CN1621411A - Recombinant targeted fusion protein for treating acquired immunodeficiency syndrome - Google Patents

Abstract

The present invention relates to gene engineering protein medicine, and is especially targeting HIV/SIV resisting protein and its coding fusion gene. The present invention also relates to CD4V1-TNF alpha m1 and CD4V1-TNF alpha m2 fusion gene, expression recombinant containing the fusion gene, engineering bacterium containing the expression recombinant, targeting CD4V1-TNF alpha m1 and CD4V1-TNF alpha m2 fusion protein expressed with the engineering bacterium and through separation and purification, and the AIDS treating use of the fusion protein.

Description

A kind of recombination targent fused protein that is used for the treatment of acquired immune deficiency syndrome (AIDS)

Technical field

The present invention relates to the engineered protein pharmaceutical field.More specifically, the present invention relates to the protein of the anti-HIV/SIV of target and its fusion gene of encoding.The invention still further relates to CD4V1-TNF α m1 and CD4V1-TNF α m2 fusion gene, comprise in the expression type recombinant chou of described fusion gene, the targent fused protein-TNF α m1 that comprises the engineering bacteria of described expression type recombinant chou, engineering bacterium expression and separation and purification thus and CD4V1-TNF α m2 and these fusion roteins any one be used for the treatment of the acquired immune deficiency syndrome (AIDS) purposes.

Background technology

Acquired immune deficiency syndrome (AIDS) (acquired immunodeficiency syndromeAIDS) is an acquired immune deficiency syndrome (AIDS), is a kind of disease of serious harm human health.AIDS is infected by human immune deficiency venereal disease poison (human immunodeficiency virus HIV) to cause.HIV mainly infects T cell, the monokaryon/huge biting and dendritic cell of expressing the CD4 molecule, and axoneure.According to the data of WHO2002, global HIV the infected surpasses 6,000 ten thousand, and wherein 1/3 has been dead.In 20 years from now on, estimate that death toll will reach 6,800 ten thousand.The first place that present global HIV/AIDS number has occupied various diseases, acquired immune deficiency syndrome (AIDS) have become the pestilence in the whole world.31 provinces accumulative totals of China in 2002 are reported HIV the infected about 890,000 examples, have reached 1,040,000 but the relevant expert estimates the actual the infected of China's AIDS, based on person between twenty and fifty, and wherein about 200,000 of death.If do not take effective measure of control, might reach 1,000 ten thousand to the infected's quantity in 2010.The infected's quantity can be controlled in 1,500,000 in 2010 if control is capable.China's AIDS is prevented and treated quite sternness of situation.

Although HAART (High Active Antiretrovirus Therapy high reactivity antiretroviral therapy) has obtained the achievement of attracting attention in developed country at present, but most of HIV the infecteds concentrate on the economically less developed region, the price limit of HAART costliness it in the application of these developing countries.Whole world HIV the infected accepts the less than 1,000,000 of antiretroviral therapy.Simultaneously there are some shortcomings that can't overcome in HAART itself, and causes the resistance of virus and viral hiding greatly, easily as the toxic side effect of HAART, and costs an arm and a leg.HAART can not thoroughly effect a radical cure acquired immune deficiency syndrome (AIDS).

Existing various treatment means comprises other various pharmacological agenies, and the research of gene therapy and therapeutic vaccine is not all achieved success.

The magnetic target therapy technology is called " biological missile " technology again, just more and more causes people's attention.A kind of mentality of designing of biological missile is monoclonal antibody and medicine, microbiotic or nucleic to be coupled at one be used from clinical treatment, and its subject matter is that target is poor; The mentality of designing of another kind of biological missile is that the utilization genetic engineering technique fusion rotein that different function section splicings is constructed is as " molecular guide " type guided missile, as artificial constructed fusion rotein TGF-PE40, IL2-PE40, CD4-PE40, CD4-TD (diphtheria toxin), be respectively applied for the treatment malignant tumour, some autoimmune disorder and acquired immune deficiency syndrome (AIDS).Clinical study is the result show, though this type of fusion rotein has good guide effect, but PE40, TD toxin protein confrontation human body are heterologous proteins, has very strong immunogenicity, produce special antibody at PE40 or TD rapidly in the inductor, antibody produces neutralizing effect lost efficacy fusion rotein.Therefore, above-mentioned these target fusion roteins none be applied to clinical treatment.

So be badly in need of low toxicity target medicine efficiently in treatment AIDS field.

Summary of the invention

In order to address the above problem, therefore, the invention provides a class fusion rotein that applies to anti-HIV/SIV, described fusion rotein is to comprise receptor polypeptides and the target fusion rotein that kills and wounds polypeptide of cytosis extremely that has that derives from human body.

The present invention relates to the cDNA of the receptor polypeptides of the gp120 of a kind of people HIV/SIV and/or gp41, its nucleotide sequence is shown in SEQ ID NO:1.The nucleotide sequence of described SEQ ID NO:1 is used for the guide effect of fusion rotein.

The invention further relates to the gp120 of a kind of people HIV/SIV and/or the receptor polypeptides of gp41, its aminoacid sequence is shown in SEQ ID NO:2.

The invention further relates to the cDNA of the varient of a kind of TNF-α, its nucleotide sequence is shown in SEQ ID NO:3.

The invention further relates to the varient of a kind of TNF-α, its amino acid is listed as shown in SEQ IDNO:4.

The invention further relates to the cDNA of the varient of a kind of TNF-α, its nucleotide sequence is shown in SEQ ID NO:5.

Described SEQ ID NO:3 and 5 nucleotide sequence are used for the killing action of fusion rotein.

The invention further relates to the varient of a kind of TNF-α, its amino acid is listed as shown in SEQ IDNO:6.

The invention further relates to a kind of targent fused protein, it is characterized in that described fusion rotein is to comprise and to have the target fusion rotein that kills and wounds polypeptide of cytosis extremely in conjunction with the receptor polypeptides of the gp120 of HIV/SIV and/or gp41.

Described receptor polypeptides is CD4 preferably, CD4V1V2 or CD4V1, perhaps their relevant segment, analogue or mutant; The described polypeptide that kills and wounds is the TNF family member who comprises TNF α, TNF β, TNF γ or derivatives thereof or its mutant; Or the TNF superfamily member, as Trail, Light or their active function zone, mutant or analogue; Or described polypeptide is pore-forming protein, complement component C9 and the particle dissolving element that derives from human body; Perhaps derive from the granzyme A of the granzyme family of human body, B, C, D, E, serine easterase; Or protein kinase C, protein kinase A, E.C. 2.7.2.4., cytopigment or DNA endonuclease; Maybe can resist polypeptide and the enzyme of bacterium, as N,O-Diacetylmuramidase, alexin; Or antioncogene and encoded protein matter thereof, as P53, and all derive from the gene with deadly cell function and coded protein or their active function zone, mutant or the analogue of human body.

Preferably, the encode gene order of described CD4V1 is the sequence shown in the SEQ ID NO:1.

Preferably, the aminoacid sequence of described CD4V1 genes encoding is the aminoacid sequence shown in the SEQ ID NO:2.

Preferably, describedly kill and wound the mutant TNF α m1 that polypeptide is TNF α, its cDNA sequence of encoding is the nucleotide sequence shown in the SEQ ID NO:3.

Preferably, the aminoacid sequence of described TNF α m1 genes encoding is the aminoacid sequence shown in the SEQ ID NO:4.

Preferably, describedly kill and wound the mutant TNF α m2 that polypeptide is TNF α, its cDNA sequence of encoding is the nucleotide sequence shown in the SEQ ID NO:5.

Preferably, the described polypeptide that kills and wounds is TNF α m2, and its aminoacid sequence is the aminoacid sequence shown in the SEQ IDNO:6.

The invention further relates to the recombinant chou pCW-CD4V1TNF α m1 of fusion gene of the cDNA of the cDNA that comprises SEQ ID NO:1 and SEQ ID NO:3.

The invention further relates to the recombinant chou pCW-CD4V1TNF α m2 of fusion gene of the cDNA of the cDNA that comprises SEQ ID NO:1 and SEQ ID NO:5.

The invention still further relates to the carrier that contains above-mentioned recombinant chou.

The invention still further relates to the eucaryon or the prokaryotic cell prokaryocyte that contain the structure plasmid vector that to express above-mentioned fusion rotein, be preferably eukaryotic cell.

The invention still further relates to the method for the above-mentioned fusion rotein of preparation, comprise: with described expression vector transformed host cell, cultivate transformant, obtain in conjunction with the receptor polypeptides of the gp120 of HIV/SIV and/or gp41 and to have the target fusion rotein that kills and wounds polypeptide of cytosis extremely comprising of reorganization.

The invention still further relates to a kind of pharmaceutical composition that contains above-mentioned fusion rotein.

The invention further relates to described fusion rotein and be used for the treatment of purposes in the medicine of acquired immune deficiency syndrome (AIDS) in preparation.

Preferably, its by kill be subjected to cell that HIV/SIV infects or in and free HIV/SIV finish.

The present invention also provides targent fused protein CD4V1-TNF α m1 and CD4V1-TNF α m2 respectively to being subjected to the HIV/SIV cells infected to have the specific killing effect and to the test cell line evidence of the neutralizing effect of free virus in the circulation of blood.

The present invention can both effectively reduce the toxicity of TNF α because target designs, and the lethal effect of TNF α can be concentrated on the cell that infected by HIV/SIV again, and the lethal effect of more effective performance fusion rotein is taken into account efficient and safety simultaneously.Warm albumen kill and wound be subjected to cell that HIV/SIV infects in, its targeted molecular CD4V1 can also in and free HIV/SIV, realize Normocellular protection.Therefore these two kinds of warm albumen all are bifunctional active polypeptide.And experimental design obtains test cell line result's support.Simultaneously, because CD4V1 and TNF α m have reduced immunogenicity to greatest extent all from human body.Moreover, between CD4V1 and TNF α m, also introduced and be rich in flexible amino acid whose one seven aminoacid sequence, can help CD4V1 and TNF α m to exercise its function with three-D space structure independently separately, and be not that heterologous protein produces immune response by immune system recognition as the human body intrinsic protein by immune system recognition separately, finally cause drug failure.

Description of drawings

Fig. 1 is the structure synoptic diagram of recombinant chou pCW CD4V1-TNF α m1.

Fig. 2 is the structure synoptic diagram of recombinant chou pCWCD4V1-TNF α m2.

Fig. 3 is recombinant chou pBS-TNF α m1, and the enzyme of pGEM-CD4V1-TNF α m1 and pCWCD4V1-TNF α m1 is cut the evaluation synoptic diagram.

Fig. 4 is warm PROTEIN C D4V1-TNF α m1 SDS-PAGE electrophorogram.Wherein

1: be inductive CD4V1-TNF α m1/DH5 α tropina not

2: be inductive CD4V1-TNF α m1/DH5 α tropina

3: be the inclusion body of CD4V1-TNF α m1/DH5 α tropina

4: for cutting the CD4V1-TNF α m1 protein sample behind the glue electroelution

Fig. 5 is the laser intensity scintigram of warm PROTEIN C D4V1-TNF α m1 SDS-PAGE glue.

Fig. 6 is the sequencing result of recombinant chou pBS-TNF α m1.

Fig. 7 is the upstream sequencing result of recombinant chou pCW-CD4V1TNF α m1.

Fig. 8 is the downstream sequencing result of recombinant chou pCW-CD4V1TNF α m1.

Fig. 9 is the sequencing result of recombinant chou pBS-TNF α m2.

Figure 10 is that fusion rotein CD4V1-TNF α m1 is to being infected the lethal effect of CEMx-174 cell by SIV.

Figure 11 is the effect of fusion rotein CD4V1-TNF α m1 to normal CEMx-174 cell.

Figure 12 is that fusion rotein CD4V1-TNF α m1 is to being infected the neutralizing effect of CEMx-174 cell by SIV.

Figure 13 is the effects (ordinate zou OD value for measure) of TNF α standard substance to infected the CEMx-174 cell by SIV.

Figure 14 is fusion rotein CD4V1-TNF α m1 influence to virus titer in fragmentation test.

Figure 15 is fusion rotein CD4V1-TNF α m1 influence to virus titer in neutralization test.

Figure 16-1,2 shows the fluorescence photo of the form of fragmentation test front and back cell, and wherein Figure 16-1 is a cell dyeing photo before the fusion rotein CD4V1-TNF α m1 fragmentation test; Figure 16-2 is a cell dyeing photo behind the fusion rotein fragmentation test, and magnification is 200X.

Figure 17-1,2 shows the fluorescence photo of the form of neutralization test front and back cell, and wherein Figure 17-1 is a cell dyeing photo before the fusion rotein CD4V1-TNF α m1 neutralization test; Figure 17-2 is a cell dyeing photo after the fusion rotein neutralization test, and magnification is 200X.

Specific embodiments

Fusion rotein is made up of targeted molecular and murderer two portions among the present invention, and the key of this target design is the specificity combination and murderer efficiently the killing and wounding target cell of realizing targeted molecular.Wherein targeted molecular is the CD4 or derivatives thereof of the gp120 specific combination on the cell that can infect with HIV or by HIV, and one is CD4V1; Murderer is tumor necrosis factor alpha (TNF α) or derivatives thereof, and wherein two kinds is TNF α m1 and TNF α m2.CD4 is the general deadman as gp120 identification target cell.Because the envelope protein gp120 that has only free HIV virus and just can be expressed virus specifically by the cell that HIV infects, thereby utilization CD4 or derivatives thereof can be realized as targeted molecular and be combined by the specificity of HIV cells infected.CD4-TNF α encoding gene and mutant thereof can be expressed the warm albumen of acquisition through eucaryon or prokaryotic system.

TNF α is a kind of monokine, is mainly produced by monocyte and scavenger cell.In addition, neutrophil leucocyte, stellate cell, endotheliocyte, smooth muscle cell also can produce TNF α.TNF α is found in 1975, is a kind of humanized's cytotoxic factor, its toxic action mainly be by with receptors bind and internalization, and then cause a series of signal transduction process, cause necrocytosis.TNF α causes two kinds of main cell responses, cell toxicant and transcriptional activation optionally.The effect of TNF α is Ca in NK and macrophage-mediated cell toxicant ²⁺Dependent/non-dependent.Cluster is assembled in TNF α tripolymer isoacceptor (mainly being P55) combination, the receptor complex internalization, and the TNF α receptor complex of internalization can transmit multiple signal.Possible mechanism is that the activation by the G albumen coupling activates Phospholipase A2 path, synthesis of prostaglandins and leukotriene.Simultaneously, the release of polyunsaturated fatty acids such as arachidonic acid and metabolism begin to produce free radical in mitochondrial electron transfer system multilated, finally pass through the oxidation and the dna degradation effect cell killing of some enzyme, fat.Information can cause the activation of transcription factor NF KappaB very soon on the other hand, and activatory NF KappaB enters nucleus, activates transcribing of series of genes.The anti-tumor activity of TNF α in known cytokine is the strongest.The kinds of tumor cells system that comprises neurofibroma, lung cancer, mammary cancer, myosarcoma and lymphoma etc. all had tangible lethal effect, so United States Government has just ratified to treat with TNF α the clinical trial application of malignant tumour as far back as 1985.But, consequently fail to become the conventional medicine of useful clinically treatment malignant tumour so far owing to the strong excessively toxic side effect of prototype TNF α.The design philosophy of our key is to think, the cell killing activity of TNF α is its basic function, from its mechanism of action, it accurately if can be imported the cell that infected by HIV/SIV, can kill the cell that is infected by HIV/SIV equally, thereby stop duplicating and breeding of HIV/SIV virus.Therefore, utilization DNA recombinant technology is built into the expression recombinant of expressing warm PROTEIN C D4V1-TNF α m1 and CD4V1-TNF α m2 with it, gives expression to warm albumen, purification renaturation then through eucaryon or prokaryotic expression system.Test cell line is the result show, two kinds of warm albumen can specially be killed the cell that is infected by HIV/SIV strongly, meanwhile, this warm proteic targeted molecular CD4V1 can also be efficiently in and free HIV/SIV, and make its inactivation.Therefore these two kinds of warm albumen all are bifunctional active polypeptide.And the normal cell that is infected by HIV/SIV is not after adding these two kinds of warm albumen, and its growth is unaffected, proves that warm albumen has good security.In contrast, prototype TNF α can not kill and wound the cell that infected by HIV/SIV, can not in and free HIV/SIV virus, further verified our design.Simultaneously, because CD4V1 and TNF α m have reduced immunogenicity to greatest extent all from human body.Therefore, two kinds of fusion roteins that the present invention makes up are used for anti-HIV/SIV good specificity and security, is a kind of potential drug that is rich in the treatment acquired immune deficiency syndrome (AIDS) of prospect.

In addition, about this fusion rotein whether immunogenicity is arranged, CD4V1 and TNF α say in principle all for people's source protein, have reduced immunogenicity as much as possible.Thereby, fusion rotein causes serious immunogenicity in view of may changing its space three-dimensional structure.These misgivings, with regard to this fusion rotein, situation may be different: the CD4V1 in this fusion rotein and TNF α m general more may be respectively with separately independently three-dimensional structure be present in the solution/blood.As CD4V1-TNF α m2, their connection peptides is held to C end from N and is Leu-Gly-Thr-Arg-lys-Arg-Lys-Pro-Val, and wherein, Leu belongs to last amino acid of C-terminal of CD4V1, is a rigidity amino acid simultaneously; Thereafter 7 amino acid all are flexible amino acid; And last 3 amino acid of this connection peptides C end belong to the N end behind 7 amino acid of N tip cut-off of former TNF α, and its 3rd amino acid Val is a rigidity amino acid.Like this, just, having constituted CD4V1 and TNF α m2 is the whole story, serves as the very big possibilities of each self-forming absolute construction of chain of living with 7 flexible amino acid therebetween with two rigidity amino acid.If be strictly to form such two three-D space structures independently, they will be the human body intrinsic protein respectively, will be unlikely to be caused drug failure by immunity system wrong identification so that induction of immunity reaction.The cytoactive test-results shows that CD4V1 and TNF α m2 have all brought into play function separately well.This result is also from the prompting of side, CD4V1 and each self-sustaining of TNF α m2 the very big possibility of original three-dimensional structure.And the Gly-Thr of the N end in the connection peptides introduces owing to having added the kpnI restriction enzyme site, and 3 amino acid are artificial introductions thereafter, count 5 amino acid, are no more than six amino acid, by generally accepted viewpoint, answer non-immunogenicity.In general, this fusion rotein is unlikely to have the intensive immunogenicity and lost efficacy in facing application.And most active poly peptide medicines of E.coli system development all have certain immunogenicity, but do not influence its clinical application.The codon that the present invention chooses synthetic is that 100 amino acid whose gene orders in the outer V1 district, variable region of born of the same parents of coding CD4 of E.coli. hobby are as targeted molecular, recombinate construction expression recombinant chou pCW-PL-SD-CD4V1TNF α m1 and pCW-PL-SD-CD4V1TNF α m2 with the derivative TNF α m1 of the potent tumor necrosis factor alpha that utilizes round pcr to obtain (TNF α) and the gene fragment of TNF α m2.

With expression type recombinant chou pCW-PL-SD-CD4V1TNF α m1 described below and pCW-PL-SD-CD4V1TNF α m2 respectively transformed into escherichia coli DH5 α obtain high expression engineering pCW-PL-SD-CD4V1TNF α m1/DH5 α and pCW-PL-SD-CD4V1TNF α m2/DH5 through screening, row SDS-PAGE glue is analyzed, and recombinate warm albumen pCW-CD4V1TNF α m1/DH5 α and pCW-CD4V1TNF α m2/DH5 account for 12% and 13.7% of bacterial protein respectively.Strain is carried out large scale culturing and is induced to pCW-PL-SD-CD4V1TNF α m1/DH5 α high expression level, extracts inclusion body, and the dissolving of 8M urea is through SDS-PAGE gel cutting back electroelution recovery albumen.Remove SDS through freezing acid acetone, through dialysis, renaturation.

Behind the protein quantification, respectively with 8,16, the concentration of 32,64,128 μ g/ml is handled the CMEx-174 cell that is infected by SIV with fusion rotein CD4V1TNF α m1.With normal CMEx-174 cell and blank sample as negative control.Observe the lethal effect of fusion rotein pair cell.The result shows: this fusion rotein can kill and wound the CMEx-174 that is infected by SIV effectively, and the kill rate of CD4-TNF α m can reach 54.9% when 128 μ g/ml.And, the CEMx-174 cell that in killing experiments, is infected by SIV separately with the effect of TNF α albumen, the growing state of discovery cell and the growing state that does not add the proteic CEMx-174 cell that is infected by SIV of TNF α do not have significant difference (P＞0.20).The lethal effect that the fusion rotein of this experimental study is described is the mediation by the target molecule, and independent TNF α albumen is not owing to there is the mediation of target molecule, thereby can't kill and wound the CEMx-174 cell of taint with SIV, preliminary identification the mechanism of action.Simultaneously, with CD4TNF α m fusion rotein respectively with 4,8, the experiment that neutralizes of the concentration of 16,32 μ g/ml, the fusion rotein of 32 μ g/ml dosage can reach 33.5% to the protection ratio of free virus, illustrates that warm albumen has provide protection to non-infected cells.Kill rate and neutralization ratio all are dose-dependence with fusion rotein, and have the titre test to support above result.By this fusion rotein of control experiment Normocellular growth there is not obvious influence simultaneously.

Test cell line shows that the fusion rotein CD4-TNF α m among the present invention is used for anti-HIV/SIV, and good specificity and security are arranged, and is a kind of potential drug that is rich in the treatment acquired immune deficiency syndrome (AIDS) of prospect.

The invention provides and comprise described antigen-4 fusion protein gene and contain this expression of gene type recombinant chou.

Preferably, the receptor polypeptides in the described fusion rotein be can with gp120 and/or gp41 specificity bonded CD4, CD4V1V2 or CD4V1, perhaps their relevant segment, analogue or mutant.

Preferably, because 100 amino acid in V1 district are enough for CD4 in conjunction with the gp120 molecule, intercepted 100 amino acid in V1 district in the CD4 total length as receptor polypeptides, its aminoacid sequence is shown in SEQ ID NO:2.

More preferably, in order to realize fusion rotein efficiently expressing in the E.coli. system, the CD4V1 gene fragment is the dna sequence dna that the E.coli. of the sudden change of synthetic has a liking for, and its dna sequence dna is shown in SEQ ID NO:1.

Preferably, described functional polypeptide is the TNF family member, as TNF α, TNF β (has another name called lymphotoxin, lymphotoxin, LT), TNF γ, or derivatives thereof or its mutant, or TNF superfamily member, as Trail (Tumor related Apoptosis inducedligand), Light (for homologous to lYmphotoxins, exhibits INducible expression, and competes with HSV GLycoprotein D for HVEM, a receptor expressed by TLymphocytes, Light; , be called HEVMLherpesvirus entry mediator ligand again) and be their active function zone, mutant or analogue.Or have pore-forming protein (human perforin, a HP that the lysing cell effect derives from human body; Pore forming protein PFP), can form the complement component C9 that complement is attacked mixture, particle dissolving plain (granulysin); Perhaps can cause the granzyme A of the granzyme family that derives from human body of apoptosis, B, C, D, E (GranzymeA, B, C, D, E), serine easterase (serine esterases); Participate in apoptotic protein kinase C (PKC) directly, protein kinase A (APK), E.C. 2.7.2.4. (caspases), cytopigment, DNA endonuclease; Can resist polypeptide and the enzyme of bacterium, as N,O-Diacetylmuramidase (Lysosome), alexin (defensin); Have the P53 of function of tumor inhibition and be their active function zone, mutant or analogue.

More preferably, wherein said functional polypeptide is mutant TNF α m1 and the TNF α m2 of TNF α.The present invention adopts dual mode to transform the structure of TNF α according to the report of pertinent literature, and the first increases by two amino acid Gly before TNF α N holds the first amino acids Val, and Thr increases by three amino acid Arg thereafter, and Gly, Pro become employing TNF α m1; It two is that N with prototype TNF α holds preceding 7 aminoacid deletion sudden change, and with Pro8, Ser9, Asp10 sports Arg8, Lys9, Arg10, obtain the TNF α m2 of attenuation synergistic, make it more to be applicable to clinical, improve its anti-tumor activity simultaneously to reduce its toxic side effect.

The invention provides the gene C D4V1-TNF α m1 of two kinds of codings anti-cancer agent targent fused protein CD4V1-TNF α m1 and CD4V1-TNF α m2, CD4V1-TNF α m2 has the nucleotide sequence of SEQ ID NO:2 and SEQ ID NO:4 respectively.

On the other hand, the invention provides the technological line of the expression type recombinant chou pCWCD4V1-TNF α m1 that comprises described gene C D4V1-TNF α m1 and CD4V1-TNF α m2 and pCWCD4V1-TNF α m2 and engineering bacteria pCWCD4V1-TNF α m1/DH5 α and pCWCD4V1-TNF α m2/DH5 α and a large amount of preparation, purifying and recombinant protein.

Further describe the present invention by the following examples, and unrestricted the present invention.

The acquisition of the gene C D4V1TNF α m1 of embodiment 1. coding recombination targent fused proteins

1.PCR primer and sequence thereof

1.1 Bo Ya company synthesizes TNF α m1 primer:

Forward primer 5 '-aaa

Gtaagaggtccaagat-3 ' 25bp contains the KpnI site

Reverse primer 5 '-aaa

Ttatcacagggcaatg-3 ' 25bp contains the BamHI site

1.2 the sequencing primer of the synthetic pCW111 of Bo Ya company:

Forward?primer?5’-AAAAAGGGCATCAAATTAAACC-3’

Reverse?primer?5’-TCGGCGATATAGGCGCCAGC-3’

The PCR reaction conditions is: 94 ℃ of sex change begin circulation after 3 minutes: 94 ℃ of sex change 30 seconds, and 56 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds.After 25 circulations, 72 ℃ were extended 10 minutes.The amplification enzyme high-fidelity Pfu that uses.Purpose fragment with in the 1.0% sepharose recovery PCR product obtains the dna fragmentation about 514bp.

2. the acquisition of mutant TNF α m1

Utilize TNF α m1 PCR primer that the TNF α cDNA that this laboratory is made up is carried out specific amplified, consequently introduce the codon of 1 KpnI site (GGTACC) and several extra coded amino acid at the 5 ' end of TNF α cDNA GGT ACCGTA AGA GGT CCA(wherein with underscore be than additional codon than prototype), this segment DNA sequence corresponding amino acid sequence is Gly ThrVal Arg Gly Pro(what be with underscore is than additional amino acid than prototype).Simultaneously, also its 3 ' end introduce 1 BamHI site ( GGA TCC) and 2 terminator codons ( UAA UGA).After the flush end PCR product that the pfu enzymatic amplification is obtained reclaims, be connected with the PBSSK flush end of cutting processing through the EcoRV enzyme.Connect product transformed into escherichia coli DH5 α, be coated on the LB agar plate that contains penbritin (100 μ g/ml), utilize blue blank screening positive clone.According to " molecular cloning experiment guide " (second edition) (J. Sa nurse Brooker, Deng. the chief editor, Jin Dongyan etc. translate, Science Press, 1992) alkaline lysis prepares the plasmid DNA of resistance clone in a small amount, and restriction analysis confirms that plasmid inserts clip size and conforms to goal gene.Because of the direction of insertion difference, obtain two kinds of products, by the BamHI single endonuclease digestion, cast out the fragment that can't cut out about 500bp and oppositely insert product, obtain order-checking recombinant chou pBS-TNF α m1.PBS-TNF α m1 enzyme is cut qualification result and is seen Fig. 3, and sequencing result is seen Fig. 6.

3. the acquisition of fusion gene CD4V1TNF α m1

Handle directed pBS-TNF α m1 forward with ApaI, KpnI double digestion and connect product, reclaim the fragment of 534bp.The CD4V1 gene order that contains the intestinal bacteria hobby from Bo Ya company synthetic pGEM-CD4 plasmid, to handle the big fragment that reclaims behind the pGEM-CD4 with ApaI, KpnI double digestion (available from the precious biotinylated biomolecule in Dalian company), be connected with the small segment of handling the back recovery with pBS-TNF α m1ApaI, KpnI double digestion, obtain recombinant chou pGEM-CD4V1TNF α m1.Recombinant chou pGEM-CD4V1TNF α m1 enzyme is cut qualification result and is seen Fig. 3.

The structure of embodiment 2. fusion gene CD4V1-TNF α m1 temperature-regulated expression type recombinant chou pCW-CD4V1TNF α m1

Use BamHI, the NdeI double digestion is handled and is handled respectively and the pCW111 carrier, reclaiming pGEM-CD4TNF α m1 enzyme cuts the big fragment of CD4 TNF 797bp fragment in the product and pCW111 and connects, obtain recombinant chou pCW-CD4V1TNF α m1, thereby the encoding gene of CD4V1TNF α m1 fusion rotein is placed temperature control promotor P _RP _LControl under.Recombinant chou pCW-CD4V1TNF α m1 enzyme is cut qualification result and is seen Fig. 3, and sequencing result is seen Fig. 7, and 8.

The acquisition of embodiment 3. coding TNF alpha-mutant TNF α m2 genes

3.1 Bo Ya company synthesizes TNF α m2 primer:

Forward primer 5 '-aaa

CgtAaacgcaagcct-3 ' contains PstI and XhoI

The site

Reverse primer 5 '-aaa

Ttatcacagggcaatg-3 ' 25bp contains the BamHI enzyme

Cut the site

3.2 the acquisition of mutant TNF α m2

Utilize TNF α m2PCR primer to plasmid pCW-CD4V1TNF α m1 is carried out specific amplified, consequently obtain the N of prototype TNF α is held preceding 7 aminoacid deletion sudden change, and with Pro8, Ser9, Asp10 sports arg8, Lys9, Arg10, the TNF α m2 of the potent sudden change of attenuation of acquisition.Simultaneously, also its 3 ' end introduce 1 BamHI site ( GGA TCC) and 2 terminator codons ( UAA UGA).The PCR product that the pfu enzymatic amplification is obtained is through BamHI and XhoI double digestion, after glass milk recovery enzyme is cut product, be connected with the PBSSK cohesive end that the XhoI double digestion is handled, obtain recombinant plasmid pBS-TNF α m2 with process BamHI, enzyme is cut qualification result and is seen Fig. 3, and sequencing result is seen Fig. 9.

The structure of embodiment 4. fusion gene CD4V1TNF α m2 temperature-regulated expression type recombinant chou pCW-CD4V1TNF α m2

With plasmid pBS-TNF α m2 behind KpnI and BamHI double digestion, glass milk reclaims about 500bp enzyme and cuts product, and handle the 5187bp enzyme that reclaims the back with pCW-CD4V1TNF α m2 through KpnI and BamHI double digestion and cut the product cohesive end and be connected acquisition temperature-regulated expression type recombinant chou pCW-CD4V1TNF α m2.

Embodiment 5. expression of fusion rotein CD4V1TNF α m1 in intestinal bacteria

1. the expression of target protein and evaluation

PCW-CD4V1TNF α m1 is transformed DH5 α, be inoculated in 5ml LB substratum then, add the penbritin that final concentration is 100mg/ml simultaneously, 32 ℃ of jolting overnight incubation.By 2% inoculum size renewed vaccination, two pipe 5ml LB/Amp+ nutrient solutions.Be cultured to OD600 32 ℃ of following joltings and be about at 0.6 o'clock.Tell a pipe then and place 42 ℃ of shaking tables, jolting is cultivated and is induced 5h; Another pipe continues at 32 ℃ of shaking culture 5h, in contrast.The centrifugal collection thalline of 4000g adds sample-loading buffer (50mmol/L Tris-HCl, pH6.8,100mmol/L DTT, 2%SDS, 0.1% tetrabromophenol sulfonphthalein, 10% glycerine), and mixing places 100 ℃ of boiling water baths to keep 5min.The centrifugal 1min of 10000g gets supernatant and carries out polyacrylamide gel electrophoresis (SDS-PAGE) analysis.Target protein matter expression amount scans in 626nm wavelength place by Chromscan3 PhastGel scanner, and the calculation expression amount.Detailed method is referring to " modern molecular biology experimental technique " second edition referring to the Lu Shengdong chief editor, 365-387 page or leaf.

2. result:

Analyze demonstration through SDS-PAGE, the target protein of pCW-CD4V1TNF α m1/DH5 alpha expression has tangible protein band to occur at about 30KD place, and does not induce sample this band not on the corresponding position, the albumen size with expect consistent, tentatively predicate target protein, see Fig. 4.Analyze through the laser scanning of SDS-PAGE gel, target protein accounts for 12.0% of total protein of cell, sees Fig. 5.

A large amount of preparations and the purifying of embodiment 6 fusion rotein CD4V1TNF α m1

1. large volume is cultivated:

Engineering bacteria pCW-PL-SD-CD4V1TNF α m1/DH5 α is inoculated in 50ml LB substratum, adds the penbritin that final concentration is 100mg/ml simultaneously, 32 ℃ of jolting overnight incubation.Be re-seeded in two bottles of 2L triangular flasks that contain 500ml LB/Amp+ nutrient solution respectively by 2% inoculum size.Be cultured to OD600 32 ℃ of following joltings and be about at 0.6 o'clock.Place 42 ℃ of continuation joltings to cultivate then and induce 5h.4000g is centrifugal, the collection thalline, places-20 ℃ of multigelations.

2. the preparation of inclusion body

2.1 washed cell:

6000g * 15 minute centrifugal collection bacterium, the wet thallus of weighing.

With 3%Triton X-100 washing thalline, stirred evenly on the agitator 30 minutes, 6000g * 15 a minute centrifugal collection consumption is 10ml-50ml damping fluid/1g wet thallus (comprising following various lavation buffer solution).

2.2 broken cell

With cell suspension, damping fluid is prepared: 50mmol/LTris.Cl (pH8.5-9.0), 2mmol/L EDTA, 100mmol/L NaCl, 0.5%Triton X-100, N,O-Diacetylmuramidase 1mg/ml with damping fluid).At room temperature stir with agitator, make the effect thickness of suspension because of N,O-Diacetylmuramidase.

2.3 supersound process

Each super making a call to 10 seconds, interval 15 seconds, 20-50 secondary action (deciding on cell concentration) is to thalline no longer till the thickness.Centrifugal then collection, 6000g * 15 minute.Aforesaid operations should carry out in frozen water, and for preventing charing, ultrasonic power is unsuitable excessive, carries out in glass beaker better, and ultrasonic probe gos deep into liquid level greater than 3 centimetres, and is good apart from cup 2 centimetres of positions, the end.

2.4 5%Triton X-100 washing

With the damping fluid precipitation that thoroughly suspends, stir and ultrasonication 30 times, referring to above-mentioned steps 2.3.Damping fluid is: 50mmol/L Tris.Cl (pH8.0), 2mmol/L EDTA, 5%Triton X-100.Centrifugal collecting precipitation, 6000g * 15 minute.

2.5 the preservation of inclusion body and dissolving:

2.5.1 dissolve inclusion body gradually with a small amount of 8M urea, to get 20 μ l solution and add 2 * sample-loading buffer, mixing boiled in the boiling water 3-5 minute.Get 20ul and carry out the SDS-PAGE electrophoretic analysis.All the other inclusion bodys can be frozen in-70 ℃.

If 2.5.2 cross column purification, should operate by following step: with containing body lysate (50mmol/LTris.Cl, pH8.0,2mmol/L EDTA, 10mMDTT, 7M Guanidinium hydrochloride) dissolving inclusion body, inclusion body concentration is about 10mg/ml.With dissolved inclusion body 12000g centrifugal after, get supernatant, obtain can upper prop sample solution.

3. the purifying of target protein

Get inclusion body solution prepared in 2.3 steps, add in equal-volume 2 * sample-loading buffer, carry out 15% SDS-PAGE electrophoresis.

3.1. gel cutting: downcut the adhesive tape and the small part sample adhesive tape at albumen Marker swimming lane place, put it into the rapid dye liquor middling speed and dyed 15 minutes, the residue gel is wrapped with preservative film and is placed 4 ℃ of refrigerators.After band is showed in the adhesive tape decolouring that speed is dyed,, downcut the gel strips that contains the purpose band portion with adhesive tape and the contrast of remaining gel.

3.2. electroelution: pack into the gel strips of downcutting and gel soak solution in the dialysis tubing and place the horizontal strip electrophoresis groove, add the protein electrophoresis wash-out and carry out constant voltage 100v electrophoresis.Behind electrophoresis 10～12h with elutriant position dialyzate electrophoresis 10～12h more also.Soak solution in the dialysis tubing is poured in the 50ml centrifuge tube, can be concentrated with appropriate amount of PEG 10000 earlier as solution is more.

3.3. the removal of trace SDS: add 5 times of freezing acid acetone-methyl alcohol albumen precipitation liquid more than the volume in centrifuge tube, 20 ℃ of precipitations are spent the night.10, centrifugal 10 minutes of 000r/m removes the supernatant collecting precipitation.The precipitation of collecting is washed 1 time with albumen precipitation liquid.

The renaturation of embodiment 7. fusion roteins

With the albumen precipitation after the washing sex change damping fluid (6mol/L Guanidinium hydrochloride, 0.1mol/L tris-HCl, PH8.6,1mmol/L EDTA, 0.05mmol/L NaCl, 10mmol/L DDT) in ice bath, change in the dialysis tubing after the dissolving, in dialysis tubing, add protein renaturation liquid (50mmol/LTris-HCl simultaneously, PH8.0,1mmol/L EDTA, 0.25mol/L NaCl, 0.25mol/L L-arginine, 5mmol/L DDT) in, in distilled water, dialyses 48 hours so that protein renaturation for 4 ℃.

Embodiment 8 recombination targent fused protein CD4V1-TNF α m1 are to the lethal effect of the CEMX-174 of SIV infection

8.1 the preparation of pattern cell:

Because HIV does not infect the CD4 positive cell of other animal, thereby does not still have a kind of people's acquired immune deficiency syndrome (AIDS) animal model so far.Only show as the change of temporary T cell quantity after the orangutan infected by HIV-1 and produce antibody, do not develop into the acquired immune deficiency syndrome (AIDS) clinical symptom.The nucleotides sequence of discovering HIV-2 and the isolating immunodeficiency virus of SIVMAC9 Asia stump-tailed macaque kind shows about 75% even higher homology, with HIV-1 45% homology is only arranged.But since SIV and HIV-1 infect, duplicate with regulation and control aspect similar substantially, the primate acquired immune deficiency syndrome (AIDS) that can cause by research SIV is as the basis of research people AIDS-like disease.Therefore this experiment employing SIV carries out preliminary experiment in vitro and reacts the anti-HIV/SIV effect in the warm albumen of target ground.

This tests employed pattern cell is CEMX-174 cell strain (laboratory animal research institute of Chinese Academy of Medical Sciences Viral Laboratory).Particularly, the CEMX-174 cell recovery that liquid nitrogen is preserved is at first transferred in the culturing bottle of the 100ml of 1640 substratum that contain 10% foetal calf serum (Tianjin Suntory Ltd.) (Gibco-BRL company, Luo Keweier, the Maryland State, the U.S.) 37 ℃, 5%CO ₂Incubator in cultivate, change liquid after 48 hours, in nutrient solution, add virus titer subsequently greater than 5120 SIV, infect the CEMX-174 cell.Because this cell is easy to infected and efficient is high, so examined under a microscope the infected situation of cell every 6-8 hour, comprises the percentage of infected cell and the form change of cell etc.Cultivation and repeated infection through 4-5 days detect efficiency of infection with immunofluorescence, average infection rate about 47%.

8.2 experimental technique and result

8.2.1MTT method is measured cytoactive

Test cell line was all estimated cytoactive by cell counting in the past, made the credible result degree not high because of its subjective factor and self many defective like this.The present invention adopts mtt assay to measure cytoactive.The commodity of MTT are called tetrazolium bromide, and the succinodehydrogenase in the viable cell plastosome can make the MTT of external source be reduced into the bluish voilet crystallization of insoluble, and is deposited in the cell, and dead cell does not have this function.Bluish voilet crystallization in dimethyl sulfoxide (DMSO) (DMSO) the energy dissolved cell.Measure its OD value with enzyme-linked immunosorbent assay instrument at 490nm wavelength place, can reflect the quantity of active cells indirectly.Within the specific limits, MTT crystallisate formation amount is directly proportional with cell quantity.So the present invention adopts MTT to measure cytoactive, improves result's objectivity, confidence level.

8.2.2 experimental result and explanation

5 dosage groups are adopted in this test, make the medicine final concentration be respectively 8,16,32,64,128 μ g/ml.In 24 porocyte culture plates, handle 1.25 * 10 respectively ⁵/ ml is reached not by the normal cell of virus infection by the CEMx-174 cell that SIV infects.For the effect of verifying fusion rotein numerator mediated by target, rather than work separately by TNF α, also designed with TNF α and acted on the cell that SIV has infected separately.As normal group, is zeroing group with 1640 substratum that do not add cell with the CEMx-174 cell of normal cultivation, and the CEMx-174 cell of the taint with SIV of handling with albumen is a test group, is control group with the CEMx-174 cell that does not add proteic taint with SIV.Draw the fluorescence per-cent of test group and control group after 48 hours by the fluorescence counting, measure cytoactive with mtt assay simultaneously, calculate the kill rate of fusion rotein.Each hole survivaling cell number of gained carries out variance analysis (analysis of variance) and T check (T-test) is analyzed, and the results are shown in Table 1,2,3.Results of statistical analysis shows P＜0.05, after the CEMx-174 cell that taint with SIV is described adds CD4V1TNF α m1, tangible lethal effect occurred, and lethal effect is enhanced basically along with the increase of dosage.Maximal dose group 128 μ g/ml wherein, the kill rate of CD4V1TNF α m1 is 54.9%.This fusion rotein of above presentation of results can kill and wound the cell that is infected by SIV, sees Figure 10.Fusion rotein CD4V1-TNF α m1 is after the CEMx-174 cytosis to taint with SIV not under 8,16,32,64, the 128 μ g/ml dosage, the upgrowth situation of observation of cell, survival condition with OD value reacting cells, growing state and the Normocellular growing state of finding cell do not have significant difference, P＞0.20.These presentation of results, fusion rotein CD4V1-TNF α m1 is to normal not the CEMx-174 cell and the nontoxicity lethal effect of taint with SIV, and the security of fusion rotein is ensured, meets the experimental design requirement, sees Figure 11.Simultaneously, after TNF α albumen acts on the CEMx-174 cell that has infected SIV separately, with the survival condition of OD value reacting cells.OD value 1.072 is a control group.There is not significant difference by T check discovery test group and control group data.See Figure 13.Kill rate=1-[(control group OD value-zeroing group OD value) * and the control group infection rate] ÷ [(test group OD value-zeroing group OD value) * test group infection rate]

Table 1. fragmentation test result

The fluorescence counting titre that grouping dosage mtt assay records is killed and wounded

Ug/ml OD value per-cent %

NC / 1.072±0.013 0％ 0 /

NC/SIV / 0.560±0.005 75.3％ 1∶1280 /

Substratum/0.061 ± 0.006 0% //

NC/SIV+ 8 0.498±0.006 72.1％ 1∶320 17.3％

CD4V1 16 0.442 ± 0.012 66.2% does not survey 32.9%

TNFαm1 32 0.421±0.015 64.0％ 1∶80 38.7％

64 0.397±0.007 61.2％ 1∶40 45.4％

128 0.363±0.004 56.1％ 1∶10 54.9％

Illustrate: 1 every group of dosage has 3 parallel holes, and each hole is measured 3 times with mtt assay

2 NC represent normal CEMx-174 cell, and the CEMx-174 cell of SIV has been infected in the NC/SIV representative, and the hundreds of proportions by subtraction of photofluorometer are infection rate

The OD value that 3 test group are measured is compared with control group (NC/SIV), and significant difference P＜0.05 is all arranged

4 Numerical Calculation Method are seen text specification

After two kinds of fusion roteins of table 2. act on the CEMx-174 cell of taint with SIV not, the influence of cell growth

The statistics inspection that grouping dosage mtt assay records

(ug/ml) OD value F check T check

NC / 1.072±0.011 / /

8 1.088±0.022 2.905 1.145

NC+ 16 1.055±0.044 4.140 0.655

CD4V1-TNFαm 32 1.093±0.035 6.578 -0.967

1 64 1.075±0.017 0.608 -0.227

128 1.078±0.069 6.586 -0.145

Illustrate: fusion rotein CD4V1-TNF α m1 is after the CEMx-174 cytosis to taint with SIV not under 8,16,32,64, the 128 μ g/ml dosage, the upgrowth situation of observation of cell, growing state and the Normocellular growing state of finding cell do not have significant difference, P＞0.20.These presentation of results, fusion rotein CD4V1-TNF α m1 is to normal not the CEMx-174 cell and the nontoxicity lethal effect of taint with SIV, and the security of fusion rotein is ensured, meets the experimental design requirement.

After table 3.TNF α albumen acts on the CEMx-174 cell that SIV infected separately, the influence of cell growth

The statistics inspection that grouping dosage mtt assay records

(ug/ml) OD value F check T check

NC/SIV / 0.370±0.011 / /

8 0.359±0.096 0.051 1.337

NC/SIV+ 16 0.359±0.096 0.132 -1.335

TNFα 32 0.370±0.022 1.730 -0.024

64 0.379±0.046 3.213 -1.352

128 0.366±0.011 0.029 0.464

Illustrate: use TNF α albumen separately behind the CEMx-174 cell to taint with SIV under 8,16,32,64, the 128 μ g/ml dosage, the upgrowth situation of observation of cell, finding the growing state of cell and not adding the proteic growing state that has infected the CEMx-174 cell of SIV of TNF α does not have significant difference, P＞0.20.This presentation of results two kinds of fusion roteins of this experimental study be mediation to the lethal effect of the CEMx-174 cell of taint with SIV by the target molecule, TNF α by in swallow and start its lethal effect behind the cell of infection.Independent TNF α albumen is not owing to there is the mediation of target molecule, thereby can't kill and wound the CEMx-174 cell of taint with SIV, meets the experimental design requirement.

Embodiment 9 recombination targent fused protein CD4V1TNF α m1 are to the provide protection of the CEMX-174 of SIV infection

9.1 the preparation of pattern cell is with 8.1

9.2 experimental technique and result

9.2.1MTT method is measured the same 8.2.1 of cytoactive

9.2.2 experimental result and explanation

With the CEMx-174 cell of normal cultivation as normal group; with 1640 substratum that do not add cell is the zeroing group; CEMx-174 cell and the SIV virion mixture handled with albumen are test group; not add proteic CEMx-174 cell and SIV virion mixture is a control group; divide 4 dosage groups, use the fusion rotein effect of 4,8,16,32 μ g/ml respectively.(experimental result sees Table 4) to remaining cell with mtt assay measures its activity with " normal CEMx-174 cell+SIV " group after 6 days with " normal CEMx-174 cell+SIV+ fusion rotein " each group, carry out statistical analysis, the result shows P＜0.05, after being described, normal CEMx-174 cytomixis SIV adds CD4V1TNF α m1, tangible neutralizing effect has appearred, suppressed the infection of cell free virus pair cell, and neutralizing effect is enhanced basically along with the increase of dosage.Wherein the protection ratio of the CD4V1TNF α m1 of maximal dose group 32 μ g/ml is 33.5%.Because the existing neutralizing effect of neutralization test effect in fact has lethal effect again, be difficult to pure neutralization ratio of definition, so react the neutralizing effect of fusion rotein with protection ratio.This fusion rotein of above presentation of results can in and free SIV, suppress SIV to the CEMx-174 cell infection, can effectively protect normal CEMx-174 cell.See Figure 12.

Protection ratio=[(test group OD value-zeroing group OD value)-(control group OD value-zeroing group OD value)] ÷ (test group OD value-zeroing group OD value)

Table 4. neutralization test result

The OD titre protection % that grouping dosage mtt assay records

(ug/ml) value

NC / 2.816±0.014 / /

NC+SIV / 1.146±0.005 1∶1280 /

1640 substratum/0.071 ± 0.004 //

4 1.302±0.002 1∶1280 12.7％

NC+SIV+ 8 1.346±0.003 1∶320 15.7％

CD4V1TNFαm 16 1.435±0.005 1∶80 21.2％

32 1.688±0.006 1∶40 33.5％

Illustrate: 1 every group of dosage has 3 parallel holes, and each hole is measured 3 times with mtt assay

2NC represents normal CEMx-174 cell, and the NC+SIV representative is mixed with the normal CEMx-174 cell of SIV particulate

The OD value that 3 test group are measured is compared with control group (NC+SIV), and significant difference P＜0.05 is all arranged

4 Numerical Calculation Method are seen text specification

The virus titer test of embodiment 10 fragmentation tests and neutralization test

Fusion rotein is killed and wounded the cell culture fluid mixing of handling back cultivation 48h, and (neutralization test will be handled 7 days) dilutes with substratum according to 10,20,40,80,160,320,640,1280 times.Get each diluent 100 μ l respectively in new aseptic 96 orifice plates, add 1.0 * 10 of equivalent ⁵/ ml CEMx-174 cell is put 37 ℃ of CO ₂Incubator (95% air, 5%CO ₂) in hatch, changed half fresh culture in per 48 hours.Observations after 7 days, the multinuclear maxicell that forms with cytogamy is that index is determined titre.Virus in (the results are shown in Table 1,4) cell culture supernatant is owing to the cell that is infected by the virus cracking behind viral proliferation is discharged in the supernatant fully.Viral load in the supernatant can be determined by the virus titer test.Under certain extent of dilution, the virus in the supernatant is diluted to below the detection sensitivity scope of titre experiment, and such dilution adds in the normal cell culture and do not cause plasmodial generation.We adopt 2 ⁿThe gradient dilution supernatant is with the method for cells infected, through cultivation after a while, whether with plasmodial generation in the culturing cell is the situation that standard is determined virus infected cell, can directly draw the result that under what extent of dilution virus concentration is lower than the detection sensitivity scope of titre test.The relative concentration of the virus that contained originally in the deducibility supernatant of taking this as a foundation, this index can be used as the objective indicator of virusology check.From titre synoptic diagram (Figure 14 and Figure 15), can find out between experimental group and the positive controls notable difference on the virus titer test-results, and be tangible dose relationship, as can be seen from the figure along with the increase of drug dose, kill and wound group and in and the papova titre descend gradually.

The fluorescence counting and the photomicrography of embodiment 11 cells infecteds

With the abundant suspendible of cell of each culture hole in fragmentation test and the neutralization test, get the 1ml cell culture fluid, centrifugal 3 minutes of 3000rpm abandons supernatant.PBS with 1ml washes once again, abandons supernatant, is adding PBS gradually, the suspendible cell, and adjusting cell concn is 2 * 10 ⁵/ ml.Get 15 μ l suspensions and drip in the ring on slide glass, dry naturally, fix 20 minutes for 4 ℃ in acetone, taking-up is dried, the antibody (the anti-SIV antibody of monkey is from the serum of taint with SIV monkey) of adding gp120,37 ℃, water-bath 30 minutes.PBS with pH7.2 washes then, and the immersion 10 minutes that is placed in one, constantly jolting therebetween.After taking out slice, thin piece, add the anti-monkey antibody of rabbit (promptly two is anti-) with 2/10000ths the blue preparation of Evens again, 37 ℃ of water-baths 30 minutes with the PBS flushing, are dried back observation under fluorescent microscope, are counted the fluorocyte number and take pictures.Observe the cell that is infected by SIV/HIV and issue yellow-green fluorescence exciting of blue light under fluorescent microscope, can see that the cell periphery is yellow-green colour, infected cells then is not yellow, is garnet.Fluorescence infection rate among fragmentation test and the protection test result sees Table 1.Fluorescence photo before and after the fragmentation test is seen Figure 16-1 and Figure 16-2.As seen the cell that is infected by SIV/HIV among Figure 16-1, and visible many cell debriss among Figure 16-2, the cell that infects for the SIV/HIV that killed and wounded by fusion rotein.Fluorescence photo before and after the protection test is seen Figure 17-1 and Figure 17-2.As seen the cell that is infected by SIV/HIV among Figure 17-1, and see among Figure 16-2 more and illustrate that cell has been subjected to effective protection of fusion rotein by the normal cell that not infected by SIV/HIV.

Sequence table

＜110〉Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences

Blood Transfusion Inst., Chinese Academy of Medical Sciences

Institute of Experimental Animals, Chinese Academy of Medical Sciences

＜120〉a kind of recombination targent fused protein that is used for the treatment of acquired immune deficiency syndrome (AIDS)

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<400>7

catatgaaaa?aagtagttct?gggtaaaaag?ggtgacaccg?ttgaactgac?ttgcaccgct 60

tcccagaaaa?agtctatcca?gttccactgg?aaaaactcca?accagatcaa?aatcctaggt 120

aaccagggtt?ccttcctgac?caaaggtccg?tctaaactga?acgatcgtgc?tgactcccgc 180

cgttctctat?gggaccaggg?taacttcccg?ctgattatca?asascctgaa?aatcgaagat 240

tccgacacct?acatctgtga?agttgaagac?cagaaagaag?aagttcaact?actggttttc 300

ggtctgggta?ccgtaagagg?tccaagatca?tcttctaaaa?ccccgagtga?caagcctgta 360

gcccatgttg?tagcaaaccc?tcaagctgag?gggcagctcc?agtggctgaa?ccgccgggcc 420

aatgccctcc?tggccaatgg?cgtggagctg?agagataacc?agctggtggt?gccatcagag 480

ggcctgtacc?tcatctactc?ccaggtcctc?ttcaagggcc?aaggctgccc?ctccacccat 540

gtgctcctca?cccacaccat?cagccgcatc?gccgtctcct?accagaccaa?ggtcaacctc 600

ctctctgcca?tcaagagccc?ctgccagagg?gagaccccag?agggggctga?ggccaagccc 660

tggtatgagc?ccatctatct?gggaggggtc?ttccagctgg?agaagggtga?ccgactcagc 720

gctgagatca?atcggcccga?ctatctcgac?tttgccgagt?ctgggcaggt?ctactttggg 780

atcattgccc?tgtgataa 798

<210>8

<211>264

<212>PRT

＜213〉people

<400>8

His?Met?Lys?Lys?Val?Val?Leu?Gly?Lys?Lys?Gly?Asp?Thr?Val?Glu?Leu

1 5 10 15

Thr?Cys?Thr?Ala?Ser?Gln?Lys?Lys?Ser?Ile?Gln?Phe?His?Trp?Lys?Asn

20 25 30

Ser?Asn?Gln?Ile?Lys?Ile?Leu?Gly?Asn?Gln?Gly?Ser?Phe?Leu?Thr?Lys

35 40 45

Gly?Pro?Ser?Lys?Leu?Asn?Asp?Arg?Ala?Asp?Ser?Arg?Arg?Ser?Leu?Trp

50 55 60

Asp?Gln?Gly?Asn?Phe?Pro?Leu?Ile?Ile?Lys?Asn?Leu?Lys?Ile?Glu?Asp

65 70 75 80

Ser?Asp?Thr?Tyr?Ile?Cys?Glu?Val?Glu?Asp?Gln?Lys?Glu?Glu?Val?Gln

85 90 95

Leu?Leu?Val?Phe?Gly?Leu?Gly?Thr?Val?Arg?Gly?Pro?Arg?Ser?Ser?Ser

100 105 110

Lys?Thr?Pro?Ser?Asp?Lys?Pro?Val?Ala?His?Val?Val?Ala?Asn?Pro?Gln

115 120 125

Ala?Glu?Gly?Gln?Leu?Gln?Trp?Leu?Asn?Arg?Arg?Ala?Asn?Ala?Leu?Leu

130 135 140

Ala?Asn?Gly?Val?Glu?Leu?Arg?Asp?Asn?Gln?Leu?Val?Val?Pro?Ser?Glu

145 150 155 160

Gly?Leu?Tyr?Leu?Ile?Tyr?Ser?Gln?Val?Leu?Phe?Lys?Gly?Gln?Gly?Cys

165 170 175

Pro?Ser?Thr?His?Val?Leu?Leu?Thr?His?Thr?Ile?Ser?Arg?Ile?Ala?Val

180 185 190

Ser?Tyr?Gln?Thr?Lys?Val?Asn?Leu?Leu?Ser?Ala?Ile?Lys?Ser?Pro?Cys

195 200 205

Gln?Arg?Glu?Thr?Pro?Glu?Gly?Ala?Glu?Ala?Lys?Pro?Trp?Tyr?Glu?Pro

210 215 220

Ile?Tyr?Leu?Gly?Gly?Val?Phe?Gln?Leu?Glu?Lys?Gly?Asp?Arg?Leu?Ser

225 230 235 240

Ala?Glu?Ile?Asn?Arg?Pro?Asp?Tyr?Leu?Asp?Phe?Ala?Glu?Ser?Gly?Gln

245 250 255

Val?Tyr?Phe?Gly?Ile?Ile?Ala?Leu

260

<210>9

<211>768

<212>DNA

＜213〉people

<400>9

catatgaaaa?aagtagttct?gggtaaaaag?ggtgacaccg?ttgaactgac?ttgcaccgct 60

tcccagaaaa?agtctatcca?gttccactgg?aaaaactcca?accagatcaa?aatcctaggt 120

aaccagggtt?ccttcctgac?caaaggtccg?tctaaactga?acgatcgtgc?tgactcccgc 180

cgttctctat?gggaccaggg?taacttcccg?ctgattatca?aaaacctgaa?aatcgaagat 240

tccgacacct?acatctgtga?agttgaagac?cagaaagaag?aagttcaact?actggttttc 300

ggtctgggta?cccgtaaacg?caagcctgta?gcccatgttg?tagcaaaccc?tcaagctgag 360

gggcagctcc?agtggctgaa?ccgccgggcc?aatgccctcc?tggccaatgg?cgtggagctg 420

agagataacc?agctggtggt?gccatcagag?ggcctgtacc?tcatctactc?ccaggtcctc 480

ttcaagggcc?aaggctgccc?ctccacccat?gtgctcctca?cccacaccat?cagccgcatc 540

gccgtctcct?accagaccaa?ggtcaacctc?ctctctgcca?tcaagagccc?ctgccagagg 600

gagaccccag?agggggctga?ggccaagccc?tggtatgagc?ccatctatct?gggaggggtc 660

ttccagctgg?agaagggtga?ccgactcagc?gctgagatca?atcggcccga?ctatctcgac 720

tttgccgagt?ctgggcaggt?ctactttggg?atcattgccc?tgtgataa 768

<210>10

<211>254

<212>PRT

＜213〉people

<400>10

His?Met?Lys?Lys?Val?Val?Leu?Gly?Lys?Lys?Gly?Asp?Thr?Val?Glu?Leu

1 5 10 15

Thr?Cys?Thr?Ala?Ser?Gln?Lys?Lys?Ser?Ile?Gln?Phe?His?Trp?Lys?Asn

20 25 30

Ser?Asn?Gln?Ile?Lys?Ile?Leu?Gly?Asn?Gln?Gly?Ser?Phe?Leu?Thr?Lys

35 40 45

Gly?Pro?Ser?Lys?Leu?Asn?Asp?Arg?Ala?Asp?Ser?Arg?Arg?Ser?Leu?Trp

50 55 60

Asp?Gln?Gly?Asn?Phe?Pro?Leu?Ile?Ile?Lys?Asn?Leu?Lys?Ile?Glu?Asp

65 70 75 80

Ser?Asp?Thr?Tyr?Ile?Cys?Glu?Val?Glu?Asp?Gln?Lys?Glu?Glu?Val?Gln

85 90 95

Leu?Leu?Val?Phe?Gly?Leu?Gly?Thr?Arg?Lys?Arg?Lys?Pro?Val?Ala?His

100 105 110

Val?Val?Ala?Asn?Pro?Gln?Ala?Glu?Gly?Gln?Leu?Gln?Trp?Leu?Asn?Arg

115 120 125

Arg?Ala?Asn?Ala?Leu?Leu?Ala?Asn?Gly?Val?Glu?Leu?Arg?Asp?Asn?Gln

130 135 140

Leu?Val?Val?Pro?Ser?Glu?Gly?Leu?Tyr?Leu?Ile?Tyr?Ser?Gln?Val?Leu

145 150 155 160

Phe?Lys?Gly?Gln?Gly?Cys?Pro?Ser?Thr?His?Val?Leu?Leu?Thr?His?Thr

165 170 175

Ile?Ser?Arg?Ile?Ala?Val?Ser?Tyr?Gln?Thr?Lys?Val?Asn?Leu?Leu?Ser

180 185 190

Ala?Ile?Lys?Ser?Pro?Cys?Gln?Arg?Glu?Thr?Pro?Glu?Gly?Ala?Glu?Ala

195 200 205

Lys?Pro?Trp?Tyr?Glu?Pro?Ile?Tyr?Leu?Gly?Gly?Val?Phe?Gln?Leu?Glu

210 215 220

Lys?Gly?Asp?Arg?Leu?Ser?Ala?Glu?Ile?Asn?Arg?Pro?Asp?Tyr?Leu?Asp

225 230 235 240

Phe?Ala?Glu?Ser?Gly?Gln?Val?Tyr?Phe?Gly?Ile?Ile?Ala?Leu

245 250

Claims (30)

1. the cDNA of the receptor polypeptides of the gp120 of a people HIV/SIV and/or gp41, its nucleotide sequence is shown in SEQ ID NO:1.

2. the receptor polypeptides of the gp120 of a people HIV/SIV and/or gp41, its aminoacid sequence is shown in SEQ ID NO:2.

3. the cDNA of the varient of a TNF-α, its nucleotide sequence is shown in SEQ IDNO:3.

4. the varient of a TNF-α, its amino acid is listed as shown in SEQ ID NO:4.

5. the cDNA of the varient of a TNF-α, its nucleotide sequence is shown in SEQ IDNO:5.

6. the varient of a TNF-α, its amino acid is listed as shown in SEQ ID NO:6.

7. targent fused protein is characterized in that described fusion rotein is to comprise and to have the target fusion rotein that kills and wounds polypeptide of cytosis extremely in conjunction with the receptor polypeptides of the gp120 of HIV/SIV and/or gp41.

8. fusion rotein according to claim 7, it is characterized in that described receptor polypeptides is CD4, CD4V1V2 or CD4V1, perhaps their relevant segment, analogue or mutant, the described polypeptide that kills and wounds is the TNF family member who comprises TNF α, TNF β, TNF γ or derivatives thereof or its mutant; Or TNF superfamily member; Or described polypeptide is pore-forming protein, complement component C9 and the particle dissolving element that derives from human body; Perhaps derive from the granzyme A of the granzyme family of human body, B, C, D, E, serine easterase; Or protein kinase C, protein kinase A, E.C. 2.7.2.4., cytopigment or DNA endonuclease; Maybe can resist polypeptide and the enzyme of bacterium, as N,O-Diacetylmuramidase, alexin; Or antioncogene and encoded protein matter thereof; And all derive from the gene with deadly cell function and coded protein or their active function zone, mutant or the analogue of human body.

9. fusion rotein according to claim 8 is characterized in that described TNF superfamily member is Trail, Light or their active function zone, mutant or analogue.

10. fusion rotein according to claim 8 is characterized in that described antioncogene is P53.

11. fusion rotein according to claim 8, the gene order of the described CD4V1 that it is characterized in that encoding is the sequence shown in the SEQ ID NO:1.

12. fusion rotein according to claim 8, the aminoacid sequence that it is characterized in that described CD4V1 genes encoding are the aminoacid sequence shown in the SEQ ID NO:2.

13. according to each described fusion rotein of claim 7-8,11-12, it is characterized in that the described mutant TNF α m1 that polypeptide is TNF α that kills and wounds, its cDNA sequence of encoding is the nucleotide sequence shown in the SEQ ID NO:3.

14. according to each described fusion rotein of claim 7-8,11-12, the aminoacid sequence that it is characterized in that described TNF α m1 genes encoding is the aminoacid sequence shown in the SEQ ID NO:4.

15. according to each described fusion rotein of claim 7-8,11-12, it is characterized in that the described mutant TNF α m2 that polypeptide is TNF α that kills and wounds, its cDNA sequence of encoding is the nucleotide sequence shown in the SEQ ID NO:5.

16. according to each described fusion rotein of claim 7-8,11-12, it is characterized in that the described polypeptide that kills and wounds is TNF α m2, its aminoacid sequence is the aminoacid sequence shown in the SEQ ID NO:6.

17. fusion rotein according to claim 13, the nucleotide sequence that it is characterized in that described fusion rotein are the nucleotide sequences shown in SEQ ID NO:7.

18. fusion rotein according to claim 14, the aminoacid sequence that it is characterized in that described fusion rotein are the aminoacid sequences shown in SEQ ID NO:8.

19. fusion rotein according to claim 15, the nucleotide sequence that it is characterized in that described fusion rotein are the nucleotide sequences shown in SEQ ID NO:9.

20. fusion rotein according to claim 16, the aminoacid sequence that it is characterized in that described fusion rotein are the aminoacid sequences shown in SEQ ID NO:10.

21. comprise the recombinant chou pCW-CD4V1TNF α m1 of the fusion gene of the nucleotide sequence shown in SEQ ID NO:7 or the SEQ ID NO:9.

22. contain the carrier of the described recombinant chou of claim 21.

23. contain the engineering bacteria of the described recombinant chou of claim 21.

24. contain the eucaryon or the prokaryotic cell prokaryocyte of the structure plasmid vector that can express the described fusion rotein of claim 7.

25. cell according to claim 24 is characterized in that described structure plasmid vector is the carrier that contains the described recombinant chou of claim 21.

26., it is characterized in that described cell is an eukaryotic cell according to claim 24 or 25 described cells.

27. prepare the method for the described fusion rotein of claim 8, comprise: with the described expression vector transformed host cell of claim 17, cultivate transformant, obtain in conjunction with the receptor polypeptides of the gp120 of HIV/SIV and/or gp41 and to have the target fusion rotein that kills and wounds polypeptide of cytosis extremely comprising of reorganization.

28. pharmaceutical composition that contains the described fusion rotein of claim 8.

29. the described fusion rotein of claim 8 is used for the treatment of purposes in the medicine of acquired immune deficiency syndrome (AIDS) in preparation.

30. purposes according to claim 29, its by kill be subjected to cell that HIV/SIV infects or in and free HIV/SIV finish.

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