CN102250863B - Gene recombination human neutraphil elastase (HNE), preparation method thereof and medicinal usage of same - Google Patents
Gene recombination human neutraphil elastase (HNE), preparation method thereof and medicinal usage of same Download PDFInfo
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- CN102250863B CN102250863B CN 201110163759 CN201110163759A CN102250863B CN 102250863 B CN102250863 B CN 102250863B CN 201110163759 CN201110163759 CN 201110163759 CN 201110163759 A CN201110163759 A CN 201110163759A CN 102250863 B CN102250863 B CN 102250863B
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Abstract
The invention discloses a gene recombination HNE, a preparation method thereof and medicinal usage of the same, belonging to the field of gene engineering. According to the invention, purified elastase is obtained through the following steps: obtaining a supernatant containing genome DNA from lysate of human neutraphil granulocyte, carrying out amplification to obtain the gene segment ELA2 of neutraphil elastase, connecting ELA2 with a cloning vector to realize conversion into escherichia coli, constructing the expression plasmid PET-28a-ELA2 of elastase, expressing elastase in escherichia coli, collecting thalli, cracking bacteria liquid with ultrasonic wave and eluting with a nickel column. According to the invention, utilization of elastase expression genes enables sufficient and stable gene recombination HNEs to be obtained, thereby providing the raw material elastase for clinical detection and experimental studies and providing specific antigens for preparation of high efficiency elastase antibodies.
Description
Technical field
The present invention relates to the genetically engineered field, specifically is the medicinal use of a kind of gene recombinant human neutrophil elastase and preparation method thereof and this Pancreatopeptidase E.
Background technology
Pancreatopeptidase E (elastase) is to examine a kind of proteolytic enzyme of neutrophil leucocyte (neutrophil granulocyte) excretory by leaflet in the inflammatory process, detects the content ability assisted diagnosis prostate gland of Pancreatopeptidase E and the situation of other accessory sex gland organ bacterial infections.
Pancreatopeptidase E can act on elastin as a subfamily of serine stretch protein family.But 6 gene expression structures and intimate Pancreatopeptidase E are arranged at present in the human body.Neutrophil leucocyte excretory Pancreatopeptidase E has the function of degraded foreign protein; Existing research shows the colibacillary outer membrane protein A of Pancreatopeptidase E degradable (ompA); Outer membrane protein A is commonly considered as the virulence factor of bacteriums such as Salmonellas; So the hydrolysis function of Pancreatopeptidase E is brought into play certain function in anti-outside route of infection.
Pancreatopeptidase E has 267 amino acid; As shown in Figure 1, the domain of Pancreatopeptidase E is analyzed, find: 7-22 amino acid is expressed one section albumen with film function; 29-242 amino acid is expressed the functional protein with serine protease, and is as shown in Figure 1.
Human neutrophil Pancreatopeptidase E (human neutrophil elastase; HNE) in pathologic processes such as the various inflammatory reactions of body, tissue injury reconstruct (like pneumonia), adult respiratory distress syndrome, pulmonary fibrosis, urgency (slowly) property injury of lung, wet lung, atherosclerosis, scleroderma, play an important role, and have the virus of prevention, the intrusion of bacterium and the function of cancer metastasis.
Pancreatopeptidase E accordings to U.S.'s National Institutes of Health standard as the index of prostatitis differential diagnosis; The prostatic fluid white blood cell count(WBC) is considered to the scorching leading indicator of diagnosis of prostate, detect struvite pelvic pain disease that Pancreatopeptidase E possibly cause the difference prostatitis and the non-inflammation pelvic pain that causes by the apparatus urogenitalis autonomic nervous dysfunction helpful.
With the human neutrophil Pancreatopeptidase E is antigen, and the preparation corresponding antibody is used for diagnosis of prostate and other accessory sex gland organ bacterial infections, detects the content of Pancreatopeptidase E.But the source that obtains neutrophil elastase from human body is limited, far can not satisfy the clinical needs that detect with test.
Summary of the invention
The present invention is exactly to be the limited problem in antigen prepd specific antibody source in order to solve with the human neutrophil Pancreatopeptidase E, and the medicinal use of a kind of gene recombinant human neutrophil elastase and preparation method thereof and this Pancreatopeptidase E is provided.
The present invention designs according to following technical scheme.
A kind of gene recombinant human neutrophil elastase is cut the expression plasmid PET-28a-ELA2 that makes up Pancreatopeptidase E with BamHI and HindIII enzyme, and is as shown in Figure 2; The nucleotide sequence of this Pancreatopeptidase E is shown in SEQUENCE LISTING.
Said gene recombinant human neutrophil elastase, this Pancreatopeptidase E has the immunogenicity of natural human neutrophil elastase.
The preparation method of said gene recombinant human neutrophil elastase, the lysate of its human neutrophil is got the supernatant that contains genomic dna; With the PCR method amplification, obtain the gene fragment ELA2 of neutrophil elastase, the competent cell of transformed into escherichia coli TOP10 behind the structure cloned plasmids; Extract DNA; Make up the PET-28a-ELA2 expression plasmid of Pancreatopeptidase E, and the expression Pancreatopeptidase E in intestinal bacteria, thalline collected; Ultrasonic degradation bacterium liquid obtains Pancreatopeptidase E behind the nickel post wash-out purifying.
The preparation method of said recombinant human neutrophil elastase, its PCR method amplimer:
Primer1: 5’-?agctgcgcgg?aggccacttc-3’
Primer2: 5’-gga?gccgt?cacca?aggctca-3’。
The preparation method of said gene recombinant human neutrophil elastase, the condition of its pcr amplification is: 94 ℃ of sex change 50 seconds, 55 ℃ of annealing 1 minute, 72 degree extended 3 minutes, circulated altogether 30 times, last 72 degree insulations 10 minutes.
The preparation method of said gene recombinant human neutrophil elastase; Its recombinant plasmid PET-28a-ELA2 is at expression in escherichia coli; The intestinal bacteria that will contain recombinant plasmid PET-28a-ELA2 are seeded in the 10ml LB substratum that contains kantlex, 37 ℃ of overnight cultures; Gained bacterium liquid is inoculated in the 1000ml LB substratum that contains kantlex, cultivates about 4h, make its A at 37 ℃
650The OD value adds sec.-propyl sulfuration galactoside during to 0.6-0.8 in bacterium liquid, making its final concentration is 0.5mM, continues to cultivate 6-8 hour; Prepare ultrasonic degradation bacterium liquid after collecting thalline.
The preparation method of said gene recombinant human neutrophil elastase; Its purified elastase is with being at 12,000 rev/mins, behind 4 ℃ of centrifugal 10min with ultrasonic degradation bacterium liquid; Deposition is suspended in by 8M urea, 10mM Tris, among the basic damping fluid 30ml of pH8.0 preparation; 12,000 rev/mins, 4 ℃ of centrifugal 10min get supernatant, and the wash-out concentration of the NaCl of use 300mM obtains the gene recombinant human neutrophil elastase of purifying.
Said gene recombinant human neutrophil elastase is as the antigen of specific antibody, the application in the efficient antibody reagent for clinical diagnosis of preparation.
The present invention of design like this utilizes the expressing gene of Pancreatopeptidase E, obtains the stable gene recombinant human neutrophil elastase of capacity, supplies raw materials for clinical detection and experimental study Pancreatopeptidase E, for preparing efficient antibody specific antigens is provided.
Description of drawings
Fig. 1 is Tryase domain (Tryp-_SPc) figure of Pancreatopeptidase E tryptase;
Fig. 2 is PET-28a-ELA2 plasmid figure;
Fig. 3 is the Pancreatopeptidase E SDS-PAGE electrophorogram of expressing;
Fig. 4 is the Pancreatopeptidase E SDS-PAGE electrophorogram of purifying;
Fig. 5 is that the Pancreatopeptidase E of purifying has the immunogenic western-blot figure of natural human neutrophil elastase.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is carried out detailed explanation.
One. the gene of human neutrophil Pancreatopeptidase E obtains
1. the lysate 1ml of end user's neutrophil leucocyte is centrifugal, and 12,000 rev/mins, 5 minutes, getting the supernatant that contains genomic dna was template, carries out pcr amplification.
Use a pair of primer to be:
Primer1: 5’-?agctgcgcgg?aggccacttc-3’
Primer2: 5’-gga?gccgt?cacca?aggctca-3’
2. be template with the genomic dna, the condition of pcr amplification is:
94 ℃ of sex change 50 seconds
Annealed 1 minute for 55 ℃
72 degree extended 3 minutes,
Circulate last 72 degree insulations 10 minutes altogether 30 times.
3.PCR product reclaims with the extracting of phenol chloroform after 1% agarose electrophoresis conclusive evidence obtains target gene.
Two. make up the cloned plasmids of human neutrophil Pancreatopeptidase E gene
1. the PCR product is reclaimed purifying with glue;
2. A-T cloning vector (like pUCm-T carrier etc.) is connected with the PCR product, the ligation system is following:
| The T cloning vector | 1μl |
| 5 * connect and cushion | 2μl |
| The T4 dna ligase | 1μl |
| The PCR product | 4μl |
| H 2O | 2μl |
Above mixture connects 12h at 16 ℃;
3. with the competent cell of the product transformed into escherichia coli TOP10 that connects, coating contains the LB flat board of penbritin, provokes the male transformant, extracts DNA in a small amount with the method for alkaline lysis, and DNA is checked order.The dna sequence dna and gene pool (genebank) reported sequence of sequencing result are carried out dna homolog property relatively, confirm the correct of cloned dna sequence, shown in SEQUENCE LISTING.
Three. the structure of expression plasmid PET-28a-ELA:
1. the gene fragment of human neutrophil Pancreatopeptidase E in the above-mentioned cloning vector is downcut with restriction enzyme BamHI and HindIII, after 1% agarose electrophoresis, cut glue and reclaim the elastin enzyme fragment;
2. above-mentioned elastin enzyme fragment is connected with PET-28a, linked system is following:
| The PET-28a carrier | 1μl |
| 5 * connect and cushion | 2μl |
| The T4 dna ligase | 1μl |
| The elastin enzyme fragment | 5μl |
| H 2O | 1μl |
Above mixture connects 12h at 16 ℃;
3. with the competent cell of the product transformed into escherichia coli DH5 α that connects, coating contains the LB flat board of kantlex, and the transformant of provoking extracts DNA in a small amount with the method for alkaline lysis;
4. with BamHI and HindIII digested plasmid DNA, confirm that cloned DNA is segmental correct, as shown in Figure 2, among the figure: Kan: kalamycin resistance encoding sequence position; LacI:lacI gene order position.
Four. the expression in intestinal bacteria
1. the intestinal bacteria inoculation that will contain recombinant plasmid PET-28a-ELA2 contains in the 10ml LB substratum of kantlex 37 ℃ of overnight cultures;
2. above-mentioned bacterium liquid is inoculated in the 1000ml LB substratum that contains kantlex, cultivates about 4h, make its A at 37 ℃
650The OD value adds sec.-propyl sulfuration galactoside (IPTG) during to 0.6-0.8 in bacterium liquid, making its final concentration is 0.5mM, continues to cultivate 6-8 hour;
3. the collection thalline, 4 ℃, 10 minutes, removes the supernatant collecting precipitation by 10,000 rev/mins;
4. above-mentioned bacterial sediment is washed once with the PBS damping fluid of pH8.0, centrifugal after, again with the PBS damping fluid of the pH8.0 deposition that suspends;
5. adding PMSF (PMSF) back ultrasonic degradation bacterium liquid, and sample thief is analyzed with SDS-PAGE, sees Fig. 3, and the arrow indication is a target protein, obtains the Pancreatopeptidase E of the about 28.6Kda of molecular weight.
Five. the separation of Pancreatopeptidase E and purifying
1. with ultrasonic degradation bacterium liquid, at 12,000 rev/mins, 4 ℃ of centrifugal 10min, with deposition be suspended in 30ml basis damping fluid (8M urea, 10mM Tris, pH8.0);
2.12 000 rev/min, 4 ℃ of centrifugal 10min get supernatant;
3. supernatant is after the affine combination of nickel post, with 100,200, the NaCl of 400mM carries out wash-out;
4. after each component is analyzed through SDS-PAGE, find out optimal wash-out concentration;
5. use the wash-out concentration purification of target Pancreatopeptidase E of the NaCl of 300mM, as shown in Figure 4, the arrow indication is a target protein, obtains the gene recombinant human neutrophil elastase.Protein Marker molecular weight (kDa) among the figure: 72,56,33,25,16,11.
Six. the immunogenicity of Pancreatopeptidase E and application
Appearance 5ug on the Pancreatopeptidase E of above-mentioned purifying is carried out the SDS-PAGE electrophoresis, and the neutrophil elastase monoclonal antibody that uses the commercially available US-BIO company of the U.S. carries out the western-blot experiment as binding antibody, sees Fig. 5, and the arrow indication is a target protein.The result shows that the Pancreatopeptidase E that the present invention obtains can have extraordinary specificity to combine with the monoclonal antibody of Pancreatopeptidase E.Experimental result shows, has similar immunogenicity according to the Pancreatopeptidase E of the inventive method preparation with the Pancreatopeptidase E of natural extract, and the higher natural elastic proteolytic enzyme of alternative cost is as the antigen for preparing efficient antibody.The research and development field that Pancreatopeptidase E that this law invention obtains and specific efficient antibody can be applicable to reagent for clinical diagnosis.Therefore, the gene recombinant human neutrophil elastase is at preparation Pancreatopeptidase E specific antibody, have application prospect as the raw material of external diagnosis reagent case.
SEQUENCE?LISTING
< 110>Tianjin Jin Sitan bio tech ltd
< 120>medicinal use of gene recombinant human neutrophil elastase and preparation method thereof and this Pancreatopeptidase E
<130> DNA
<160> 1
<170> PatentIn?version?3.1
<210> 1
<211> 3956
<212> DNA
<213> 2?Ambystoma?laterale?x?Ambystoma?jeffersonianum
<220>
<221> 1
<222> (1)..(3956)
<223>
<400> 1
gcacggaggg?gcagagaccc?cggagcccca?gccccaccat?gaccctcggc?cgccgactcg 60
cgtgtctttt?cctcgcctgt?gtcctgccgg?ccttgctgct?ggggggtgag?tttttgagtc 120
caacctcccg?ctgctccctc?tgtcccgggt?tctgttccca?cctctccata?gagggcccca 180
ccagtgtggg?tccctcatcc?tcacagggga?ggtgccagct?gggacaagga?gaccagaaga 240
gactgaggtt?ctgagcggtg?aagccaccac?caggagccca?gagttggggt?ttgaaaaccg 300
gggagggggg?gggggtcgca?ggtcgccctc?tgggttcaag?tccaggtctg?tctgtgcctt 360
ggaggggcac?cgtggggagg?tccctttgcc?tctccgtgcc?tcagtttcct?catctgaaca 420
acaggggtgc?gaacggcccc?gatcccgtgg?gttcccggtg?ggggatcccg?tgggttcccg 480
gtgggggatc?ccgtgggttc?ctggtggggg?atccagaggc?cccgtggccg?ggaggggaca 540
ggctccttgg?caggcactca?gcacccgcac?ccggtgtgtc?cccaggcacc?gcgctggcct 600
cggagattgt?ggggggccgg?cgagcgcggc?cccacgcgtg?gcccttcatg?gtgtccctgc 660
agctgcgcgg?aggccacttc?tgcggcgcca?ccctgattgc?gcccaacttc?gtcatgtcgg 720
ccgcgcactg?cgtggcgaat?gtgtgagtag?ccgggagtgt?gcgcgcccgg?ctcggacccc 780
gcgtcccggt?ctgtgaggtg?ggtgggggga?ggccggggcc?ggggctgctg?gcgggggggg 840
gtccgtccag?ggcccgcggg?gcccctcgag?caccttcgcc?ctcaggcccg?tcgccggatg 900
gggacgacaa?ggcgcggctg?agccccgacc?cccggggccg?cccctgagcc?ccgcctctcc 960
ctccccggca?gaaacgtccg?cgcggtgcgg?gtggtcctgg?gagcccataa?cctctcgcgg 1020
cgggagccca?cccggcaggt?gttcgccgtg?cagcgcatct?tcgaaaacgg?ctacgacccc 1080
gtaaacttgc?tcaacgacat?cgtgattctc?caggtgccgc?cgggcggggc?ggggggcgca 1140
ggggcggagg?ccagaggcct?ggggagggtg?gaggctgcga?cggaggggcg?cgtcggggcc 1200
gctcgtgggg?acctggggtg?gcatcgtggg?ctgggtggtc?ccctctccgc?gcctcggtct 1260
gcacctctgt?gaaacgggaa?aatacccgcc?atgggccgtt?gaggggttaa?atgagatcct 1320
gcagggaggc?cccgatctgc?tgtcaatcaa?caaacttact?gagaagggag?gccccgatct 1380
gttgtcaatc?aacaaactta?ctgagaaggg?aggccccgat?ctgttgtcaa?tcaacaaact 1440
tactgagaag?ggaggccccg?atctgctgtc?aatcaacaaa?cttactgaga?agggaggccc 1500
cgatctgttg?tcaatcaaca?aacttactga?gaagggaggc?cccgatctgc?tgtcaatcaa 1560
caaacttact?gagaagggag?gccccgatct?gttgtcaatc?aacaaactta?ctgagaaggg 1620
aggccccgat?ctgctgtcaa?tcaacaaact?tactgagaag?ggaggccccg?atctgttgtc 1680
aatcaacaaa?cttactgaga?agggaggccc?cgatctgctg?tcaatcaaca?aacttactga 1740
gattctttgt?gtctctccat?tcaccagtcc?tgtggcccag?ggcaggggcc?gcctctgtct 1800
ttgggaaaag?gggcaaaagt?ccccaccttt?ccacccctgt?ccgcggcttg?cagttctggt 1860
tatttcctgg?gcgccgggcc?ccgtggctca?ggcctgtcat?cccagcactt?tgggaggctg 1920
aggcgggtgg?atcacgaggt?caggtgttcg?agaccagcct?gagcaacata?gtgaaacccc 1980
gtctctacta?aaatacaaaa?aaaaaaaatt?agccgagtgt?ggttgtgggt?gcctgtaatg 2040
ccaactactc?aggaggctga?ggaaggagaa?tcgcttgaac?cccggaggcg?gagattgcag 2100
tgagctgaga?tcacaccact?gcactccagc?ctgggtctca?aaaaaaaaaa?aaaagattcc 2160
tccctgggaa?gggttagagg?gagagtttcc?ttgtcactaa?gttttctcat?agctctcacc 2220
cagtgcagtg?gcgcgatcgc?agctcactac?agcctccatc?tcctgggctc?aagccaccct 2280
ctcagcttgg?aatggggggt?agctggaacc?acaggtgcca?ccacgtgggt?ccaccacgtc 2340
tggctaatat?atatatatac?acacacacat?acatatatta?taaataataa?atatatattt 2400
tatttaaata?aaatatataa?tatttataat?tattttataa?ttataataat?atttatataa 2460
ttataaatat?catttataat?tataatattt?attattttat?aaaataataa?atataaaata 2520
tataaaaata?tttttataaa?ataataaaaa?tatatatata?cacacatata?tatatatttt 2580
ttgagacaag?tctcgctctg?tcgcccaggc?tggagcgcag?tggcacaatc?tcagctcact 2640
gcaacctccg?cctcccaggt?tcaagcgatt?ctcctgcctc?agcctcccag?gtagctggga 2700
ctacaggcgc?ccgccaccac?gcctggctaa?tttttggtat?tgttagtaga?gacggggttt 2760
aaccatgtta?gccaggatgg?tcttgatctc?ctgacctttt?gattggccca?cctcagcctc 2820
ccaaaatgct?gggattatag?gcgtgagcca?ccgcacctgg?caattttttt?ttattatttt 2880
tgtagacatg?gggctttgcc?acattgccca?ggctggtctt?gaatgcctgg?cctcaagtga 2940
tcctcctgcc?tcgccctccc?aaagtgctgg?gcttacaagc?atgagccacc?gcgcccggct 3000
gtagtttttt?tgttaactga?gcacctactg?cttcctgcac?tcaagccaca?tccagggaca 3060
acctccaacg?ccctgagcct?tggtgacggc?tcccactcta?cagatgggga?aaccgaggct 3120
tgccttgggg?agcagagtgt?ggggtgggta?tcctgccctg?caggatccca?gaaccacagt 3180
ggaacctgag?atggggaaac?tgaggcccgg?agaggggagg?gtcatcatca?ctgccccgtg 3240
tgacgcgctg?acgatctgtc?cccaccgcca?cagctcaacg?ggtcggccac?catcaacgcc 3300
aacgtgcagg?tggcccagct?gccggctcag?ggacgccgcc?tgggcaacgg?ggtgcagtgc 3360
ctggccatgg?gctggggcct?tctgggcagg?aaccgtggga?tcgccagcgt?cctgcaggag 3420
ctcaacgtga?cggtggtgac?gtccctctgc?cgtcgcagca?acgtctgcac?tctcgtgagg 3480
ggccggcagg?ccggcgtctg?tttcgtacgt?gccctgggtg?tccctctgct?ccccacccgc 3540
tcccagcccg?gactgcagca?acaggcaccg?tggctagacc?ctaggaggga?cttcccaacc 3600
ctgacaggcg?gcgggcaggt?gggcagggcc?tcgcagtcca?gcttccccac?cttgtctgcc 3660
tccacagggg?gactccggca?gccccttggt?ctgcaacggg?ctaatccacg?gaattgcctc 3720
cttcgtccgg?ggaggctgcg?cctcagggct?ctaccccgat?gcctttgccc?cggtggcaca 3780
gtttgtaaac?tggatcgact?ctatcatcca?acgctccgag?gacaacccct?gtccccaccc 3840
ccgggacccg?gacccggcca?gcaggaccca?ctgagaaggg?ctgcccgggt?cacctcagct 3900
gcccacaccc?acactctcca?gcatctggca?caataaacat?tctctgtttt?gtagaa 3956
Claims (6)
1. the preparation method of a gene recombinant human neutrophil elastase is characterized in that: the lysate of human neutrophil, get the supernatant that contains genomic dna; With the PCR method amplification, obtain the gene fragment ELA2 of neutrophil elastase, the competent cell of transformed into escherichia coli TOP10 behind the structure cloned plasmids; Extract DNA; Make up the pET-28a-ELA2 expression plasmid of Pancreatopeptidase E, and, collect thalline in the expression in escherichia coli Pancreatopeptidase E; Ultrasonic degradation bacterium liquid obtains Pancreatopeptidase E behind the nickel post wash-out purifying;
The primer of said PCR method amplification:
Primer1: 5’-?agctgcgcgg?aggccacttc-3’
Primer2: 5’-gga?gccgt?cacca?aggctca-3’。
2. according to the preparation method of the said gene recombinant human neutrophil elastase of claim 1; It is characterized in that: the condition of pcr amplification is: 94 ℃ of sex change 50 seconds, and 55 ℃ of annealing 1 minute, 72 degree extended 3 minutes; Circulate last 72 degree insulations 10 minutes altogether 30 times.
3. according to the preparation method of the said gene recombinant human neutrophil elastase of claim 1; It is characterized in that: recombinant plasmid pET-28a-ELA2 is at expression in escherichia coli; Be that the intestinal bacteria that contain recombinant plasmid pET-28a-ELA2 are seeded in the 10ml LB substratum that contains kantlex 37 ℃ of overnight cultures; Gained bacterium liquid is inoculated in the 1000ml LB substratum that contains kantlex, cultivates 4h at 37 ℃, when making its A650 OD value to 0.6-0.8, in bacterium liquid, add sec.-propyl sulfuration galactoside, making its final concentration is 0.5mM, continues to cultivate 6-8 hour; Prepare ultrasonic degradation bacterium liquid after collecting thalline.
4. according to the preparation method of the said gene recombinant human neutrophil elastase of claim 1; It is characterized in that: purified elastase is 12 with ultrasonic degradation bacterium liquid; 000 rev/min; Behind 4 ℃ of centrifugal 10min, deposition is suspended in by 8M urea, 10mM Tris, among the basic damping fluid 30ml of pH8.0 preparation; 12,000 rev/mins, 4 ℃ of centrifugal 10min get supernatant, and the wash-out concentration of the NaCl of use 300mM obtains the gene recombinant human neutrophil elastase of purifying.
5. according to the preparation method of the said gene recombinant human neutrophil elastase of claim 1, it is characterized in that: the nucleotide sequence of this Pancreatopeptidase E is shown in SEQUENCE LISTING sequence 1.
6. according to the preparation method of the said gene recombinant human neutrophil elastase of claim 1, it is characterized in that: this Pancreatopeptidase E has the immunogenicity of natural human neutrophil elastase.
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|---|---|---|---|---|
| CN101918547A (en) * | 2007-12-04 | 2010-12-15 | 普罗特昂治疗公司 | Elastin zymoprotein of reorganization and its production and use |
| CN101974555A (en) * | 2010-08-20 | 2011-02-16 | 中国农业大学 | Genetic engineering bacterium for expressing elastase 2B and construction method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101918547A (en) * | 2007-12-04 | 2010-12-15 | 普罗特昂治疗公司 | Elastin zymoprotein of reorganization and its production and use |
| CN101974555A (en) * | 2010-08-20 | 2011-02-16 | 中国农业大学 | Genetic engineering bacterium for expressing elastase 2B and construction method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 王瑞,高向东.人中性粒细胞弹性蛋白酶及其抑制剂的研究.《药学进展》.2005,第29卷(第8期),350-354. * |
| 马国尔,郑金旭.中性粒细胞弹性蛋白酶研究现状.《江苏大学学报(医学版)》.2006,第16卷(第3期),262-263. * |
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