CN1580265A - T-20 fusion protein, and its preparing method and use - Google Patents

Abstract

The invention relates to the expression, cultivation and purification technique of T-20 interfusion albumen in variety of yeast strains. In the end, the pharmaceutical preparation as outcome, high purity and regrouped T-20 interfusion albumen, can be used to cure AIDS.

Description

T-20 fusion rotein, Preparation Method And The Use

Technical field

The present invention relates to T-20 fusion rotein, Preparation Method And The Use.Particularly, the present invention has realized the expression of T-20 fusion rotein in methanol yeast, and has set up the method for protein purification, and feasible production in enormous quantities, cheaply becomes possibility, for the clinical application of T-20 fusion rotein is laid a good foundation.

Background technology

HIV-1 invasion host cell has two important steps: acceptor identification and film merge.The HIV-1 at first special receptor protein of recognition of host cell surface also combines with it, complicated conformational change has taken place in virus and host cell surface then, cause the HIV-1 film to merge mutually, make the HIV-1 core particle enter in the protoplasma of host cell with the host cell plasma membrane.

Similar with other retrovirus, the membrane glycoprotein of HIV-1 is synthetic on the rough surfaced endoplasmic reticulum of host cell with the form of polyprotein precursor molecule.The env gene of coding membrane glycoprotein precursor is positioned at virus genomic later half district, and expression product is the polypeptide chain of one section 88kD.This polypeptide chain enters endoplasmic and by glycosylation, makes molecular weight reach 160kD in building-up process, thereby is called as gp160.Gp160 is anchored on the endoplasmic reticulum with single transmembrane peptides section, and forms tripolymer soon after synthetic.

In Golgi complex, the trimerical glycosyl of gp160 is modified to more complicated form, and each gp160 molecule is all cut off by the proteolytic ferment of host cell.Form two mature proteins that molecular weight is respectively 120kD and 41kD, be called gp120 and gp41, gp120 is enclosed in gp41 tripolymer surface, and mutually combining with non covalent bond forms the gp120/gp41 complex body.In addition, also may form disulfide linkage between gp120 and the gp41, make composite structure more firm.

Sophisticated gp120/gp41 tripolymer arrives the host cell plasma membrane by the big vesica of Golgi complex excretory, participates in the parcel to the HIV-1 virion.

The first step of HIV-1 invasion is that gp120 combines with the CD4 acceptor molecule of cell surface.The calmodulin binding domain CaM of gp120 and CD4 mainly is the C4 district, and C3 district and C5 district have also participated in and the combining of CD4.(Smith D H, Byrn R A, Marsters S A, et al.Science, 1987 such as Smith; 238:1704) find that also solubility CD4 (sCD4) also can combine with gp120, gp120 split away off from viromembrane, thus in and the infection of HIV-1.But owing to neutralization reaction can not take place in most of HIV-1 strains are, thereby sCD4 can not be as a kind of medicine.

The cell invasion of HIV-1 also needs the participation of co-receptor.Find that very early HIV-1 can not invade the mouse cell of having integrated people CD4.But after the CD4-cell and mouse CD4+ cytogamy with the people, HIV-1 then can enter hybrid cell.In addition, specific HIV-1 strain can only be infected a few class people CD4+ cells, but can not infect the people CD4+ cell of other types.The invasion that these experiments all illustrate HIV-1 also need the human cell surface other have tissue-specific cofactor.The interaction of co-receptor and gp120 is very complicated.It is generally acknowledged that gp120 has formed the binding site of co-receptor with after CD4 combines through conformational change.(Atchison R E, Gosling J, Monteclaro F S, et al.Science, 1996 such as Atchison; Analysis revealed 274:1924), the HIV-1 invasion needs gp120 and co-receptor to form a kind of structure of complexity, and never only is the combination of simple site.In a single day this structure forms, and gp120 and gp41 are broken away from, thereby has started next process of HIV-1 invasion host cell: film merges.

HIV-1 can not only make its film host cell membrane merge, and can mediate infected cell and CD4+ cell on every side merges, the formation synplasm.What participate in the formation of virus-cytogamy or synplasm directly is the film exterior lateral area of gp41.This zone is followed successively by from the N end melts film peptide (fusion peptide), spiral section 1 (H1), immunity ring district (IM), spiral section 2 (H2).1997, on Cell and Nature magazine, delivered the X ray measuring result of gp41 film outside main region (H1 and H2) crystalline structure respectively.In crystalline structure, three sections H1 spirals are parallel to central authorities and form core, around being centered around to three sections H2 spiral antiparallels, form the hexa-atomic bundle of spiral.This confirms that gp41 exists with tripolymer.

T-20 takes from the membranin gp41 film exterior domain C-terminal sequence of virus, has 36 amino acid, and it is the membrane fusion inhibitor of hiv virus.Clinical trial shows that independent or compound medication all has the obvious treatment effect to acquired immune deficiency syndrome (AIDS).491 volunteers from North America and Brazil participate in the clinical trial of T-20, and before this, on average everyone has used 12 kinds of anti-AIDS drugs, and their average virus load is 100,000/ μ l, and the T cell counting is 80/ μ l.In the experiment, everyone uses 3 to 5 kinds of pharmacological agenies.One group of medicine that only uses listing, one group is used the listing medicine to add T-20.After 6 months, use among the experimenter of T-20 37% virus load drop to detection less than level (being lower than 400/ μ l), and control group has only 16%.20% virus load drops to and is lower than 50/ μ l among the experimenter of use T-20, and control group has only 7%.The experimenter's of T-20 T cell average increment 76/ μ l, and control group has only increased by 32/ μ l.Other has 504 also to participate in clinical experiment from Europe and Australian volunteer, and before this, all experimenters once made and heal with medicine, and failed at last, and their average T cell count is 98/ μ l.After 6 months, use to add among the experimenter of T-20 treatment 28% virus drop to detection less than level (being lower than 400/ μ l), and control group has only 14%.The experimenter's of T-20 T cell count average increment 65/ μ l, and control group has only increased by 38/ μ l.

The mechanism that T-20 suppresses the film fusion also is not very clear.Has similarity according to gp41 and influenza virus fusion rotein hemagglutinin, someone has proposed " hair clip precursor intermediate theory " in 1993, be that HIV is when the film fusion takes place, H1 helix-coil structure is from three Yuans intrafascicular being drawn out of H2 spiral, and become unbound state, T-20 takes advantage of the occasion to be incorporated into H1, has stoped the formation of later hairpin structure and film to merge.But recent discovers, there is not the specific binding site of T-20 in H1, also has the T-20 land in the H2 zone; Simultaneously, T-20 also has membrane responsiveness, and the T-20 that the C-terminal octyl group is modified can better finish the film location, shows higher retarding effect, so the retarding effect of T-20 may be also relevant with membrane responsiveness.

Fc refers to comprise the molecule or the sequence of the non-antigen-binding portion thereof sequence of antibody, and preferred people source can be from any isoform, as IgG, IgA, IgM, IgE, IgD.Fc compares T-20 with the fusion rotein of T-20 and has higher stability, and the transformation period in vivo is longer.

And the methanol yeast expression system is the efficient expression system that grew up in recent years, than intestinal bacteria, mammalian cell and insect cell expression system,, the methanol yeast expression system is easy to advantages such as purifying, expression amount height, cost be lower but having good expression may command secreting, expressing, albumen.

We are cloned into T-20 and Fc gene in the expression vector simultaneously, transform methanol yeast, have screened the high expression level bacterial strain, have groped fermentation, purifying process then, have realized the scale operation of T-20 fusion rotein, for its clinical application is laid a good foundation.

Summary of the invention

An object of the present invention is to provide the fusion rotein of T-20 and Fc.

Another object of the present invention provides the production method for preparing the T-20 fusion rotein with methanol yeast expression T-20 fusion rotein and separation and purification.This method comprises:

(a) will the encode nucleotide sequence of fusion rotein of T-20 and Fc operationally is connected in intestinal bacteria, methanol yeast, CHO or insect expression vector, obtains T-20 fusion rotein recombinant expression vector;

(b) change the recombinant expression vector in the step (a) over to the host bacterium, screening obtains to express the engineering bacteria of T-20 fusion rotein;

(c) engineering bacteria in the condition bottom fermentation culturing step (b) that is fit to described expressing fusion protein;

(d) purifies and separates goes out described fusion rotein from the fermentation culture product of step (c).

This method comprises that also in described step (a) before, the T-20 gene that obtains with chemosynthesis is that template is carried out PCR, and obtains the Fc gene with the RT-PCR method.

Another object of the present invention provides the pharmaceutical preparation of the T-20 fusion rotein of acquisition.

The present invention also provides the said medicine preparation to be used to prevent and treat the purposes of acquired immune deficiency syndrome (AIDS).

T-20 fusion rotein described in the present invention refers to the albumen that T-20 or its variant, fragment and Fc or its variant, fragment merge.In a preferred embodiment, described T-20 fusion rotein is the R1-L-R2 form.In another preferred embodiment, T-20 fusion rotein of the present invention is the R1-R2 form.Wherein R1 is Fc albumen, its variant or fragment, and R2 is T-20 albumen, its variant or fragment, and L is a joint.Described T-20 albumen or its variant, fragment are selected from following:

(a) the T-20 sequence shown in the SEQ ID NO:1;

(b) aminoacid sequence X-Y, X wherein, Y is any residue that comprises position 1-36 shown in Figure 1, and X＜Y, particularly 1-36;

Described Fc albumen is selected from following:

(A) aminoacid sequence shown in the SEQ ID NO:2;

(B) aminoacid sequence of inferior part (A) has the different aminoacids that replaces or lack in following one or more positions, and numbering wherein adopts the numbering of SEQ ID NO:2:

I. one or more cysteine residues;

Ii. one or more tyrosine residuess;

Iii. disappearance or with the Serine of L-Ala the position of substitution 5;

Iv. disappearance or with the L-glutamic acid of glutamine the position of substitution 20;

V. disappearance or with the L-glutamic acid of L-Ala the position of substitution 130;

Vi. disappearance or with the Methionin of L-Ala the position of substitution 105;

Vii. the disappearance or the Methionin of the position of substitution 107;

Viii. lack the aminoacid sequence of N end 1-17 position;

Ix. replace or lack one or more residues to eliminate the Fc receptor binding site;

X. replace or lack one or more residues to eliminate complement Clq binding site;

Xi. the combination of inferior part i-x;

Described joint is selected from following:

(a) ala-(ala) n-ala, n can be the integer between 1-4;

(b) gly-(gly) n-gly, n can be the integer between 0-4;

(c)gly-pro-gly；

(d)gly-gly-pro-gly-gly；

(e)val；

(f)tyr-val；

(g) ser-gly-(gly) 6-gly; And

(h) any combination of inferior part (a)-(g).

The present invention also provides the nucleotide sequence of a kind of T-20 of coding and Fc fusion rotein, as everyone knows, one seed amino acid can have multiple codon coding, the present invention provides proteic nucleotide sequence of a kind of coding T-20 (SEQ ID NO:3) and the coding proteic nucleotide sequence of Fc (SEQ ID NO:4) respectively, but in certain embodiments, can suddenly change to the nucleic acid codon according to actual needs, particularly select the methanol yeast preference codon for use, but the aminoacid sequence of the T-20 fusion rotein that coding obtains is constant.

The methanol yeast of indication comprises that pichia pastoris phaff (pichia pastoris), pichiamathonolica etc. can utilize the yeast of methyl alcohol as sole carbon source among the present invention.The expression vector that uses among the present invention can be selected various known carriers for use, as business-like carrier: pHIL-D2, pPIC9, pPICZ α, pPIC3.5K etc.Change recombinant expression vector over to host cell by methods such as electricity conversion or protoplast transformation, utilize appropriate condition screening recon, can abduction delivering T-20 fusion rotein.

Among the present invention, engineering bacteria is cultivated at the appropriate condition bottom fermentation and be may further comprise the steps:

After the engineering strain activation of screening, insert preparation seed liquor in the nutrient solution (for example BMGY, YPD nutrient solution), be seeded in the fermentor tank after the overnight incubation, fermentation is basic medium (a for example basic salt culture medium) with substratum, fermentation condition is controlled to be pH4.0-8.0, preferred pH4.5-7.0, the best is pH5.0,28 ℃-37 ℃ of temperature, the best is 30 ℃, and dissolved oxygen is controlled at more than 30%.Cultivate and begin feed supplement after 4-24 hour, add glycerine or glucose etc., when thalline OD value reaches high-density (OD ₆₀₀Be 15-200), add inductor (as methyl alcohol) abduction delivering, control pH3.0-7.0, preferred pH3-4.0, the best is pH3.0, induces end in 12-84 hour.

Among the present invention, purifies and separates goes out the T-20 fusion rotein and comprises following main points from tunning:

Collect tunning, the T-20 fusion protein expression products is present in nutrient solution or the thalline; In thalline, then need break bacterium as expression product; Obtain product (comprising Protein A affinity column chromatography, ion column chromatography, oppositely chromatography, dialysis, ultrafiltration or gel-filtration etc.) through the multistep purifying; With SDS-PAGE and RP-HPLC check, obtain purity greater than 95% T-20 fusion rotein.

Prove conclusively, T-20 is safely and effectively in clinical application.Therefore, this fusion rotein is safe and is suitable for drug use.

The T-20 fusion rotein that adopts the inventive method to make demonstrates external anti-fusion-activity.The method that the present invention prepares the T-20 fusion rotein is simple, rate of recovery height, and injury protein activity not, strong operability.

Contain the pharmaceutical preparation that polypeptide that the inventive method obtains and conventional carrier and auxiliary agent are formed, the pharmaceutical preparation that comprises the polypeptide that only useful the inventive method obtained, or with the pharmaceutical preparation that the polypeptide that the inventive method was obtained is formed with traditional assistant agent and additive as activeconstituents, comprise injection, oral preparations, contain formulation and lung, air flue, nasal cavity administrated preparation and other parenterai administration compositions.

Injection contains active polypeptide of the present invention, preferably stores with aseptic freeze-dried thing.Before administration, mix with suitable isotonic solution, isotonic solution comprises the isotonic water system that traditional being suitable for injected or injected, wherein contain the common additive such as stablizer and the chaotropic agent that are useful on injection liquid, preferred physiological saline or the isotonic solution by the damping fluid furnishing.

Oral preparations contains active polypeptide of the present invention, comprises solids composition, liquid composition and spray composite by the perception method preparation.Said composition is to contain active polypeptide of the present invention to mix with at least a inert diluent commonly used.Said composition also can add other additives outside the inert diluent in reality generally speaking: as lubricant (for example Magnesium Stearate), suspension agent, disintegrating agent (as the glycolic acid cellulose calcium), stablizer (for example lactose), solubility promoter (for example L-glutamic acid, aspartic acid), sweeting agent, perfume compound and sanitas etc.

Contain formulation and lung, air flue, nasal cavity administrated preparation and other parenterai administration compositions and contain active polypeptide of the present invention, comprise that active polypeptide of the present invention and common additive such as inert diluent, stablizer, emulsifying agent, lubricant etc. mix, and with the spray of currently known methods preparation, liniment, suppository, ointment etc.

The T-20 fusion rotein can be used for prevention and treatment acquired immune deficiency syndrome (AIDS), as: use separately and same AZT, DDC, DDI, D4T, 3TC, Na Weila product (Nevirpine), Delavirdine (Delavirdine), forelock Wen Nawei (Efavinavor), Sequanavir, come Dun Nawei (Ritonavir), Yin Dinawei (Indinavir), nail (unit of length) Fen Nawei (Nelfinavir), An Pilenawei (Amprenavir) to wait the arbitrary combination use of compositions.

Description of drawings

Fig. 1 shows the structure synoptic diagram of recombinant plasmid 1 of the present invention.

Fig. 2 shows the expression of T-20 fusion rotein of the present invention in methanol yeast.

Fig. 3 shows the anti-fusion-activity of T-20 fusion rotein of the present invention.

Fig. 4 shows the structure synoptic diagram of recombinant plasmid 2 of the present invention.

Fig. 5 shows the synoptic diagram of recombinant plasmid 3 of the present invention." Ampicillin " expression ammonia benzyl resistant gene among the figure, " α factor " expression " alpha factor ".

Fig. 6 shows the synoptic diagram of recombinant plasmid 4 of the present invention.

Embodiment

Embodiment 1: expression vector is that the engineering bacteria of pPICZ α C makes up

This example utilizes the EasySelectTM Pichia expression system of Invitrogen company to express the T-20 fusion rotein.Utilize pPICZ α C to be expression vector, with T-20 gene and Fc gene clone behind the alpha factor signal peptide gene.

1.1 the structure of the clone of gene and recombinant plasmid 1 (Fig. 1)

Design and synthesize T-201, T-202, T-203, T-204, Fc1, Fc2 (SEQ ID NO:5,6,7,8,9,10) is with T-201, T-202, T-203, T-204 is a primer, pcr amplification T-20 gene order, and wherein 5 ' end and 3 ' end are introduced Kpn I and Xba I site respectively.With Fc1, Fc2 is primer, is that the template synthetic first chain cDNA is a template with extractive total RNA from peripheral blood lymphocyte, pcr amplification Fc gene, and 5 ' end and 3 ' end are introduced Cla I and Eco RI site respectively.The PCR condition is: 94 ℃ 1 minute, 52 ℃ 1 minute, 72 ℃ 1 minute 30 seconds, totally 35 take turns circulation.The PCR product reclaims rear clone to the pGEM-T carrier, obtains recombinant plasmid pGEM-T-T-20 and pGEM-T-Fc, is defined as the gene order of T-20 and Fc through order-checking.

Cut expression plasmid pPICZ α C with Cla I and Eco RI, be connected with Fc gene fragment after Cla I and Eco RI handle then, transformed into escherichia coli TOP10F ' (available from Invitrogen company), coating contains on the less salt LB flat board of 25ug/ml ZeocinTM.The screening transformant obtains recombinant plasmid, cuts recombinant plasmid with Kpn I and Xba I then, is connected with Fc gene fragment after Xba I handles with Kpn I, and transformed into escherichia coli TOP10F ' is coated with and contains 25ug/ml Zeocin ^TMLess salt LB flat board on, the screening transformant obtain recombinant plasmid 1.Identical through dna sequence analysis with implementation sequence.

1.2 recombinant plasmid 1 transforms host bacterium X-33

A large amount of extracting recombinant plasmids 1 with Sac I linearizing, with the dehydrated alcohol precipitation, dry after 70% washing with alcohol then, are dissolved in (concentration is about 1 μ g/ μ L) in an amount of TE solution.Electricity transforms the X-33 host bacterium for preparing then, is coated on the YPDS screening culture medium that contains 100ug/ml ZeocinTM, cultivates 3 days, and obtains yeast transformant for 30 ℃.

1.3 expression screening

From transformant, select 20 mono-clonals, be inoculated into respectively in the BMGY substratum of 25ml, 30 ℃ are cultured to OD600 to 5.0, and centrifugal collection thalline is suspended to abduction delivering in the BMMY substratum of 150ml, added 1.5ml methyl alcohol in per 24 hours, abduction delivering 72 hours, SDS-PAGE is carried out in sampling, sees that the obvious expression band is arranged about 27kd, be the T-20 fusion rotein, screening high expression level bacterial strain is engineering bacteria (Fig. 2).

1.4 anti-fusion-activity is measured

The T-20 fusion rotein has anti-fusion-activity, can reduce plasmodial formation, can detect its activity by measuring plasmodial formation amount.The Hela cell H1-J.C53 cell that HIV infects was cultivated 24 hours at DMEM, and EDTA handles cell makes its wash-out standby.Do not infect the H1-J.C53 cell and cultivate the different extent of dilution T-20 DMEM solution of adding after 24 hours in 96 orifice plates, ultimate density is that 0-100 μ M does not wait, and bathes altogether 20 minutes.Add the HIV cells infected after handling then, about 50 in every hole, negative control only adds substratum, and 37 ℃ in CO ₂Incubator was cultivated 18-24 hour.Fixed cell is used 0.5% violet staining, and three-dimensional dissecting microscope is counted synplasm, and synplasm is few more, and the anti-fusion faculty of expression T-20 is strong more.The synplasm quantity that does not add T-20 is 100%.Test-results shows that the T-20 fusion rotein has good anti-fusion faculty (Fig. 3).

Embodiment 2: expression vector is the structure of the engineering bacteria of pPIC9

This example uses the Invitrogen Pichia pastoris of company expression system to express the T-20 fusion rotein.

2.1 the structure of recombinant plasmid 2 (Fig. 4)

Design and synthesize primer T-205, T-206, Fc3, Fc4 (SEQ ID NO:11,12,13,14), introduce XhoI restriction enzyme site and yeast signal peptide cleavage site (KEX2 enzyme recognition site) among the primers F c3, the Fc4 primer is introduced Sna BI, and T-205 primer and T-206 introduce Sna BI and Not I site respectively.With pGEM-T-Fc is template, is primer with Fc3, Fc4, pcr amplification Fc gene, and electrophoresis reclaims gene fragment, cuts with XhoI and Sna BI enzyme and handles and reclaim.Cut the pPIC9 plasmid with XhoI and Sna BI enzyme simultaneously, electrophoresis reclaims the back and is connected with the Fc gene fragment that Sna BI handles with XhoI, and transformed into escherichia coli DH5 α (available from Invitrogen company) screens transformant and gets recombinant plasmid pPIC9-T-20 then.Being template then with pGEM-T-T-20, is primer with T-205, T-206, pcr amplification T-20 gene, and electrophoresis reclaims gene fragment, cuts with Sna BI and Not I enzyme and handles and reclaim.Cut the pPIC9-Fc plasmid with Sna BI and Not I enzyme simultaneously, electrophoresis reclaims back and Sna BI and is connected with T-20 gene fragment after the Not I processing, transformed into escherichia coli DH5 α is coated on the LB flat board of the penbritin that contains 100ug/ml then, and the screening transformant gets recombinant plasmid 2.Dna sequence analysis proof gene order is correct.

2.2 recombinant plasmid 2 transforms host bacterium GS115

A large amount of extracting recombinant plasmids 2 are used the SalI linearizing, with the dehydrated alcohol precipitation, dry after 70% washing with alcohol then, are dissolved in (concentration is about 1 μ g/ μ L) in an amount of TE solution.Electricity transforms the GS115 host bacterium for preparing then, is coated on the MD screening culture medium, cultivates 3 days, and obtains yeast transformant for 30 ℃.

2.3 expression screening

Select 20 mono-clonals to be inoculated into respectively the 3ml MGY substratum from the MD flat board, 30 ℃ of cultivations add 27ml MM substratum after 48 hours induces, and expresses 72 hours, and sampling is carried out SDS-PAGE and detected, and the bacterial strain of screening high expression level is an engineering strain.

Embodiment 3: expression vector is the structure of the engineering bacteria of pMET α A

This example uses the Invitrogen Pichia methonolica of company expression system to express the T-20 fusion rotein.

3.1 the structure of recombinant plasmid 3

With the recombinant plasmid 2 among Xho I and the Not I double digestion embodiment 2, reclaim T-20 and Fc antigen-4 fusion protein gene fragment, be connected with the pMET α A plasmid that Not I enzyme is cut processing with Xho I, transformed into escherichia coli DH5 α, be coated on the LB flat board of the penbritin that contains 100ug/ml, the screening transformant gets recombinant plasmid 3 (Fig. 5).

3.2 recombinant plasmid 3 transforms host bacterium PMAD11

A large amount of extracting recombinant plasmids 3 with Apa I linearizing, with the dehydrated alcohol precipitation, dry after 70% washing with alcohol then, are dissolved in (concentration is about 1 μ g/ μ L) in an amount of TE solution.Electricity transforms the PMAD11 host bacterium for preparing then, is coated on the MD screening culture medium, cultivates 3 days, and obtains yeast transformant for 30 ℃.

3.3 expression screening

Select the BMDY substratum that 20 mono-clonals are inoculated into 50ml respectively from the MD flat board, cultivate OD for 30 ℃ ₆₀₀Be 6.0, centrifugal collection thalline is re-suspended to abduction delivering in the BMMY substratum of 5ml, per methyl alcohol of adding 50ul in 24 hours, and abduction delivering 72 hours, sampling SDS-PAGE, screening high expression level bacterial strain is an engineering bacteria.

Embodiment 4: expression vector is the structure of the engineering bacteria of pGAPZ α A

This example is used the continuous expression carrier pGAPZ α vector expression T-20 fusion rotein of Invitrogen company.

4.1 the structure of recombinant plasmid 4

With the recombinant plasmid 2 among Xho I and the Not I double digestion embodiment 2, reclaim T-20 and Fc antigen-4 fusion protein gene fragment, be connected with the pGAPZ α A plasmid that Not I enzyme is cut processing with Xho I, transformed into escherichia coli TOP10F ', coating contains 25ug/ml Zeocin ^TMLess salt LB flat board on, the screening transformant obtain recombinant plasmid 4 (Fig. 6).

4.2 recombinant plasmid 1 transforms host bacterium X-33

A large amount of extracting recombinant plasmids 1 with Avr II linearizing, with the dehydrated alcohol precipitation, dry after 70% washing with alcohol then, are dissolved in (concentration is about 1 μ g/ μ L) in an amount of TE solution.Electricity transforms the X-33 host bacterium for preparing then, and coating contains 100ug/ml Zeocin ^TMThe YPDS screening culture medium on, cultivated 3 days, and obtained yeast transformant for 30 ℃.

4.3 expression screening

From transformant, select 20 mono-clonals, be inoculated into respectively in the YPD substratum of 50ml, 30 ℃ of culture expression 72 hours, sampling SDS-PAGE, screening high expression level bacterial strain is an engineering bacteria.

Embodiment 5: the fermentation of engineering bacteria

The engineering strain that obtains among the embodiment 1 that will deposit certainly is inoculated in the BMGY substratum 30 ℃ and cultivates after 24 hours, be inoculated in the fermentor tank as seed liquor, fermention medium is the solution that basic medium adds trace element, 30 ℃ of leavening temperatures, pH5.0, the control dissolved oxygen is not less than 30%, and thalli growth begins to add glycerine after 24 hours, simultaneously pH is lowered to 3.0.Thalline OD reaches at 200 o'clock begins to add the methanol induction expression.Initial methyl alcohol addition is controlled to be every liter per hour 1-2ml, increases every liter per hour subsequently gradually to 15-20ml, keeps oxygen dissolving value greater than 30% by regulating stirring velocity and air flow, feeds pure oxygen in case of necessity.Continuous induction is cultivated after 72 hours and is gathered in the crops nutrient solution, centrifugal collection supernatant, 4.0 ℃ of preservations.

Embodiment 6: the purifying of tunning

The fermented liquid that embodiment 5 obtains is regulated pH to 7.4～7.6 with phosphate buffered saline buffer, ultra-filtration membrane with MWCO20KD is concentrated into 1/5 of original volume, last sample is to Protein A Sepharose FF affinity column, after the target protein combination, with the monitoring of 280nm Ultraviolet Detector down, the phosphate buffered saline buffer with 0.1mol/L pH7.6 washes to baseline.With the acetate buffer wash-out target protein of 0.1mol/L pH3.5, collect elution peak, regulate about pH to 7.0 with phosphate buffered saline buffer.Sampling analysis, SDS-PAGE show that purity of protein is greater than 95%.

Sequence table

＜110〉Shanghai Fuchun Zhongnan Biotechnology Co., Ltd

＜120〉T-20 fusion rotein, Preparation Method And The Use

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＜213〉artificial sequence

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<222>(1)..(36)

＜223〉the proteic aminoacid sequence of T-20

<400>1

Tyr?Thr?Ser?Leu?Ile?His?Ser?Leu?Ile?Glu?Glu?Ser?Gln?Asn?Gln?Gln

1 5 10 15

Glu?Lys?Asn?Glu?Gln?Glu?Leu?Leu?Glu?Leu?Asp?Lys?Trp?Ala?Ser?Leu

20 25 30

Trp?Asn?Trp?Phe

35

<210>2

<211>232

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＜213〉artificial sequence

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＜223〉the proteic aminoacid sequence of Fc

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Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala

1 5 10 15

Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro

20 25 30

Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val

35 40 45

Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val

50 55 60

Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln

65 70 75 80

Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln

85 90 95

Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala

100 105 110

Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro

115 120 125

Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr

130 135 140

Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser

145 150 155 160

Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr

165 170 175

Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr

180 185 190

Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe

195 200 205

Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys

210 215 220

Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys

225 230

<210>3

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＜213〉artificial sequence

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＜223〉the proteic encoding sequence of T-20

<400>3

tacaccagcc?tgatccactc?cctgattgaa?gaatctcaga?accagcaaga?aaagaacgaa 60

atgtggtcgg?actaggtgag?ggactaactt?cttagagtct?tggtcgttct?tttcttgctt 120

caagaactgc?tggagctgga?taaatgggca?tctctgtgga?actggtttta?atgagttctt 180

gacgacctcg?acctatttac?ccgtagagac?accttgacca?aaattact 228

<210>4

<211>1398

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉the proteic encoding sequence of Fc

<400>4

gagcccaaat?cttgtgacaa?aactcacaca?tgcccaccgt?gcccagcacc?tgaactcctg 60

ctcgggttta?gaacactgtt?ttgagtgtgt?acgggtggca?cgggtcgtgg?acttgaggac 120

gggggaccgt?cagtcttcct?cttcccccca?aaacccaagg?acaccctcat?gatctcccgg 180

ccccctggca?gtcagaagga?gaaggggggt?tttgggttcc?tgtgggagta?ctagagggcc 240

acccctgagg?tcacatgcgt?ggtggtggac?gtgagccacg?aagaccctga?ggtcaagttc 300

tggggactcc?agtgtacgca?ccaccacctg?cactcggtgc?ttctgggact?ccagttcaag 360

aactggtacg?tggacggcgt?ggaggtgcat?aatgccaaga?caaagccgcg?ggaggagcag 420

ttgaccatgc?acctgccgca?cctccacgta?ttacggttct?gtttcggcgc?cctcctcgtc 480

tacaacagca?cgtaccgtgt?ggtcagcgtc?ctcaccgtcc?tgcaccagga?ctggctgaat 540

atgttgtcgt?gcatggcaca?ccagtcgcag?gagtggcagg?acgtggtcct?gaccgactta 600

ggcaaggagt?acaagtgcaa?ggtctccaac?aaagccctcc?cagcccccat?cgagaaaacc 660

ccgttcctca?tgttcacgtt?ccagaggttg?tttcgggagg?gtcgggggta?gctcttttgg 720

atctccaaag?ccaaagggca?gccccgagaa?ccacaggtgt?acaccctgcc?cccatcccgg 780

tagaggtttc?ggtttcccgt?cggggctctt?ggtgtccaca?tgtgggacgg?gggtagggcc 840

gatgagctga?ccaagaacca?ggtcagcctg?acctgcctgg?tcaaaggctt?ctatcccagc 900

ctactcgact?ggttcttggt?ccagtcggac?tggacggacc?agtttccgaa?gatagggtcg 960

gacatcgccg?tggagtggga?gagcaatggg?cagccggaga?acaactacaa?gaccacgcct 1020

ctgtagcggc?acctcaccct?ctcgttaccc?gtcggcctct?tgttgatgtt?ctggtgcgga 1080

cccgtgctgg?actccgacgg?ctccttcttc?ctctacagca?agctcaccgt?ggacaagagc 1140

gggcacgacc?tgaggctgcc?gaggaagaag?gagatgtcgt?tcgagtggca?cctgttctcg 1200

aggtggcagc?aggggaacgt?cttctcatgc?tccgtgatgc?atgaggctct?gcacaaccac 1260

tccaccgtcg?tccccttgca?gaagagtacg?aggcactacg?tactccgaga?cgtgttggtg 1320

tacacgcaga?agagcctctc?cctgtctccg?ggtaaatgaa?tgtgcgtctt?ctcggagagg 1380

gacagaggcc?catttact 1398

<210>5

<211>37

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>5

gatgaattct?acaccagcct?gatccactcc?ctgattg 37

<210>6

<211>60

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>6

gcctgatcca?ctccctgatt?gaagaatctc?agaaccagca?agaaaagaac?gaacaagaac 60

<210>7

<211>60

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>7

ccagttccac?agagatgccc?atttatccag?ctccagcagt?tcttgttcgt?tcttttcttg 60

<210>8

<211>36

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>8

cgctctagat?cattaaaacc?agttccacag?agatgc 36

<210>9

<211>47

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>9

gtatctctcg?agaagagagg?ctgaagcaga?gcccaaatct?tgtgaca 47

<210>10

<211>45

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>10

gatgaattct?ttacccggag?acagggagag?gctcttctgc?gtgta 45

<210>11

<211>29

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>11

tacgtataca?ccagcctgat?ccactccct 29

<210>12

<211>30

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>12

gcggccgctt?taaaaccagt?tccacagaga 30

<210>13

<211>47

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>13

tctctcgaga?aaagagaggc?tgaagctgag?cccaaatctt?ctgacaa 47

<210>14

<211>37

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>14

tacgtattta?cccggagaca?gggagaggct?cttctgc 37

Claims (10)

1. method that produces the T-20 fusion rotein, this method comprises:

(a) will the encode nucleotide sequence of fusion rotein of T-20 and Fc operationally is connected in intestinal bacteria, methanol yeast, CHO or insect expression vector, obtains T-20 fusion rotein recombinant expression vector;

(b) change the recombinant expression vector in the step (a) over to the host bacterium, screening obtains to express the engineering bacteria of T-20 fusion rotein;

(c) engineering bacteria in the condition bottom fermentation culturing step (b) that is fit to described expressing fusion protein;

(d) purifies and separates goes out described fusion rotein from the fermentation culture product of step (c).

2. the method for claim 1 is characterized in that, in described step (a) before, the T-20 gene that obtains with chemosynthesis is that template is carried out PCR, and obtains the Fc gene with the RT-PCR method.

3. method as claimed in claim 1 is characterized in that, described T-20 fusion rotein is R1-L-R2 and R1-R2 form, and wherein R1 is Fc albumen, its variant or fragment, and R2 is T-20 albumen, its variant or fragment, and L is a joint.

4. method as claimed in claim 3 is characterized in that, described T-20 albumen or its variant, fragment are selected from following:

(a) the T-20 sequence shown in the SEQ ID NO:1;

(b) aminoacid sequence X-Y, X wherein, Y is any residue that comprises position 1-36 shown in Figure 1, and X＜Y, particularly 1-36;

5. method as claimed in claim 3 is characterized in that, described Fc albumen is selected from following:

(A) aminoacid sequence shown in the SEQ ID NO:2;

(B) aminoacid sequence of inferior part (A) has the different aminoacids that replaces or lack in following one or more positions, and numbering wherein adopts the numbering of SEQ ID NO:2:

I. one or more cysteine residues;

Ii. one or more tyrosine residuess;

Iii. disappearance or with the Serine of L-Ala the position of substitution 5;

Iv. disappearance or with the L-glutamic acid of glutamine the position of substitution 20;

V. disappearance or with the L-glutamic acid of L-Ala the position of substitution 130;

Vi. disappearance or with the Methionin of L-Ala the position of substitution 105;

Vii. the disappearance or the Methionin of the position of substitution 107;

Viii. lack the aminoacid sequence of N end 1-17 position;

Ix. replace or lack one or more residues to eliminate the Fc receptor binding site;

X. replace or lack one or more residues to eliminate complement Clq binding site;

Xi. the combination of inferior part i-x.

6. method as claimed in claim 3 is characterized in that, described joint is selected from following:

(a) ala-(ala) n-ala, n can be the integer between 1-4;

(b) gly-(gly) n-gly, n can be the integer between 0-4;

(c)gly-pro-gly；

(d)gly-gly-pro-gly-gly；

(e)val；

(f)tyr-val；

(g)ser-gly-(gly)6-gly；

(h) any combination of inferior part (a)-(g).

7. a T-20 fusion rotein is characterized in that, it is R1-L-R2 and R1-R2 form, and wherein R1 is Fc albumen, its variant or fragment, and R2 is T-20 albumen, its variant or fragment, and L is a joint.

8. the nucleotide sequence of coding claim 7 described T-20 fusion rotein.

9. the pharmaceutical preparation that contains the described T-20 fusion rotein of claim 8.

10.T-20 the purposes of fusion rotein is characterized in that, is used to prepare the medicament of prevention or treatment acquired immune deficiency syndrome (AIDS).

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