CN1240830A - 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
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Abstract
The present invention discloses a gene sequence able to be expressed effectively in colibacillus and constructs effecient expression plasmid pHX-hIFN a-2b and human interferon a-2b as production bacteria of colibcillus. It has very high productive value and can be used to prepare more medicines and other products.
Description
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.
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.
The present invention is that the following embodiment that passes through implements:
Description of drawings in the invention:
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-F IIF III
The structure of Fig. 5, M13mp18-F IF IIF III
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/Hind?III+EcoR?I
2. plasmid
3.EcoR?I
4.Bg1?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.
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 IFN 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/Hind III), F
III(Hind III/Sal I).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
II. and M13mp18-F
III, with the F that cuts out
IIThe M13mp18-F that fragment and enzyme are cut
IIIConnect, the clone cuts, screens with BamH1 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 h I 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 EcoR I and Sal I 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 EcoR I and Sal I 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 the 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.
Bacterial classification stability: (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 (2)
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, described with the synthetic gene efficient expression vector pLY4 that packs into according to claim 1, the people's gene engineering Interferon Alpha-2b of the engineering bacteria preparation of structure can be used for injection, the exterior-use capsule medicine production is used and the application of other products.
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| Application Number | Priority Date | Filing Date | Title |
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| CN99113732A CN1113964C (en) | 1999-05-25 | 1999-05-25 | High-efficiency expression of artificially synthetic interferon alpha-2b gene in colibacillus |
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| 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 |
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| Publication Number | Publication Date |
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| CN1240830A true CN1240830A (en) | 2000-01-12 |
| CN1113964C CN1113964C (en) | 2003-07-09 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102367441A (en) * | 2011-06-20 | 2012-03-07 | 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 |
| CN109553659A (en) * | 2018-11-26 | 2019-04-02 | 上海华新生物高技术有限公司 | A kind of cell-penetrating peptides and transdermal interferon |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU648214B2 (en) * | 1991-12-31 | 1994-04-14 | Lucky Limited | Recombinant gene coding for human alpha interferon and expression vector thereof, etc. |
-
1999
- 1999-05-25 CN CN99113732A patent/CN1113964C/en not_active Expired - Lifetime
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102367441A (en) * | 2011-06-20 | 2012-03-07 | 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 |
| 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 |
| CN109553659A (en) * | 2018-11-26 | 2019-04-02 | 上海华新生物高技术有限公司 | A kind of cell-penetrating peptides and transdermal interferon |
| 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 |
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| Publication number | Publication date |
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| CN1113964C (en) | 2003-07-09 |
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Granted publication date: 20030709 |