CN102268093A - Fusion protein of hepatic-targeted peptide and human interferon a2b, and preparation method and application thereof - Google Patents

Abstract

The invention discloses a fusion protein of hepatic-targeted peptide and human interferon a2b, and a preparation method and application thereof. The nucleotide sequence of the fusion protein of hepatic-targeted peptide and human interferon a2b provided in the invention is described by SEQ ID NO. 1, and the amino acid sequence coded by the nucleotide sequence is described by SEQ ID NO. 2. According to in vitro tests, the fusion protein provided in the invention has obvious activity of resisting HBV and hepatocyte targeting. Since characteristics of both hepatic-targeted peptide of plasmodium circumsporozoite protein CSPI-plus and human IFNa2b are combined into the fusion protein in the invention, the fusion protein can be used for preparation of novel hepatic-targeted antiviral drugs and is of profound significance for lowering down morbidity and mortality of liver diseases caused by infection of HBV. According to the invention, the prepared fusion protein has a high expression level and is easy to purify, production period for the fusion protein is short, and low cost is realized; therefore, the invention has important application prospects and is of practical significance.

Description

A kind of liver target peptide and human interferon a2b fusion rotein and its production and application

Technical field

The present invention relates to biological technical field, be specifically related to a kind of liver target peptide and human interferon a2b fusion rotein and its production and application.

Background technology

Hepatitis B virus (hepatitis B virus, HBV) for having a liking for hepatovirus, HBV infects the situation that has become global harm at present, in 6,000,000,000 populations of the whole world, about 2,000,000,000 people had HBV to infect experience, 3.5 hundred million people are the chronic HBV infection person, about 80% patient has liver cell in various degree impaired, and may develop into liver cirrhosis and liver cancer.The number of dying from the hepatitis B relative disease every year is up to more than 100 ten thousand, and WHO has infected HBV and classified the whole world ten one of the most common deadly cause of disease greatly as.China belongs to the high popular district of HBV, and general crowd HBsAg carrying rate is up to 7.18%, and this not only has a strong impact on people's health, also is a serious social concern simultaneously.

Vast amount of clinical proves, HBV is long-term existence and constantly duplicate the continuously active that causes hepatic disease and development is the basic reason that causes disease progression in vivo, so antiviral therapy is chronic hepatitis B (chronic hepatitis B, CHB) Zhi Liao a key.The antiviral that is used at present hepatitis B clinically mainly contains interferons, nucleosides (acid) analogue and immunomodulator.1. nucleosides (acid) analogue is brought into play anti-HBV effect by the activity that suppresses HBV-DNA reversed transcriptive enzyme and DNA polymerase, comprise lamivudine (Lamivudine, 3TC), Entecavir (Entecavir, ETV), Adefovir (Adefovir, ADV) etc.Because advantages such as it is easy to use, patient's tolerance is good are widely used in treating CHB patient.But along with the life-time service of nucleosides (acid) class medicine, after the generation of resistance HBV and the drug withdrawal knock-on etc. become a problem that can not be ignored, thereby limited its clinical application.2. immunomodulator comprises Zadaxin, immune ribonucleic acid, HBV specific transfer factor and traditional Chinese medicine etc., and they can improve the specific immunity of human body to HBV, by the target cell that identification and destruction HBV infect, removes HBV.But the application of immunomodulator in CHB at present also lacks specific aim, and curative effect is satisfied not enough, still remains further to be studied.

(Interferon IFN) at a plurality of links performance such as the duplicating of HBV gene, expression antivirus actions, can also transfer host's immunologic function to Interferon, rabbit, forms the antiviral activity of double mechanism, becomes one of choice drug of CHB antiviral therapy.Difference according to cell source, molecular structure, physico-chemical property and the biologic activity of IFN, it is divided into alpha-interferon (IFN α), beta-interferon (IFN β) and gamma-interferon (IFN γ) three types, each type is formed amino acid whose difference according to it can be divided into a plurality of hypotypes again, as IFN α 1b, IFN α 2b etc.IFN α 2b is first anti-HBV medicine of FDA approval, and through the clinical application of decades, its treatment hepatitis B has obtained abundant affirmation.Yet as one of choice drug of anti-HBV, IFN α 2b exists shortcomings such as dosage is big, curative effect is not ideal enough, toxic side effect is big, has limited its widespread use in clinical.This major cause be since HBV mainly at liver cell endoparasitism and duplicating, IFN α 2b diffusion degraded easily in vivo causes average relatively tissue distribution during treatment, fails to form effective antivirus action concentration at liver.In order to reach antiviral effect, drug dose often will strengthen several times, tens times, and the increasing of drug dose has also increased the weight of its untoward reaction, is easy to generate resistance HBV again, thereby affects the treatment.At this problem, carried out the targeted therapy research of IFN in recent years, utilize modes such as liposome, mediated monoclonal antibody and asialoglycoprotein receptor mediation specifically IFN to be transported to target organ or target cell.Targeted therapy has that specificity is good, selectivity is strong, can reduce drug dose and administration number of times, and reduces toxic side effect and improve advantages such as curative effect of medication, thereby might become in the HBV treatment of infection one of effective means.Yet, owing to be subjected to the influence of various factors such as kind, size and specificity of carrier, though the target of IFN research at present has certain target, remain be difficult to reach the accurate target of cell levels, easily by deficiencies such as body immune system removings.Therefore, further seek efficient special targeting vector, explore the novel targeted IFN of design and seem extremely important.

Infectious when press the mosquito bite host, (Sporozoite SPZ) just strides across the liver sinusoid Kupffer with blood flow after several minutes, initiatively adhesion, invades liver cell rapidly to enter the plasmodium sporozoite of capillary vessel.Discover, this process relates to one deck table of SPZ surface coverage by albumen---circumsporozoite protein (Circumsporozoite protein, CSP), thus plasmodium at first utilizes the identification of itself and surface of hepatocytes acceptor interaction, sticks to surface of hepatocytes.By intravenous injection, the CSP of reorganization just can be incorporated into surface of hepatocytes in several minutes, and it is efficient and special liver cell targeted to show that CSP has.CSP contains 400 amino acid approximately, is made of the conservative I district of N end, central iteron and the conservative II district of C end.In recent years studies show that the conservative I district of CSP N end can with the acceptor of surface of hepatocytes---heparan sulfate proteoglycan (heparan sulfateproteoglycans, HSPGs) combination specifically, show CSP I district except that comprising conserved sequence KLKQP, the heparin sulfate binding sequence is also contained in the upstream of conserved sequence, and this peptide section that contains KLKQP and heparin sulfate binding sequence is called I-plus.Ancsin etc. studies confirm that CSP I-plus with saturated mode heparin-binding and heparin sulfate gel column, and suppress combining of recombinant C SP and heparin gel.Robertson etc. are fixed on Macrogol Ester plastid top with CSP I-plus and are prepared into and contain the CSP liposome, thereby experiment in vivo and vitro shows that all it can be specifically in conjunction with HSPGs target hepatic tissue.After the mouse vein administration, containing CSP liposome 15 minutes can remove from the mouse peripheral blood apace, and quantitative analysis shows that concentration that mouse liver contains the CSP liposome is heart, kidney and lungs hundreds of times, is 12 times of spleen.Above result of study prompting CSP I-plus is a good targeting vector.

Summary of the invention

The objective of the invention is to according to above shortcomings in the prior art, and, provide a kind of new liver targeted interferon IFN-CSP based on the anti-HBV effect of efficient and the special liver cell targeted and IFN of CSP I-plus.Utilize that liver target peptide CSP I-plus is efficient special liver cell targetedly made the IFNa2b liver that leads it in the liver enrichment, thereby improved the specificity that interferon therapy HBV infects, reduce the whole body consumption, reduce toxic side effects, improve amount effect ratio.

Another purpose of the present invention is to provide a kind of new fusion gene, the CSP I-plus gene splicing that derives from plasmodium circumsporozoite protein in resisting at the C of people IFN α 2b gene end and get, and is transformed the two and to be made the fusion gene expression amount be greatly enhanced.

A further object of the invention is to provide the application of above-mentioned liver target peptide and human interferon a2b fusion rotein.

Above-mentioned purpose of the present invention is achieved by the following technical programs:

Based on the anti-HBV effect of efficient and the special liver cell targeted and IFN of CSP, the present invention adopts plasmodium falciparum CSP I-plus as guide molecule, utilizes gene engineering method that itself and IFN α 2b are merged, preparation liver targeted interferon IFN-CSP.Utilize that CSP I-plus is efficient special liver cell targetedly made the IFNa2b liver that leads it in the liver enrichment, thereby improved the specificity of interferon therapy chronic HBV infection, reduce the whole body consumption, reduce toxic side effects, improve amount effect ratio.The present invention has creatively explored one and has utilized parasitic some molecule to treat the new way of human diseases, targeted therapy mode with present Interferon, rabbit compares as utilizing researchs such as liposome, mediated monoclonal antibody and asialoglycoprotein receptor mediation simultaneously, have the accurate active target that can reach cell levels and be difficult for by characteristics such as body immune system removings, thereby have extraordinary application prospect, will bring certain economic benefits and social benefit to society.

A kind of liver target peptide and human interferon a2b fusion rotein, its nucleotide sequence are shown in SEQ ID NO:1, and this nucleotide sequence coded aminoacid sequence is shown in SEQ ID NO:2.

The preparation method of the present invention's above-mentioned liver target peptide and human interferon a2b fusion rotein is characterized in that comprising the steps:

(1) dna sequence dna of liver target peptide and human interferon a2b gene is transformed, utilized SOE-PCR method gene fusion construct;

(2) make up recombinant expression plasmid;

(3), obtain the engineering bacteria of expressed fusion protein, fermentation culture with the recombinant expression plasmid transformed host cell;

(4) purifies and separates obtains fusion rotein IFN-CSP.

As a kind of preferred version, among the above-mentioned preparation method, gene fusion construct is according to the aminoacid sequence of human interferon a2b and the aminoacid sequence of plasmodium falciparum liver target peptide in the step (1), adopt the codon of intestinal bacteria preference, nucleotide sequence by computer aided design (CAD) liver targeted interferon IFN-CSP gene, whole gene is divided into 16 overlapped oligonucleotide segments, adopts the overlapping extension PCR method of improvement to make up fusion gene with nucleotide sequence shown in SEQ ID NO.1.Further, during PCR used primer sequence shown in SED ID NO:3 ~ 19.

As a kind of preferred version, among the above-mentioned preparation method, used expression vector is preferred but be not limited to prokaryotic expression carrier pET32a during the described structure recombinant expression plasmid of step (2).

As a kind of preferred version, among the above-mentioned preparation method, expressive host bacterium described in the step (3) is preferred but be not limited to intestinal bacteria.

The present invention adopts the codon of intestinal bacteria preference, by the nucleotide sequence of computer aided design (CAD) liver targeted interferon IFN-CSP gene according to the aminoacid sequence of people IFNa2b and the aminoacid sequence of plasmodium falciparum CSP I-plus.Whole gene is divided into 16 overlapped oligonucleotide segments, adopt overlapping extension PCR (splice-overlap extension-PCR, SOE-PCR) technique construction has the fusion gene of nucleotide sequence shown in SEQ ID NO:1, called after fusion gene IFN-CSP(Fig. 1 ~ 2).This fusion gene cloning is gone into the pMD20-T carrier, carry out PCR, double digestion, order-checking evaluation.The result shows that IFNa2b and CSP I-plus sequence, the order that splices and combines and direction are entirely true in the fusion gene.Associated biomolecule information science analysis tools such as ProtParam, the ProtScale that CDD program in the utilization NCBI server and http://expasy.org website provide, NPS are carried out forecast analysis to physicochemical property, functional domain, hydrophobicity, the secondary protein structure of fusion rotein.The result shows that IFN-CSP is a cationic protein, contain 184 amino acid, wherein ((Arg+Lys) content is respectively 25 and 27 to acidic amino acid residue for Asp+Glu) and alkaline amino acid residue, molecular weight is 21481.7 Da, theoretical iso-electric point is 8.33, instability index is 53.91, and fat-soluble index is 81.63, and GRAVY index (amphipathic index) is-0.537.Has typical IFN superfamily conserved domain simultaneously with this albumen of CDD database analysis on the NCBI.Carry out the hydrophilic/hydrophobic analysis with ProtScale and show that this proteic hydrophilic amino acid quantity is greater than hydrophobic amino acid.NPS analyzes and shows that this albumen secondary structure is the primary structure element with alpha-helix, random coil and β-corner.

The prokaryotic expression plasmid of further gene fusion construct carries out respectively recombinant plasmid I FN-CSP/pMD20-T and the prokaryotic expression carrier pET-32a that successfully constructs on the basis of fusion gene of successfully having recombinated KpnI and HindThe III double digestion also connects with T4 DNA ligase enzyme.Shaking bacterium extraction plasmid through the positive clone of bacterium colony PCR preliminary evaluation carries out KpnI/ HindThe III double digestion identifies that the result shows the recombinant plasmid warp KpnI/ HindThe III double digestion goes out and expection band of the same size (Fig. 3), and through the dna sequencing analysis, no base mispairing determines that finally recombinant expression plasmid IFN-CSP/pET32a successfully constructs.

The reorganization prokaryotic expression plasmid that successfully constructs is transformed the host bacterium E. coliBL21 (DE3), utilize isopropyl-(Isopropyl-β-D-thiogalactopyranoside, IPTG) abduction delivering, adopt sodium lauryl sulphate-poly amic acid gel electrophoresis (Sodium Dodecyl Sulfate-Polyacrylamine gel electrophoresis, SDS-PAGE), western blotting (Western Blot) method is carried out Analysis and Identification to expression product, the result shows that expressed proteins is target protein Trx-6His-IFN-CSP, shows expressing fusion protein success (referring to accompanying drawing 4).Respectively the conventional expression condition that influences exogenous gene expression (inducing temperature, induce thalline initial concentration, inductor concentration, induction time) is optimized, finds that IFN-CSP/pET32a recombinant plasmid optimum expression condition in e. coli bl21 (DE3) host bacterium is: inducing temperature is that 37 ℃, starter bacteria concentration are that OD600 is about 0.6, the final concentration of inductor IPTG is that 0.8 mM, induction time are 5 hours.Because the target protein of abduction delivering has 6 * His label, can combine and hang on the pillar with 6 * His ligand specificity on the HisTrap HP affinity column, other foreign proteins can not combine and penetrate with affinity column, thereby reach effect of separating purification.Again respectively with phosphate buffered saline buffer (PBS) the wash-out target protein that contains 100 mM, 300 mM, 500 mM imidazoles, the Fractional Collections elutriant, the SDS-PAGE analytical results shows, contain fusion rotein hardly in the PBS elutriant of 100 mM imidazoles, contain a large amount of fusion roteins in the PBS elutriant of 300 mM imidazoles, contain a spot of fusion rotein in the PBS elutriant of 500 mM imidazoles.Purified target protein cuts the label protein except that Trx-6His with the enteropeptidase enzyme, remove enteropeptidase with EKapture Agarose post affinity capture from reaction mixture, enzyme is cut mixture and is obtained fusion rotein IFN-CSP(referring to accompanying drawing 4 again after HisTrap HP post is further purified).

CSP-IFN and normal mouse liver, the heart, spleen, lung and the nephridial tissue section of different concns are hatched altogether, with Anti-IFN-HRP is that antibody carries out the immunohistochemical methods test, and the result shows that recombination fusion protein IFN-CSP can combine specifically with the normal mouse liver cell.With the HepG2.2.15 cell is HBV cells infected model, adding the nutrient solution that contains high, medium and low different concns CSP-IFN respectively cultivates, ELISA detects HBsAg, the HBeAg concentration in the cell conditioned medium liquid, and the result shows that CSP-IFN can significantly suppress the secretion of HBsAg, HBeAg.

Compared with prior art, the present invention has following beneficial effect:

Liver target peptide CSP I-plus and IFNa2b that the present invention will derive from plasmodium circumsporozoite protein in resisting first merge, utilize CSP I-plus efficient special liver cell targeted with the IFNa2b liver that leads, make it in the liver enrichment, thereby improved the specificity of interferon therapy chronic HBV infection, reduce the whole body consumption, reduce toxic side effects, improve amount effect ratio.The present invention has creatively explored one and has utilized parasitic some molecule to treat the new way of human diseases, targeted therapy mode with present Interferon, rabbit compares as utilizing researchs such as liposome, mediated monoclonal antibody and asialoglycoprotein receptor mediation simultaneously, have the accurate active target that can reach cell levels and be difficult for by characteristics such as body immune system removings, thereby have extraordinary application prospect, will bring certain economic benefits and social benefit to society.Specifically beneficial effect of the present invention is summarized as follows:

1. adopt the new fusion gene of SOE-PCR technique construction first, in suitable expression system, express obtaining fusion rotein.Fusion rotein of the present invention combines the performance of two kinds of albumen/peptides that merged, utilize CSP I-plus efficient special liver cell targeted with the IFNa2b liver that leads, improved the specificity of interferon therapy chronic HBV infection, reduce the dosage of interferon therapy hepatic diseases, reduce its toxic side effect and resistance, open up new road for developing clinical novel hepatic targeting drug.

2. the present invention peptide section and people IFNa2b that creatively will derive from plasmodium falciparum CSP merges, and provides new thinking for utilizing parasitic some molecule to treat human diseases.

3. provide construction of fusion protein concrete, complete technical scheme.The present invention is according to intestinal bacteria preference codon, to liver target peptide and interferon gene modification and codon optimized, not only improved the expression efficiency of fusion rotein in intestinal bacteria, can also avoid the restriction of partial monopoly, compare with traditional polypeptide synthesis method and from the method for natural resource extraction polypeptide, preparation method of the present invention has broken through the confinement of only extracting or obtain with the amino acid synthetic method in the past polypeptide from natural resource.Simultaneously the experimental installation of needed substratum and fermentation usefulness all has cheap and advantage easy handling, makes in enormous quantities, scale production cheaply becomes possibility, has good application prospect.

4. fusion rotein of the present invention is a pharmaceutical grade protein, can be eliminated by modes of action such as degraded, drainage and receptor-mediated endocytosis, thereby can not accumulate in vivo, and human body is had no side effect.

Description of drawings

Fig. 1 is fusion gene reorganization synoptic diagram;

Fig. 2 is the reorganization of fusion gene IFN-CSP.Wherein, M is dna molecular amount standard (DL 2000); 1-4 is a first step pcr amplification product; 5 total length fusion gene IFN-CSP for reorganization;

Fig. 3 identifies for bacterium colony PCR and the double digestion of reorganization prokaryotic expression plasmid IFN-CSP/pET32a.Wherein, M is that (DL 2000 for dna molecular amount standard; DL10000); 1 is the PCR evaluation of recombinant plasmid IFN-CSP/pET-32a; 2 are Kpn I, HindIII double digestion recombinant plasmid I FN-CSP/pET-32a;

Fig. 4 is that SDS-PAGE and the Western-blotting of fusion rotein IFN-CSP identifies.A figure is that expression, purifying, the enzyme of target protein Trx-6His-IFN-CSP cut the acquisition with fusion rotein IFN-CSP.Wherein, M is the protein molecule quality standard; The 1-2 representative E. coliBL21/pET32a-IFN-CSP induces preceding and induces the back whole bacterial protein; 3 representatives E. coliInclusion body after the BL21/pET32a-IFN-CSP carrying out ultrasonic bacteria breaking; 4 represent target protein Trx-6His-IFN-CSP behind the purifying; On behalf of target protein Trx-6His-IFN-CSP enzyme, 5 cut the back mixture; On behalf of the Trx-6His-IFN-CSP enzyme, 6 cut the IFN-CSP albumen of back purifying; On behalf of the Trx-6His-IFN-CSP enzyme, 7 cut the Trx label of back in the elutriant; B figure is that the Western-blotting of target protein Trx-6His-IFN-CSP identifies.Wherein, M is the protein molecule quality standard; 1-2 is respectively E. coliBL21/pET32a-IFN-CSP induces preceding and induces the back whole bacterial protein.

Embodiment

Further explain the present invention below in conjunction with embodiment, but embodiment does not do any type of qualification to the present invention.The experimental technique of unreceipted actual conditions among the embodiment, can be with reference to this area ordinary method condition, method and condition described in " the molecular cloning experiment guide " write as J. Sa nurse Brooker (Sambrook) etc., or carry out according to the condition that the manufacturer that manufactures a product advises.What the present invention adopted is conventional commercial carrier and expressive host bacterium, and this area can substitute other prokaryotic vectors of use and not give unnecessary details in an embodiment one by one, but can not therefore limit the scope of the invention.

The transformation of embodiment 1 gene, the reorganization of fusion gene IFN-CSP and the structure of expression plasmid thereof

Present embodiment utilizes the pET expression system of Novagen company to express fusion gene IFN-CSP, is expression vector with pET32a (+) plasmid, and host cell is intestinal bacteria.

The transformation of 1 gene and fusion gene IFN-CSP reorganization (Fig. 1-2)

The aminoacid sequence of the people IFNa2b (NP_000596) that announces according to NCBI and the aminoacid sequence of plasmodium falciparum CSP I-plus (CAA33421), adopt the codon of intestinal bacteria preference, by the nucleotide sequence of computer aided design (CAD) liver targeted interferon IFN-CSP gene.Whole gene is divided into 16 overlapped oligonucleotide segments, and with Primer Premier 5.0 biosoftwares design primer P1-P16, primer sequence is shown in SEQ ID NO:3 ~ 19.Its middle and upper reaches P1 introduces KpnI restriction enzyme site, downstream P16 introduce terminator codon and Hind IIIRestriction enzyme site.Adopt improvement SOE-PCR technique construction to have the fusion gene of nucleotide sequence shown in SEQ ID NO.1, concrete grammar is as follows:

(1) the first step pcr amplified fragment IC 1., IC 2., IC 3., IC 4.

1. with amplified fragments IC is example, and all the other are similar.In 0.2 ml Eppendorf pipe, add the following table ingredients listed item by item:

5×PrimeSTAR Buffer（Mg2+ plus）	10.0μl
		dNTP Mixture (2.5mmol/L each)	4.0μl
Sterile H2O	27.5μl
		P1 Primer (10 μM/L)	2.0μl
P2 Primer (10 μM/L)	2.0μl
		P3 Primer (10 μM/L)	2.0μl
P4 Primer (10 μM/L)	2.0μl
		PrimeSTAR HS DNA Polymerase (2.5units/μl)	0. 5μl
Total volume	50.0μl

The Eppendorf pipe is inserted in the DNA amplification instrument, and reaction conditions is: 98 ℃ of 10 sec, 68 ℃ of 30 sec circulates 10 times.Adopt the TIANgel Midi Purification Kit purifying PCR product of Beijing TIANGEN company.

(2) SOE-PCR for the second time, by the three-step approach gene fusion construct, concrete grammar is as follows: the first step with PCR reclaim product IC 1., IC 2., IC 3., 4. IC be template, since IC 1., IC 2., IC 3., 4. IC all have the chain of overlapping between the adjacent segment, thereby the extension by overlapping chain in amplified reaction is stitched together four fragment genes, the antigen-4 fusion protein gene of synthetic total length.Second step increased in a large number with primer P1 and P16 again.The 3rd step was that fusion gene adds Poly A tail with TaKaRa Taq polysaccharase, was beneficial to next step TA clone.PCR reaction system and condition are as follows:

In 0.2 ml Eppendorf pipe, add the following table ingredients listed item by item:

5×PrimeSTAR Buffer（Mg2+ plus）	10.0μl
		dNTP Mixture (2.5mmol/L each)	4.0μl
Sterile H2O	25.5μl
		IC①	2.0μl
IC②	2.0μl
		IC③	2.0μl
IC④	2.0μl
		PrimeSTAR HS DNA Polymerase (2.5units/μl)	0. 5μl
Total volume	48.0μl

The Eppendorf pipe is inserted in the DNA amplification instrument, and reaction conditions is: 98 ℃ of 10 sec, 68 ℃ of 40 sec circulates 10 times.Taking out the Eppendorf pipe adds

P1 Primer (10 μM/L)	1.0μl
		P16 Primer (10 μM/L)	1.0μl

The Eppendorf pipe is inserted in the DNA amplification instrument, and reaction conditions is: 98 ℃ of 10 sec, 68 ℃ of 40 sec circulates 30 times.Taking out the Eppendorf pipe adds

TaKaRa Taq (5units/μl)

0. 25μl

The Eppendorf pipe is inserted in the DNA amplification instrument, and 72 ℃ are extended 20min.Reclaim purified pcr product with TIANgel Midi Purification Kit.The purpose fragment cloning is gone into pMD 20-T carrier, obtain recombinant plasmid I FN-CSP/pMD 20-T, difference transformed into escherichia coli DH5 α, after the blue hickie screening of process, bacterium liquid PCR evaluation, enzyme are cut evaluation and dna sequencing analysis, show that to obtain in the fusion gene IFNa2b and CSP I-plus sequence, the order that splices and combines and direction entirely true, illustrate that fusion gene recombinates successfully.

The structure of 2 integrative gene expression vectors (Fig. 3)

Recombinant plasmid I FN-CSP/pMD 20-T and expression vector pET32a are used restriction enzyme respectively KpnI and HindIII is carried out double digestion, connects with the T4 ligase enzyme then and spends the night, and connects product and transforms E.coliBL21 transforms bacterial classification and is applied to the flat board that contains penbritin, cultivate 16 ~ 18h after, carry out bacterium liquid PCR after picking list bacterium colony is cultivated, enzyme is cut and identified and the dna sequencing checking that the result shows that fusion gene expression plasmid IFN-CSP/pET32a successfully constructs.

The preparation (Fig. 4) of embodiment 2 fusion rotein IFN-CSP

The IFN-CSP/pET32a plasmid is transformed the expressive host bacterium that has prepared E.coliBL21(DE3) competent cell, through ammonia benzyl resistance screening, the positive single bacterium colony of picking IFN-CSP/pET32a (peptone of 1wt% in the 5ml LB liquid nutrient medium, the yeast extracting powder of 0.5wt%, the sodium-chlor of 85 mmol/L), 37 ℃, 220rpm jolting 8 ~ 12h are inoculated into this bacterium liquid in the fresh LB liquid nutrient medium in the 1:100 ratio then, 37 ℃, 220rpm jolting to OD600 about 0.6 o'clock, the IPTG that adds final concentration and be 1.0mmol/L induces 4 ~ 6h.Centrifugal collection mycetocyte adds 1 * SDS-PAGE Buffer(50mM Tris-HCl pH6.8,100mM DTT, 2%SDS, 0.1wt% tetrabromophenol sulfonphthalein, 10wt% glycerine), boil 5min, centrifugal, get the supernatant application of sample.Make the concentrated glue of 6 volume %, the separation gel of 15 volume %, electrophoretic voltage: concentrate glue 80V, separation gel 120V.After electrophoresis finishes, gel carries out coomassie brilliant blue staining, decolouring, scanning analysis, and because of fusion rotein contains 184 amino acid, theoretical molecular is about 21.48KD, and carrier pET32a has the Trx label protein, and the target protein that molecular weight is about 17kDa so expression should be about 39kDa.SDS-PAGE result shows: induce the back thalline to have obvious band of expression to conform to expection, target protein mainly is that the form with inclusion body is present in the ultrasonic precipitation of splitting behind the bacterium.Because target protein contains 6 * His label of pET32a coding, adopting mouse source His monoclonal antibody is an anti-Western Blotting that carries out, the result show after inducing contain that the expression of recombinant plasmid bacterial strain has a specific band at the 39kDa place and not inductive do not have, confirmed that further expressed proteins is the target protein that has the His label.Respectively the conventional expression condition that influences exogenous gene expression (inducing temperature, induce thalline initial concentration, inductor concentration, induction time) is optimized, to improve the expression level of target protein.Transformed bacteria is inoculated in the LB substratum that contains 100 μ g/ml penbritins, and 37 ℃ of shaking culture are spent the night.Next day, according to the ratio of volume ratio 1:100 incubated overnight bacterium liquid is joined shaking culture in the 250 ml triangular flasks of LB substratum that 50 ml contain 100 μ g/ml penbritins is housed, grew to OD600 at about 0.2,0.4,0.6,0.8,1.0,2.0 o'clock at cell concentration respectively, adding inductor IPTG respectively is that 0.2,0.4,0.6,0.8,1.0,1.2 mmol are placed under 37 ℃, 34 ℃, 31 ℃ and 28 ℃ of culture temperature and carry out abduction delivering to final concentration, and respectively at inducing back 2 h, 4 h, 6 h, 8 h, 10 h, 12 h to receive bacterium.Expression product is through 15% SDS-PAGE electrophoresis detection, and the result shows that IFN-CSP/pET32a recombinant plasmid optimum expression condition in e. coli bl21 (DE3) host bacterium is: inducing temperature is that 37 ℃, starter bacteria concentration are that OD600 is about 0.6, the final concentration of inductor IPTG is that 0.8 mM, induction time are 6 hours.

By best abduction delivering condition abduction delivering gene engineering recombinant bacterium 1000ml, the thalline of centrifugal collection abduction delivering carries out carrying out ultrasonic bacteria breaking, 4 ℃ of 14 centrifugal 30min of 000rpm, collecting precipitation.With solubilization of inclusion bodies liquid (50mmoL/L Tris – HCl buffer, 8 mmoL/L urea, PH8.0) dissolving inclusion body precipitation.Because the target protein of abduction delivering has 6 * His label, can combine thereby can utilize the HisTrap HP of GE healthcare and protein purification system to carry out affinitive layer purification with 6 * His ligand specificity.With sex change liquid centrifugal after the gained supernatant cross the HisTrap HP purification column of pre-equilibration, carry out linear elution afterwards with nonspecific proteins on the Binding buffer flush away pillar, the miaow concentration range is 0 ~ 500mM, the wash-out cumulative volume is 10 times of post bed.The purity of analyzing purified product Trx-6His-Mdc-hly through the gel scanning analysis system is about 95%.Carry out the gradient dialysis and progressively remove denaturing agent under 4 ℃, the recombinant protein that obtains is cut 16 h with enzyme under the reorganization Enterokinase room temperature.Protein mixed solution after enzyme is cut is removed residual enteropeptidase with Enterokinase Capture Kit, and filtrate is removed N end Trx label after HisTrap HP post, collects and penetrates liquid, concentrates with U-Tube concentrator ultrafiltration desalination, and-80 ℃ of preservations are standby.

The external pharmacodynamics of embodiment 3 fusion rotein IFN-CSP of the present invention and to the selectively targeted effect of liver cell research

(1) the external pharmacodynamic study of IFN-CSP: with the HepG2.2.15 cell is HBV cells infected model, select for use the nutrient solution that contains different CSP-IFN concentration gradients to cultivate HepG 2.2.15 cell, changed the nutrient solution that contains medicine, and collected the 3rd, 6,9 day supernatant liquor respectively in per 3 days.HBsAg in the cell conditioned medium liquid, HBeAg adopt the ELISA method to detect, and measure its A value with microplate reader, and conversion draws sample concentration according to standard substance A value.The result shows that CSP-IFN can significantly suppress the secretion of HBsAg, HBeAg.

(2) IFN-CSP is to the selectively targeted effect of liver cell: get paraffin-embedded mouse normal hepatocytes, kidney, the heart, lung, spleen tissue respectively, and after the conventional dewaxing aquation, the microwave antigen retrieval; 3% superoxol incubated at room, 10 min, PBS washes 3 times, adds 37 ℃ of sealings of normal sheep serum l h, and the recombination fusion protein IFN-CSP that adds the different concns gradient puts wet 4 ℃ in box spend the night (blank PBS); Add Anti-IFN-HRP (the 2% MPBS dilution of 1:500 dilution, the PBS that contains 2% skim-milk) puts wet box and hatch l h for 37 ℃, add DAB colour developing liquid chamber temperature colour developing 5-10 min after the PBS washing, 1% Hematorylin is redyed, transparent and the resinene mounting of dehydration back dimethylbenzene, all tissue slicies carry out HE dyeing simultaneously.On SmartScape 2002 biomicroscope image analysis systems, carry out image analysis.The result shows that recombination fusion protein CSP-IFN can combine specifically with the mouse normal liver cell.Wherein the brown yellow granule of normal liver cell surface generation is big, is to be dispersed in distribution; And be the tiny brown yellow granule that point-like is dispersed in distribution on a small quantity in that lung, spleen, kidney, the heart are rarely seen.

SEQUENCE?LISTING

 

＜110〉Guangdong Pharmaceutical University

 

＜120〉a kind of liver target peptide and human interferon a2b fusion rotein and its production and application

 

<130>

 

<160> 18

 

<170> PatentIn?version?3.2

 

<210> 1

<211> 552

<212> DNA

＜213〉fusion rotein IFN-CSP nucleotide sequence

 

<400> 1

tgtgatctgc?ctcaaaccca?cagcctgggt?agccgtcgta?ccttgatgct?cctggcacag 60

 

atgcgtcgta?tctctctttt?ctcctgcttg?aaggaccgtc?atgactttgg?atttccacag 120

 

gaggagtttg?gcaaccagtt?ccaaaaggct?gaaaccatcc?ctgtcctcca?tgagatgatc 180

 

cagcagatct?tcaatctctt?cagcacaaag?gactcatctg?ctgcttggga?tgagaccctc 240

 

cttgacaaat?tctacactga?actctaccag?cagctgaatg?acctggaagc?ctgtgtgatt 300

 

cagggggtgg?gggtgacaga?gactccactg?atgaaggagg?actccattct?ggctgtgcgt 360

 

aaatacttcc?aacgtatcac?tctctatctg?aaagagaaga?aatacagccc?ttgtgcctgg 420

 

gaggttgtcc?gtgcagaaat?catgcgttct?ttttctttgt?caacaaactt?gcaagaaagt 480

 

ttacgtagta?aggaagacaa?cgagaaatta?cgtaaaccaa?aacataaaaa?attaaagcaa 540

 

ccagcggatg?gt 552

 

 

<210> 2

<211> 184

<212> PRT

＜213〉fusion rotein IFN-CSP aminoacid sequence

 

<400> 2

 

CDLPQTHSLG?SRRTLMLLAQ?MRRISLFSCL?KDRHDFGFPQ?EEFGNQFQKA?ETIPVLHEMI 60

QQIFNLFSTK?DSSAAWDETL?LDKFYTELYQ?QLNDLEACVI?QGVGVTETPL?MKEDSILAVR 120

KYFQRITLYL?KEKKYSPCAW?EVVRAEIMRS?FSLSTNLQES?LRSKEDNEKL?RKPKHKKLKQ 180

PADG 184

 

 

<210> 3

<211> 29

<212> DNA

<213> P1

 

<400> 3

ggaattccat?atgtgtgatc?tgcctcaaa 29

 

 

<210> 4

<211> 58

<212> DNA

<213> P2

 

<400> 4

gtgccaggag?catcaaggta?cgacggctac?ccaggctgtg?ggtttgaggc?agatcaca 58

 

 

<210> 5

<211> 58

<212> DNA

<213> P3

 

<400> 5

accttgatgc?tcctggcaca?gatgcgtcgt?atctctcttt?tctcctgctt?gaaggacc 58

 

 

<210> 6

<211> 58

<212> DNA

<213> P4

 

<400> 6

ggttgccaaa?ctcctcctgt?ggaaatccaa?agtcatgacg?gtccttcaag?caggagaa 58

 

 

<210> 7

<211> 58

<212> DNA

<213> P5

 

<400> 7

caggaggagt?ttggcaacca?gttccaaaag?gctgaaacca?tccctgtcct?ccatgaga 58

 

 

<210> 8

<211> 58

<212> DNA

<213> P6

 

<400> 8

agtcctttgt?gctgaagaga?ttgaagatct?gctggatcat?ctcatggagg?acagggat 58

 

 

<210> 9

<211> 58

<212> DNA

<213> P7

 

<400> 9

ctcttcagca?caaaggactc?atctgctgct?tgggatgaga?ccctccttga?caaattct 58

 

 

<210> 10

<211> 58

<212> DNA

<213> P8

 

<400> 10

aggcttccag?gtcattcagc?tgctggtaga?gttcagtgta?gaatttgtca?aggagggt 58

 

 

<210> 11

<211> 58

<212> DNA

<213> P9

 

<400> 11

ctgaatgacc?tggaagcctg?tgtgattcag?ggggtggggg?tgacagagac?tccactga 58

 

 

<210> 12

<211> 58

<212> DNA

<213> P10

 

<400> 12

ggaagtattt?acgcacagcc?agaatggagt?cctccttcat?cagtggagtc?tctgtcac 58

 

 

<210> 13

<211> 58

<212> DNA

<213> P11

 

<400> 13

gctgtgcgta?aatacttcca?acgtatcact?ctctatctga?aagagaagaa?atacagcc 58

 

 

<210> 14

<211> 58

<212> DNA

<213> P12

 

<400> 14

aacgcatgat?ttctgcacgg?acaacctccc?aggcacaagg?gctgtatttc?ttctcttt 58

 

 

<210> 15

<211> 58

<212> DNA

<213> P13

 

<400> 15

cgtgcagaaa?tcatgcgttc?tttttctttg?tcaacaaact?tgcaagaaag?tttacgta 58

 

 

<210> 16

<211> 44

<212> DNA

<213> P14

 

<400> 16

cgtaatttct?cgttgtcttc?cttactacgt?aaactttctt?gcaa 44

 

 

<210> 17

<211> 55

<212> DNA

<213> P15

 

<400> 17

aagacaacga?gaaattacgt?aaaccaaaac?ataaaaaatt?aaagcaacca?gcgga 55

 

 

<210> 18

<211> 30

<212> DNA

<213> P16

 

<400> 18

ccgctcgaga?ccatccgctg?gttgctttaa 30

Claims (9)

1. liver target peptide and human interferon a2b fusion rotein is characterized in that its nucleotide sequence shown in SEQ ID NO:1, and this nucleotide sequence coded aminoacid sequence is shown in SEQ ID NO:2.

2. the preparation method of claim 1 described liver target peptide and human interferon a2b fusion rotein is characterized in that comprising the steps:

(1) dna sequence dna of liver target peptide and human interferon a2b gene is transformed, utilized SOE-PCR method gene fusion construct;

(2) make up recombinant expression plasmid;

(3), obtain the engineering bacteria of expressed fusion protein, fermentation culture with the recombinant plasmid transformed host cell;

(4) purifies and separates obtains liver target peptide and human interferon a2b fusion rotein.

3. according to the preparation method of claim 2 described liver target peptide and human interferon a2b fusion rotein, it is characterized in that gene fusion construct is according to the aminoacid sequence of human interferon a2b and the aminoacid sequence of plasmodium falciparum liver target peptide in the step (1), adopt the codon of intestinal bacteria preference, nucleotide sequence by computer aided design (CAD) liver targeted interferon IFN-CSP gene, whole gene is divided into 16 overlapped oligonucleotide segments, adopts the overlapping extension PCR method of improvement to make up fusion gene with nucleotide sequence shown in SEQ ID NO.1.

4. according to the preparation method of claim 3 described liver target peptide and human interferon a2b fusion rotein, it is characterized in that whole gene is divided into 16 overlapped oligonucleotide sheets has no progeny, primer sequence used during PCR is shown in SED ID NO:3 ~ 19.

5. according to the preparation method of claim 2 described liver target peptide and human interferon a2b fusion rotein, it is characterized in that step (3) described when recombinant expressed used expression system be prokaryotic expression system.

6. according to the preparation method of claim 2 described liver target peptide and human interferon a2b fusion rotein, it is characterized in that the expressive host bacterium is intestinal bacteria described in the step (3).

7. a recombinant vectors is characterized in that described recombinant vectors contains the nucleotide sequence of claim 1 described liver target peptide and human interferon a2b fusion rotein.

8. a host cell is characterized in that described host cell contains the described recombinant vectors of claim 7.

9. claim 1 described liver target peptide and the human interferon a2b fusion rotein application in the medicine of preparation anti-hepatitis B virus infective.

Images