CN1408862A - Artificial synthetic beta-interferon gene and its expression carrier - Google Patents

Abstract

The present invention is a kind of artificial synthetic beta-interferon gene. Based on the effect of gene structure on foreign gene expression and the preference of codon, human IFN-beta Ser17 is designed and synthesized via gene engineering technological process. A kind of recombinant plasmid carrier bBV220 containing the nucleotide sequence of the present invention and converted colibacillus DH5-alpha are also disclosed. The colibacillus DH5-alpha can raise the expression level of the gene, is suitable for industrial production in large scale, has the preservation number of CCTCC No.M201035 and has an expression amount accounting for 30% of total strain protein. The present invention establishes a fermentation, denaturation, renaturation and purification process and this creates the condition for mas sproduction of IFN-beta Ser17.

Description

Artificial synthetic beta-interferon gene and expression vector thereof

The technical field this patent is a kind of with genetic engineering technique production people IFN-β Ser ¹⁷Method, international Patent classificating number is C12N.

The activity of prior art IFN-β relates to the adjusting of non-specific humoral immunization and the immune response of anti-virus infection.IFN-β can increase the antigenic expression of I class HLA and blocking-up antigenic expression of II class HLA under IFN γ stimulates.IFN-β can stimulate the NK cell activity, thereby the same cytotoxic reaction that increases dependence and antibody.The suppressor T cell activity that is caused by some stimulator also can be activated by IFN-β.IFN-β can increase low-affinity IgE acceptor synthetic ( CD23) in the activated monocyte, IFN-β can induce mopterin (neopterin).The concentration that IFN-β also can increase the serum β2Wei Qiudanbai suppresses the expression of some chondriogen with selectivity.IFN-β also is proved to be has the cell proliferation of inhibition activity to the various kinds of cell cording of setting up from solid tumor.

The external IFN-β that adopts treats some disease, and existing division is as follows:

1. property wart, i.e. pointed condyloma (condylomata acuminata) can be behind the most of knurl body of surgical removal, and part or whole body use, and prevent its recurrence, and certain curative effect is arranged.

2. treatment hepatitis C and relevant liver cancer gap IFN treatment can suppress the HCV related neoplasms in postoperative recurrence (Ikeda etc. 2000).(1999) such as Terranova R have studied alpha-interferon, and interferon beta and lymphoblastoid Interferon, rabbit are to the curative effect of senile hepatitis C, because the hepatitis C more than 60 years old, its pathologic process can be accelerated.The result shows that alpha-interferon is best, and its remission rate can reach 75%; Interferon beta is 53.8%; The lymphoblastoid Interferon, rabbit is 60%; But the side reaction minimum of interferon beta does not almost have, and dimension balance curative effect and toxic side effects are treated senile hepatitis C, first-selected interferon beta.

3. the treatment multiple sclerosis can reduce recurrence with IFN-β treatment multiple sclerosis (MS), delays maimed process.

Human's gene is an eukaryotic gene, if do not do any transformation, expression amount is very low in intestinal bacteria (belonging to prokaryotic organism), is not suitable for carrying out extensive and commercial production.Because the potential applicability in clinical practice of interferon-, lot of domestic and foreign producer is all at development heart new method and new technology.

Summary of the invention the present invention is that the above-mentioned weak point that overcomes prior art is improved, the interferon-gene that the synthetic codon changes, and be inserted into expression plasmid, transformed into escherichia coli again, improve this expression of gene level, be suitable for large-scale commercial production.

Purpose of the present invention can reach by following technical measures:

In view of the merger of codon different with intestinal bacteria to the hobby of amino acid code, the present invention design synthesized contain intestinal bacteria hobbies codon can produce IFN-β Ser ¹⁷Gene, but the amino acid that does not change product forms and puts in order, and improves gene at colibacillary expression level.This gene is packed among the custom-designed efficient expression vector pBV220, and be transformed in the bacillus coli DH 5 alpha, obtain high expression level, expression amount accounts for more than 30% of whole cell total protein, and the engineering bacteria after the conversion is intestinal bacteria E.Coli-DH5 α-pBV220-IFN-β Ser ¹⁷, deposit number: CCTCC NO.M201035 (Chinese typical culture collection center, China).The present invention simultaneously also provides the purification technique that efficiently expresses of an interferon-.The ultrasonic degradation bacterium, obtain pure inclusion body after, inclusion body is with after SDS cracking, sec-butyl alcohol extracting, going up Sephacryl S-100 purifying, greater than 95%, specific activity is greater than 2.0 * 10 through SDS-PAGE and HPLC purity checking for product ⁷IU/ml.20 amino acid sequence analysis proofs of N-terminal are in full accord with the sequence of theoretical prediction.It is equally matched with external like product that biological function is measured.Therefore, the present invention has the meaning of scale operation.

Synoptic diagram of the present invention is as follows:

The people IFN-β Ser of Fig. 1, synthetic design ¹⁷Gene and amino acid sequence corresponding;

Nucleotide sequence distinct cases contrast figure in nucleotide sequence and the natural human beta-interferon in Fig. 2, the synthetic human dna sequence dna of the present invention, NNN represents codon inequality;

Fig. 3, synthetic IFN-β Ser ¹⁷The sequencing of gene;

Fig. 4, IFN-β Ser ¹⁷The structure of expression plasmid;

Fig. 5, IFN-β Ser ¹⁷Enzyme cut and identify and amplification and the evaluation of PCR;

The tomographic map of Fig. 6, Sephacryl S-100;

Fig. 7, IFN-β Ser ¹⁷SDS-PAGE electrophoresis calibrating;

General each the row protein content table of Fig. 8, each stage SDS-PAGE figure of interferon-separation and purification;

Fig. 9, IFN-β Ser ¹⁷The HPLC calibrating;

Respectively analyse luminosity peak-data table in Figure 10, the efficient liquid phase chromatographic analysis.

Embodiment is described in detail technical characterictic of the present invention by following example.

Materials and methods

(1) restriction enzyme, Taq enzyme, dna molecular amount standard are all available from Boehringer.Microbial culture yeast extract, peptone are available from Britain Oxford.Inorganic salt reagent is homemade analytical pure

(2) bacterial classification and plasmid.Bacillus coli DH 5 and plasmid pBV220 are the applicant and preserve, and send the preservation of Wuhan DSMZ simultaneously, and preserving number is CCTCC NO.M201035。

(3) preparation of plasmid, competent preparation, the conversion of DNA and the screening of transformant, ordinary methods such as the hydrolysis of restriction enzyme are equal, referring to＜molecular cloning 〉.

(4) SDS is available from Serva, and Sephacryl S-100 is available from Pharmacia company, and RPML-1640 is available from Nihon Pharmaceutical Co., Ltd..

(5) fermentation of engineering bacteria is cultivated under 30 ℃ of conditions, abduction delivering under 42 ℃ of conditions.

(6) preparation of inclusion body and purifying.Bacterium is through ultrasonication, SDS cracking, sec-butyl alcohol extracting, SephacrylS-100 chromatography purification.IFN-β Ser ¹⁷Activity test method, adopt folliculus oral membrane inflammation virus (VSV)-people amnion cell system that goes down to posterity to carry out.

The concrete steps of implementing are described below respectively:

The coding human gene of design, synthetic a kind of synthetic is characterized in that:

、,：ATG TCT TAT AAT CTT CTG GGT TTC CTG CAG CGT TCT TCT AAC TTC CAG TCT CAGAAA CTG CTG TGG CAG CTG AAC GGT CGT CTG GAA TAC TGC CTG AAA GAC CGT ATGAAC TTC GAC ATC CCG GAA GAA ATC AAA CAG CTG CAG CAG TTC CAG AAA GAA GACGCT GCT CTG ACC ATC TAC GAA ATG CTG CAG AAC ATC TTC GCT ATC TTC CGT CAGGAC TCT TCT TCT ACC GGT TGG AAC GAA ACC ATC GTT GAA AAC CTG CTG GCT AACGTT TAC CAC CAG ATC AAC CAC CTG AAA ACC GTT CTG GAA GAA AAA CTG GAA AAAGAA GAC TTC ACC CGT GGT AAA CTG ATG TCT TCT CTG CAC CTG AAA CGT TAC TACGGT CGT ATC CTG CAC TAC CTG AAA GCT AAA GAA TAC TCT CAC TGC GCT TGG ACCATC GTT CGT GTT GAA ATC CTG CGT AAC TTC TAC TTC ATC AAC CGT CTG ACC GGTTAC CTG CGT AAC TAA

People IFN-β Ser ¹⁷Be made up of 165 amino acid, the synthetic gene adds terminator codon totally 501 bp, the synthetic back of gene with the method for PCR 5 of gene ' with 3 ' respectively add EcoRI and BamHI restriction enzyme site.Select colibacillary preference codon for use, the synthetic gene can be efficiently expressed in intestinal bacteria, produce a large amount of people IFN-β Ser ¹⁷, and do not change its aminoacid sequence.

The codon that is changed is seen Fig. 2 synopsis, is the codon at the intestinal bacteria preference for having changed of sign with NNN.

Design, a kind of expression plasmid that contains nucleotide sequence of the present invention of structure.

The structure of described plasmid vector has used plasmid vector pBV220, and it is by P _RP _LSeries connection strong promoter, Cits857 temperature sensitive aporepressor gene, the coding human's of described synthetic nucleotide sequence, transcription termination signal makes up and forms, and above structure makes the carrier of structure become efficient expression vector.

Design, transform a kind of transformed into escherichia coli, it is characterized in that:

With the recombinant plasmid vector transformed host cell that the present invention makes up, the present invention adopts intestinal bacteria as the host.

The plasmid vector transformed into escherichia coli DH5 α that is built into the present invention, and obtain high expression level; The intestinal bacteria that transform have carried out preservation by China typical culture collection center in Wuhan City Wuhan University; Its deposit number is: CCTCC NO.M201035.

Described e. coli expression product exists with the form of inclusion body, and inclusion body is with after SDS cracking, sec-butyl alcohol extracting, going up Sephacryl S-100 purifying, and purity is greater than 95%, and specific activity is greater than 2.0 * 10 ⁷IU/ml.

Embodiment 1:IFN-β Ser ¹⁷Gene synthetic

One, the synthetic design of gene:

8 used segment length of HuIFN β DNA splicing are about the synthetic DNA fragment of 80bp:

1. βL15′-ATGTCTTATAATNNNNNNNNNNNNNNNNNNN CCAGTCTCAGAAACTGCTGT-3′

2. βL25′- CCAGTCTCAGAAACTGCTGTNNNNNNNNNNNNNNNNNNNNNA CTTCGACATCCCGGAAGAA-3′

3. βL35′- ACTTCGACATCCCGGAAGAANNNNNNNNNNNNNNNNNNNNN TCTACGAAATGCTGCAGAAC-3′

4. βL45′- TCTACGAAATGCTGCAGAACNNNNNNNNNNNNNNNNNNNNN GAAACCATCGTTGAAAACCTG-3′

5. βR13′- CTTTGGTAGCAACTTTTGGACNNNNNNNNNNNNNNNNNNN CCTTCTTTTTGACCTTTTTCTTC-5′

6. βR23′- CCTTCTTTTTGACCTTTTTCTTCNNNNNNNNNNNNNNNNNN AATGATGCCAGCATAGGACG-5′

7. βR33′- AATGATGCCAGCATAGGACGNNNNNNNNNNNNNNNNNNNNNC ACAACTTTAGGACGCATTG-5′

8. βR43′- CACAACTTTAGGACGCATTGNNNNNNNNNNNNNNNNNNNTGGACGCATT-5′

Above-mentioned dna fragmentation represents in the sequence that underscore is partly represented the complementary lap-joint of sequence.

According to design, the end of L4 and R4 has 20 base complementrities, and this links to each other two chains, and in the PCR reaction system, two chains are complementary portion template generation each other chain extension not.Reaction conditions: L4 5 μ L (0.1OD/ μ l), R4 5 μ L (0.1OD/ μ l), 10 ℃ of TaKaRa La buffer 10 μ l, dNTP (25mmol/L) 16 μ l, H ₂O 63 μ l, reaction system is totally 100 μ l.After 94 ℃ of constant temperature 5min, 55 ℃ of constant temperature add TaKaRa LA Taq enzyme 1 μ l, constant temperature 5min, 72 ℃, 5min. can obtain two complementary long-chains being spliced by L4 and R4, and L4R4 by name is the template of next step PCR reaction.

Three times the PCR reaction chain prolongs.With back in like manner, according to the design, L4 and L3, L2, L1 all have the tumor-necrosis factor glycoproteins of 20bp, R4, R3, R2, R1 have the tumor-necrosis factor glycoproteins of 20bp successively.Utilize these primers,, promptly get aim sequence according to following three-step reaction.PCR reaction 1:L3 (0.01OD/ μ l) 1 μ l, R3 (0.01OD/ μ l) 1 μ l, L4R4 1 μ l, 10 * TaKaRa La buffer10 μ l, dNTP (25mmol/L) 10 μ l, TaKaRa LA Taq enzyme 1 μ l, H ₂O 76 μ l, reaction system is totally 100 μ l, 94 ℃ of C, 30s, 55 ℃ of C30s, 72 ℃ of C, 30s, totally 30 circulations.Product is called L3L4R4R3.PCR reaction 2:L2 (0.01OD/ μ l) 1 μ l, R2 (0.01OD/ μ l) 1 μ l, L3L3R4R3 1 μ l, 10 * TaKaRaLa buffer10 μ l, dNTP (25mmol/L) 10 μ l, TaKaRa LA Taq enzyme 1 μ l, H ₂O 75 μ l, reaction system is totally 100 μ l, 94 ℃ of C, 30s, 55 ℃ of C30s, 72 ℃ of C, 30s, totally 30 circulations.Product is called L2L3L4R4R3R2.PCR reaction 3:L1 (0.01OD/ μ l) 1 μ l, R1 (0.01OD/ μ l) 1 μ l, L2L3L4R4R3R2 1 μ l, 10 * TaKaRa La buffer10 μ l, dNTP (25mmol/L) 10 μ l, TaKaRa LA Taq enzyme 1 μ l, H ₂O 75 μ l, reaction system is totally 100 μ l, 94 ℃ of C, 30s, 55 ℃ of C30s, 72 ℃ of C, 30s, totally 30 circulations.Product is called L1L2L3L4R4R3R2R1, is aim sequence after enzyme is cut.Fig. 5 illustrates IFN-β Ser ¹⁷Enzyme cut and identify and amplification and the evaluation of PCR; Wherein 1. being recombinant plasmid EcoRI+BamHI double digestion, 2. is pcr amplification product, 3. 4. is molecular weight Marker for PCR identifies product.

DNA chain overlap joint and the synoptic diagram that prolongs are as follows:

First DNA chain overlap joint and chain extension

Three step PCR reaction chains prolong

Final chain extension reaction product

L1L2L3L4R4R3R2R

Two, the order-checking of Hai Boya company is served in the evaluation of synthetic gene product.

Embodiment 2: fermentation culture

Choose single colony inoculation in the 3mlLB substratum, cultivate 10hr for 30 ℃, be inoculated in 300mlLB30 ℃ then and cultivate 10hr, this reaches and is fermentation seed liquid, and fermention medium: LB cultivation Kiev adds M ₉Salt, referring to (" molecular cloning "), penbritin concentration is 50 μ g/mL, stream added after 50% glucose was sterilized separately, the pH value is set at 7.0, adopts pid control law to regulate the pH value with strong aqua, and stirring and air flow are regulated according to dissolved oxygen (DO), to guarantee that DO is not less than 20%, 30 ℃ of cultivation and is warmed up to 42 ℃ of inducing culture 4 hours after 4-5 hour.PH control method, pO are adopted in adding of glucose respectively ₂Control method, concentration control method.

Embodiment 3: purifying

Fermented liquid is through 4,000RPM is centrifugal, and thalline is washed 3 times with TE (10mM Tris-HCl 1mM EDTA pH 7.0), and it is resuspended to add 5 times of TE liquid, ultrasonication under the condition of ice bath (200W/200V), 5 times repeatedly, each 1 minute at interval, 5,000RPM removed supernatant in centrifugal 30 minutes, inclusion body with isopyknic sec-butyl alcohol extracting, is gone up Sephacryl S-100 with containing 2%SDS, 10Mmdtt, 150mMPBS cracking then.

Embodiment 4:IFN-β Ser ¹⁷The evaluation of product

a，SDS-PAGE

Fig. 7 is IFN-β Ser ¹⁷SDS-PAGE electrophoresis rating diagram, show the IFN-β Ser behind the purifying ¹⁷Be single band; Fig. 8 is each row protein content table of described evaluation SDS-PAGE collection of illustrative plates;

B, the HPLC calibrating

Fig. 9 is IFN-β Ser ¹⁷The HPLC rating diagram shows its main peak IFN-β Ser ¹⁷Content greater than more than 95%; Figure 10 respectively analyses luminosity peak-data table in the described efficient liquid phase chromatographic analysis;

C, the N-terminal analysis

20 the amino acid whose qualification results and the theoretical prediction result of N-terminal are in full accord.Its sequence is: Ser Tyr AsnLeu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln Ser Gln Lys Leu Leu;

D is than the evaluation of living

Measure the biologic activity of Interferon, rabbit by cytopathic-effect inhibition assay, with the ratio of protein content greater than 2.0 * 10 ⁷IU/mg.

Claims (5)

1.β-DNA,：：ATG TCT TAT AAT CTT CTG GGT TTC CTG CAG CGT TCT TCT AAC TTC CAG TCT CAGAAA CTG CTG TGG CAG CTG AAC GGT CGT CTG GAA TAC TGC CTG AAA GAC CGT ATGAAC TTC GAC ATC CCG GAA GAA ATC AAA CAG CTG CAG CAG TTC CAG AAA GAA GACGCT GCT CTG ACC ATC TAC GAA ATG CTG CAG AAC ATC TTC GCT ATC TTC CGT CAGGAC TCT TCT TCT ACC GGT TGG AAC GAA ACC ATC GTT GAA AAC CTG CTG GCT AACGTT TAC CAC CAG ATC AAC CAC CTG AAA ACC GTT CTG GAA GAA AAA CTG GAA AAAGAA GAC TTC ACC CGT GGT AAA CTG ATG TCT TCT CTG CAC CTG AAA CGT TAC TACGGT CGT ATC CTG CAC TAC CTG AAA GCT AAA GAA TAC TCT CAC TGC GCT TGG ACCATC GTT CGT GTT GAA ATC CTG CGT AAC TTC TAC TTC ATC AAC CGT CTG ACC GGTTAC CTG CGT AAC TAA

2. one kind contains the recombinant plasmid vector of nucleotide sequence according to claim 1.

3. recombinant plasmid vector according to claim 2 is characterized in that:

Used plasmid vector pBV220, it is by P _RP _LSeries connection strong promoter, Cits857 temperature sensitive aporepressor gene, the coding human's of described synthetic nucleotide sequence, transcription termination signal makes up and forms, and above structure makes the carrier of structure become efficient expression vector.

4. intestinal bacteria that transform by the described plasmid vector of claim 2.

5. host according to claim 4 is characterized in that described colibacillary deposit number is:

CCTCC NO.M201035。

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