CN1113964C - High-efficiency expression of artificially synthetic interferon alpha-2b gene in colibacillus - Google Patents
High-efficiency expression of artificially synthetic interferon alpha-2b gene in colibacillus Download PDFInfo
- Publication number
- CN1113964C CN1113964C CN99113732A CN99113732A CN1113964C CN 1113964 C CN1113964 C CN 1113964C CN 99113732 A CN99113732 A CN 99113732A CN 99113732 A CN99113732 A CN 99113732A CN 1113964 C CN1113964 C CN 1113964C
- Authority
- CN
- China
- Prior art keywords
- gac
- ctg
- leu
- tct
- aga
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention discloses high-level expression of gene order in escherichia coli and constructs the high-level expression of plasmid pHX-hIFNa-2b and human interferon alpha-2b escherichia coli production bacterium. The present invention has the advantage of high productive value and can be used for producing various medicinal preparations and other products.
Description
Technical field
The present invention relates to hereditary biological field, be particularly related to a kind of efficient expression system of Interferon Alpha-2b gene in intestinal bacteria of synthetic, contain Interferon Alpha-2b expression of gene plasmid and the engineering bacteria and the application of this human interferon alpha-2 b in the medicine of preparation anti-virus infection of this synthetic.
Background technology
The artificial Interferon Alpha-2b of genetically engineered (rhIFN α-2b) multiple virus infection is had curative effect preferably, even, result of treatment is arranged also to some tumour, particularly leukemia.If can reduce production costs, cheap rhIFN α-2b medicine is provided in a large number, will produce very high social benefit and economic benefit.
Summary of the invention
The invention provides one than better rhIFN α-2b expression system in the past.It is by selecting the codon of intestinal bacteria preference for use, and with computer Simulation the minimum free energy of formation of translation initiation district and Interferon Alpha-2b gene 5 ' secondary structure, above-mentioned two factors have been taken all factors into consideration, designed and synthesized the human interferon alpha-2 b gene order, with its efficient expression vector pLy4 that packs into, be built into pHX-hIFN α-2b expression plasmid.The genetic engineering interferon production bacterium that makes up gained is one and can efficiently expresses people's gene engineering Interferon Alpha-2b system, have very high industrialization productive value, will be used to produce the drug manufacture of preparations such as human interferon alpha-2 b injection liquid and human interferon alpha-2 b application capsule.
Feature of the present invention is to be to design the gene order of human interferon alpha-2 b at escherichia coli expression, the pedestrian worker that goes forward side by side is synthetic, made up the expression plasmid pHX-hIFN α-2b of human interferon alpha-2 b, make up the human interferon alpha-2 b intestinal bacteria and produced bacterium, and carried out efficiently expressing of human interferon alpha-2 b, expression amount reaches about 30% of bacterial protein, has very high productive value.And the proof engineering bacteria is stable.
Description of drawings
Fig. 1, synthetic hIFN α-2b gene DNA order.
Fig. 2, FI, the segmental splicing of FII, FIII and clone.
The synthetic electrophorogram of fragment PCR in Fig. 3, hIFN α-2b gene.
M：pGEM?DNA?Marker
The structure of Fig. 4, M13mp18-FIIFIII.
The structure of Fig. 5, M13mp18-FIFIIFIII.
Fig. 6, PUC18-hIFN α-2b.
Fig. 7-1 gene sequencing 5 '-→
Fig. 7-2 gene sequencing 3 '-→
The structure of Fig. 8, pHX-hIFN α-2b expression plasmid.
Fig. 9, pHX-hIFN α-2b plasmid enzyme restriction is identified figure.
1. λ/HindIII+EcoRI
2. plasmid
3. EcoR?I
4. Bgl?II
5. EcoR?I/Bgl?II
6. EcoR?I/Sal?I
Figure 10, pHX-IFN α-2b 50 generation cleavage maps
1.λ/Hind?III?Marker
2.pHX-hIFN α-2b the 10th generation restriction enzyme mapping
3.pHX-hIFN α-2b the 20th generation restriction enzyme mapping
4.pHX-hIFN α-2b the 30th generation restriction enzyme mapping
5.pHX-hIFN α-2b the 40th generation restriction enzyme mapping
6.pHX-hIFN α-2b the 50th generation restriction enzyme mapping
7.λ/Hind?III?Marker
Figure 11, pHX-hIFN α-2b 50 generation protein expression SDS-PAGE electrophorograms
M:SDS-PAGE lower molecular weight standard protein
1.pHX-hIFN α-2b the 10th generation protein expression
2.pHX-hIFN α-2b the 20th generation protein expression
3.pHX-hIFN α-2b the 30th generation protein expression
40.pHX-hIFN α-2b the 40th generation protein expression
50.pHX-hIFN α-2b the 50th generation protein expression
Culture condition: M9 substratum, 30 ℃ spend the night, 42 ℃ induce.
Embodiment
The present invention has selected the codon of intestinal bacteria preference for use, and with computer Simulation the translation initiation district
The present invention has selected the codon of intestinal bacteria preferences for use, and with computer Simulation the minimum free energy of formation of translation initiation district and I FN α-2b gene 5 ' end secondary structure, take all factors into consideration above-mentioned two factors and designed complete synthesis human interferon alpha-2 b (hIFN α-2b) gene order (seeing Fig. 1-1 and Fig. 1-2) and synthetic assembling route.
1, synthetic positive and negative two chains with hIFN α-2b gene of small segment resolve into 16 part eclipsed small segments in regular turn, synthesize with dna synthesizer respectively.
2,16 small segments of middle fragment assembly synthetic are divided into three groups in order, connect into three middle fragment (F through PCR respectively
I, F
II, F
III), length is respectively: F
I-200bp, F
II-100pb, F
III220bp, their ends separately all contain restriction enzyme site: F
I(EcoR I/Bgl II.Bam H I), F
II(Bam H I.Bgl II/HindIII), F
III(HindIII/SalI).Cut by terminal restriction enzyme site enzyme separately respectively, and be cloned among M13mp18 or the M13mp19, measure DNA sequence, therefrom filter out the M13mp18-F that contains correct sequence
I, M13mp19-F
IIAnd M13mp18-F
IIIIn the simple journey (see figure 2) of segmental splicing.The PCR electrophorogram is identified (see figure 3).
3, M13mp18.F
II.F
IIIStructure with Hind III and Pvu I respectively enzyme cut M13mp19-F
IIAnd M13mp18-F
III, with the F that cuts out
IIThe M13mp18-F that fragment and enzyme are cut
IIIConnect, the clone cuts, screens with BamHl and Sall enzyme, obtains M13mp18-F
II.F
III. make up simple journey (see figure 4).
4, M13mp18F
I.F
II.F
IIIStructure cut M13mp18-F with Bam hI and Sal I enzyme
II.F
IIIObtain F
II.F
IIIFragment is with the M13mp18-F that cuts through same enzyme
IBe connected, clone, cut screening, obtain M13mp18-F with EcoRI and SalI enzyme
I.F
II.F
IIIMake up simple journey (see figure 5).
5, the structure of PUC18-hIFN α-2b is cut M13mp18-F with EcoRI and SalI enzyme
I.F
II.F
III, gained F
I.F
II.F
IIIFragment is connected with the PUC18 that cuts through same enzyme.Obtain PUC18F
I.F
II.F
IIIThis plasmid is cut with Bgl II enzyme, connects again, promptly obtains PUC18-hIFN α-2b (see figure 6).Dna sequencing conclusive evidence synthetic hIFN α-2b gene conforms to coding correct (seeing Fig. 7-1, Fig. 7-2, Fig. 1-1 and Fig. 1-2) with design.
The structure of embodiment 2 pHX-HIFN α-2b expression plasmid
Efficient expression vector pLY4 is provided by Liu Xinyuan professor laboratory, and this is the temperature-induced type high-expression vector that Liu Xinyuan professor laboratory makes up.The structure data is consulted " Chinese science " (B collects), 1995,25 (10): 1063-1070 in detail.From PUC18-hIFN α-2b, cut out hIFN α-2b gene clone in pLY4 with EcoR I and Sal I, obtain expression plasmid pHX-hIFN α-2b (see figure 8).This plasmid efficiently expresses in intestinal bacteria, and expression level can reach about 30% of bacterial protein.Plasmid size and enzyme are cut evaluation figure (see figure 9).
1, host bacterium: large intestine bar figure DH5 α
2, the conversion of plasmid pHX-hIFN α-2b:
Be inoculated in the LB test tube from the dull and stereotyped picking bacterium of fresh large intestine bar DH5 α LB, 37 ℃ of overnight incubation are transferred in triangular flask again, continue to shake cultivation 2-2.5 hour.Culture was put ice bath after 10 minutes, and centrifugal collection thalline, and suspend with the calcium chloride solution of precooling, ice bath be after 20 minutes, and centrifugal collection thalline, resuspending be in the calcium chloride solution of precooling, and was sub-packed in the Eppendorf pipe, refrigerated standby.
Get the bacterium liquid that a pipe calcium chloride was handled, add plasmid pHX-hIFN α-2b, ice bath is after 20 minutes, and 42 ℃ of water-baths of transposition 2 minutes are coated with an amount of bacterium liquid in the LB culture dish that contains penbritin (Amp), and 37 ℃ of overnight incubation promptly obtain the transformed bacteria bacterium colony.
3, the screening of transformed bacteria:
Transform bacterium colony from the dull and stereotyped picking of going up of fresh conversion, be inoculated in respectively in the test tube that contains penbritin selection substratum, 37 ℃ of shaking culture are spent the night, respectively centrifugal collection thalline.Use alkaline lysis, thalline is suspended, sodium hydroxide cracking, sodium-acetate neutralization, centrifugal gained supernatant liquor is used phenol-chloroform extracting respectively, ethanol sedimentation, centrifugal recovery plasmid DNA.After the plasmid DNA that reclaims was dissolved in TE respectively, with EcoR I/Sal I double digestion, agarose electrophoresis filtered out the sample that contains 500bp left and right sides small segment.
The bacterium sample that carries recombinant plasmid with the screening gained is inoculated in the LB substratum that contains penbritin respectively, 30 ℃ of overnight incubation.Transfer in the M9 substratum, cultivate after 2 hours for 30 ℃, 42 ℃ are continued to cultivate centrifugal collection thalline 4 hours.
The part thalline has obvious band of expression with SDS-PAGE electrophoretic examinations whole bacterial protein at the 15KD place, accounts for about 30% of bacterial protein.
Another part thalline guanidine hydrochloride dissolution is used WISH cell, is basic detection system with VSV, presses cytopathic-effect inhibition assay and measures biological activity, obtains positive findings.
Above presentation of results, the people's gene work α-2b Interferon, rabbit production bacterium that makes up gained is a system that can efficiently express people's gene engineering α-2b Interferon, rabbit, has suitability for industrialized production and is worth.
(pHX-IFN α-2b) extracts the plasmid DNA in the thalline to the bacillus coli DH 5 alpha (CCTCC No.M99002) that is built into aforesaid method after LB penbritin inclined-plane switching 50 times, and identify restriction enzyme mapping identical with former bacterial classification (Figure 10) with restriction enzyme (EcoR I/Sal I).SDS-PAGE figure shows that 50 generation expression levels are not seen significant difference (Figure 11).Above presentation of results this project bacterium is stable.
Claims (4)
1, the efficient expression system of an artificial synthetic Interferon Alpha-2b gene in intestinal bacteria is characterized in that:
(1) the human interferon alpha-2 b gene order that designs and synthesizes:
1 10
Met?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?ArgGAG?GAA?TTC?ATG?TGT?GAC?CTG?CCG?CAG?ACC?CAC?TCT?CTG?GGT?TCT?CGT?CGTCTC?CTT?AAG?TAC?ACA?CTG?GAC?GGC?GTC?TGG?GTG?AGA?GAC?CCA?AGA?GCA?GCA
20 30Thr?Leu?Met?Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?LeuACC?CTG?ATG?CTG?CTG?GCT?CAG?ATG?CGT?CGT?ATC?TCT?CTG?TTC?TCT?TGC?CTGTGG?GAC?TAC?GAC?GAC?CGA?GTC?TAC?GCA?GCA?TAG?AGA?GAC?AAG?AGA?ACG?GAC
40Lys?Asp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?PheAAA?GAC?CGT?CAC?GAC?TTC?GGT?TTC?CCG?CAG?GAA?GAG?TTC?GGT?AAC?CAG?TTCTTT?CTG?GCA?GTG?CTG?AAG?CCA?AAG?GGC?GTC?CTT?CTC?AAG?CCA?TTG?GTC?AAG
50 60Gln?Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?PheCAG?AAA?GCT?GAA?ACC?ATC?CCG?GTT?CTG?CAC?GAA?ATG?ATC?CAG?CAG?ATC?TTCGTC?TTT?CGA?CTT?TGG?TAG?GGC?CAA?GAC?GTG?CTT?TAC?TAG?GTC?CTC?TAG?AAG
70 80Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu?LeuAAC?CTG?TTC?TCT?ACC?AAA?GAC?TCT?TCT?GCT?GCT?TGG?GAC?GAA?ACC?CTG?CTGTTG?GAC?AAG?AGA?TGG?TTT?CTG?AGA?AGA?CGA?CGA?ACC?CTG?CTT?TGG?GAC?GAC
90Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu?AlaCTG?GAC?AAA?TTC?TAC?ACC?GAA?CTG?TAC?CAG?CAG?CTG?AAC?GAC?CTG?GAA?GCTGAC?CTG?TTT?AAG?ATG?TGG?CTT?GAC?ATG?GTC?GTC?GAC?TTG?CTG?GAC?CTT?CGA
100 110Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys?Glu?AspTGC?GTT?ATC?CAG?GGT?GTT?GGT?GTT?ACC?GAA?ACC?CCG?CTG?ATG?AAA?GAA?GACACG?CAA?TAG?GTC?CCA?CAA?CCA?CAA?TGG?CTT?TGG?GGC?GAC?TAC?TTT?CTT?CTG
120 130Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu?Tyr?Leu?LysTCT?ATC?CTG?GCT?GTT?CGT?AAA?TAC?TTC?CAG?CGT?ATC?ACC?CTG?TAC?CTG?AAAAGA?TAG?GAC?CGA?CAA?GCA?TTT?ATG?AAG?GTC?GCA?TAG?TGG?GAC?ATG?GAC?TTT
140Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg?Ala?Glu?Ile?MetGAA?AAG?AAA?TAC?TCT?CCG?TGC?GCT?TGG?GAA?GTT?GTT?CGT?GCT?GAA?ATC?ATGCTT?TTC?TTT?ATG?AGA?GGC?ACG?CGA?ACC?CTT?CAA?CAA?GCA?CGA?CTT?TAG?TAC150 160Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser?Leu?Arg?Ser?Lys?GluCGT?TCT?TTC?TCT?CTG?TCT?ACC?AAC?CTG?CAG?GAA?TCT?CTG?CGT?TCT?AAA?GAAGCA?AGA?AAG?AGA?GAC?AGA?TGG?TTG?GAC?GTC?CTT?AGA?GAC?GCA?AGA?TTT?CTTTAAATT
(2) synthetic gene is packed into efficient expression vector pLY4 becomes pHX-hIFN α-2b expression plasmid, makes up the genetic engineering interferon alpha-2b engineering bacteria (CCTCC Mo.99002) of gained.
2, the Interferon Alpha-2b expression of gene plasmid pHX-hIFN α-2b that contains synthetic as claimed in claim 1.
3. the engineering bacteria that contains expression plasmid pHX-hIFN α-2b as claimed in claim 2, its preserving number are CCTCC NO.99002.
4. the application of human interferon alpha-2 b as claimed in claim 1 in the medicine of preparation anti-virus infection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN99113732A CN1113964C (en) | 1999-05-25 | 1999-05-25 | High-efficiency expression of artificially synthetic interferon alpha-2b gene in colibacillus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN99113732A CN1113964C (en) | 1999-05-25 | 1999-05-25 | High-efficiency expression of artificially synthetic interferon alpha-2b gene in colibacillus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1240830A CN1240830A (en) | 2000-01-12 |
| CN1113964C true CN1113964C (en) | 2003-07-09 |
Family
ID=5276891
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN99113732A Expired - Lifetime CN1113964C (en) | 1999-05-25 | 1999-05-25 | High-efficiency expression of artificially synthetic interferon alpha-2b gene in colibacillus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1113964C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102367441B (en) * | 2011-06-20 | 2014-09-17 | 安徽安科生物工程（集团）股份有限公司 | Preparation method for recombinant human interferon alpha-2b free of methionine |
| CN109553659B (en) * | 2018-11-26 | 2022-06-21 | 上海华新生物高技术有限公司 | Cell penetrating peptide and transdermal interferon |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1073977A (en) * | 1991-12-31 | 1993-07-07 | 株式会社乐喜 | The purifying of coding human's recombination, expression vector and this Interferon, rabbit |
-
1999
- 1999-05-25 CN CN99113732A patent/CN1113964C/en not_active Expired - Lifetime
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1073977A (en) * | 1991-12-31 | 1993-07-07 | 株式会社乐喜 | The purifying of coding human's recombination, expression vector and this Interferon, rabbit |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1240830A (en) | 2000-01-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2024002149A1 (en) | Recombinant type-iii collagen and method for preparing same | |
| CN1363654A (en) | Genetically engineered bacteria and process for preparing insulinotropic hormone secretion peptide GLP-1 (7-36) | |
| CN104402975B (en) | Anti-aging small peptide and preparation method thereof | |
| CN1158304C (en) | Antibacterial frog skin peptide derivative | |
| CN1113964C (en) | High-efficiency expression of artificially synthetic interferon alpha-2b gene in colibacillus | |
| CN113880941B (en) | Recombinant humanized IxIII collagen, expression strain and application thereof | |
| CN1304422C (en) | Non-amidated omega-conotoxin VIIA and its prepn process and application | |
| CN1305902C (en) | Method for preparing human interleukin 29 and recombinant IL-29 engineering strain | |
| CN101875691B (en) | Scorpion arialgesic antitumoral peptide mutant and preparation method thereof | |
| CN105218659B (en) | Human BMP-7 mature peptide and expression thereof | |
| CN87102497A (en) | Alpha interferon analogue | |
| CN104829691B (en) | Smoothing wrinkle small peptide and preparation method thereof | |
| CN107177614A (en) | A kind of method for improving canine recombinant interferon gamma fusion protein antiviral activity | |
| CN100516217C (en) | Artificial synthesised scorpion chloride ion neurotoxin gene-rBmK CTa | |
| CN101928711B (en) | Chicken interferon-alpha gene by total gene synthesis and protein expression | |
| CN101870976B (en) | Gene synthesis duck alpha interferon gene and protein expression | |
| CN1487083A (en) | Grouper insulin-like growth factor II gene, carrier and recombinant strain containing the gene and application thereof | |
| KR100729830B1 (en) | Expression vector with Penetratin as a fusion partner to enhance transdermal delivery of recombinant proteins | |
| CN1408863A (en) | Human interleukin-10 gene sequenc and E coli containing the said gene sequence | |
| CN1057564C (en) | Microbe capable of producing active recombinant phycobiliprotein and its preparing method | |
| CN1274823C (en) | Production of human interleukin 288 and recombined IL-288 engineering bacteria | |
| FI82074C (en) | Process for the preparation of human leukocyte interferon alfa-2 | |
| CN1059929C (en) | Human thymosin alpha-source and preparing method thereof | |
| CN1693458A (en) | Production method of human interleukin 28 A and recombination IL-28A engineering bacteria | |
| CN1199993C (en) | N-terminal deletion lipocyte complement related protein and its preparation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CX01 | Expiry of patent term | ||
| CX01 | Expiry of patent term |
Granted publication date: 20030709 |