CN1664105A - MIP gene clone, recombination, expression and protein purifying method for Legionlla pneumophila - Google Patents
MIP gene clone, recombination, expression and protein purifying method for Legionlla pneumophila Download PDFInfo
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- CN1664105A CN1664105A CN 200410006363 CN200410006363A CN1664105A CN 1664105 A CN1664105 A CN 1664105A CN 200410006363 CN200410006363 CN 200410006363 CN 200410006363 A CN200410006363 A CN 200410006363A CN 1664105 A CN1664105 A CN 1664105A
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- mip
- legionella pneumophilia
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- bacterium
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 19
- 101150075489 mip gene Proteins 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 16
- 102000004169 proteins and genes Human genes 0.000 title abstract description 5
- 238000005215 recombination Methods 0.000 title abstract description 3
- 230000006798 recombination Effects 0.000 title abstract description 3
- 238000001042 affinity chromatography Methods 0.000 claims abstract description 4
- 208000007764 Legionnaires' Disease Diseases 0.000 claims description 38
- 241000589248 Legionella Species 0.000 claims description 37
- 241000894006 Bacteria Species 0.000 claims description 22
- 239000013612 plasmid Substances 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 11
- 239000013604 expression vector Substances 0.000 claims description 8
- 238000001556 precipitation Methods 0.000 claims description 7
- 239000013599 cloning vector Substances 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- 230000008521 reorganization Effects 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 230000003321 amplification Effects 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 5
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 5
- 230000000968 intestinal effect Effects 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 239000002953 phosphate buffered saline Substances 0.000 claims description 5
- 238000005342 ion exchange Methods 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 2
- 230000003115 biocidal effect Effects 0.000 claims description 2
- 239000004202 carbamide Substances 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 238000001890 transfection Methods 0.000 claims description 2
- 230000015556 catabolic process Effects 0.000 claims 1
- 238000006731 degradation reaction Methods 0.000 claims 1
- 238000003759 clinical diagnosis Methods 0.000 abstract description 3
- 241000588724 Escherichia coli Species 0.000 abstract description 2
- 101000976909 Legionella pneumophila Outer membrane protein MIP Proteins 0.000 abstract 1
- 241001597008 Nomeidae Species 0.000 abstract 1
- 239000000427 antigen Substances 0.000 abstract 1
- 102000036639 antigens Human genes 0.000 abstract 1
- 108091007433 antigens Proteins 0.000 abstract 1
- 230000001939 inductive effect Effects 0.000 abstract 1
- 102100026934 Mitochondrial intermediate peptidase Human genes 0.000 description 11
- 239000000047 product Substances 0.000 description 10
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 238000013016 damping Methods 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 238000005336 cracking Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 241000589242 Legionella pneumophila Species 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 108010064696 N,O-diacetylmuramidase Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- -1 isopropyl- Chemical group 0.000 description 1
- 229940115932 legionella pneumophila Drugs 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention relates to the method of colony, recombination, expressing and purifying to the MIP gene of the Legionella pneumophila macrophage infectivity potentiator and the special primer; and the application of MIP protein and its analogue and derivant in the clinical diagnosis and treatment for the patient infected with Legionalla Neomaphalla. The invention is characterized in that: preparing the MIP gene by means of PCR with the got Legionalla Neomaphalla MTP genom DNA, restructuring the said gene and colonizing it to the bacillus coli expressing carrier, inducing the expression and extracting MTP protein, and getting high-purity protein by using the method of affinity chromatography, which can be applied in the further clinical diagnosis for the the patient infected with Legionalla Neomaphalla and in the detecting for the antigen and antibody of Legionalla Neomaphalla.
Description
The present invention relates to express after the reconstruct of legionella pneumophilia MIP gene in the genetically engineered field, purification process, the particularly reorganization of legionella pneumophilia MIP gene, expression and purifying; Application aspect infection of legionella clinical diagnosis, treatment.
Albumen for stable acquisition capacity, be used to study proteic anti-efficiently MIP antibody for preparation simultaneously, the present inventor is by the method for gene recombination, obtained reorganization legionella pneumophilia MIP gene, in order to strengthen its antigenicity, the contriver stablizes, has expressed efficiently legionella pneumophilia MIP albumen simultaneously.
The method that the purpose of this invention is to provide above-mentioned legionella pneumophilia MIP gene, expression and protein purification.
Technical scheme of the present invention is: clone, reorganization, the proteic method of expression and purification legionella pneumophilia MIP may further comprise the steps:
(1), the gene of clone legionella pneumophilia MIP, with the gene of PCR method amplification MIP;
(2), the MIP gene with above-mentioned amplification is connected structure legionella pneumophilia MIP gene clone plasmid with cloning vector;
(3), above-mentioned cloned plasmids subclone is advanced expression vector, the plasmid DNA transfection intestinal bacteria after will connecting again;
(4), expressing human legionella pneumophilia MIP gene
A, antibiotic LB plate is contained in the above-mentioned intestinal bacteria shop that has a legionella pneumophilia MIP expression carrier, plate is placed 37 ℃ of incubator incubated overnight;
Select a bacterium colony on b, the slave plate, put into dress LB substratum (containing microbiotic) culturing bottle, shake in the case at 37 ℃ and cultivate, survey bacterium liquid A
650The OD value is to stop in 1.0 o'clock cultivating;
C, add IPTG in bacterium liquid, making its final concentration is 0.5mM, continues to cultivate 6-8 hour;
D, results bacterium;
(5), purifying legionella pneumophilia MIP albumen:
A, above-mentioned bacterial precipitation is washed once with the phosphate buffered saline buffer of pH8.0; After centrifugal, the phosphate buffered saline buffer suspension with pH8.0 precipitates again;
B, cracking bacterium, the centrifuging and taking supernatant liquor;
C, preliminary purification legionella pneumophilia MIP;
D, carry out purifying with ion-exchange and nickel column chromatography.
The sequence of primer special of the present invention is
Primer 15 ' AAGGATAAGTTGTCTTATAG 3 '
Primer 25 ' AAGCTTCTGTCCATCCTGGGA 3 '
Below in conjunction with accompanying drawing embodiments of the invention are described further.
Fig. 1 is the protein electrophoresis (SDS-PAGE) of legionella pneumophilia MIP genetic expression
Embodiment:
One, the acquisition of legionella pneumophilia MIP gene
1, get 1 milliliter of legionella pneumophilia, centrifugal, 12,000 rev/mins, 3 minutes, remove supernatant liquor, with 0.3 milliliter of Tris damping fluid suspension bacterial sediment, put 100 the degree water in 10 minutes, centrifugal 12000 rev/mins 5 minutes.
Get and ask liquid (containing legionella pneumophilia genes group DNA) to make pcr amplification for template.
2, the design primer is as follows:
Primer 15 ' AAGGATAAGTTGTCTTATAG 3 '
Primer 25 ' AAGCTTCTGTCCATCCTGGGA 3 '
3, be masterplate with above-mentioned DNA, pcr amplification, the PCR reaction conditions is:
94 ℃ of sex change 50 seconds,
Annealed 1 minute for 55 ℃,
72 ℃ were extended 1 minute and 30 seconds,
Totally 35 circulations, last 72 ℃ of insulations 10 minutes
4, the PCR product is after the preliminary conclusive evidence of 1% agarose electrophoresis, and phenol/chloroform extracting is reclaimed;
Two, make up the legionella pneumophilia genes cloned plasmids
1, the PCR product is reclaimed purifying with glue.
2, A-T cloning vector (any cloning vector is all passable, as T-easy, pGEX etc.) is connected with the PCR product, the ligation system is as follows:
A-T cloning vector 1ul
5x connects damping fluid 2ul
T4 dna ligase 1ul
PCR product 4ul
H
2O 2ul
Above mixture was 16 ℃ of ligations 12 hours;
3, will connect product Transformed E .Coli JM109 competent cell, coating contains penbritin LB plate, and the picking positive colony prepares plasmid DNA with alkaline lysis, and plasmid DNA is checked order.Sequencing result and gene pool (genbank) reported sequence are carried out dna homolog relatively, confirm that gene order is correct.
Three, the structure of expression plasmid pQE30-MIP:
1, the gene fragment of legionella pneumophilia in the above-mentioned cloning vector is used under restriction enzyme BamHI and the HindIII double digestion,, reclaimed the legionella pneumophilia genes fragment through 1% agarose electrophoresis;
2, the pQE30 expression vector (expression vector also can be other carrier such as pET, pGST etc.) that said gene fragment and same enzyme are cut connects, and the ligation system is as follows:
PQE30 (BamHI, HindIII enzyme cut the back) 1ul
5x connects damping fluid 2ul
T4DNA ligase enzyme 1ul
Legionella pneumophilia MIP gene segment 5ul
H
2O 1ul
Above mixture was 16 ℃ of ligations 12 hours
3, will connect product and transform the M15 competent cell, coating kantlex and penicillin LB plate, picking positive colony;
4, prepare plasmid DNA with alkaline lysis, plasmid DNA is identified recombinant plasmid pQE30-MIP with BamHI and HindIII double digestion contain legionella pneumophilia MIP gene segment.
Four, the expression of pQE30-legionella pneumophilia MIP gene in intestinal bacteria:
What 1, will filter out has recombinant plasmid pQE30-MIP base engineering bacteria inoculation 10ml LB (containing kantlex and penbritin) in vitro, and 37 ℃, the 300r/min shaking table is cultivated, and surveys bacterium liquid A
650The OD value stops during for 0.6-0.8 cultivating.
2, above-mentioned 10ml bacterium liquid is put into the 1000ml LB substratum culturing bottle of (containing kantlex and penbritin) is housed, shake in the case at 37 ℃ and cultivate, survey bacterium liquid A
650The OD value when its OD value is 0.6-0.8, adds IPTG (isopropyl-) and induces in bacterium liquid, making its final concentration is 0.5mM, continues to cultivate 4-6 hour;
3, collect bacterium,, 4 ℃, centrifugal 20 minutes, remove supernatant liquor, collecting precipitation with 5000 rev/mins.With the phosphate buffered saline buffer of the pH8.0 precipitation that suspends, and add PMSF (phenylmethylsulfonyl fluoride) immediately to final concentration 1mM, be put in-20 ℃ and freeze moltenly, and sample thief analyzes with SDS-PAGE, as shown in Figure 1.
Five, the proteic separation and purification of legionella pneumophilia MIP
1, make the bacterium cracking with sonioation method (also can use the N,O-Diacetylmuramidase cracking process), then under 4 ℃ of conditions 12, the centrifugal 10min of 000r/min, precipitation be resuspended in 30ml basis damping fluid (8M urea, 10mM Tris, pH8.0), 12, the centrifugal 20min of 000r/min gets supernatant liquor;
2, supernatant liquor is through DEAE ion-exchange and Ni-NTG-His post affinity chromatography: the DEAE ion exchange column carries out wash-out with the NaCl that basal liquid adds 100mM, 200mM, 400mM respectively.Each component is analyzed through 12.5%SDS-PAGE.The component that contains desirable proteins is carried out Ni-NTG-His post affinity chromatography, adds 15nM, 30mM, 60mM, 120mM, 220mM imidazoles wash-out with basal liquid respectively.The each component of collecting is made 12.5%SDS-PAGE to be analyzed.
As shown in Figure 2, the albumen of purifying presents a band through the SDS-PAGE electrophoretic analysis.
In the above-described embodiments, the structure of expression plasmid can also respectively have primer with corresponding two restriction endonuclease sites of expression vector with 5 of a pair of upstream and downstream ' end, DNA is a template with legionella pneumophilia MIP cloned plasmids, and the PCR product is carried out purifying with ordinary method; With two corresponding restriction enzymes PCR product and expression vector are carried out enzyme and cut PCR product and expression vector after cutting with the ordinary method purifying enzyme; Be connected with expression vector with the PCR product of T4 ligase enzyme purifying.
Claims (2)
1, the method for a kind of expression and purification legionella pneumophilia MIP gene and reorganization legionella pneumophilia MIP is characterized in that may further comprise the steps:
(1), the gene of clone legionella pneumophilia MIP, with the gene of PCR method amplification legionella pneumophilia MIP;
(2), the legionella pneumophilia MIP gene with above-mentioned amplification is connected structure legionella pneumophilia MIP cloned plasmids with cloning vector;
(3), above-mentioned legionella pneumophilia MIP cloned plasmids subclone is advanced expression vector, the plasmid DNA transfection intestinal bacteria after will connecting again;
(4), express legionella pneumophilia MIP albumen.
A, the above-mentioned intestinal bacteria inoculation that has a legionella pneumophilia MIP expression carrier is contained antibiotic LB plate, plate is placed 37 ℃ of incubator incubated overnight;
Select a bacterium colony on b, the slave plate, inoculate in LB substratum (the containing microbiotic) bottle, shake in the case at 37 ℃ and cultivate, survey bacterium liquid A
650ODValue stops during for 0.6-0.8 cultivating;
C, add IPTG in bacterium liquid, making its final concentration is 0.5mM, continues to cultivate 4-6 hour;
D, results bacterium;
(5), purifying legionella pneumophilia MIP albumen.
A, above-mentioned bacterial precipitation is washed once with the phosphate buffered saline buffer of pH8.0, centrifugal after, again with the phosphate buffered saline buffer of the pH8.0 precipitation that suspends;
B, ultrasonic degradation bacterium, centrifugal, precipitation is added 8M urea suspend, centrifugal, get supernatant liquor;
C, the ion-exchange of purifying human and Ni-NTG-His post affinity chromatography are carried out purifying.
2, expression and purification reorganization legionella pneumophilia MIP gene according to claim 1 and method, it is characterized in that: the gene of described PCR method amplification legionella pneumophilia MIP and used primer are: primer 15 ' AAGGATAAGTTGTCTTATAG, primer 25 ' AAGCTTCTGTCCATCCTGGGA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100063632A CN1322128C (en) | 2004-03-01 | 2004-03-01 | MIP gene clone, recombination, expression and protein purifying method for Legionlla pneumophila |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100063632A CN1322128C (en) | 2004-03-01 | 2004-03-01 | MIP gene clone, recombination, expression and protein purifying method for Legionlla pneumophila |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1664105A true CN1664105A (en) | 2005-09-07 |
| CN1322128C CN1322128C (en) | 2007-06-20 |
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|---|---|---|---|
| CNB2004100063632A Expired - Lifetime CN1322128C (en) | 2004-03-01 | 2004-03-01 | MIP gene clone, recombination, expression and protein purifying method for Legionlla pneumophila |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101545010A (en) * | 2009-05-11 | 2009-09-30 | 北京海康基因芯片开发有限公司 | Method for detecting infectious disease pathogens and kit |
| CN104277114A (en) * | 2013-07-09 | 2015-01-14 | 许颖 | New fusion protein and legionella pneumophila vaccine prepared from new fusion protein |
| CN107419006A (en) * | 2017-05-19 | 2017-12-01 | 中山大学 | The CPA primer sets and its detection method of a kind of quick detection legionella pneumophilia |
-
2004
- 2004-03-01 CN CNB2004100063632A patent/CN1322128C/en not_active Expired - Lifetime
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101545010A (en) * | 2009-05-11 | 2009-09-30 | 北京海康基因芯片开发有限公司 | Method for detecting infectious disease pathogens and kit |
| CN101545010B (en) * | 2009-05-11 | 2014-07-16 | 海康生命科技有限公司 | Method for detecting infectious disease pathogens and kit |
| CN104277114A (en) * | 2013-07-09 | 2015-01-14 | 许颖 | New fusion protein and legionella pneumophila vaccine prepared from new fusion protein |
| CN107419006A (en) * | 2017-05-19 | 2017-12-01 | 中山大学 | The CPA primer sets and its detection method of a kind of quick detection legionella pneumophilia |
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| Publication number | Publication date |
|---|---|
| CN1322128C (en) | 2007-06-20 |
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