and 37 ℃, enzyme was cut 1 hour.See 3 of accompanying drawing 3.Enzyme is cut product and is separated with affinity column SNBC 3S NTA Resin. collect the elution peak penetrate peak and NTA-0. merge this two kinds of components, the proteolytic enzyme of 31KD removed with the ultrafiltration pipe of 10K, again with the ultrafiltration pipe of 1～5K to DCD-1L desalination and concentrated.See 4 of accompanying drawing 3.MALDI-TOF/TOF-MS measures the molecular weight of reorganization DCD-1L.The results are shown in accompanying drawing 4.The iso-electric point of reorganization DCD-1L is measured in isoelectrofocusing.Protein concentration is measured with the Lowry method.
The anti-streptococcus aureus CMCC of embodiment 3 Dermcidin-1Ls (B) 26003 effects
Golden yellow grape ball CMCC (B) 26003 is inoculated in the 20mLLB substratum, is cultured to OD600nm=0.4～0.5.Collect thalline, after washing 2 times with 10mM PBS (pH7.4), use 10mM pH6.5, the PBS of 100mM NaCl is diluted to 2 * 106/mL. with bacterium and adds 100 μ L bacterium liquid in the reorganization DCD-1L of different concns, 37 ℃, acts on 4 hours, dilute 10～50 times, get 100 μ L and add in the 10mL soft agar, mixing is layered on the LB flat board.37 ℃, overnight incubation.After bacterium colony grows, number bacterium colony numbers.Reorganization DCD-1L anti-microbial activity formula: (colony number of the colony number/control group of survival) * 100.The results are shown in accompanying drawing 5.
The embodiment 4 enterobacteria CMCC of the Dermcidin-1L Chinese People's Anti-Japanese Military and Political College (B) 44102 effects
Intestinal bacteria CMCC (B) 44102 is inoculated in the 20mLLB substratum, is cultured to OD600nm=0.4～0.5.Collect thalline, after washing 2 times with the PBS of 10mM pH7.4, use 10mM pH6.5, the PBS of 100mM NaCl is diluted to 2 * 106/mL. with bacterium and adds 100 μ L bacterium liquid in the reorganization DCD-1L of different concns, 37 ℃, acts on 4 hours, dilute 10～50 times, get 100 μ L and add in the 10mL soft agar, mixing is layered on the LB flat board.37 ℃, overnight incubation.After bacterium colony grows, number bacterium colony numbers.Reorganization DCD-1L anti-microbial activity formula: (colony number of the colony number/control group of survival) * 100.The results are shown in accompanying drawing 6.
The anti-Candida albicans CMCC of embodiment 5 Dermcidin-1Ls (B) 9800 effects
Candida albicans CMCC (B) 9800 is inoculated in the 20mL Gause I substratum, is cultured to OD450nm=0.4～0.6.Collect thalline, after washing 2 times with the PBS of 10mM pH7.4, with 10mM PBS (pH6.5,100mM NaCl) bacterium is diluted to 2 * 106/mL. and in the reorganization DCD-1L of different concns, adds 100 μ L bacterium liquid, 37 ℃, act on 4 hours, dilution 10-50 doubly, get 100 μ L and add in the 10mL soft agar, mixing is layered on the LB flat board.37 ℃, overnight incubation.After bacterium colony grows, number bacterium colony numbers.Reorganization DCD-1L anti-microbial activity formula: (colony number of the colony number/control group of survival) * 100.The results are shown in accompanying drawing 7.
The nucleotides sequence tabulation
＜110〉East China Normal University
＜120〉a kind of genetic engineering bacterium and structure side thereof of efficiently expressing recombinant human sweet gland antibacterial peptide
Method and application
＜130〉specification sheets, claims
<160>4
<170>PatentIn?version?3.2
<210>1
<211>84
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＜213〉artificial sequence
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tcttctttgt?tggagaaggg?tttggacggt?gctaagaagg?tgttggtggt?ttgggtaagt
60
tgggtaagga cgctgttgag gact
84
<210>2
<211>85
<212>DNA
＜213〉artificial sequence
<400>2
caaaacagag?tccaaaacgt?ccttaacgtc?gtgaacagca?cccttaccaa?cagactccaa
60
gtcctcaaca gcgtccttac ccaac
85
<210>3
<211>40
<212>DNA
＜213〉artificial sequence
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ggcggatcca tcgaaggtcg ttcttctttg ttggagaagg
40
<210>4
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<212>DNA
＜213〉artificial sequence
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ccggaattct cacaaaacag agtcc
25
Claims (3)
1. the genetic engineering bacterium of an efficiently expressing recombinant human sweet gland antibacterial peptide, it is characterized in that, this genetic engineering bacterium is the e. coli bl21 (DE3) that carries recombinant plasmid, and described recombinant plasmid is to contain the plasmid pET32a (+) that Dermcidin-1L is the DCD-1L gene.
2. the construction process of the described genetic engineering bacterium of claim 1 is characterized in that, the concrete operations step:
The amplification of the first step DCD-1L dna sequence dna
Entrust the following base sequence of biotech company's chemosynthesis of specialty: DCD-1L a-Dermcidin-1L 5 ' end 85bp single stranded DNA sequence, DCD-1L b--Dermcidin-1L 3 ' end 85bp single stranded DNA sequence, P5--5 ' holds primer, introduce BamHI restriction enzyme site and FactorXa site, P3--3 ' holds primer, introduce EcoRI restriction enzyme site and terminator codon TCA
DCD-1L?a(85bp)：
5’-TCTTCTTTGTTGGAGAAGGGTTTGGACGGTGCTAAGAAGG TGTTGGTGG
TTTGGGTAAG?TTGGGTAAGG?ACGCTGTTGA?GGACT-3’
DCD-1L?b(85bp)：
5’-CAAAACAGAGTCCAAAACGTCCTTAACGTCGTGAACAGCACCCTTACCAA
CAGACTCCAA?GTCCTCAACA?GCGTCCTTAC?CCAAC-3’
BamHI
P5：5’-GGCGGATCC
ATCGAAGGTCGTTCTTCTTTGTTGGAGAAGG-3’
Factor?Xa
EcoRI
P3：5’-CCGGAATTC
TCACAAAACAGAGTCC-3’
End
DCD-1L a and DCD-1L b be template each other, add Ex Taq archaeal dna polymerase, carry out pcr amplification, obtain double-stranded DNA, being complete DCD-1L gene fragment, is template with the double-stranded DNA, adds primer P5 and P3 and carries out pcr amplification again, reaction product reclaims with DNA GelExtraction Kit, obtains the DCD-1L dna fragmentation;
The second step structure contains the DCD-1L dna fragmentation of the genetic engineering bacterium of Dermcidin-1L gene order with two kinds of restriction enzymes double zyme cutting the first steps acquisitions, described two kinds of restriction enzymes are BamHI and EcoRI, purifying reclaims small segment, with same two kinds of restriction enzymes double zyme cutting plasmid pET-32a (+), thereby purifying reclaims big fragment, the small segment and the big fragment that connect above-mentioned recovery, the recombinant plasmid pET-32a-DCD-1L that must contain the DCD-1L dna fragmentation, pET-32a-DCD-1L is transformed into bacillus coli DH 5 alpha with recombinant plasmid, make and contain the Dermcidin-1L gene order, it is the genetic engineering bacterium of DCD-1L dna sequence dna, entrust the biotech company of specialty to check order this genetic engineering bacterium, confirm that this genetic engineering bacterium contains complete reorganization DCD-1L gene fragment;
The 3rd step made up the genetic engineering bacterium that efficiently expresses Dermcidin-1L
Genetic engineering bacterium extracting recombinant plasmid pET-32a-DCD-1L from second step built is transformed into e. coli bl21 (DE3), the genetic engineering bacterium of the Dermcidin-1L that obtains efficiently expressing.
3. method of producing Dermcidin-1L with the described genetic engineering bacterium of claim 1 is characterized in that the concrete operations step:
The first step liquid culture
The described genetic engineering bacterium that efficiently expresses Dermcidin-1L of claim 1 is cultured to OD in the LB liquid nutrient medium
600nm=0.4～1.0 o'clock, the adding final concentration was that the I PTG of 0.4～1.0mM carried out abduction delivering 3～5 hours, produces and accumulate the Dermcidin-1L fusion rotein of solubility expression;
The Dermcidin-1L fusion rotein that the second step purifying makes
Centrifugal collection thalline is with the resuspended thalline of damping fluid, after the carrying out ultrasonic bacteria breaking, centrifugal collection supernatant, carry out affinity chromatography, the fusion rotein component of collector's sweet gland antibacterial peptide is that the Millipore Amicon Ultra-15 ultrafiltration pipe of 5KD is concentrated into 2～8mg/mL with albumen with molecular weight cut-off;
The 3rd step preparation recombinant human sweet gland antibacterial peptide
With Factor Xa enzymolysis Dermcidin-1L fusion rotein, 23 ℃ of following cracking 2～5 hours, termination reaction is carried out affinity chromatography once more, collects and penetrates the peak, and lyophilize obtains recombinant human sweet gland antibacterial peptide.
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| DCD-1L在毕赤酵母中的克隆和表达. 赖玉平等.中国生物工程杂志，第24卷第2期. 2004 * |
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