CN1020752C - Vaccines and immunoassays for acquired immune deficiency syndrome - Google Patents

Abstract

The invention relates to recombinant viruses which direct the expression of peptides or proteins similar to epitopes of the adenopathy virus LAV and human T lymphocyte leukaemia virus HTLV III, which is the causative agent of the acquired immunodeficiency syndrome AIDS. These viruses can be formulated in the form of vaccines containing live or inactivated viruses or in polyvalent vaccines for protecting against infection caused by LAV/HTLV III. Peptides and proteins similar to epitopes of this virus are also described. They can be prepared using recombinant DNA techniques or by chemical synthesis and used as immunogens in subunit vaccines or as antigens in diagnostic immunological assays.

Description

Vaccines and immunoassays for acquired immune deficiency syndrome

1. FIELD OF THE INVENTION

The present invention relates to severally can express some peptide and proteinic virus.It is relevant that the antigenic determinant of these peptides and protein and lymphadenopathy virus (LAV) and human body T-chronic myeloid leukemia virus (HTLV-III) and LAS (LAS) and Acquired immunity lack the pathogeny factor of levying (AIDS).

The virus that can express the LAV/HTLV III related peptides that causes protective immune response that the present invention relates to can be used as immunogen and is used at the virus vaccines preparation of LAS or AIDS or the preparation of polyvalent vaccine.In fact the infective virus that the present invention relates to is bred in the host but is not caused disease, thereby the virus vaccines preparation that can be used for living prolongs immunostimulation, and causes actual immunizing power.

The invention still further relates to some peptide and protein relevant with LAV/HTLV III antigenic determinant, peptide that this is specific or protein can be in LAS or AIDS subunit vaccine preparation or polyvalent vaccine preparation as immunogen or be used for the diagnostic immunoassay of LAS or AIDS as antigen.These peptides and protein can obtain by the DNA recombinant technology in any carrier host system or obtain by chemical synthesis process.Thereby, the invention still further relates to structure to DNA new sequences and carrier, these carriers and DNA comprise plasmid DNA and parasitize the viral DNA of human body, animal or insect, perhaps phage, they can instruct LAV/HTLV III related peptides and protein expression in appropriate host, thus can be from corresponding host cell above-mentioned peptide and the protein of purifying.The present invention has also described chemosynthesis LAV/HTLV III related peptides and method of protein.

A specific embodiment of the present invention uses the recombinant chou vaccinia virus to prepare the related protein of LAV/HTLV III capsid or core.Coding LAV/HTLV III capsid glycoprotein or proteic env of nuclear structure or gagDNA order are inserted into respectively can be instructed in the cowpox carrier that LAV/HTLV III gene expresses in appropriate host.The LAV/HTLV III capsid associated protein that produces by recombined vaccinia virus can be used as antigen and immunogen and can induce body fluid or immunity that cell is regulated for the human primate in Asia.The LAV/HTLV III gag related protein that is produced by the recombinant chou vaccinia virus is to have immunoreactive protein, and it has contained the major antigen determinant of eukaryotic protein.

In virus vaccines, the recombined vaccinia virus that can express LAV/HTLV III capsid associated protein can use separately, perhaps uses together with the cowpox recombinant chou of expressing other LAV/HTLV III associated protein (as nuclear structure albumen).On the other hand, the LAV/HTLV III associated protein that produces of recombinant virus can purifying or is synthetic and be used as immunogen in the subunit vaccine preparation with chemical method.Because LAV/HTLV III associated protein is regarded as " exotic " in the host animal body, thereby the immunne response that takes place to be regulated by body fluid and/or cell is to resist this protein or its molectron.Because the existence of this immunne response, utilize suitable vaccine preparation can protect the host avoid the LAV/HTLV III with postoperative infection.

An alternative embodiment of the invention, recombinant baculovirus (Autogroapha californica nu-clear polyhedrosis virus or Ac NPV) is used to prepare the associated protein of LAV/HTLV III capsid and nuclear.The DNA sequence of coding capsid protein or proteic env of nuclear structure or gag is inserted into baculovirus vector respectively, and this carrier can instruct LAV/HTLV III expression of gene in appropriate host.The LAV/HTLV III albumen that recombinant baculovirus produces has antigenicity through radioimmunoprecipitation and ELISA mensuration proof.

The present invention also provides LAV/HTLV III antigenic product, has general significance aspect medical.It comprises uses specific peptide of the present invention and protein in immunoassay as reagent, as using in ELISA test and radioimmunoassay as diagnostic reagent, these experiments can be determined in blood sample, body fluid, the tissue etc. has narrow spectrum antibody to the LAV/HTLV III.In addition, these reagent also are being instruments that use value is arranged aspect the pathogenesis of describing the LAV/HTLV III.

2. background of invention

2.1 HIV (human immunodeficiency virus)

It is that a kind of serious immunodeficiency that causes mainly due to the shortage of patient's cell adjusting immunne response ability is levied that acquired immune deficiency is levied.（Gottlieb，M.et al.，1981，N.Engl.J.Med.305：1425;Masur，J.et al.，1981，N.Engl.J.Med.305：1431）。Now lifting two examples describes this sick clinical symptom: the early stage that (a) is called lymph node syndrome (LAS), it is characterized by chronic lymphoglandula and levy, oligoleukocythemia and peripheral blood helper (OKT4 cell) reduce and to cause the normal circumference helper T lymphocyte and to suppress the lymphocytic ratio of T (OKT4:OKT8) and reverse to 0.1 and further descend with the deterioration of the state of an illness from 2; (b) immunodeficiency symptom is characterized as the minimizing of OKT4 cell and the reverse of normal OKT4 and OKT8 ratio, the repeated infection that the lymphocyte absolute quantity reduces and mainly caused by Pneumocystis carinii; The state of an illness for most of this later stage of case finally causes death.The deterioration phenomenon of lymphoma and Kaposi also appears in some patient.Also there is not method can cure or treat this disease at present.

Suffer from various types of epidemic datas of this disease according to the patient, pathogenesis is a contagium (contagia).The evidence that following three groups provide will prove the paathogenic factor of AIDS effectively, be a kind of retrovirus (retrovirus) that a kind of tropism is arranged for helper T lymphocyte.

(a) R.C.Gallo and work together in national health research institute from AIDS and initial stage AIDS patient isolates a kind of morbific retrovirus of cell (retrovirus) (HTLV III) (Gallo that makes on one's body, R.C.et al., 1984, Science 224:500; Popovic, M.et al., 1984, Science 224:497) they have also detected the antibody of anti-HTLV III in the AIDS patient serum.

(b) L.Montagnier and work together and reveal cervical lymph node disease in Pasteur's Institute from a bit table and the patient who has suffered from the AIDS disease under a cloud isolates a kind of retrovirus (retrovirus) (LAV/HTLV III) of T-lymphatic vessel on one's body.(Barre-Sinoussi, F., et al., 1983, Science 220:868), this group equally also obtains antibody (Kalyansraman, the V.S. of anti-LAV/HTLV III from AIDS patient serum, et al., 1984, Science225:321).Even they also from one because of isolating LAV/HTLV III (Feorino, P.M., et al., 1984, Science 225:69) the lymphocyte of having accepted the patient that donating blood of AIDS patient catch the AIDS disease.

(c) J, Levy and colleagues thereof isolate communicable retrovirus (retrovirus) (term is called sick relevant retrovirus of AIDS or ARV) (Levy, J.A., et al. from AIDS patient's peripheral mononuclear cells, 1984, Science 225:840).

Although three kinds of viruses are separated independently of one another, perhaps they belong to common retrovirus subgroup (subgroup) (Levy, J.A., et al., 1984, Science 225:840), is referred to as the LAV/HTLV III at this.

The general structure of retrovirus is for to be wrapped in by the lipoid layer that comprises capsid outside the core of a nucleoglucoprotein, and capsid formed in the cell blastogenesis stage.What implant capsid and stay the outside is glycoprotein by encoding viral.These materials determined viral host type and and the acceptor of permissive cell appearance produce specificity and react.It is believed that neutralizing antibody combines with capsid glycoprotein and stops interaction between itself and the cell surface receptor.(PP.534-535, The Molecular Biology of Tumor Viruses, ed.J.Tooze, 1973, Cold Spring Harbor Laboratory; PP.226-227 and 236-237 in, RNA Tumor Viruses, ed.R.Weiss, N.Teich, H.Varmus, and J.Coffin, 1982, Cold Spring Har-bor Laboratory.) under LAV/HTLV III particular case, evidence suggests, be present in the lymphocytic T of a class T ₄Antigen is the component part of Virus receptors or acceptor.（Dalgeish，A.G.，et al.，1984，Nature 312：763;Klatzmann，D.，et al.，1984，Na-ture 312：767）。

Fig. 1 represent for the rna gene group of LAV/HTLV III.Wherein there are three genes usually to mention.The internal structure albumen (core protein) of gag genes encoding virus is also determined viral generic specificity antigen.

The virus particle that the pol genes encoding is relevant with ThermoScript II.Env genes encoding viral glycoprotein.This genome of region representation with sor and 3'-orf mark partly comprises open reading frame, but its function is not also understood at present.

2.2 vaccine

Make at present in many ways with prevention and treatment virus infection.Comprise that use can cause vaccine, chemical agent and the Interferon, rabbit of immunne response.The method of traditional preparation process vaccine has the inactivation of viruses of use or uses two kinds of attenuated virus.Inactivation of viruses is that a kind of harmless biological agent has kept the ability that causes immunity simultaneously again.These " killed " virions cause the infection that after this immunne response is drawn by live virus with opposing after injecting the host.Yet crucial can not accomplish to kill all virions when being to use inactivated vaccine, even and if can accomplish this point because the virus of deactivation is irreproducible in the host, the immunization time is too short, often needs to repeat vaccination.In addition, inactivation process may change viral protein and immunocompetence is descended.

Morbific ability that virus strain has been passed through perform toxic attenuation effect substantial loss.A kind of method that makes viral attenuation is to make the virus growth and/or the cultivation of going down to posterity continually in cell cultures under unusual condition.Select the virus mutant that virulence lowers then, this mutant has kept the ability of induction of immunity reaction.It can be bred in the host and make the immunization time remaining for a long time because of attenuated virus, so often more satisfactory as immunogen.But be to use vaccine alive also to have some problems, wherein the most worrying is whether the reduction of virulence is enough.

Adopt the way and the preceding method of subunit vaccine to contrast.This method relates to the immune response that the proteins react of immunology related substances is only arranged with those.Have concerning many that the glycoprotein of encoding viral contains the antigenic determinant that can induce neutralizing antibody the capsid virus.For example, the glycoprotein of LaCrosse virus (Gonzalez-Scarano, F., Shope, R.E., Calisher, C.E.and Nathanson, N., 1982, Virobogy 120:42.), neonatal calf diarrhea virus (calf Diarrhea virus) (Matsuno, S.and Inouye, S., 1983, Infection andImmunity 39:155) venezuelan equine encephalomyelitis virus (Mathews, J.H.and Roehrig, J.T., 1982, JImm.129:2763), Punta Toro virus (Dalrym-ple, J.M., Peters, C.J., Smith, J.F andGentrg M.K., 1981, ln " duplicating of negative strand virus ", D.H.L.Bishop and R.W.Compans, eds., P.167.Elsevier, New york), mouse white corpuscle virus (steeves, R.A., strand, M.and Augst, J.T., 1974, J.Virol.14:187), and mouse mammary tumor virus (Massey., R.J.and Schochetman, G., 1981, Virology 115:20). an advantage of subunit vaccine is for having got rid of incoherent viral material.

Vaccine often is aided with adjuvant.Adjuvant helps to improve tolerance and immune level, and the immunogenic dosage of use will lack when using more separately.The mechanism of action of adjuvant is complicated, and is also not known fully.Its effect may postpone the release of antibody and the degraded of antibody simultaneously for stimulating the activity of phagolysis and endoplasmic reticulum system.The example of adjuvant have following these, Freund's adjuvant (fully or not exclusively), adjuvant 65 (contains peanut oil, mannide-olein and aluminum stearate), Propiram Ni Keduo alkylol cpd L-121, Avridine, and the inorganics gel of aluminium hydroxide or aluminum phosphate or alum etc.The Freund's adjuvant has not re-used the mankind, becomes a kind of potential carcinogens because of it contains non-biochemical metabolism mineral oil.

2.2.1 recombinant DNA technology and vaccine virus

The recombinant DNA technology that is used for producing subunit vaccine comprises that molecular cloning and clone's virogene can cause the protein that the immunity neutralization is replied in suitable carriers is expressed in the host animal body.And any genetic information of having got rid of other the above-mentioned particular proteins of non-expression, the host is not subjected to the influence of complete virus because the danger that does not have to infect.

Recently, the someone proposes a kind of for preparing the method that subunit vaccine is very useful.(Mackett, M., Smith, G.L.and Moss, B., 1982, ProcNatl.Acad.Sci79:7415-7419; Mackett, M., Smith, G.L.and Moss, B., 1984, J.Virol.49:857-864; Panicali, D.and Paoletti, E., 1982, Proc.Nat'l.Acad.Sci.79:4927-4931). this method comprises uses vaccinia virus as carrier, external gene is inserted its karyomit(e), and make it to express particular proteins.The vaccinia virus of reorganization imports host animal after inserting alien gene, makes the host produce immunne response for the product of recombinant gene expression.Because the recombined vaccinia virus of living can be used as vaccine, so this method has possessed the common advantage of subunit vaccine and living vaccine.

Vaccinia virus comprises the anti-chain DNA karyomit(e) of a linearity, have about 187 kilobasas to and in the tenuigenin of infected cells, duplicate.In these viral cores, comprise a complete transcriptase system (comprising that cap forms, and methylates and the poly adenase).These systems cause that virus infection is necessary.After having lost nuclear, these viruses just lost infection ability.Vaccinia virus transcribe that control sequence (promotor) can be transcribed by the rna polymerase promoter of cowpox but can not be by the rna polymerase promoter of host cell.

Have only when the cowpox promotor is bonded on the encoding histone dna sequence dna of alien gene, the foreign DNA of recombined vaccinia virus could be expressed.Plasmid vector also is called the insertion carrier and is fabricated, and mosaic gene is inserted vaccinia virus.

One type insertion carrier is made up of following each several part, (a) promotor that contains the vaccinia virus of transcription initiation site; (b) the restriction endonuclease cloning site that is positioned at the downstream of transcribing the beginning site of several uniquenesses is to insert exogenous dna fragment.(c) be positioned at the lateral nonessential vaccinia virus DNA of promotor and cloning site, (as the TK gene), can guide mosaic gene to insert virus genomic corresponding non-important area, (d) bacterium replication origin and the antibiotics resistance mark that duplicates and select in intestinal bacteria.Mackett is described this type of carrier example.（Mackett，M.，Smith，G.L.and Moss，B.，1984，J.Virol.49：857-864）。

Recombined vaccinia virus inserts plasmid by the recombinant bacteria that will contain foreign gene and adds in advance by the cell of vaccinia virus infection, obtains through the transfection effect.In infected cells homologous recombination taking place and makes allogenic gene insert viral genome.Infected cells can be passed through immunological technique, DNA plaque hybridization or the selection of subsequently can isolating recombinant virus carrying out gene sifted out.These cowpox recombinant chous have kept their basic function and infectivity and can hold the foreign DNA of about 35,000 bases.

Expression of exogenous gene can be measured (as immuno-precipitation, radioimmunoassay or immunoabsorption) by enzyme process or immunoassay.The membrane glycoprotein of the natural appearance of infecting through recombinant vaccinia of cell synthetic is through glycosylated, and may be transported to cell surface can carry out the method that multiple copied is cloned by the method for use strong promoter or to an independent gene, obtains the expression of higher degree.

2.2.2 recombinant DNA technology and baculovirus

The someone proposes a kind of method recently, has very big meaning for the preparation of recombinant protein.(Pennock et al., 1984, Mol.cell Biol.4:399; Smith et al., 1983, J.Virol.46:584) this method is used baculovirus vector to express and is inserted its genomic foreign gene.After this baculovirus recombinant chou imports suitable host cell, just can express its foreign gene.

The archetype of baculovirus is Autogroapha californica nuclear polyhedral virus (Ac NPV).Ac NPV genome comprises a double-stranded cyclic DNA that contains 128 kilobasas, this genomic gene map has several restriction sites (Smith, G.E.and Summers, M.D., 1978, Virology 89:517) virus is duplicated in the nuclear of infected cells.Produce the promptly inaccessible or non-inaccessible virion of two kinds of morphology of virus behind the wild-type Ac NPV cells infected.Inaccessible virion is called polyhedral albumen parcel by a kind of, and this polyhedron albumen is encoded by polyhedron gene.(consulting Virol.131:561-565,1983). the virion of non-obturation can be observed from cells infected at an easy rate by opticmicroscope.

When the promotor of recombinant baculovirus with after the dna sequence dna of the proteins encoded of foreign gene is connected, the foreign DNA of this recombinant baculovirus could be expressed.Plasmid vector also is called the insertion carrier and is fabricated, and will embed gene and insert Ac NPV.One type insertion carrier is made up of following each several part, (a) an Ac NPV promotor that contains transcription initiation site; (b) being positioned at of several uniquenesses transcribed the limiting acid endo enzyme cloning site that begins the downstream, site, to insert exogenous dna fragment.(c) be positioned at lateral nonessential Ac NPVDNA(of promotor and cloning site such as polyhedron gene) can insert viral karyomit(e) by guiding gene.(d) bacterium replication origin and the antibiotics resistance mark that duplicates and select in intestinal bacteria.People such as Miyamota are described the example of examples of such carriers.（1985，Mol.Cell.Biol.5：2860）。

Recombinant baculovirus can obtain by the recombinant bacteria plasmid and the common transfectional cell of baculovirus DNA that will insert foreign gene.Homologous recombination takes place in infected cells and make foreign gene can insert viral genome.In case foreign gene has inserted polyhedron gene, just can not produce inaccessible virion again, thereby can come out the reorganization plaque select by the way of observing the plaque that produces owing to the shortage occlusor.Infected cells also can be passed through immunological technique, DNA plaque hybridization or subsequently the method for the gene Selection of isolating recombinant virus screen.The recombinant chou of baculovirus has kept their basic functions and infection ability.

Expression of exogenous gene can detect (for example immuno-precipitation, radioimmunoassay, or immunoblotting) by enzyme process or immunoassay.By using strong promoter or, can improving the degree of expression to the multiple copied cloning process of individual gene.

3. summary of the invention

Instruct the peptide relevant or the virus of protein expression to be narrated with LAV/HTLV III antigenic determinant.The present invention expresses the recombinant virus that can induce the antigenic determinant of protective immune response relevant with the LAV/HTLV III, can be made into the virus vaccines preparation, is used for protecting people to resist the infection of LAV/HTLV III.In the embodiments of the invention, be used among the host not pathogenic but in infected host, can express infective virus that has of the antigenic determinant relevant with the LAV/HTLV III, the preparation of the virus vaccines that preparation is lived.

The present invention also relates to relevant peptide with the antigenic determinant of LAV/HTLV III or protein.This induces the material that produces the protection immunne response to can be used in subunit vaccine or polyvalent vaccine protects people in case the infection of LAV/HTLV III.In addition, peptide of the present invention or protein can be used the diagnostic detection of what AIDS or LAS.Peptide of the present invention or protein can produce in any host cell expression carrier system and can be separated, and these comprise, as by the animal of suitable recombinant virus infection or insect cell; By recombinant plasmid, the microorganism of the bacterium class of coemid or phage transfection is by the yeast of recombinant plasmid transformed.The present invention provides method simultaneously, and technology and being used to is expressed the structure of the DNA of coding LAV/HTLV III epitopes gene information.In addition, polypeptide and albumen mass-energy involved in the present invention are synthesized by chemical method.

(see embodiment for details) in embodiments of the invention coding LAV/HTLV III capsid glycoprotein or the proteic gene order of core texture (being exactly the env or the gag gene order of LAV/HTLV III) is inserted in the plasmid so that the vaccinia virus promotor is positioned at the 5' position of the initial methionine(Met) sequence (ATG) of LAV/HTLV III gene order, makes the establishment of inserting gene be positioned at the side of cowpox thymidine kinase (TK) dna sequence dna.These plasmid transfections are to going in the cell with the wild-type vaccinia virus infection in advance, and like this, the chimeric LAV/HTLV III env of the insertion of side or the TK gene that the gag gene is recombined to vaccinia virus go in proper order to allow to be arranged in TK.These cells dissolved and cause virus on the TK cell, form plaque.Recombinant virus forms the plaque ability by them and selects on these cells in the presence of the 5-bromodeoxyuridine, also can by it with carry out DNA-DNA as LAV/HTLV III capsid or LAV/HTLV III gag sequence through suitable radiolabeled probe and hybridize and select.Just hybridizing plaque and be purified expansion, producing LAV/HTLV III capsid glycoprotein or the proteic ability of core texture thereby recombinant virus is used to measure them.The protein proof of expressing by recombined vaccinia virus relevant with LAV/HTLV III capsid has antigenicity and immunogenicity, and has the ability that causes in the inferior human primate that body fluid and cell are regulated immunity.Produce by recombined vaccinia virus with LAV/HTLV III gag proteins associated matter be the immunoreactive protein that has that contains real core protein major antigen determinant.

In the another embodiment of the present invention, LAV/HTLV III env or gag are inserted in proper order and go in the plasmid so that the baculovirus polyhedron promoter can be positioned at the 5' position of the initial methionine(Met) order (ATG) of LAV/HTLV III gene behind the 3' end polyhedron DNA.These plasmids go to cell with the shaft-like DNA cotransfection of wild-type, make chimeric LAV/HTLV III gene be arranged in and are recombined to the other Ac NPV side in proper order that the baculovirus polyhedron gene goes.Cell is dissolved and form virus plaque, owing to there is not the recombinant virus of capsid, and available range estimation screening or carry out the DNA-DNA screening by hybridization with radiolabeled LAV/HTLV III env or LAV/HTLV III gag probe.The reorganization plaque is purified and enlarges, and the recombinant virus of generation screens recombinant virus by the immuno-precipitation of generation LAV/HTLV III associated protein ability and the method for sds page analysis.The infected cells solute detects it surveys LAV/HTLV III antibody in serum ability with the ELISA method.

4. description of drawings

Fig. 1 represents the provirus genome structure of complete LAV/HTLV III.Hatching partly is open reading frame.The narrow spectrum antigen of this regional code group, ThermoScript II and capsid protein, respectively by gag, pol and env representative.Cross-hauling is partly represented overlapping open reading frame.Numeral expression on the figure is from the base pair number as the cap number of sites downstream of virus transcription starting point.Restriction site Bg, the Bgl II; Ec, the EcoR I; Hn, the Hind III; Kp, the Kp-n I; Ss, the Sst I is come mark.

Fig. 2 is illustrated in the Nucleotide program in the LAV/HTLV III specificity zone (EcoRT to Sstl) among the plasmid pRS-3DNA, and complete LAV/HTLV III capsid gene (nucleic acid 5766 to 8349) is included in the insertion portion of LAV/HTLV III.Be used for PV-env1, the restriction site during PV-env2 and PV-env5 make up indicates.Also indicated complete capsid gene amino-acid sequence simultaneously by the nucleotide sequence data-speculative.

Fig. 3 diagrammatizes the structure of the plasmid that includes a LAV/HTLV III capsid protein coded sequence, and this capsid protein coded sequence is connected on the downstream of a vaccinia virus promotor.The box indicating of LAV/HTLV III capsid encoding sequence, the dark-coloured box indicating of cowpox promotor.The nucleotide sequence of the connection portion of cowpox promoter region and LAV/HTLV III capsid coded sequence is represented in the below of figure.The initiator codon and the reading frame of the mosaic gene of line part representative supposition.Three and the tetramino acid of attention in the translation order (Pro-Val) of reorganization pv-env2 is corresponding with the 43 and 44 amino acid of LAV/HTLV III capsid encoding sequence.

Fig. 4 diagrammatizes the structure of the plasmid that contains the complete LAV/HTLV III capsid protein coded sequence that is inserted in a vaccinia virus promotor downstream.The dark-coloured box indicating of vaccinia virus promotor.The plasmid pv-env5 that contains complete LAV/HTLV III capsid coding region is that two steps of branch are set up.The 5' of coding region and 3' partly are cloned into respectively at first and are formed on aminoterminal among the pGS20 is the pv-env1 of LAV/HTLV III capsid gene and the pv-env2 that comprises LAV/HTLV III capsid gene at carboxyl terminal.As shown in the figure, this two part is in Stu I position recombine.

Fig. 5 graphic representation lacks the vaccinia virus promotor downstream that is inserted in of conversion film order, includes the plasmid structure of LAV/HTLV III capsid protein encoding sequence.Plasmid pv-env7 is set up in two steps.The 5' of pv-env5 partly is inserted into and forms a middle interstitial granules-pv-env5/26 among the P26, and interstitial granules provides an assembling translation termination order (TAA) to form the env gene of a brachymemma in this.Then with the brachymemma of TAA terminated env be inserted into pG20 in proper order to set up pv-env7, wherein cowpox TK gene order is positioned at the side of the env gene of brachymemma.

Fig. 6 represents the structure and the selection of vaccinia virus recombinant.Solid line is represented thymidine kinase (TK) gene of vaccinia virus.This TK gene also is present among the plasmid pv-env5DNA, still the involved insertion gene disruption that cowpox 7.5K promotor (dark-coloured square frame) and LAV/HTLV III capsid coding region (blank square frame) are arranged of TK gene in plasmid.After cell was infected by cowpox, the recombinant plasmid that contains the TK gene that is interrupted by LAV/HTLV III capsid gene was directed to the cell that has infected.So insert the lateral TK of gene and occurred in sequence reorganization and LAV/HTLV III capsid gene is imported the vaccinia virus genome being positioned at.The virus that contains LAV/HTLV III capsid gene that reorganization produces is TK ^-.

Fig. 7 represents to contain the feature of the genome structure of LAV/HTLV III env gene recombination vaccinia virus.

Fig. 7 A represents the Restriction Enzyme analysis of cowpox recombinant chou v-env5 and v-env5NY and relevant parent's cowpox strain (WR and V-NY) thereof.The DNA fragment that is produced by the hydrolysis of Restriction Enzyme Hind III coagulates in the electrophoresis separated at agarose, demonstrate colour band behind ethidium bromide staining.

Fig. 7 B represent by the restriction fragment of separating is transferred on the nitrocotton and with LAV/HTLV III capsid is had in proper order the Southern blot that obtains behind the probe hybridization of narrow spectrum nick translation.

Fig. 8 A represents being analyzed by the Western immunoblot that cowpox-LAV/HTLV III env recombinant chou expressed protein is carried out.The protein in following source on 7-15% gradient SDS-polyacrylamide gel, separate and electrotransfer to nitrocellulose paper: LAV/HTLV III viral protein (LAV/HTLV III); The cell of wild-type vaccinia virus infection (WTvv); Non-infected cells (mock); By recombinant virus v-env2(v-env2), or v-env5(v-env5) cell that infects.Protein can detect by peroxidase and anti-IgG antibodies reaction way for AIDS patients serum's immunoreactivity.Gp150, gp110 and gp41 represent the product of LAV/HTLV III env gene.Molecular weight is represented with kilodalton.

The Western immunoblot that Fig. 8 B is illustrated in two kinds of cells by cowpox-LAV/HTLV III env recombinant chou expressed protein analyzes.Deutero-protein reacts to nitrocotton paper and with the pooled serum that comes from LAV/HTLV III seropositivity individuality with SDS-polyacrylamide gel electricity arteries and veins (SDS-PAGE) separation and electrotransfer from BSC-40 or Hela cell.

The cell of hurdle 1 and 5 expression simulated infections; The cell of hurdle 2 and 6 wild-type vaccinia virus infections; The cell that hurdle 3 and 7 expressions are infected by v-env5, the cell that hurdle 4 and 8 expressions are infected by v-env2.Gp150, gp110 and gp41 represent LAV/HTLV III-env gene product.In order to ¹²⁵The albumin A of I mark is measured immunoreactive protein matter.

Fig. 9 A represents the result with the expressed albumen radioimmunoprecipitation assay of the cell of wild vaccinia virus (WTvv) or recombined vaccinia virus (v-env2 and v-env5) infection.Protein is used after infecting 10-12 hour ³⁵The S-methionine mark.Human serum of labelled protein and contrast (N) or AIDS patients serum (I) reaction in the cellular lysate, immunocomplex is precipitated by streptococcus aureus (Staphylococcus aureus) albumin A.The albumen of immunoprecipitation separates in the 15%SDS-polyacrylamide gel electrophoresis, and detects with photofulorography.Molecular weight is represented with kilodalton.

Fig. 9 B represent for cowpox of the present invention-LAV/HTLV III recombinant chou expressed with ³The protein of H-glucosamine mark carries out the result of radioimmunoprecipitation assay.No matter the Hela cell is to simulated infection (hurdle 1 and 5), or by the vaccinia virus infection of wild-type (hurdle 2 and 6) or by v-env5(hurdle 3 and 7) or v-env2(hurdle 4 and 8) all using of infecting ³H-glucosamine mark.Pooled serum (hurdle 5-8) by adding normal human's serum (hurdle 1-4) or the seropositive individuality of LAV/HTLV III and albumin A make the molten born of the same parents' thing of cell generation immunoprecipitation.Immunoprecipitation albumen separates with SDS-PAGE again.

Fig. 9 C represents with the analytical results of " pulse-chase " radioimmunoprecipitation for the LAV/HTLV III capsid protein of being expressed by cowpox-LAV/HTLV III recombinant chou.The Hela cell infects with wild-type vaccinia virus v-env5 or v-env2, ³⁵The S methionine mark, and with following the trail of the substratum cleaning.In the following timed interval, cell is cleaned, and dissolving with the pooled serum generation immunoprecipitation of LAV/HTLV III seropositivity individuality, and separates with SDS-PAGE: 0 hour ( hurdle 1,7,13); Hour 0.5 ( hurdle 2,8,14); 1 hour ( hurdle 3,9,15); 2 hours (hurdle 4,10,16); 6 hours ( hurdle 5,11,17) and 12 hours ( hurdle 6,12,18).Hurdle M provides and uses C ¹⁴The mark of the molecular weight standard of mark.

Fig. 9 D is provided in the cell and reaches in the radioimmunoprecipitation assay result who is undertaken by the albumen relevant with LAV/HTLV III capsid in the substratum of the cell of recombinant vaccinia of the present invention-LAV/HTLV III virus infection.No matter the Hela cell simulated infection (hurdle 1), or wild-type vaccinia virus (hurdle 2), v-env5(hurdle 3) or v-env2(hurdle 4) infect, all use ³⁵The S methionine(Met) comes mark.Cell is separated from substratum and is molten broken.The pooled serum generation immunoprecipitation of molten born of the same parents' thing of cell (pellet) and substratum (supe) each and LAV/HTLV III seropositivity individuality.Immunoprecipitation albumen separates with SDS-PAGE.Fig. 9 E provides reach the radioimmunoprecipitation assay result who is undertaken by the albumen relevant with LAV/HTLV III capsid in the substratum of the cell of recombinant vaccinia of the present invention-LAV/HTLV III virus infection in cell.Reorganization v-env comprises complete LAV/HTLV III shell coded sequence, and the LAV/HTLV III capsid coded sequence that v-env7 comprises shortage is striden film (anchor) zone.No matter the Hela cell simulated infection (hurdle 1), or by wild-type vaccinia virus (hurdle 2), v-env5(hurdle 3) or v-env2(hurdle 4) infect, all use ³⁵The S methionine(Met) comes mark.Cell is separated from substratum and is molten broken.The pooled serum that molten born of the same parents' thing of cell (pellet) and substratum (supernatant liquor) add LAV/HTLV III seropositivity individuality makes it to take place immunoprecipitation.Immunoprecipitation albumen separates with SDS-PAGE.

Figure 10 provides the Western immunoblot that carries out for the serum sample through the mouse of recombinant vaccinia-LAV/HTLV III virus immunity to analyze.Mouse is by v-env5 or the immunity of v-env2 recombined vaccinia virus.After 8 week, use separated by SDS-PAGE and electrotransfer to the LAV/HTLV III viral protein on the nitrocellulose paper react with serum sample.The anti-rat immune globulin of goat is attached on the phospholipase of alkalescence, in order to measure the LAV/HTLV III albumen that those can be discerned by mouse serum.Hurdle a to e provides the individual serum sample that takes out of mouse of having inoculated v-enV5 from five, and hurdle f to k then provides the serum sample of having inoculated the mouse individuality of v-enV2 from five.The pooled serum that obtains from the C57B16J mouse (NMS) to LAV/HTLV III seropositivity individuality (AIDS) and non-immunity is assigned to as positive and negative contrast.LAV/HTLV III capsid glycoprotein gp150, the position of gp110 and gp41 is pointed out.

Figure 11 provides the histogram of the seroconversion of the macaque that has inoculated recombined vaccinia virus v-env5.Four monkeys are scratched method inoculation 2 * 10 with skin ⁸The v-env5 of pfu in addition four then inoculate 2 * 10 ⁷The v-env5 of pfu.An animal is inoculated 2 * 10 ⁷The simple gD recombinant chou (v-HSVgD1) of pfu contrast cowpox bleb. inoculate after ten weeks for the first time, except that animal NO.81, award all animals and use the second time 2 * 10 ⁸The inoculation of the same virus of pfu.Gather inoculation preceding (blank square frame); The four stars phase behind the primary vaccination (shade line square frame); Serum sample with the four stars phase (filled box) after the inoculation for the second time.Adopt the LAV/HTLV III virus of purifying to measure seroconversion by ELISA as the method for target antigen.

Figure 12 provides a Western IDA that carries out for the macaque serum sample through cowpox-LAV/HTLV III recombinant virus v-env5 immunity of as above Figure 11 description.Gather before the primary vaccination (hurdle pre) and inoculate four stars after date serum sample the second time.React with LAV/HTLV III virion protein behind diluted 50 times of the serum, this virion protein separates and is fixed on the nitrocellulose membrane by electrotransfer through SDS-PAGE, and electrotransfer is with two kinds of capsid glycoprotein of the most effective a kind of mensuration gp110(Figure 12 A) and gp41(Figure 12 B) method.The LAV/HTLV III albumen of being discerned by macaque serum is by measuring anti-human immunoglobulin of goat and alkaline phosphatase bonded method.Pooled serum from LAV/HTLV III seropositivity individuality (AIDS) is used for positive control.Real LAV/HTLV III virus particle (AIDS) is used as a kind of contrast.

Figure 13 has described by cowpox NY-LAV/HTLV III recombinant virus, the Western IDA of the chimpanzee serum sample of v-env5NY immunity.Two chimpanzees are with 5 * 10 ⁸The v-env5NY of pfu carries out subcutaneous injection, and another animal is injected with the v-HSVgD1NY of same dosage simultaneously, and it is the single viral gD recombinant chou of setting up from homology cowpox-NY of cowpox bubble rash.All animals have carried out injection for the second time in eight weeks behind initial injection.Collected immune preceding serum sample (hurdle 1), the serum solution sample in two weeks (hurdle 3) after eight weeks behind the initial immunity (hurdle 2) and the immunity for the second time.Serum dilute after 50 times take out partly with separate by SDS-polyacrylamide gel electricity arteries and veins and and be fixed on LAV/HTLV III virion albumen test on the nitrocellulose membrane through electrotransfer, adopt best to the gp41 detection scheme.The goat-anti human immunoglobulin detection of protein by the LAV/HTLV III of chimpanzee serum identification by linking to each other with alkaline phosphatase.The serum of collecting LAV/HTLV III seropositivity individuality is as positive control.

Figure 14 has described the nucleotide sequence in the single-minded zone of LAV/HTLV III (SstI to KpnI) that occurs in plasmid pKS-5DNA, whole LAV/HTLV III gag gene (Nucleotide 340 to 1871) is included in the LAV/HTLV III inset.The restriction site that is used for the pA-gag1 establishment is marked.The whole amino-acid sequence that the data of nucleotide sequence can be reasoned out from the gag gene is also illustrated.

Figure 15 is that the plasmid pAC-gag1 that comprises the LAV/HTLV III gag protein code order in whole insertion AcNPV promotor downstream makes up diagram.The pAC610 cloning vector comprises a nucleotide sequence corresponding with the polyhedron gene promotor simultaneously, and contains the 5' leader sequence and be positioned at the 3' polyhedron order that the cloning site of transcription initiation site downstream portion interrupts.The cloning site that is positioned at position-8 is: EcoR I, Sst I, Sma I (Xma I), BamH I, Xba I and Pst I.The Sal I, AccI and Hinc II are not single.There are not Kpn I or Bgl II site.

Figure 16 has described reorganization AcNPV(Ac-gag1) establishment and screening.The box indicating polyhedron promoter of band shade and the polyhedron gene that 5' leader sequence filled box is represented baculovirus.Portion gene is present among the plasmid pAc-gaglDNA, is blocked by LAV/HTLV III gag password area (hollow square frame).The recombinant plasmid dna and the common transfectional cell of wild-type AcNPV DNA that comprise the part of the polyhedron gene that is interrupted by LAV/HTLV III gag gene (pAc-gag1).The reorganization that occurs in the polyhedron order of inserting the other position of gene side imports LAV/HTLV III gag gene order in the AcNPV genome.

Figure 17 has described from the Northern spot analysis of the RNA of the Spodoptera frugiperda cell extraction that is infected by the Ac-gag1 of wild-type AcPNY and use LAV/HTLV III gag specific molecular probe.

Figure 18 has described the result of a kind of radioimmunoprecipitation assay of expressed protein in the cell that the baculovirus Ac-gag1 that is recombinated infects.Respectively in back 24 hours of infection (hurdle 1,2), when 48 hours (hurdle 3,4) and 72 hours (hurdle 5,6) with [ ³⁵S] methionine mark protein 2 hours.Labelled protein in the cell lysis thing reacts with the human serum ( hurdle 1,3,5) or the AIDS patients serum ( hurdle 2,4,6) that compare usefulness, and with streptococcus aureus (Staphylococcus aureus) albumin A immunocomplex is precipitated.The albumen of immunoprecipitation carries out electrophoretic separation with the 10%SDS polyacrylamide gel, and available photofluorography is measured.Molecular weight unit is that kilodalton calculates.

Figure 19 provides proteinic " pulse-chase " radioimmunoprecipitation assay result for LAV/HTLV III gene-correlation.This LAV/HTLV III gene-correlation protein is taken from the cell that AcNPV infects of recombinating through the present invention.Spodoptera frugiperda(sf9) cell infected by Ac-gag1 and back 24 hours of infection with pulse labeling method mark 5 minutes.Cell is cultivated 2 hours (hurdle 3,4), 4 hours (hurdle 5,6) and 8 hours (hurdle 7,8) with the perfect medium washing and with complete culture medium culturing 0 hour (hurdle 1,2) then.During these, cell experiences washing respectively, and molten born of the same parents reach and pooled serum or normal human's serum ( hurdle 1,3,5,7) of LAV/HTLV III seropositivity individuality ( hurdle 2,4,6,8) carry out the immunoprecipitation process.Protein separates with SDS-polyacrylamide gel electrophoresis process.

Figure 20 illustrates five kinds and comprises the structure that is attached at the plasmid on the vaccinia virus 7.5K promotor with the LAV/HTLV III genome that contains all lengths of gag coded sequence, and these five kinds of plasmids are pv-gag1, pv-gag2, pv-gag3, pv-gag4 and pv-gag5.The pGS62 cloning vector that uses in these structures and pGS20 similar substantially (referring to Fig. 3), different is the EcoRI site that has a uniqueness in the downstream, SmaI position of pGS20.

Figure 21 represents for each by recombinant vaccinia LAV/HTLV III virus (v-gag1NY, v-gag2NY, v-gag3NY, v-gag4NY and v-gag5NY), carry out the Western immunoblot of expressed protein in the cell that parent vaccinia virus (v-NY) and simulation (MOCK) cells infected simulation (MOCK) is infected) result that analyzes.LAV/HTLV III virus particle (LAV/HTLV III) sample of purifying can be used as positive control.It is 1/10th infected that the BSC-40 cell that merges has.Infection can continue 12 hours, and collecting cell is gone molten broken more during this period.The albumen of all cells is separated by SDS-PAGE, is fixed on the soluble cotton by electrotransfer.Film can react with following substances:

(1) AIDS patients serum (Figure 21 B) can measure it with the anti-human normal immunoglobulin of the goat that combines alkaline phosphatase.

(2) can detect it with the anti-rat immune globulin of the goat that is incorporated into alkaline phosphatase at the mouse monoclonal antibody that is defined as gag albumen p25 and p18 (Figure 21 A and 21C).

Figure 22 represents the structure of plasmid pAc-env5, and this plasmid includes the coded sequence of the complete LAV/HTLV III shell that is inserted in AcNPV polyhedron promoter downstream.Figure 15 has represented the pAc610 cloning vector the preceding.

Figure 23 represents the analytical results that carries out for albumen expressed in the cell that has infected recombinant baculovirus Ac-env5 with radioimmunoprecipitation.Cell of simulated infection (MOCK) and parent baculovirus strain (AcNPV) are listed among the figure in contrast.To the expressed albumen of infected sf9 cell, after infecting 28 hours, use ³⁵ S methionine mark 2 hours.Labelled protein in the molten thing of the born of the same parents of cell and AIDS patients serum or at the reaction of the mouse monoclonal antibody of gp110 or gp41 precipitate immunocomplex with streptococcus aureus (Staphylococcus aureus) albumin A.Immunoprecipitation albumen separates with SDS-PAGE and detects with photofluorography.

5. invention is carefully stated

Present invention is directed at and produce peptide or the proteic virus relevant with LAV/HTLV III antigenic determinant.Also be directed to simultaneously these peptides or the albumen that can adopt DNA recombination method or chemical synthesis to produce.Among the present invention these may cause the virus of protective immune response or peptide, albumen, can be as comprising in the various vaccine preparations that immunogen in the polyvalent vaccine preparation is with the infection of the cause of disease material LAV/HTV III that prevents LAS and AIDS disease.In addition, peptide that the present invention relates to or albumen can be used as the specific antibody that detects LAV/HTLV in the patient body in the immunology diagnosis analytical method.Peptide that immunological method of the present invention is used or albumen should have antigenicity (can with patient LAV/HTLV III antibody response); but needn't possess immunogenicity (can cause immunne response) in addition, the antigen in the immunology diagnosis also needn't cause the protective immune response reaction in vivo.

An embodiment has been to use the DNA recombinant technology among the present invention, and the nucleotide sequence of coding LAV/HTLV III antigenic determinant is inserted in the expression vector, and this carrier instructs the expression of LAV/HTLV III order in appropriate host cell.These expression vector one host cell systems can be used for external produce polypeptide or the albumen relevant with the LAV/HTLV III, and like this, gene product just can be come out by purifying from cell culture.Can cause that the peptide of protective immune response or albumen can be used as the immunogen in the subunit vaccine preparation.Moreover, immunogenicity is arranged among the present invention or only have antigenic peptide and albumen just can detect the specific antibody of LAV/HTLV III in the patient body as the antigen in the immunodiagnosis analysis.Another layer meaning of producing about peptide and albumen among the present invention is: by the nucleotide sequence that comprises in antigen expressed and/or immunogenic LAV/HTLV III related peptides and the proteic recombinant chou, thereby can infer the amino-acid sequence of peptide and protein product.These peptides and albumen can be synthetic with chemical method, and are used for subunit vaccine preparation (if immunogenicity is arranged), or as the antigen (if tool antigenicity and/or immunogenicity) in the immunodiagnosis.

When virus as expression vector, when instruct expressing the immunogen relevant, virally itself just can constitute vaccine with the LAV/HTLV III antigenic determinant that can cause the defensive immunity reaction.Can be used to prepare the living vaccine that the entity immunity is provided to the non-pathogenic infectious recombinant virus of host.Perhaps, can prepare the virus vaccines of deactivation by " killing " virus.In addition, can also prepare the polyvalent vaccine that contains two or more LAV/HTLV III epitopes, or all be a LAV/HTLV III antigenic determinant, and the antigenic determinant of other pathogenic things all can prepare.

Just convenient for narration, method of the present invention can be divided into following several stages: (a) gene or the broken segmental separation of gene of coding LAV/HTLV III viral protein.(b) gene or gene fragment are inserted expression vector.(c) identification and expression vector are grown in the host, and expressing gene can be in host system duplicates expression.(d) mensuration of the evaluation of gene product and purifying (e) product immunological competence.(f) vaccine preparation.

Described the structure of recombined vaccinia virus and baculovirus in the inventive embodiments, these viruses contain LAV/HTLV III capsid gene, instruct and the Ia protein expression of LAV/HTLV III capsid protein in by the tissue culture cells of recombinant virus infection.In addition, the recombined vaccinia virus that contains LAV/HTLV III gag gene and the structure of baculovirus have been described among another embodiment of invention.Gag gene is wherein instructing in the tissue culture cells that is subjected to recombinant virus infection and the relevant protein expression of LAV/HTLV III nuclear structure protein immunization.Yet composition described herein, method is not limited in the proteic recombinant virus of expressing LAV/HTLV III capsid or gag gene-correlation and makes up, the structure that also can be used for any expression vector system recombinant chou produces and the relevant polypeptide of the sick any pathogenic thing antigen of AIDS.

For the purpose of clear, entire method will be from LAV/HTLV III capsid and the two aspect discussion of gag gene.Same technology also can be used for similar pattern and sets up expression vector, produces and the relevant polypeptide of any LAV/HTLV III albumen, and the polypeptide of relevant virus.These albumen are including but not limited to LAV/HTLV III gene product, as gag, and pol and env gene, and be called sor so far at least four kinds, yar, 3'-orf, the subsidiary gene of art or trs (referring to, Fish-er et al., 1986, Science233:655-659)

5.1 the gene of coding LAV/HTLV III viral protein or the separation of gene fragment

The separation of LAV/HTLV III gene relates to and at first isolates the dna fragmentation that contains required gene order (as shell or gag).As previously mentioned, the LAV/HTLV III has a rna gene group, so the gene corresponding D NA of coding LAV/HTLV III can obtain with following any method.(a) carry out cDNA clone (b) Poly(A that contains by the RNA that from purified virus, separates by obtaining from LAV/HTLV III cells infected) RNA carry out the genomic dna that cD-NA clone (c) clone comes out from LAV/HTLV III cells infected purifying.After this, the LAV/HTLV III DNA here just is meant the DNA of the gene of coding LAV/HTLV III.

In order to produce LAV/HTLV III dna fragmentation, LAV/HTLV III DNA can shear at some special sites with various restriction enzymes.Can under the condition that manganese exists, use deoxyribonuclease (DNase) that DNA is cut into fragment; Also can shear DNA, for example use sound wave with physical method.Linear then dna fragmentation just can be separated with normalized technology by its size, comprising the electric arteries and veins of (but being not limited thereto method) agarose and polyacrylamide gel, and technology such as column chromatography.

If (restricted) enzyme does not destroy the generation antigenic capacity of protein gene product, any or merge several restriction enzymes all can be in order to cut the LAV/HTLV III dna fragmentation that contain capsid or gag order.For example, a proteinic antigenic site can contain 7-14 the amino acid of having an appointment, and the albumen as capsid polypeptide precursor (about 97,000 dalton) size just contains many discontinuous antigen sites so.Consider overlapping order wherein again, secondary and tertiary structure, some courses of processing, as acidylate, glycosylation, phosphorylated, the antigen position may be contained several thousand like this.Yet the polypeptide gene of the part capsid of many sections order may be coded in same antigen position again.The result is, many restriction enzymes will combine in order to obtaining dna fragmentation, and these fragments can instruct generation to contain the distinctive amino-acid sequence of capsid of different antigenic determinants when being inserted into appropriate carriers in the time.

After producing dna fragmentation, can realize by the whole bag of tricks the discriminating of the dna fragmentation that contains required LAV/HTLV III gene.At first, can measure order with respect to the genomic dna fragmentation of whole LAV/HTLV III, compare according to the amino-acid sequence of prediction and the amino-acid sequence of capsid protein or gag nucleoprotein respectively then, determine to contain the fragment of capsid protein gene or gag gene order.Secondly, in case after whole genome was determined in proper order, big open reading structure can be from 5' to the 3' serialization.Because the genome of all retroviruss of measuring is up to now arranged, and all is 5'gag-pol-env-3', the position of the most contiguous 3' end is the capsid gene coding most probably in the big open reading structure, and the most contiguous 5' end is the gag genes encoding probably.The 3rd, the discriminating of special gene can realize by the identification and the homology of other known retroviruss, or by nucleic acid hybridization analysis, if perhaps the known words of order are by order relatively.

On the other hand, the available mRNA of segmental discriminating that contains capsid or gag gene selects to carry out the method separation complementary mRNA of LAV/HTLV III dna fragmentation to hybridize in this process.The external translation product of the mRNA that separation is obtained carries out immunoprecipitation analysis to differentiate mRNA, so also identified the complementary LAV/HTLV III dna fragmentation that contains capsid gag protein gene order.At last by from LAV/HTLV III cells infected, separating the absorption of polysome, the mRNA that selects specificly, antibody so that fixing anti-capsid or gag protein instruct comes the polysome of self-absorption with the mRNA(that selects) do template can synthesize the capsid protein of penetrating property mark cDNA(complementary DNA is arranged).Like this, containing radiolabeled mRNA or cDNA just can be used as probe and is used to identify the LAV/HTLV III dna fragmentation that contains capsid or gag gene order.The method of separating capsid or gag protein gene is perhaps produced the mRNA complementary DNA (cDNA) of coding capsid or gag gene also including, but not limited to chemosynthesis gene order itself (if order is known).

When identify and separate finish after, contain we the LAV/HTLV III dna fragmentation of interested order be inserted into a cloning vector earlier, for example go in a plasmid cloning vector, this carrier will change appropriate host cell over to repetition DNA, so just can produce many copies of our interested LAV/HTLV III order.This process is that LAV/HTLV III dna fragmentation is attached on the cloning vector, and this carrier contains complementary sticky end.If but in the cloning vector not with LAV/HTLV III dna fragmentation on during the corresponding position of complementary restriction site, the end of dna molecular just needs through processing so, this processing is modified and is comprised the generation blunt end, its method is the single stranded DNA of digestion distal portion, perhaps fill and terminal single stranded DNA complementary DNA section, but just tack binding of end like this.In addition, all available method that nucleotide sequence is attached on the DNA end in the site of any needs obtains (joint), and these joints can comprise the oligonucleotide of energy recognition sequence on special chemical synthetic coding restriction site.According to additive method, carrier after the shearing and LAV/HTLV III dna fragmentation can be processed with the method that adds the homopolymer rear.

In conjunction with the recombinant DNA molecules transformed host cell of isolating gene or cDNA or synthetic DNA order, can produce many copies of gene.Like this,, from transformant, isolate recombinant DNA molecules, just can obtain a large amount of genes, if necessary, can also fetch the insertion gene by separating in the recombinant DNA molecules of separating as long as cultivate transformant.

If final purpose is that gene is inserted virus expression carrier such as vaccinia virus or adenovirus, can be modified with LAV/HTLV III gene bonded recombinant DNA molecules, so that gene is at virus order side.Like this, the cell producer reorganization that is infected by the virus, gene just can insert in the viral genome.

Whole LAV/HTLV III genome is now by Wain-Hobson(Wain-Hobson, S.et al, 1985, Cell 40:9) etc. clone and order-checking.One of them clone is meant that the LAV/HTLV III genome order dna fragmentation that comprises one 9.2 kilobasas injects the λ J19 of the Hind III position of λ L47.1.

A useful especially λ-J19 subclone that contains LAV/HTLV III shell gene is pRS-3, it comprises 3, the EcoRI of 840 base pairs is to the LAV/HTLV III nucleotide sequence fragment of SstI, and this fragment is inserted between the EcoRI and SstI position of pUc18.The specific DNA of the LAV/HTLV III that contains among the pRS-3 is from the section between the SstI position of 9129 Nucleotide in the 5289th Nucleotide EcoRI site to the in LAV/HTLV III genome.(Wain-Hobson, S., et al., 1985, Cell 40:9): see Fig. 2, wherein described LAV/HTLV III nucleotide sequence contained among the pRS-3.Yet because the degeneracy of Nucleotide sequence of coden, also can be used for reaching the purpose of clone LAV/HTLV III gene among the present invention as the encode DNA of same amino-acid sequence of other that describe among Fig. 2.These include but are not limited to: contain the DNA sequence of all or part shell nucleotide sequence, as shown in Figure 2 these orders can be encoded of the same race or amino-acid residue (as the amino acid of identical polar) that function is suitable just different codon caused a change of hiding replacing.

The λ J19 subclone of the useful especially LAV/HTLV of containing III gag gene is pKS-5 and pSS-5.The pKS5 plasmid contains among the pUc18 from SstI to KpnI3, the LAV/HTLV III fragment of 148 base pairs.The specific DNA of LAV/HTLV III among pKS-5 system comes from the interior SstI site to 3 from 224 Nucleotide of LAV/HTLV III genome, the KpnI site of 372 Nucleotide.The pSS-5 plasmid comprises among the pUC18 SstI to segmental 5.1 thousand base pairs of SalI LAV/HTLV III.The specific DNA of LAV/HTLV III among pSS-5 system from the LAV/HTLV III genome from the section between the SalI site of SstI site to 5331 Nucleotide of 224 Nucleotide.(Wain-Hobson, S., et al., 1985.Cell 40:9) sees Figure 14, wherein described the LAV/HTLV III nucleotide sequence in pKS-5 and pSS-5.But because the degeneracy of sequence of coden Nucleotide, as shown in Figure 14, the DNA sequence of other coding same amino acid orders is also in the present invention available, the gag gene of clone LAV/HTLV III.These include but not limited to contain the DNA sequence of all or part capsid nucleotide sequence.As shown in figure 14, these different codons of the same race or amino-acid residue (for example amino acid of identical polar) that function is suitable that can be encoded in proper order replace.So just, cause a change of hiding.

5.2LAV/HTLV III encoding histone order

Insert expression vector.

Coding LAV/HTLV III albumen such as capsid gag or wherein the nucleotide sequence coding of a part be inserted into suitable expression, promptly one possesses and transcribes, the carrier of encoding histone order ability is inserted in translation.Various host-vector system can be used for the expression of encoding histone order, and they include, but are not limited to the cells of mamma animals system that virus (as vaccinia virus, adenovirus etc.) infects; The insect cell system that virus (as baculovirus) infects; Microorganism is as the yeast that contains yeast vector or use phage DNA, the bacterium that plasmid DNA or Ke Si DNA transform.The expression factor of these carriers according to their ability to express with characteristic and different.By applied host-vector system, available each suitable transcription and translation key element.For example, when in the cells of mamma animals system, cloning, can use the promotor of separating by the cells of mamma animals genome (as mouse metallothionien promotor), or isolating promotor (as vaccinia virus 7.5k promotor) in the virus of in cell, growing.Recombinant DNA promotor that produce or that synthetic technology obtains also can be used for participating in the transcription of insertion sequence.

Effective translation of inserting the encoding histone order needs specific start signal equally.These signals comprise ATG initiator codon and contiguous order thereof.When whole LAV/HTLV III capsid or gag gene comprise that it self initiator codon and contiguous order thereof are inserted in the suitable expression, just no longer need to add separately the translation control signal.When a part of having only capsid or gag coded sequence is inserted into, the translation control signal of external source must be provided, comprise the ATG initiator codon.In addition, initiator codon must be coordinated to guarantee the translation of whole insertion sequence mutually with the reading framework of protein coding gene.The translation control signal of these external sources and initiator codon can be that various sources are come, and natural or synthetic all can.

Any method that aforesaid dna fragmentation is inserted in the method for carrier all can be used for setting up the expression vector that contains a mosaic gene, and this mosaic gene is by suitable transcribing/translate control signal and encoding histone is formed in proper order.These methods can comprise extracorporeal recombinant DNA and synthetic technology, and reorganization (gene recombination) in the body.

Selected vaccinia virus and baculovirus as expression vector in embodiments of the present invention.But the present invention is not limited in and uses vaccinia virus and baculovirus.As previously mentioned, operable expression vector includes but not limited to following carrier or their derivative: human or animal's virus is as vaccinia virus or adenovirus; Insect viruses such as baculovirus; Yeast vector; It is some other or the like that phage vector and plasmid and coemid dna vector also have.

When adenovirus is used as expression vector, the binding of control complex body is transcribed-translated to LAV/HTLV III gene and an adenovirus, for example, back promotor and three leader sequences.Then this mosaic gene is inserted in the adenoviral gene group by reorganization in external or the body.Be inserted into virus genomic nonessential zone (as area E 1 or E3) and will produce great-hearted recombinant virus, it can be expressed and LAV/HTLV III proteins associated in the host cell that infects.At present, two strains (4 types and 7 types) of existing adenovirus vaccine of being got permission and using as the military personnel.They are the initial carriers of expressing LAV/HTLV III gene of electing as.

In addition, also will select host cell strain, it needs to regulate the expression of insertion sequence, maybe can modify and process the product of mosaic gene by desired special shape.The expression that starts from certain promotor can be enhanced owing to the existence of certain inductor (as zinc and calcium ion for the effect of metallothionein promotor), so the proteic expression of LAV/HTLV III of genetically manipulated is under control.If alien gene clone's protein product is lethal to host cell, it is very important that this control just seems.In addition, the modification of protein product (as glycosylation), processing (as shearing) is also very important to proteic function.Different host cells is the processing modified mechanism behind the distinctive protein translation of tool all.By selecting suitable clone or host system can guarantee the proteic correct modification and the processing of exogenous gene expression.

Among two embodiment of the present invention, we are connected LAV/HTLV III shell sequence of coden (being respectively the whole of order and two kinds of forms of its part) and the 7.5k promotor of LAV/HTLV III gag sequence of coden and vaccinia virus respectively, have formed mosaic gene in various plasmids.Mosaic gene in these plasmids is placed in the side of the additional vaccine virus order of vaccinia virus Tk dna homolog.The structure of mosaic gene relates to and uses natural and synthetic Nucleotide, these Nucleotide is control signals that coding LAV/HTLV III env or LAV/HTLV III gag transcribe in proper order and translate.By recombinating in the body between plasmid vector and the vaccinia virus gene pool homology TK zone, these mosaic genes are imported the vaccine virus expression vector then.These recombinant viruses that contain mosaic gene produce relevant albumen of LAV/HTLV III capsid or the relevant albumen of LAV/HTLV III gag as expression vector.

In other embodiments of the invention, we are connected the polyhedron promoter of LAV/HTLV III shell sequence of coden and LAV/HTLV III gag sequence of coden and Autographa Californica nuclear polyhedral virus (A cNPV) respectively, have formed mosaic gene in various plasmids.Mosaic gene both sides in these plasmids are additional A cNPV orders.The structure of mosaic gene relates to the use of natural nucleotide, the control signal of these nucleotide codings LAV/HTLV III gag or env order transcription and translation.By recombinating in the body between plasmid vector and the A cNPV genome homologous dna, mosaic gene is imported A cNPV expression vector then.The recombinant virus of these mosaic genes is as expression vector; To produce albumen relevant or the albumen relevant with LAV/HTLV III gag with LAV/HTLV III shell.

5.3 the evaluation of the recombinant chou of the carrier of reproducible and expression insertion gene

The expression vector that contains external source insertion gene fragment can be identified by three approach commonly used.(a) appearance of DNA-DNA hybridization (b) " mark " gene function or the expression of disappearance (c) insertion sequence.Article one, approach, the appearance of inserting the expression vector foreign gene, the available DNA-DNA that inserts the probe of dna homolog order with external source that contains is hybridized and is measured.The second approach, recombinant vectors/host system can be at some because some " mark " gene function that the insertion of foreign gene causes in the carrier (be exactly the thymidine kinase vigor, antibiotic resistance, transform phenotype, formation of baculovirus occlusor or the like) occur or the basis that disappears on be called evaluation and screening.For example, if when LAV/HTLV III gene is inserted in the carrier marker gene order, comprises the LAV/HTLV III and insert segmental recombinant chou and can identify by the forfeiture of marker gene function.Three approach, recombinant expression vector can be identified by the exogenous gene expression product of measuring recombinant chou.Can be in physics, measure on the basis of the functional property aspect of immunology or gene product.

In case a special recombinant DNA molecules obtains identifying and separate that available several method is bred, and depends on whether such recombinant chou constitutes self-replacation unit's (replicon).A self-replacation unit, such as plasmid, virus, cells etc. can self-reproduction under cellular environment that is fit to and growth conditions.In order to breed, the recombinant chou that lacks self-replacation unit must be integrated with the molecule with this replication unit.For example, some expression plasmid carrier that changes the host over to need combine to guarantee the stably express of breeding and recombination with cell chromosome.In case set up appropriate host system and growth conditions, recombinant expression vector just can be bred and a large amount of preparation.

Among the embodiment who is described in detail in example of the present invention, the insertion gene that comprises the env of LAV/HTLV III or gag sequence of coden is inserted in the TK gene in the vaccinia virus genome, and virus is transformed into TK then ^-, just lost the ability of viral generation thymidine kinase.Such recombinant chou can utilize them containing a kind of TK of making of 5-bromodeoxyuridine ⁺Cell-lethal but do not make TK ^-Energy for growth on the substratum of the nucleoside analog of cell-lethal is selected.Use LAV/HTLV III env or LAV/HTLV III gag specific probe,, recombinant chou is carried out further determining by DNA-DNA hybridization.TK ^-Recombinant virus separates by the plaque purifying.Virus strain can obtain from infected tissue culture cells.

In the embodiments of the invention that in example, go through, the env of LAV/HTLV III or the gene fragment section of gag sequence of coden are inserted in the chromosomal polyhedron gene of Ac NPV, thereby make virus be transformed into the phenotype of non-obturation.These recombinant chous can be observed out their and lack inaccessible virion, thus selected come out.Recombinant chou utilizes LAV/HTLV III env or gag specific molecular probe further to be identified by Northern spot analysis method.Recombinant virus separates by the plaque purifying, can obtain virus strain from infected tissue culture cells.

5.4 the identification of gene expression product and purifying

After recombinant chou with LAV/HTLV III gene is identified, just can analyze the product of this gene.Can analyze product according to the characteristic of product physics, immunity or function.Immunoassay has the meaning of particularly important, because of its final purpose be with gene product and the recombinant virus that can express this product is used for vaccine preparation and/or in immunoassay diagnostics as antigen.

Many antiserum(antisera)s can be used for analyzing the immunoreactivity of this product, they including, but not limited to: take from LAS or patient's AIDS the serum and the antiserum(antisera) of the anti-LAV/HTLV III of polyvalent virus, virus capsid protein, or comprise the analogue that bacterial system produces by the nuclear core protein immunization original molecule of gag genes encoding, perhaps contain the synthetic peptide of LAV/HTLV III shell antigenic determinant, perhaps examine core antigen.Peptide of describing among the present invention and proteic identification are based on following two requirements: the first, only in the metainfective cell of recombinant virus, produce with LAV/HTLV III proteins associated.The second, LAV/HTLV III proteins associated will have immune response to AIDS patient serum, or with the antibody of various anti-LAV/HTLV shells or nuclear core albumen or their analogue, derivative immune response is arranged.

Albumen must have immune response activity, and no matter its part from whole gene order or gene order still links together from two or more and instructs the gene of synthetic fusion roteins.This reactive behavior can show by the immunological technique of standard, as the method with radioimmunoprecipitation assay, radioimmunoassay competition or immunodotting (immunoblots).

After LAV/HTLV III proteins associated is identified out, can its separation and purification be come out with standard method, this this method comprises chromatography (reaching the column chromatography of pressing the molecular size design as ion-exchange, affinity chromatography), centrifugal, dissolubility difference, or the used standard technique of other separation and purification albumen.

On the other hand, in case after the relevant proteic Identification of Fusion Protein of LAV/HTLV III that is produced by recombinant chou that immune response activity arranged was come out, the amino-acid sequence in this albumen can be inferred out by the nucleotide sequence of mosaic gene in the recombinant chou.So this albumen just can synthesize by known standard chemical process.(as: seeing Hunkapiller, M.et al., 1984, Nature 310:105-111)

No matter these peptides in the embodiment of the invention are from recombinant DNA technology or from chemical synthesis, all include, but not limited to as Fig. 2 or the described all or part of amino-acid sequence of Figure 14, also comprise such a case, promptly amino-acid residue is replaced by the suitable amino-acid residue of function in the order, and causes that an asymptomatic order changes.As, one or more amino-acid residues are replaced by the suitable amino-acid residue of another function (identical as polarity) in the order, and this just causes a so-called asymptomatic variation.A certain amino acid whose substituent can be organized under this amino acid other members that are and select in the order.For example, nonpolar amino acid (hydrophobic) comprises L-Ala, leucine, Isoleucine, Xie Ansuan, proline(Pro), phenylalanine, tryptophane and methionine(Met).The neutral pole acidic amino acid comprises glycine, Serine, Threonine, halfcystine, tyrosine, l-asparagine and glutamine.Positively charged (alkalescence) amino acid comprises arginine, Methionin, Histidine.Electronegative (acidity) amino acid comprises aspartic acid, L-glutamic acid.

5.5 determining of recombinant chou product immunity vigor

The immunological competence of the product that the LAV/HTLV III is relevant can be determined by such method: after promptly monitoring purified albumen or synthetic peptide or protein immunization, and the immunne response of experimental animal.When LAV/HTLV III proteins associated was expressed by the infective recombinant virus of tool, recombinant virus itself can make the experimental animal immunity.Experimental animal can comprise mouse, rabbit, chimpanzee, finally is human subject.Import immunogenic method can comprise oral, intradermal injection, intramuscular injection, peritoneal injection, intravenous injection, subcutaneous injection, nasal cavity, or the immunization route of other any standards.The immunne response of test body can be analyzed (a) with the immune mottle of immune absorption analysis (ELISA) (immunoblots) of known technology such as desmoenzyme, radioimmunoassay sedimentation analysis or the like by three kinds of approach, and the resultant immune serum that analysis obtains is to the reactive behavior of real LAV/HTLV III virus antigen.(b) immune serum in vivo in and the infectious ability of LAV/HTLV III.(Robert-Guroff, M, 1985, Nature316:72-74) (be intended to measure anti-shell antibody), and (c) prevention LAV/HTLV III infection and/or alleviate by the infection symptoms of the animal of immunity.（Francis，D.P，1984，Lancet 2：1276-1277：Gujdusek，D.C.）1985，Lancet 1：55-56）。

5.6 vaccine preparation

The purpose of this embodiment of the present invention is a kind of vaccine of preparation, and the immunogen system in this vaccine is relevant with LAV/HTLV III epitope; Perhaps this vaccine contains a kind of recombinant virus, and the immunogen of this expression of recombinant virus evokes aversion response to LAV/HTLV III virus infection, with prevention LAS or AIDS disease.In addition, the polyvalent vaccine preparation can be prepared into and have the relevant immunogenic determinant of a plurality of LAV/HTLV III.Such vaccine is including, but not limited to following situation: the vaccine that contains the relevant decision base of LAV/HTLV III shell separately or with other LAV/HTLV III decision base, as common use of the relevant decision base of gag.The polyvalent vaccine that has comprised the epitope of env and gag coding so simultaneously, in the expansion that prevents the AIDS disease highly significant.When two groups of patients produce antibody at capsid antigen bunch, wherein do not show the symptom patient seem by contrast produce more anti--gag antibody.（Science，1986，233：419）。About also showing patient AIDS, research do not show that in early days the symptom stage produces the antibody of anti-capsid protein and core protein (gag) simultaneously.Along with increasing the weight of of the state of an illness, after symptom displayed, anti--gag antibody reduced, anti-simultaneously shell antibody quantity keep previous level (Science, 1986,223:282).The inclusion that contains the relevant epitope of gag in vaccine formulation acquires a special sense for the immunoprophylaxis and the immunotherapy of LAV/HTLV III relative disease.One embodiment of the present of invention have been done special description to the preparation of this inclusion.

Polyvalent vaccine can be made into the recombinant virus that contains LAV/HTLV III gene product and/or express the mosaic gene product.These also can be mixed for LAS with other antigen or AIDS is sick and the prevention of other diseases.Next be that virus formulation is given an example.

5.6.1 virus vaccines preparation

No matter be to prepare with the recombinant viral vaccine or the recombinant viral vaccine of deactivation of living.How selection depends on the character of expressing the basic recombinant virus of LAV/HTLV III related antigen decision.When recombinant virus has infectivity to the immune host of quilt but can not cause a disease.The vaccine of living is preferentially selected for use because it in the host, breed can prolong to take place similar type that the nature clinical symptom infects and amount stimulation, thereby provide substantial long-time immunity.The recombinant virus with infectivity that imports host animal can be by its associated protein of insertion genetic expression LAV/HTLV III, thereby stimulates the antigenic immunne response of LAV/HTLV III.Challenge has under the situation of provide protection to follow-up LAV/HTLV III in this immunne response, and the recombinant virus itself of living can be used as the vaccine to the AIDS viral communication.Such recombinant virus product can be used in the preparation of (as natural reservoir (of bird flu viruses)) system in external (as tissue culture cells) and the body.In this vaccinia virus recombinant is particularly useful.Conventional preparation method and Dryvax can be used the recombinant viral vaccine preparation of living.(example is seen Vaccinic Viruses and Factors for Vaccina Antigens, Ed, and Quinnan, G.V., Elsevier, 1985, PP, 109-116).

The multivalence live-virus vaccine can prepare with a kind of or some recombinant viruses with several LAV/HTLV III of expression related antigen decision base of infectivity.The antigenic determinant vaccine of LAV/HTLV III also can comprise the virus of expressing the organism antigenic determinant that causes other diseases, and for example vaccinia virus (can admit the foreign DNA of about 35 kilobasas) is designed to contain the coded sequence of LAV/HTLV III capsid and gag related antigen determinant.Virus also may be encoded except that the antigenic determinant of LAV/HTLV III.Recombinant virus like this itself also can be used as immunogen in polyvalent vaccine.Vaccinia virus or other viral mixtures, their each genetic expression that can instruct the different antigenic determinants for different LAV/HTLV III and/or other pathogenic organisms bodies to encode can be used in the polyvalent vaccine.

No matter whether recombinant virus is to infectious by the host of immunity, can prepare the preparation of inactivated vaccine.Inactivated vaccine is " dead " in some sense, and formaldehyde treated is used in their infectivity destroyed usually.Under ideal state, the infectivity of virus has been destroyed but has not influenced capsid or the coat protein with virus immunity originality.Be the preparation inactivated vaccine, a large amount of recombinant viruses must be grown in cultivation so that enough relevant antigen is provided.The inactivation of viruses mixture of expressing different antigenic determinants can be used to prepare polyvalent vaccine.In some cases, aforesaid method is good than adopting the vaccine formulation of living, because the problem of having avoided interfering with each other between the live virus to bring.In either case, the recombinant virus of deactivation or virus mixture should be prepared to strengthen it to antigenic immunne response with suitable adjuvant.Suitable adjuvant comprises, but not and be limited to: inorganic gel such as lead hydroxide, surfactant such as lysolecithin, Propiram Ni Keduo does not have alcohol, polyanion, the peptide class, oiliness emulsion, and potential adjuvant of human body such as BCG(bacille calmette-Guerin) and dialister bacterium (corynebacterium parvum).

Have many methods can import above-mentioned vaccine preparation, they comprise oral and by approach such as intracutaneous, muscle, peritoneal cavity, vein, subcutaneous or nasal cavity contacts, but are not limited in these.When the virus recombinant vaccine of in preparation, utilize living, then can adopt parent's wild-type virus natural infection approach, wherein parent's wild-type virus in vaccine formulation in order to the preparation recombinant virus.

5.6.2 inferior monomer vaccine preparation

Remove virus vaccines in subunit's preparation, LAV/HTLV III associated protein itself is as immunogen (and can be polyvalent).As previously mentioned, subunit vaccine only comprises and makes the host obtain immune necessary related immune material.The LAV/HTLV III associated protein of correspondingly, purifying from recombinant chou can be expressed LAV/HTLV III antigenic determinant.These recombinant chous have comprised the virus infection culturing cell of expressing LAV/HTLV III antigenic determinant as previously mentioned, bacterium transformant, yeast transformant or by the insect of virus infection (referring to 5.2,5.3 and 5.4 joints).In an alternative embodiment of the invention, LAV/HTLV III related peptides or associated protein can be synthetic with chemical process.Thereby its acid order of these peptides or proteinic ammonia can be derived from the mosaic gene nucleotide sequence that instructs its expression.(referring to 5.4 joints).

No matter immunogen be purify from recombinant chou or chemosynthesis, final product all is adjusted to suitable concentration and adds suitable vaccine adjuvant and packages spare then.Suitable adjuvant includes but are not limited to: inorganic gel such as aluminium hydroxide; Tensio-active agent such as lysolecithin, Pu Lulannikeshi polyvalent alcohol, polyanion, peptide, oily emulsion and human body adjuvant such as BCG(bacille Calmette-Guerin with potential utility value) and dialister bacterium.（corynebacteri-um parvum）。Immunogen and liposome share or link as vaccine preparation with polysaccharide or basic his polymer in vaccine preparation.

Peptide or albumen that the LAV/HTLV III is relevant are a kind of haptens, that is to say, because of its energy selectivity and the reaction of its isobody, and can not excite immunne response, so this molecule has antigenicity and disimmune.This haptens can be connected on a carrier or the immune molecule, for example with covalence key; A kind of high molecular weight protein such as serum albumin with make this haptens have immunogenicity after haptens combines.This hapten-carrier can be made into a kind of vaccine and use.

5.6.3 passive immunization and anti-idiotype antibody

Antibody with one or more antigenic determinants of the active immunity of carrying out with virus or the subunit vaccine anti-LAV/HTLV III that different is forms by control early stage can make the host obtain the short-term protection.Thereby above-mentioned vaccine preparation can be used to produce antibody and be used for passive immunization therapy.Preferablely in the medicine use the human body immune protein, because allogenic immunoglobulin (Ig) will evoke the immunne response to external source immunogen composition.This kind passive immunization provides a kind of basis of urgent protection for without immunity and emitting the people who contacts patient's AIDS danger in hospital or other health organs.On the other hand, these antibody capables are used to prepare the antibody of anti-specific gene, and this anti-specific gene antibody can be used as antigen, can evoke immunne response at LAV/HTLV III antigenic determinant.

5.7 immunoassay

Peptide relevant with the LAV/HTLV III of the present invention and protein can be used as antigen in immunoassay, with the antibody of anti-LAV/HTLV III in the various tissues that detect patient and the blood in body fluid and blood bank and the hospital.For this reason, antigen of the present invention can be used for known any method of immunity in this technical field, and these methods include, but is not limited to utilize the competition and the non-competing assay method of following cited technology: mensuration, CA, complement fixation(CF) mensuration, immunoradiometric assay, fluorescence immunoassay, a-protein immunoassay and immunoelectrophoresis mensuration etc. are penetrated in radiation immunity mensuration, the immunoassay of enzyme linked immunological absorption mensuration (ELISA) " sandwich ", precipitin reaction, GDP reaction, immunity expansion.

Immunoassay of the present invention can be used for checking whether the blood among blood bank and the patient once was exposed in the LAV/HTLV III and monitors the patient who is curing LAS or AIDS.According to the present invention, any peptide and the protein relevant with the antigen of LAV/HTLV III all can be used for immunoassay.These antigens include, but are not limited to and following gene product env, gag, peptide and protein that pol is relevant also include, but are not limited at present other four kinds of gene: sor-tat of name just, 3'-orf and art(or trs simultaneously) relevant peptide and the protein of product.In a special embodiment of the present invention, one group of antigen can be in immunoassay, are used to detect the profile of antibody of different determinants of patient's anti-LAV/HTLV III.In the another embodiment of the present invention, can the patient who inoculate vaccine preparation of the present invention be exercised supervision, to find out that this patient is later on whether cruelly among the LAV/HTLV III.Under this class situation, used antigen peptide preferably of the present invention or protein in the immunoassay, but described peptide and protein should not be the immunogens that patient inoculates used vaccine preparation, because these individuals that inoculated are seropositivity for special determinant used in the vaccine preparation, the result be cause testing positive, and no matter whether be exposed among the virus.For example, if a patient inoculates with the determinant relevant with LAV/HTLV III film of the present invention, what this patient should use any other so carries out the inspection that the LAV/HTLV III infects with non-capsid LAV/HTLV III proteins associated matter, to determine whether there is the antibody special to the LAV/HTLV III among the patient.Therefore, can check whether the patient who has inoculated the determinant relevant with capsid exists LAV/HTLV III cAg, and perhaps other LAV/HTLV III antigen includes but not limited to special antibody such as sor and 3'-orf gene product.

Among the another embodiment, peptide relevant with the LAV/HTLV III of the present invention and protein are used to detect and measure the existence and the quantity thereof of the LAV/HTLV III-HTLV III encoded protein matter in patient's the blood, serum etc. in the competition immunoassay is arranged.

6, embodiment: cowpox shell recombinant chou

In following each example, various plasmid vectors will be built to such an extent that contain mosaic gene, are the genomes that is inserted into vaccinia virus by the body reorganization comprising these mosaic genes that contain cowpox promoter region and LAV/HTLV III shell coded sequence of transcribing the LAV/HTLV III film coded sequence below the control sequence that are positioned at relevant acne poison.These recombinant virus are identified and purified, and viral raw material is prepared by infected tissue culture cells.Proved that the relevant protein of immunoreactive LAV/HTLV III shell is produced by these recombined vaccinia virus external.These recombinant chous are tested on one's body laboratory animal, bring out the ability of the immunne response of neutral or protectiveness to understand them, and they are as the possibility of the vaccine of AIDS resisting.To each step of this enforcement of the present invention be described in detail below.

6.1. the program of total step

6.1.1 cell and virocyte

America green monkey kidney cell (BSC-40 cell, from the BSC-1 cell, ATCCNo, a kind of continuous system of the cercopithecus aethiops cell that obtains among the CCL26) be from R. Kang Di (R.Condit New York, Buffalo, the department of biochemistry associate professor of State University of New York) locates to obtain, and at the improved Eagle's medium (DMEM of Dole's pendant section (Dulbecco), Gibco, GrandIsland, NY) breeding also adds 10% N of cleer and peaceful consumption of fetal blood and respectively is the penicillin and the Streptomycin sulphate of every milliliter 100 unit in above-mentioned substratum.Human body 143TK cell (Rhim, J.Set al., 1975, Intl.J.Cancer 15:23-29) be from the rich (M.Botchan that looks into of M., the California, Berkeley, the professor of University of California's molecular biosciences system) locate to obtain, and in the above-mentioned substratum of the 6-bromodeoxyribouridine (BUdR) that is added in an amount of 25 mcg/ml, breed.

People's diploid cell (MRC-5) is to take from U.S. typical case culture sample (ATCCNO.CCL171), and with the used same medium of BSC-40 in breed.

Vaccinia virus (WR strain, ATCC NO, VR-119) be to obtain from R. Kang Di, increase in containing the BSC-40 cell of following culture solution then, this culture solution is made up of following ingredients: the dissociate Streptomycin sulphate of penicillin+100 units per ml of calf serum+100 units per ml of DMEM+5% gamma-globulin.TK ^-Recombinant chou is selected from the 143TK in the same substratum that contains 25 mcg/ml bromodeoxyribouridines (BUdR) ^-Cell, and the enterprising line space spot of the same clone purifying in the DMEM culture solution, also contain 1% noble's agar (DIFCO.Detroit MICH) in the described solution, the 5% the third albumen calf serum that dissociates, concentration respectively is the penicillin and the Streptomycin sulphate of 100 units per ml, and 25 mcg/ml bromodeoxyribouridines.(PBS, every liter contains 8 gram NaCl to the virus starting material, 0.2 gram KCl, 1.5 gram NaH in phosphate buffered saline(PBS) ₂PO ₄With 0.2 gram K ₂HPO ₄) middle dilution, above-mentioned phosphate-buffered saline is added with 1mM MgCl ₂With 0.01% bovine serum albumin (PBSAM).

The vaccinia strain of New York board of health is (Mariet-ta, what PA) purification obtained in the antismallpox vaccine of Xiao Shouing (Dryvax Lot321501G) the commodity manufacturing from Huai Erfu (Wyeth) laboratory.Antismallpox vaccine is to see below with PBSAM() dilution, on the BSC-40 cell, carry out the plaque purifying in succession three times again.Virus starting material (hereinafter referred to as v-NY) are to purify through this patch isolatingly to make on the BSC-40 cell, and are used to prepare recombinant virus.The recombinant virus starting material that obtain from v-NY are placed in the MRT-5000 cell and breed.

6.1.2.DNA preparation, restriction and modification

Except as otherwise herein provided, the used all method of following operation is all as document (Maniatis et al., 1982.Molecular Cloning, Cold Spring Harbor Larboratory) relative section is described like that in: the preparation of plasmid DNA (pp.86-96), the restrictive diges-tion of DNA (pp.98-106) and the purification (pp.157-161 and p.170) of restricted fragment from the sepharose of low melting glass, the alkaline phosphatase enzyme reaction (pp.133-134) of e. coli dna polymerase Klenow fragment reaction (pp.107-114) little Roll and the reaction conditions of ligase enzyme reaction (p.146), otch is translated the preparation (pp.109-112) and the DNA-DNA hybridizing method (pp.324-325) of probe.

6.2 contain the structure of the plasmid vector of the vaccinia virus promoter region that is connected with LAV/HTLV III ENV gene.

Following segment has been described and has been contained the structure that anterior locations is the various plasmid vectors of the vaccinia virus LAV/HTLV III capsid gene of transcribing control sequence, and these chimeric order sides and TKDNA join.After, these plasmid recombinant carriers are used for LAV/HTLV III shell coded sequence is inserted by the back of in vivo recombinating the genome of vaccinia virus.

What narrate in following segment is, LAV/HTLV III capsid coded sequence is purified from PRS-3, and PRS-3 is the subclone (Wain-Hobson of λ J19, S., et al., 1985, Cell 40:9) and the downstream, cowpox 7.5k promoter region (Mackett, M., the Smith that insert among the PGS20 to be comprised, G.L.and Moss, B., 1984, J.Virol.49,857-864), to make up pv-env1, pv-env2, pv-env5 and pv-env7.Each structure in these structures, mosaic gene (being about to cowpox 7.5k promoter region is connected in the special nucleotide sequence of LAV/HTLV III) side and cowpox TK gene order join.

As previously mentioned, one 3840 base pair EcoRI to SstI fragment of the LAV/HTLV III DNA inset that comprised by the λ J19 of EcoRI of being cloned into pUC18 and SstI position of pRS-3 is formed.It is by the SstI restricted fragment of J19 being cloned into the SstI position of pUC18, and with creation pBT-1, the latter digests with EcoRI and reconnects.This DNA is used to change intestinal bacteria (E.coli), and plasmid PRS-3 is separated.The special DNA of LAV/HTLV III that PRS-3 comprised be in LAV/HTLV III genome from EcoRI position to the SstI position that is positioned at Nucleotide 9129 that is positioned at Nucleotide 5289 (wain-Hobson, S., et al., 1985, Cell40:9); See Fig. 2.

6.2.1 contain the structure of the plasmid vector of the vaccinia virus promoter region that is connected with 3 coded sequences of LAV/HTLV III ENV gene

5 microgram pRS-3 plasmid DNA are separated on the sepharose of the fragment that is produced degree when 1% low melt with restriction enzyme KpnI digestion fully.Isolate one 2.68 kilobase then to (Kbp) fragment, and purified.This fragment contains the Nucleotide that comprises order (5889-8349) and is numbered 5889 to 8572 LAV/HTLV III one distinctive DNA, and described order is the C-terminal portions coding of LAV/HTLV III coat protein.

With above-mentioned 1 microgram fragment with mix with the linearizing 0.5 microgram pGS20DNA of restriction enzyme BamHI in advance.In the presence of oligodeoxynucleotide joint, allow these two kinds of segmental anastomosiss in 4 ℃ temperature, carry out 24 hours by the 5'-CATGGTG-3'-OH of 0.6 microgram 5'-GATCCACCATGGTAC-3'-OH and 0.3 microgram.These joints are used for: (a) will change into the BamHI sticky end to (Kbp) segmental KpnI sticky end from 2.68 kilobase that the pRS-3 plasmid DNA obtains, the BamHI sticky end complementation of the latter and the pGS-20DNA that cut; (b) in proper reading frame frame, provide a rotaring intertranslating start order (ATG) about the required nucleotide sequence of effective translation of LAV/HTLV III shell gene order and the mRNA that transcribes.Link that mixture is used for that transformed into escherichia coli bacterial strain MC1000. inserts orientation to the plasmid DNA that has the resistance transformant from ampicillin and obtain and at the BamHI that connects contact, the test of the regenerated at NcoI and KpnI position.Fixed structure such as Fig. 3 of this needed plasmid pv-env-2, wherein corresponding to nucleotide coding be 5889-8572(as shown in Figure 2) the carboxyl encoding part of LAV/HTLV III shell gene be to be positioned at downstream, 7.5k cowpox promoter region.For the initial ATG that the DNA joint is supplied with, LAV/HTLV III shell is positioned at the proper reading frame frame in proper order.

6.2.2. contain the structure of the plasmid vector of the vaccinia virus promoter region that is connected LAV/HTLV III env genes encoding order 5' end

The plasmid DNA of 5 microgram pRS-3 is complete with the digestion of AVa II restriction enzyme, and the fragment that is produced is separated on the sepharose of 1% low melting glass.Isolate one 0.82 kilobase then to fragment, and purified.This fragment contains Nucleotide and is numbered 5671 to 6490(as shown in Figure 2) LAV/HTLV III one distinctive order.This is included as the order of the N-terminal part coding in the capsid protein matter, and (Nucleotide is numbered 5766 to 6490 and 95 base pairs of the 5' near-end untranslated sequences of LAV/HTLV III.Above-mentioned fragment is handled with e. coli dna polymerase Klenow fragment in the presence of excessive deoxyribonucleotide triphosphate.The passivity fragment of gained is coupled on the pGS20DNA of 0.5 microgram, and the latter uses the SmaI linearizing in advance, and handles with the alkaline phosphatase (CIAP) of little Roll.The binding process was carried out 16 hours under 12 ℃ of temperature.Connect mixture and be used to transform big change coli strain MC1000.Carry out the test that relevant vaccinia virus is transcribed the LAV/HTLV III insertion orientation of control sequence to have the plasmid DNA that obtains the resistance transformant from ampicillin.The fixed structure of needed plasmid pv-env1 shows as Fig. 4, wherein corresponding to Nucleotide numbering 5671 to 6490(as shown in Figure 2) the amino encoding part of the LAV/HTLV III shell gene that contains himself initial ATG be to be positioned at downstream, cowpox 7.5k promoter region.

6.2.3 contain the plamid vector construction of the vaccinia virus promoter region on the whole coded sequence that is connected LAV/HTLV III env gene

The plasmid DNA of 2 microgram pv-env1 restriction enzyme StuI, PvuI and XhoI complete digestion.The fragment that is produced is separated on the sepharose of 1% low melting glass.Thereby pv-env1 produces 24 kilobase to fragment with StuI and PvuI cutting, and one of them fragment comprises the 5' part of LAV/HTLV III, and another then contains pGS20; And XhoI will contain 4 kilobase of pGS20 fragment will be cut into less fragment, and 4 kilobase that therefore contain 5' part in vaccinia virus transcriptional control element and the LAV/HTLV III shell coded sequence can be identified StuI and PvuI fragment.Then this fragment is separated, purified and make it be attached to 6.5 kilobase that produced by the StuI of pv-env2 and PvuI restriction digestion on the fragment.Connect mixture and be used for transformed into escherichia coli bacterial strain MC1000.Select then ampicillin is had the resistance transformant.To the regeneration of the plasmid DNA that obtains from each transformant test StuI and PvuI restriction site, and whether parent's 4 kilobase are to existing fragment with 6.5 kilobase.Fig. 4 illustrates needed plasmid pv-env5, it contain corresponding to Nucleotide be numbered 5766 to 8349(as shown in Figure 2), with the whole casing gene of vaccinia virus transcriptional control element downstream banded LAV/HTLV III.

6.2.4 contain structure with the plasmid vector that lacks the vaccinia virus promoter region that the LAV/HTLV III env gene of striding film (ANCHOR) order is connected

Plasmid pv-env5(10 microgram) with the complete digestion of Hind III, the fragment that is produced is separated on the sepharose of 1% low melting glass.3 kilobase to the 5' promotor of the env-coded sequence that contains cowpox 7.5K promotor and LAV/HTLV III are purified to fragment, handle with the Klenow enzyme then, to insert sticky end.Then this fragment is attached to already on carrier (cloning vector) p26 that clones with EcoRI digestion and the multidigit point of using the alkaline phosphatase (CIAP) of Klenow enzyme and little Roll to handle successively.The connection effect at the insertion Hind III position of pv-env5 and the insertion EcoRI position of plasmid p26 has produced one and the in-phase terminator codon of env coded sequence.This intermediate structure is called pv-env5/26.

Plasmid pv-env5/26DNA is used for transformed into escherichia coli MC1000, and is increased and purify.It digests it with BamHI and HPaI then, and the fragment that is produced is separated on 1% sepharose.Then 2.1 kilobase that contain LAV/HTLV III shell coded sequence are separated fragment, then be attached to again earlier on the plasmid pGS20 that digested with BamHI and SmaI.The plasmid pv-env7 that is produced contains the vaccinia virus 7.5k promoter region that is attached on the peculiar order of LAV/HTLV III, and described order is numbered 7698(Fig. 5 from 96 base pairs of env initiator codon top until Nucleotide) the Hind III position located.Therefore, this plasmid lack the LAV/HTLV III env gene of being inferred anchor (Ahchor) in proper order.

6.3 contain characteristic and the structure of the recombinant chou vaccinia virus of chimeric LAV/HTLV III env gene

Two kinds of parent strains of vaccinia virus are used to constitute recombinant virus, and promptly kind of the V-NY(that derives of the bacterial strain preserved of research on standard bacterial strain WR and New York board of health sees 6.1.1).Chimeric LAV/HTLV III env is inserted into the vaccinia virus genome in proper order and realizes by reorganization in the body, this is that the vaccinia virus sequential cipher of the mosaic gene side of pv-env5 and pv-env7 and thymidine kinase (TK) gene joins and just makes it to become possibility owing to plasmid pv-env2.These plasmids are introduced the TK that can be made among the cell of vaccinia virus infection on the plasmid recombinates in proper order and between the genomic DNA sequence homologous of vaccinia virus.The insertion of mosaic gene is because dual group result in flanking sequence.This class recombinant chou will have the mosaic gene that inserts in the cowpox TK gene, therefore, be TK on phenotype ^-These TK ^-The reorganization physical efficiency is selected it is grown in the substratum that adds BUdR, and wherein BUdR is to TK ⁺Cell is lethal, but to TK ^-Cell does not have lethal effect.The existing report of this general theory of law (Mackett, M., Smith, G.L.and Moss, B., 1984.J.Virol.49,857-864)

6.3.1 contain the structure of the recombinant chou vaccinia virus of chimeric LAV/HTLV III env gene

The plate of 100 millimeters sizes fills 80% African green monkey kidney cell (BSC40 strain) that merges and infects with vaccinia virus (WR bacterial strain), and wherein infection multiplicity (moi) is 0.05.After cultivating 2 to 4 hours under 37 ℃, cover cells infected with the DNA of plasmid pv-env2 or pv-env5 and the coprecipitate of calcium phosphate.These throw outs are by with 0.5 milliliter of 2XDNA-CaCl ₂Solution dropwise adds (the 2XDNA-CaCl for preparing in 0.5 milliliter of 2XHe BS solution ₂Solution is that 20 microgram plasmid DNA are dissolved in 0.5ml concentration is to form in the 0.25M calcium chloride solution; Contain 16 milligrams of sodium-chlor in every milliliter of 2XHe BS solution, 0.74 milligram of Repone K, the dihydrate of 0.25 milligram sodium hydrogen phosphate, 2 milligrams of glucose and 10 milligrams of HEPES, PH is 7.08).At room temperature place 30 minutes to form the DNA-calcium phosphate coprecipitation.Precipitation covered after 4 hours, cell cleans once with 1XHe BS, in the 1XHe of 2 milliliter of 15% glycerine BS solution, cultivated 3 minutes then, temperature is 37 ℃, then clean once with 1XHe BS again, use 10 milliliters of growth mediums (DMEM+10% calf embryo serum+100 units per ml penicillin+100 units per ml Streptomycin sulphates) to cultivate again.Two days later, obtain cells infected, and collect (4 ℃, 2000 * g centrifugal 10 minutes) with centrifugal separation.Virus preparing raw material method is that these cells are suspended among 1 milliliter of PBSAM again, carries out then that secondary freezes and thaw cycle, then carries out three times 15 seconds sonication again.

Recombinant chou is selected as follows: making extent of dilution is 10 ^-30.1 milliliter of above-mentioned viral starting material solution deposition fill on the human body 143TK cell of fusion at 60 millimeters plates, cover (with every disc gage) with 5 milliliters of DMEM solution that contain following ingredients: 1%Nobel agar (Difco, Detroit, MICH), 5% calf serum, 25 microgram DMEM/ milliliter BUdR.Through two days the deposition after, cell contains nutrient agar 0.01% as above-mentioned same composition and adds toluylene red it is dyeed by covering 2 milliliters (with every disc gages), dye and collected plaque in back first day, and it is suspended among 0.5 milliliter of PBSAM again, the aliquot of viral suspension (0.25 milliliter), being used for being seeded in diameter in substratum (the DMEM+10% calf embryo serum+100 units per ml penicillin+100 units per ml Streptomycin sulphates+25 mcg/ml BUdR) infection of selecting is 16 millimeters fusion 143TK in the pond ^-Cell.Cells infected is collected by centrifugation, and then is suspended in the 100 microlitre PBS solution that contain 0.5 mg/ml trypsinase and 0.2 mg/ml ethylenediamine tetraacetic acid (EDTA) (EDTA).Cell is cultivated down at 37 ℃ and was made its dissolving in 30 minutes, carries out three sonications then, each 20 seconds.Adopt many ponds to filter menifold (multi-well filtering manifold); Make cell lysates be collected on the nitrocellulose membrane (Schleicher and Schuell, Arlington, MA).By DNA-DNA hybridization (referring to Mackett, M., Smith, G.L and Moss, B., 1982, Proc.Natl.Acad.Sci.79 7415-7419) can determine whether to exist in these samples the distinctive DNA sequence of LAV/HTLV III env.Otch is translated (nick-translation) prepared plasmid pRS-3DNA with phosphorus-32 mark and is used as hybridization probe.The recombinant chou that described hybridization probe is had a positive hybridization under selective conditions (substratum that contains BUdR) at 143TK ^-Carry out plaque on the cell again and purify twice, again under non-selective condition, on the BSC-40 cell, purify once then.After confirming by DNA-DNA hybridization at last, get that three purification patches prepare viral starting material on the BSC-40 cell, and use it for later characterization.

The structural representation of recombinant virus joins in LAV/HTLV III shell gene order (env) side in downstream, cowpox 7.5k promoter region (p →) and the TK DNA sequence in the cowpox genome among the figure as shown in Figure 6.The viral starting material that obtain from reorganization between vaccinia virus genome and the plasmid pv-env5 are called v-env5, and it comprises complete LAV/HTLV III shell gene.Herein in the described specific implementation method of embodiment, v-env5 contains 96 base pairs of whole shell genes and 5-near-end untranslated sequences and 223 base pairs of 3'-near-end untranslated sequences.The viral starting material that obtain from pv-env2 are called v-env2, and it comprises most LAV/HTLV III shell gene (being the KpnI fragment), but lack the part order of 42 the initial amino acid codings that are used to LAV/HTLV III coat protein.Because the signal sequence of the supposition of LAV/HTLV III coat protein is to be arranged in 49 initial amino acid of described protein, then v-env2 should produce a kind of protein that does not have signal sequence, can expect that therefore the protein relevant with the LAV/HTLV III that is produced by described recombinant virus can not be transported on the film.In contrast, comprise complete LAV/HTLV III env v-env5 in proper order and should produce a kind of protein that can be transported to film.The viral starting material that obtain from pv-env7 are called env7, and it comprises the whole N-end of LAV/HTLV III env shell gene, but lack the order in downstream, Hind III position.Because what have in this structure to be inferred does not stride film (anchor) in proper order, people can expect that the protein relevant with the LAV/HTLV III that is produced by described recombinant chou is oozed by branch and enter in the growth medium.This character is to help the proteinic purification of this kind, to use it to make diagnostic reagent or to be used for preparation as subunit vaccine.

6.3.2 the restriction figure of cowpox LAV/HTLV III shell recombinant chou

Respectively from parent's vaccinia strain WR and v-NY, and obtain DNA among their derive kind of v-env5 and the v-env5NY and separate (Espos-ito et al by the method for having described already, 1981, J.Virol.Methods, 2:175-180), with restriction enzyme Hind III it is digested, the fragment that is produced is dissolved on 0.7% sepharose separates.Show typical restriction figure (Fig. 2 A) by the DNA that obtains among the parent plant WR, and as described before (Defilippes, 1982, J.Virol.43:136-149) the restriction figure of .v-NY is similar, but has any different with the figure of WR.The most tangible difference is Hind III B and the segmental length of C aspect, and these fragments only are the genomic terminal fragments of vaccinia virus.Recombinant chou v-env5 and v-env5NY do not have the Hind III J fragment of their parent plants; The substitute is and observe two new segment, size is respectively 5.4 kilobase to right with 3.2 kilobase.This discovery is consistent with the result who LAV/HTLV III shell coded sequence is inserted the dual group of process that is positioned at the segmental cowpox TK gene of Hind III J.Owing in the LAV/HTLV order, there is a Hind III position, produce two fragments by inserting.This result has also confirmed the orientation with respect to the LAV/HTLV III insertion of vaccinia TK gene.

6.3.3 the Southern blot of cowpox LAV/HTLV III shell recombinant chou analyzes

Analyze by Southern blot and to have confirmed two new segmental identity of Hind III in the restriction digestive process of recombinant virus genomes, producing.As only being transferred on the nitrocellulose membrane in sepharose separated DNA fragment shown in Fig. 7 A, hybridizing to then has narrow spectrum fracture to translate on the probe to LAV/HTLV III shell in proper order.Shown in Fig. 7 B, only find hybridization in to fragment in 5.4. and 3.2 kilobase.This result has confirmed the genome structure of recombinant virus shown in Figure 6.

6.4 by in the tissue culture cell of recombinant chou vaccinia virus infection with the expression of LAV/HTLV III shell proteins associated matter.

The recombinant chou vaccinia virus of carrying chimeric LAV/HTLV III env gene has proved can express the protein relevant with the LAV/HTLV III behind the cell during infected tissue cultivates.In addition, find that also these protein can carry out immune response with AIDS patient's serum.

6.4.1 utilize immune patch method differentiate by the cell expressing of recombinant chou vaccinia virus infection with LAV/HTLV III shell proteins associated matter

100 millimeters plate fill and merge the BSC-40 cell, make it be subjected to derive kind of v-env5 or v-env2 of wild-type vaccinia virus or its recombinant chou and infect, and infection multiplicity is 10.Course of infection was carried out 12 hours, then wanted harvested cell at 12 hours, cleaned once with PBS then, by centrifugal collection.Then again the cell piller that infects is suspended in (Laemmli, V.K, 1970, nature 227:680) in 1 milliliter of Laemmli sample buffer, ebuillition of heated 4 hours is so that its dissolving.Whole cell protein electrophoretic method in one whole part 75 microlitre cytolysates separate on the gradient SDS-polyacrylamide gel that makes it at 7-15%.In addition, organize in contrast with a LAV/HTLV III virus particle sample of having been purified and a simulated infection cytolysate.The inclusion of gel by electrotransfer to nitrocellulose membrane.Earlier film was cultivated 30 minutes in 5 milliliters of PBS solution that contain 5% alipoidic milk power (milk) then, temperature is a room temperature, then by PBS, 5% alipoidic milk power with take from that AIDS patient's serum (heat 1: 1000 extent of dilution of inactivated serum) forms, temperature also is the solution for continuous cultivation 2 hours of room temperature.Then film cleans 5 times with the PBS solution that contains 0.05% polysorbas20 (polyoxyethylene sorbitol monolaurin), cleans once with independent PBS solution more afterwards.Film after the cleaning at room temperature contains 1% normal goats serum (heat inactivated serum) with 5 milliliters, and is added with the PBS water culture 2 hours of 1: 3000 anti-human immunoglobulin's one horseradish peroxidase binding substances of dilution goat.The PBS solution that described film contains 0.05% polysorbas20 again cleans 5 times, and uses TBS(0, and 5MNa Cl+20m MTris-HCl PH7.5) cleans once.The horseradish peroxidase binding substances that is combined on the film can detect with following method: make chloro-naphthol staining reagent and film in the dark act on 10 minutes, temperature is that (the chloro-naphthol staining reagent mixes two kinds of solution of A, B and prepares room temperature before use, wherein consisting of of solution A: 20 milliliter of 30% cold methanol+60 milligram 4 chloro-1-naphthols; Solution B is: 60 microlitres, 30% cold hydrogen peroxide+100 milliliter TBS).Be combined on the film, can combine by the anti-human immunoglobulin's horseradish peroxidase of it and goat binding substances with staining reagent with protein that antibody in the AIDS human serum reacts and detect.

This analytical results shows the A as Fig. 8, shows among the figure that the v-env5 of vaccinia virus recombinant has produced one and comprised three kinds of proteinic families, and wherein said protein can be specifically plays immune response with AIDS patient's serum.And the electrophoretic mobility of this proteinoid is similar to the real LAV/HTLV III sugar-protein gp150 that is considered to by the env genes encoding, gp110 and gp41(Robey, and W.G.et al., 1985, Science228:593-595).In the cell of mimic cells infected or wild-type vaccinia virus infection, all do not produce these protein.The recombinant chou v-env2 that lacks the 5' proximal sequential produces the protein shortened, but still can play immune response with the AIDS human serum, wherein mention the 5' proximal sequential and be to the initial methionine(Met) of the supposition of LAV/HTLV III membrane protein and 42 initial amino acid and play the coding effect.By inference, translating of the order of the env among the recombinant chou v-env2 is because (vide infra) that the AUG codon that used joint is transcribed in proper order in this structure recombinant chou starts.

In second experiment, two clones (BSC-40 and Hela) infect with v-env5 and v-env2.The peculiar protein of LAV/HTLV III in the cells infected lysate is to measure by following western immune spot-ing.

Merge individual layer BSC-40 or Hela cell and infect with recombinant chou v-env5 or v-env2, infection multiplicity (moi) has 50 plaque shaping units for each cell.Infected back 12 hours, cell cleans secondary with phosphate buffered saline(PBS), and then is suspended in the Laemmli sample buffer, heated and boiled 5 minutes.Protein in the cells infected makes it to react with the pooled serum of taking from the seropositive individuality of LAV/HTLV III to nitrocellulose membrane with sodium lauryl sulphate-polypropylene amine (SDS-PAGE) electrophoretic separation, electrotransfer again.(Amersham, a-protein MA) can be used for detection can play immunoreactive protein with the iodine-125 mark according to chloramine-t method (according to the specification sheets of manufacturers).In Fig. 8 B, hurdle 1 and 5 comprises the protein of taking from the simulated infection cell; Hurdle 2 and 6 comprises the protein of taking from wild-type vaccinia virus infection cell; Hurdle 3 and 7 comprises the protein from the v-env5 cells infected; Hurdle 4 and 8 comprises the protein from the v-env2 cells infected.The LAV/HTLV III virion protein matter of purifying through saccharose gradient is used as control group (LAV/HTLV III); Coat protein gp150, the position of gp110 and gp41 all as shown in the figure.Molecular weight standard is represented (kD) with kilodalton.

In the cell that v-env5 infects, can detect with the pooled serum of taking from the seropositivity individuality and play immunoreactive three kinds of main protein (Fig. 8 B intermediate hurdles 3 and 7).As calculated, these proteinic molecular weight are 150KD, 120KD and 41KD, and with LAV/HTLV III shell sugar-protein gp150, the molecular weight of gp110 and gp41 is similar.

Recombinant virus v-env2 lacks the signal sequence of generally acknowledging of LAV/HTLV III env, is respectively 99KD but can produce three kinds of molecular weight at least, and 68KD and 40KD can play immunoreactive polypeptide (seeing Fig. 8 intermediate hurdles 4 and 8).

6.4.2 utilize immuno-precipitation differentiate by the cell expressing of recombinant chou vaccinia virus infection with LAV/HTLV III shell proteins associated matter

Below described immune precipitation determination prove, can be synthesized by the cell of recombinant chou vaccinia virus infection of the present invention and can be specifically immunoreactive protein take place with AIDS patient's serum.

100 millimeters plates fill and merge the BSC-40 cell, make it be subjected to the wild-type vaccinia virus, or its recombinant chou v-env5, and perhaps v-env2 infects, and infection multiplicity is 10.After infecting 9.5 hours, with no methionine(Met) do not replenish serum DMEM replacing growth medium, infected back 10 hours, with 2 milliliters contain 100 microcurie/milliliters [ ³⁵S]-the no methionine(Met) DMEM of methionine(Met) replaces above-mentioned substratum, and this radio-labeling process carried out 2 hours under 37 ℃ of temperature.When labeling process finished, cell cleaned once with PBS, collects with centrifuging then.The cell pellet is suspended in 1 milliliter of dissolving damping fluid with following compositions: 1%NP-40(polyoxyethylene (9) P-tert octyl phenol), 0.5% Sodium desoxycholate .0.1M NaCl, 0.01MTris-HCl, PH7.4 and 1mMEDTA, with cell lysates in the miniature centrifuge tube of Eppendorf centrifugal 1 minute, make it clarification again.

The heat-inactivated human serum of 5 microlitres that to take from contrast crowd or AIDS patient is added in the cell lysates of 100 microlitres, to carry out immunoprecipitation.Cultivation is after 1 hour down at 4 ℃, and (Pansorbin Cell Calbiochem-Behring Corp., Lajolla CA), continue down to cultivate 1 hour at 4 ℃ to add the golden glucose spheres mycetocyte of 60 microlitre activatory.In the miniature centrifuge tube of Eppen-dorf centrifugal 30 seconds then, to collect immunoprecipitation complex, temperature is 4 ℃, then with containing 1M NaCl, 0.1%NP-40,0.01MTris-HCl and PH is that 7.4 solution cleans 1 time, uses R IPA damping fluid [10mMTris-HCl, PH7.2 again, 0.15MNaCl, 1% Sodium desoxycholate, 1%Triton X100(polyoxyethylene (9-10) P-tert octyl phenol), 0.1% sodium lauryl sulphate].Immunoprecipitate through washing is suspended in the Laemmli sample buffer of 50 microlitres once more, and ebuillition of heated 1 minute was used the miniature centrifuge tube of Eppen-dorb centrifugal 1 minute then.The protein of staying behind the immunoprecipitation in the supernatant liquid carries out electrophoresis at the 15%SDS-polyacrylamide gel.Behind the electrophoresis, to gel-colored, then handle (at room temperature handling 30 minutes), carry out photofulorography after the drying with the 1M sodium salicylate with sodium salicylate with coomassie basket look dyestuff.

This analytical results as shown in Figure 9, it shows that recombinant chou v-env5 is synthetic and can with the AIDS patients serum immunoreactive gang protein take place specifically.These proteinic apparent molecular weights are 160,140,120,42 and 40 kilodaltons (KD), (Robey, W.G.et al., 1985 roughly conform to the apparent molecular weight of the sugar-protein relevant with shell of the LAV/HTLV III of having reported already, Science 228:593-595). this proteinoid neither is present in simulated infection cell or the wild-type vaccinia virus infection cell, also can be by contrast human serum immunoprecipitation.In the v-env2 cells infected, having produced a kind of apparent molecular weight is the terminal protein that removes of 95KD, and is discerned by the AIDS patients serum.This LAV/HTLV III env proteins associated matter of removing end is started by the AUG codon of joint sequence coding probably, and described joint is positioned at the 5' near-end of this recombinant chou LAV/HTLV III inset in proper order.

6.4.3 the tritium-glycosamine labelled protein relevant that cowpox-LAV/HTLV III recombinant virus produces with LAV/HTLV III shell

The radioimmuno-precipitation assay that describes below shows that cowpox of the present invention-LAV/HTLV III recombinant virus can produce the glycosylation coat protein.

Hela cell or quilt are simulated infection (seeing Fig. 9 B, hurdle 1 and 5), or respectively by wild-type vaccinia virus (hurdle 2 and 6), recombinant chou v-env5(hurdle 3 and 7) or v-env2(hurdle 4 and 8) infect, infection multiplicity is the 50pfu/ cell.In 4 to 16 hours, (concentration is 23 microcurie/milligrams, amounts to 0.25 microcurie, Amersham) is added in the substratum with tritium-glycosamine after infecting.Cell is containing 0.1MNa Cl, 0.01MTris-HCl, PH7.4,1mMEDTA, 1%NP-40(polyoxyethylene (9) P-tert-octyl phenol then with phosphate buffered saline washing 2 times) and the damping fluid of 0.5% Sodium desoxycholate in dissolve.The equal portions cell lysates, or with normal human's serum (hurdle 1-4) or with mix from the pooled serum in the seropositive individuality of LAV/HTLV III (hurdle 5-8).Immune-reactive protein matter is then separated with SDS-PAGE with the fixedly staphylococcus glucose coccus cell precipitation that has a-protein then, and detects with photofulorography.

With glucosamine labelled with radioisotope result shown in the hurdle 7 of Fig. 9 B, it shows that the protein that makes with the v-env5 recombinant chou just as LAV/HTLV III coat protein, glycosylation has taken place also.The difference of glycosylation pattern can be in order to explain the nuance of electrophoresis swimming degree between the protein that obtained by recombinant chou and the LAV/HTLV III virus particle glycoprotein.

As can estimating, in the v-env2 cells infected, do not observe the glycosylation (Fig. 9 B, hurdle 8) of N connection with tritium-glucosamine from the protein that lacks signal peptide.

6.4.4 the pulse-tracking immunoprecipitation relevant that cowpox-LAV/HTLV III recombinant virus produces with LAV/HTLV III shell

Someone proposes, and the gp150 of LAV/HTLV III is a kind of precursor, can obtain outside protein gp110 and transmembrane protein gp41 from described this precursor." pulse-tracking " described below immune precipitation determination shows from the molecular weight of cowpox-LAV/HTLV III recombinant chou manufacturing and is respectively 150KD, 120kD and 41kD protein have the gp150 of a kind of and real LAV/HTLV III, the similar precursor that gp110 and gp41 proposed-product relation.

Merge individual layer Hela cell and use wild-type vaccinia virus (seeing Fig. 9 C, hurdle 1-6), recombinant chou v-env5(hurdle 7-12 respectively), or v-env2(hurdle 13-18) infect, infection multiplicity is the 50pfu cell.After infecting in 10.5 hours, nodal cell is with 100 microcurie/milliliters ³⁵(greater than 800 Curie/millimole, Amersham) mark is 15 minutes for the S-methionine(Met).Then wash once with 2 milliliters of warm in advance tracking substratum (the improved Eagle's medium of Dulbecco+3 mg/ml L-methionine(Met)+5% calves clear+100 units per ml penicillin+100 mcg/ml Streptomycin sulphates), add 1 milliliter of identical substratum again, get back in the incubator then.In the different time, pair cell washs and dissolves, and described in previous 6.4.3, and the protein of cellular products uses the pooled serum from LAV/HTLV III seropositivity individuality to carry out immunoprecipitation.The protein of immunoprecipitation separates with SDS-PAGE again, and detects with photofulorography.Each tracking time length is as follows: 0 hour (hurdle 1,7,13); Hour 0.5 (hurdle 2,8,14); 1 hour (hurdle 3,9,15); 2 hours (hurdle 4,10,16); 6 hours (hurdle 5,11,17) and 12 hours (hurdle 6,12,18).The result as shown in Figure 9, hurdle 7-12 represents to be respectively 150kD by the molecular weight that recombinant chou makes, the protein of 125kD and 41kD, its precursor one product relation and real LAV/HTLV III gp150, the precursor one product relationship consistency of gp110 and gp41.In the Hela cell, the proteinic processing of 150kD seem slow and efficient low because pulse-mark six hours, in the 150kD protein less than tracked 120kD and the 41kD protein of arriving of 50% radioactivity.PRELIMINARY RESULTS shows that this processing is more effective in the human peripheral blood cell of some type that infects with same recombinant virus.These experiments also show, the env order that lacks among the env-2 of signal sequence of real LAV/HTLV III emv can be expressed not modified 87kD precursor (780 amino acid), the latter is formed to a 99kD intermediate, and is cut into 68 and 40kD polypeptide (Fig. 9 C, hurdle 13-18).

6.4.5 in the growth medium of the cell that is infected by cowpox-LAV/HTLV III recombinant virus with the existence of LAV/HTLV III shell proteins associated matter.

Radioimmuno-precipitation assay described below proves, the immunoreactive albumen mass-energy that has that is produced by the recombinant chou vaccinia virus of the present invention is expressed with a pattern that is similar to real LAV/HTLV III shell glycoprotein and processed.

The individual layer Hela cell that merges or with simulated infection (seeing Fig. 9 D, hurdle 1), or use wild-type vaccinia virus (hurdle 2), recombinant chou v-env5(hurdle 3 respectively) or v-env2(hurdle 4) infection, infection multiplicity is the 20pfu/ cell.Be 100 microcurie/milliliters with concentration behind the cell infection ³⁵(greater than 800 Curie/millimole, Amersham) mark is 10 to 12 hours for the S-methionine(Met).Then remove substratum when mark finishes, centrifugation is 2 minutes under 12,000 * g, clarifies, and uses the pooled serum from the seropositive individuality of LAV/HTLV III to carry out immunoprecipitation then.The protein that carries out the cells infected of immunoprecipitation with same serum is presented at " ball " (Pellet) on the plate of mark, and the protein that obtains from substratum then shows with on the plate of " Supe " mark.

As desired, also in the cell culture medium that infects, detect (Fig. 9 D, hurdle 3) by the 120kD protein of recombinant chou manufacturing to the gp110 of LAV/HTLV III.These results prove that recombinant virus v-env5 can express LAV/HTLV III env gene, and can produce immunoreactive protein, and the latter is carried out processing by a pattern that is similar to real LAV/HTLV III shell glycoprotein.

As desired, in the cell culture medium that v-env2 infects, do not detect the polypeptide relevant (Fig. 9 D, passage 4) with LAV/HTLV III shell to the protein that lacks signal peptide yet.

On the other hand, a kind of molecular weight of cell expressing that infected by v-env7 is the terminal protein that goes of 130kD.This protein lacks terminal 217 amino acid of C-of the anchor order that contains LAV/HTLV III coat protein.This protein (gp130) of end that goes is secreted in growth medium, than gp110 or its precursor gp150(Fig. 9 E) much effective.Gp130 is not processed into gp110 effectively simultaneously, removes in the terminal molecule even cutting part still is present in.

6.5LAV/HTLV the immune efficacy of III env cowpox recombinant virus.

The recombinant virus that carries chimeric LAV/HTLV III env gene has been proved to be able to bring out the antibody response that the human primates of two kinds of strain mouse and a kind of Asia tackles the LAV/HTLV III.

6.5.1 the immune efficacy of cowpox LAV/HTLV III env cowpox recombinant chou in mouse.

For identifying by the immunogenicity of recombinant chou vaccinia virus v-env5 and v-env2 expressed protein.Following experiment shows that these recombinant virus have the ability of bringing out the immunne response of all main glycoprotein of LAV/HTLV III.

Two kinds of strain mouse, wherein a kind of is inbred lines (C57B16J), another kind is outbreeding system (ICR), all uses recombinant virus v-env2 or v-env5 inoculation.All animals are in 5-7 age in week all in when inoculation.What adopt is following four kinds of vaccination ways: footpad inoculation, the inoculation of tail cut, intranasal vaccination and intraperitoneal inoculation.Wherein footpad inoculation is that palmula injects 25 microlitres (5 * 10 behind every mouse ⁶Pfu) recombinant virus, the inoculation of tail cut then are with the skin friction of a furcation area pin with the tail bottom, inject 10 microlitres (2 * 10 then on the surface of scratching ⁷Pfu) recombinant virus is placed in the mouse nose, allows mouse inhalation vaccination thing.As for peritoneal injection, be with 10 microlitres (2 * 10 ⁷Pfu) recombinant virus injects the peritoneal cavity of mouse.All viral starting material and thinner all prepare by 6.1 methods.The serum sample of two mouse is gathered every two weeks in the inoculation back, and carries out assay determination with enzyme linked immunological absorption assay method (ELISA) that describes below and West-ern spot analysis method.

6.5.1.1 seroconversion by the usefulness cowpox-LAV/HTLV III env recombinant chou mice immunized of enzyme linked immunological absorption method proof.

The enzyme linked immunological absorption determination data of 6 all serum samples is generalized into table 1.Recombinant chou v-env2 and the v-env5 seroconversion rate of C57B16J mouse are respectively 100% and 95%.And for the IRC mouse, wherein the v-env2 seroconversion rate is 44%; V-env5 then is 100%, and it is the highest to obtain the mean value that complete seroconversion rate and enzyme linked immunological absorption tire in by afterbody cut method mice immunized.

Table 1 is with carrying chimeric LAV/HTLV III

The mice serum transformation efficiency * of the recombinant virus inoculation of env gene

Inoculation back mice serum transformation efficiency

Recombinant virus vaccination ways ICR C57B16J

V-env2 footpad inoculation 3/3 4/5

The tail cut inoculates 3/3 4/5

Intranasal vaccination 0/3 4/5

The intraperitoneal inoculation does not do 3/5

V-env5 footpad inoculation 4/4 5/5

The tail cut inoculates 3/3 5/5

Intranasal vaccination 3/3 4/5

The intraperitoneal inoculation does not do 5/5

* seroconversion is measured by the enzyme linked immunological absorption method hereinafter, represents with the mouse number and the ratio of the interior inoculation of group total mice of each group seroconversion.

The ELISA data obtain with following method: purified already and the LAV/HTLV III virus particle of deactivation with carbonate buffer agent (50nM yellow soda ash, PH9.6) dilution, be added to 96-well droplet plate (100 microlitres/well, each well contain the LAV/HTLV III of 0.2 microgram deactivation) then.Under 4 ℃ of temperature, place and spend the night, so that cohesive process is carried out.Unconjugated protein is extracted out, and each well is used the 200 microlitre PBS solution washings that contain 5% alipoidic milk power earlier, and then washs with 4% sucrose solution of 300 microlitres.Excessive sucrose solution is extracted out, made culture plate at room temperature dry (1 hour).And then the PBS solution that 50 microlitres contain 2.5 microliters of mouse serum samples is added in each well, make it 37 ℃ of reactions 1 hour down.When cultivate finishing, with the PBS solution well-flushing that contains 0.05% tween 20 (polyoxyethylene sorbitol glycerine-moon silicon ester) 5 times.Anti-mouse horseradish one superoxide enzyme conjugates (solution of PBS+0.05% tween 20 carries out 1: 5000 dilution) with the goat of 50 microlitres is added in each well again, makes it 37 ℃ of reactions 45 minutes down.After washing 5 times, add 100 microlitres citric acid-O-phenylenediamine one peroxide substrate with the PBS solution that contains 0.05% tween 20.The following process preparation of substrate: the dihydrate and the 0.537 gram sodium hydrogen phosphate crystal (Sodium Phosphat Dibasi Crystal) of 0.294 gram sodium are dissolved in 10 ml waters, pH value are adjusted to 5.0 with hydrochloric acid; Add 2 microlitres, 30% hydrogen peroxide and 4 milligrams of O-phenylenediamines before use.Culture plate at room temperature is placed on the dark place and cultivated 30 minutes.Adding 100 microlitre concentration then in each well is 1.3 normal sulfuric acid, and reaction is stopped, and 490 millimicrons of absorbancys of locating the assaying reaction mixture.

The result of table 1 is with the ratio of seropositive mouse number with the mouse sum of being inoculated.6 weeks of inoculation back are collected serum sample, with ELISA it are analyzed.The serum sample of 5 inoculation mouse is as control group.The average ELISA of control group tire for ICR and be respectively 0.021 ± 0.005 and 0.017 ± 0.010 for the C57B16J mouselet, the ELISA that takes from the serum sample of the mouse that is subjected to immunization tires than a control group Senior Three above standard deviation, and these serum samples are be evaluated as the positive.

6.5.1.2 the mouse generation that transforms through serum resists the antibody of real LAV/HTLV III shell glycoprotein to prove by the Western immune spot measuring.

In order to prove whether the mouse with the recombinant virus inoculation produces the antibody that tackles real LAV/HTLV III capsid glycoprotein, taking from the serum sample of immunized mice measures by following Western immune spot-ing: 5 to 7 the week age inbred lines male mouse (C57B16J, the Jackson laboratory) is to carry out immunization, promptly is bifurcated the pin place of scratching and applies 10 microlitres and include 2 * 10 in tail bottom by the afterbody cut ⁷The inoculum of pfu recombinant chou vaccinia virus.8 weeks after inoculation, in the bloodletting of animal posterior orbit sinus, and blood plasma kept freezing state, till using, take out the equal portions serum sample, add 0.2%NP-40 and 30% alipoidic milk power, with 50 times of phosphate buffered saline (PBS) dilutions, make it then and the qualitative response of LAV/HTLV III virion protein, the latter is separated by SDS-PAGE, and is fixed on the nitrocellulose membrane by the electrotransfer method.The LAV/HTLV III protein of being discerned by these serum is to use the anti-mouse immuning ball protein with alkaline phosphatase bonded goat to detect.

8 all serum samples of being got on one's body with the mouse of the C57B16J group of recombinant virus inoculation at afterbody cut place the results are shown in Figure 10.The animal of useful recombinant virus inoculation all produce the antibody that can react with LAV/HTLV III shell glycoprotein gp41.Some uses v-env5(Figure 10, and the hurdle is the animal of immunization a), and its serum also can be discerned gp150 and gp110, and this just shows that described recombinant virus can bring out the immunne response of anti-all main glycoprotein of LAV/HTLV III.

6.5.2 the immunogenicity of cowpox-LAV/HTLV III recombinant chou v-env5 in the inferior mankind's primates.

In two kinds of human primate species macaques in Asia (Macaca fascicularies) and chimpanzee, immunogenicity to cowpox-LAV/HTLV III recombinant chou v-env5 is studied: following each trifle is described result prove, v-env5 can bring out the immunity of body fluid and cell participation at described two species.

6.5.2.1 humoral response with the macaque of v-env5 immunization

9 long-tail crust macaques comprise that young macaque to the macaque that grows up, is used to study the immunogenicity of cowpox-LAV/HTLV III recombinant chou v-env5.In advance all animals have all been done generaI investigation,, also do not had T-parent's lymph venereal disease poison of monkey or the HIV (human immunodeficiency virus) of monkey to prove conclusively the antibody that does not tackle vaccinia virus and LAV/HTLV III in its body, and anti-these viral antibody.Four monkeys are with 2 * 10 ⁸The v-env5 inoculation of pfu then inoculates 2 * 10 for other four ⁷The identical virus of pfu.In addition, also has an animal inoculation pvaccination 2 * 10 ⁷The contrast recombinant chou vaccinia virus of pfu dosage (the simple property of cowpox-bleb gDI) recombinant chou V-HSg DI).All animals all inoculate with cutaneous scarification.Before the inoculation, and postvaccinal the 4th, the 6th and the 8th week gather serum and heparinization blood sample.Through ten weeks, again all animals are carried out the inoculation second time after inoculation first, used virus is identical with last time, and dosage is 2 * 10 ⁸Pfu.In whole experiment, all animals all show the self-healing skin injury and the normal physiology indication of typical vaccine infection.Adopt ELISA and Western spot assay method analysing body fluid to reply.

In order to make elisa assay, the serum sample will be with containing 3% alipoidic milk power and 0.2%NP40(polyoxyethylene (9) P-tert-octyl phenol) 50 times of PBS solution dilutions, make it then to react with the LAV/HTLV III virion protein matter that has been fixed in the droplet well.Except that second kind of used antibody be with the anti-human body antibody of horseradish peroxidase bonded goat, identical described in ELISA operation steps herein and the 66.5.1.1 joint.

All animals were having only two animal seroconversion after the inoculation all by seroconversion as far as possible for the first time after result shown in Figure 11 proved the immunization second time.

Produced what antibody for conclusive evidence is subjected to the animal of immunization, the serum sample of gathering in 4 weeks after the immunization is analyzed with the Western spotting method for the second time.Optimum detection for two kinds of shell glycoprotein gp41 and gp110 adopts two kinds of schemes.For optimum detection gp41, serum is with being added with 30% 50 times of PBS solution dilutions not implementing pasteurellaceae disinfectant milk and 0.2%NP-40, then at room temperature with the LAV/HTLV III virion protein qualitative response of having been purified 1 hour that is fixed on the nitrocellulose membrane.Then film is with the PBS solution washing that is added with 0.2%NP-40, again with 2000 times of the PBS buffering dilutions that is added with 0.2%NP-40, and makes it and (Zymed CA) reacts with the anti-human immunoglobulin's antibody of alkaline phosphatase bonded goat.And in order to detect gp110 serum with being added with 3% 50 times of PBS solution dilutions not implementing pasteurellaceae disinfectant milk, then at room temperature with the LAV/HTLV III virion protein qualitative response of having been purified 1 hour that is fixed on the nitrocellulose membrane.Then film is with the PBS solution washing that is added with 0.05% tween 20, again with 2000 times of PBS damping fluid dilutions, and make it and with the anti-human immunity protein antibodies of alkaline phosphatase bonded goat (Zymed, CA) reaction.More than in arbitrary method, the final washing behind the antibody response is for the second time all used and is contained 0.1 molconcentration Tris(pH9.5), 0.1 molconcentration sodium-chlor and 5 millimole concentration magnesium chloride solutions.With antigen bonded antibody on the film be to detect as follows: make film and contain 0.1 molconcentration Tris(PH9.5), 0.1 molconcentration sodium-chlor, 5 millimole concentration magnesium chlorides, 0.33 mg/ml bromo-chloro-indoles an aromatic plant metioned in ancient books phosphoric acid salt and 0.17 mg/ml nitroblue tetrazolium father-in-law solution react.Finish at filter paper and the reaction of above-mentioned chromogen, then water cleans, and takes a picture behind air drying again.

Shown in Figure 12 B, all animals that are subjected to secondary inoculation all show the intensive antibody response to gp41.In addition, wherein four animals also produce the antibody that carries out kickback with gp110.In a word, the result shown in Figure 12 A has proved that recombinant chou v-env5 can bring out macaque and produce the distinctive antibody of LAV/HTLV III shell.

6.5.2.2 in the immunne response of regulating with the cell of the macaque of v-env5 immunization

Adopt the Ficoll-hypaque centrifugal separation, after second time intradermal immunization, separate peripheral blood lymphocyte (PBL) the blood of 4 all resulting macaque heparinizations, wherein inoculate for the second time used inoculum or v-env5, or the herpes simplex virus glycoprotein D (V-HSVgdl) of the cowpox recombinant virus of contrast expression.From only with v-env5 inoculation once macaque 81 and never be subjected to also to isolate peripheral blood lymphocyte the macaque blood of immunization.These peripheral blood lymphocytes are suspended in the RPMI1640 substratum of the deactivation normal human serum that is supplemented with 10% heat (GIBCO, Grand island, New York), and in the end volume is to take out 1 * 10 in 0.1 milliliter the substratum then ⁵Peripheral blood lymphocyte puts it in the well of 96 well round bottom culture plates.Adding 0.1 milliliter of v-env5(that contains the deactivation of ultraviolet (UV) light in each well is 1 * 10 before the UV-light deactivation ⁶The pfu/ milliliter), or unspoilt LAV/HTLV III (1 mg/ml is about 1 * 10 ⁵TCID ₅₀) substratum, wherein the LAV/HTLV III is to adopt twice sucrose gradient centrifugation, the supernatant liquor of the cem cell that infects from the LAV/HTLV III is purified and is obtained, and described cem cell is to belong to the negative T chronic myeloid leukemia of HLA DR system.Stimulated back six days, every well PBL is with the tritium-TdR(tritium-thymidine of 1 microcurie, New England Nuclear, Boston, MA) mark is 6 hours, takes out cell then, measure the Cpm(Curie of tritium-TdR/minute), can obtain the mean value of 4 identical experiment wells thus.To be incorporated into by the cpm of irritation cell, tritium-TdR can calculate stimulation index divided by being incorporated into the not cpm of irriate cell.

Table 2

The LAV/HTLV III capsid glycoprotein of expressing with the cowpox recombinant virus carries out the proliferation response that the LAV/HTLV III of the PBL of gained after the immunization is brought out to macaque

Be used for virus to the PBL stimulation

The viral LAV/HTLV III V-env5 of macaque immunity

Numbering inoculation cpm* cpm* SI* cpm* SI*

67 v-env5 1.586 7.465 4.7 60.415 38.1

68 v-env5 2.245 9.075 4.0 28.638 12.8

74 v-env5 1.585 4.732 3.0 21.487 13.6

75 v-env5 581 8.645 14.9 37.847 14.1

76 v-env5 2.479 5.987 2.5 36.657 14.8

80 v-env5 1.077 13.922 12.9 24.752 23.0

81 v-env5 965 3.985 4.1 40.572 42.0

82 v-env5 2.581 8.580 3.3 46.847 18.1

73 v-HSVgD1 612 553 1.0- 56.217 91.9

26 do not have 2.911 1.822 1.0-, 2.240 1.0-

27 do not have 1.228 1.072 1.0-2.532 2.0

* the cpm that is incorporated into ³H-TdR

The * stimulation index

Macaque as shown in table 2, that all were subjected to immunization comprises and only accepts No. 81 macaques of a V-env5 inoculation that they all produce some lymphocytes, and these cells stimulate the generation specificity to reply to external LAV/HTLV III.In contrast, accepted No. 73 macaques of cowpox-herpes simplex virus recombinant chou V-HSVgD1, its lymphocyte only responds to the vaccinia virus stimulation and stimulates reactionless to the LAV/HTLV III.The macaque that is not subjected to immunization does not in contrast produce the lymphocyte that can do reaction to above-mentioned arbitrary antigen.And, carry out the macaque of immunization with v-env5, its lymphocytic reaction is in size with identical with No. 73 macaques of V-HSVgD1 inoculation.These results show, express LAV/HTLV III env gene by the recombinant virus in macaque and can not be suppressed at the immune response that the T cell participates in external or the body.

Stimulate for which part lymphocyte of macaque of determining to be subjected to immunization can be subjected to the LAV/HTLV III, specially check the performance of the PBL of irriates with the IL-2 acceptor that is present in activated T cell or B cell of understanding with two colour developing immunofluorescences.CD4 and CD8 antigen are to be present among the T cell, and CD20(Bp35) antigen is to be present among the B cell.The result proves shown in the table 3, and the PBL that is stimulated by virus of the nearly all IL-2 of being expressed as acceptor also shows Ze CD4 or CD8 antigen, and 1.5% to 2% co expression CD20(Bp35 is arranged) antigen.This result shows that the lymphocyte of irriate is the T cell.

Table 3

Stimulate the IL-2 on the resulting PBL of macaque of inoculation from the LAV/HTLV III capsid glycoprotein of expressing with LAV/HTLV III or recombinant chou vaccinia virus

CD20(Bp35) antigenic expression

Macaque stimulates the thin IL-2R+ cell of PBL IL-2R+ common

Total % CD4 CD9 CD20(Bp35 of the viral born of the same parents of numbering)

74 v-env5 27.7 48.6 52.2 2.0

80 v-env 25.6 62.4 40.0 NT

74 LAV/HTLVⅢ 14.7 50.0 59.0 1.9

80 LAV/HTLVⅢ 12.0 58.0 38.0 1.5

80 virus-free 0.3 0.3-0.3-0.3-

The result is obtained by following mode in the table 3: PBL dyes simultaneously, dialogue iuntercellular plain-2 acceptor (IL-2R) phycoerythrin bonded monoclonal antibody 2A3(Becton-Dickin-son, Inc., Mountain View, CA) dye, to CD20 Seattie with fluorescein bonded monoclonal antibody 1F5(Gene tic Systems Corp, WA) dye, with to CD8 with G10-1(Gene tic Systems Corp Seattle, WA) dye, perhaps to CD4 T4a(OrthoDiagnostics, Baritan N.J.) dyes.Used antibody saturation concentration, be to determine (Becton-Dickinson by the lymphocyte with the flow microfluorometer (FMF) analysis that has improved FACS III sorter is carried out titration, Mountain View, CA). two look quantitative analyses are carried out (Ledbetter, J.A., et al. by the method for former description, 1984, Perspectives in Immunogenetics and Histcompatibility, 6,119-129).The scattering door at forward angle and right angle adjusted to comprise lymphoblast and a large amount of small lymphocytes.Shown numerical value is the total percentage ratio of IL-2R+ cell, and IL-2R+ cell expressing CD4, CD8 or CD20(Bp35) percentage ratio.

After having confirmed that the T cell is subjected to antigenic stimulation, produced the lymphokines that comprises IL-2, they can promote T cell and B cytodifferentiation and/or hyperplasia, and the killer cell of energy active natural, and killer cell can be dissolved the cell of the various virus infectiones that comprised HIV (human immunodeficiency virus).Therefore we will understand the T cell of accepting the inoculation macaque whether produce IL-2 after with the stimulation of LAV/HTLV III.

Two experiments have been carried out for this reason.In experiment 1, from for the second time with v-env5 or carry out intradermal by the V-HSVgD1 that WR vaccinia strain constitutes and inoculate in the heparinization blood of macaque in 4 weeks and isolate PBL.And in experiment 2, from for the first time with 2 * 10 ⁵The v-env5 of pfu, V-HSVgDI or v-env5 recombinant virus carry out isolating PBL in the macaque heparinization blood after intradermal inoculated for 4 weeks, and wherein said recombinant virus is that the vaccine virus strain (v-env5NY) by New York board of health constitutes.PBL is suspended in the RPMI1640 substratum that adds 10% heat-inactivated normal human's serum, again with 2 * 10 ⁵PBL puts into the well of 96 well round bottom culture dish.The v-env5(that adds the UV-light deactivation then in well is 1-10 before the UV-light deactivation ⁶Pfu/ milliliter or the LAV/HTLV III (1 mcg/ml) of being purified.Stimulated back two days, and from same well, obtained supernatant liquid, and verify they support the relevant CTI-2 cell of IL-2 (provide by doctor S.Gillis, ImmunexCorp., Seattle, hyperplasia ability WA), so to wash 24 hours before measuring, to remove IL-2.During last 6 hours that cultivate with supernatant liquid, allow cell use ³The H-TdR mark is measured then and to be incorporated into cell ³The amount of H-TdR.By the typical curve that influence gained of test human body IL-2 recombinant chou (providing), can calculate the IL-2 activity unit that exists in the supernatant liquid to CTLL-2 cell proliferation by doctor Gillis.The table III is listed experimental result.

Table 4

Inoculated PBL in the macaque blood with the post-stimulatory inoculation of capsid glycoprotein of LAV/HTLV III HIV (human immunodeficiency virus) or with the IL-2 that PBL produced of the recombinant chou macaque that shows as AIDS virus coat glycoprotein with what LAV/HTLV III or recombinant chou vaccinia virus were expressed

Experiment 1: the IL-2(units per ml in the supernatant liquid)

The virus of macaque immunostimulation PBL

The virus-free LAV/HTLV III v-env5 of numbering inoculation

67 v-env5 0 28.0 96.5

68 v-env5 0 16.0 124.0

74 v-env5 0 9.0 52.0

73 v-HSVgDI 0 0 188.0

26 do not have 000

Experiment 2:

03 v-env5 0 14.4 55.8

05 v-env5NY 0 16.8 33.3

49 v-env5NY 0 7.1 26.7

52 v-env5NY 0 2.4 27.6

59 v-HSVgDI 0 0 27.6

26 do not have 000

27 do not have 000

The result of the experiment 1 in the table 4 shows, be subjected to the PBL that obtains in the macaque blood of twice of v-env5 immunization, after v-env5 or LAV/HTLV III stimulated, its supernatant liquor contained IL-2 again, this is can bring out the CTLL-2 hyperplasia by them to be confirmed, the latter is the relevant clone of a kind of IL-2.Equally, as show shown in the experiment 2 of III, be subjected to the v-env5 immunization once be numbered 03 macaque, and with same recombinant chou (V-env5NY, see 6.3 joints) " Smallpox Vaccine " bacterial strain carry out all three macaques of one shot immunity, the PBL through LAV/HTLV III or v-env5 stimulation that is taken out detects IL-2 in the clear liquid thereon.In contrast to this, only after stimulating, just produce IL-2 with v-env5 from the PBL that 73 and 59 macaque gets that is numbered that is subjected to cowpox-HSVgD-1 recombinant virus immunization, and with not producing IL-2 after the stimulation of LAV/HTLV III.In addition, be numbered 26 and 27 the macaque that is not subjected to immunization, but after LAV/HTLV III or the stimulation of recombinant chou vaccinia virus, all do not produce the IL-2 of detected level.Owing at first be that the T cell that has auxiliary/induced activity just produces IL-2 after being subjected to antigenic stimulation, these results prove, are subjected to have helper cell really in the macaque of recombinant virus immunization, and the latter can discern the LAV/HTLV III.Except these T cells that produce IL-2 produce one of the antibody that tackles LAV/HTLV III capsid antigen may act in the B cytodifferentiation, in effector cell's's (if can kill the virose T lymphocyte of viral pair cell or the natural killer cell of cells infected) differentiation and/or process of expansion, may relate to the T cell that these produce IL-2.

6.5.2.3 be subjected to the humoral response of the chimpanzee of V-ENV5NY immunization

Two immature chimpanzee acceptance belong to the intradermal inoculation of the v-env5NY of v-env5 vaccine strain, and inoculum dosage is 5 * 10 ⁸Pfu.The cowpox herpes simplex virus gD recombinant chou that animals received is tired equally, i.e. the corium of V-HSVgD1 NY inoculation, the latter is made of as V-env5NY identical parent's cowpox bacterial strain (V-NY).For the first time 8 weeks after the immunization, all animals are all accepted inoculation for the second time, and dosage is the same.Gather a serum sample every two weeks after the immunization, and measure the distinctive antibody of LAV/HTLV III with ELISA and Western spotting method, in whole experiment, all animals all show the typical self-healing skin injury of vaccinia virus infection, and have normal physiology indication.

The ELISA method that chimpanzee serum is used is and used identical of serum of macaque.Result shown in the table 5 shows, two laboratory animal (124 and 149) are 8 weeks after immunization for the first time, and seroconversion has taken place, and its antibody horizontal continues to raise after accepting for the second time immunization.Control animal (134) keeps seronegativity.

Table 5

Accept the elisa assay result of serum sample of the chimpanzee of recombinant chou vaccinia virus immunization

The ELISA reading of LAV/HTLV III-peculiar serum antibody

No. 149 chimpanzees of No. 124 chimpanzees of No. 134 chimpanzees of taking a sample

Time V-HSVgDINY V-env5NY v-env5NY

0.084 ± 0.015 0.100+0.005 0.123+0.025 in advance draws blood

For the first time connect 0.085 ± 0.010 0.185 ± 0.020 0.403 ± 0.027

8 weeks after kind

For the second time connect 0.075 ± 0.012 0.237 ± 0.017 0.523 ± 0.073

2 weeks after kind

The special antibody of LAV/HTLV III that detected in being subjected to the immunization animal for checking is anti-capsid glycoprotein, and the spy has done analysis with the Western spotting method to same serum.Used method is suitable for gp41 antibody most, and analyzes used identical with serum of macaque.Result shown in Figure 13 proves that these two the special antibody of inoculating of the LAV/HTLV III that animal produced are anti-shell glycoprotein really, and these antibody horizontals raise after the immunization in the second time.

6.5.2.4 accept the immunne response that the cell of the chimpanzee of v-env5NY immunization participates in

Save the immunne response that cell that isolated peripheral blood lymphocyte (PBL) in the described same animal blood is used to verify these animals participates in 6.5.2.3.From carrying out intradermal inoculation 4 weeks of back with recombined vaccinia virus for the first time,, from heparinization blood, isolate PBL by the Eicoll-hypaque centrifugation.Then PBL is seeded in the RPMI1640 substratum that is added with 10% heat-inactivated normal human's serum and penicillin/streptomycin in the 96 well culture dish, wherein every well contains 1 * 10 ⁵Cell.Then add unspoilt LAV/HTLV III (5 mcg/ml) or LAV/HTLV III shell glycoprotein (1 mcg/ml) in well, the latter is isolating from the LAV/HTLV III of having purified with lens phytohemagglutinin chromatography.After six days, be combined in tritium-thymidine in the cell with liquid flashing counting measuring.

As shown in table 4, the lymphocyte of two animals (124 and 149) that is subjected to the v-env5NY immunization is hyperplasia with coat protein (env) hormesis of LAV/HTLV III virus particle or purification.By comparison, No. 134 animals accepting the inoculation of cowpox-herpes simplex virus recombinant chou v-HSgD1 NY do not produce the lymphocyte that can discern the LAV/HTLV III.

Table 6

The immunne response that participates in the cell of the chimpanzee of cowpox group body inoculation

Engage tritium-thymidine of PBL

The virus that chimpanzee stimulates

The numbering immunization does not have antigen LAV/HTLV III env

124 v-env5NY 2482 37，132 26，370

149 v-env5NY 375 20，292 27，115

134 NY 367 1，987 367

64 do not have 452 567 222

72 do not have 737 2,417 905

After the PBL of all chimpanzees stands human body mixing xenogeneic PBL behind phytohaemagglutinin (PHA, 2 mcg/ml) or the X-x ray irradiation x and stimulates, all show and look out high-caliber tritium-thymidine combination, its numerical value is respectively 38,870 to 109,790cpm and 42,172 to 12,067cpm.Accept the chimpanzee of v-env5 or v-HSVgDINY immunization, its PBL demonstrates has the intensive proliferation response to vaccinia virus; And be not subjected to the PBL hyperplasia not of the chimpanzee of immunization with the stimulation of vaccinia virus.

In a word, result shown in table 1-6 and Figure 11-13 shows that (a) recombinant chou vaccinia virus v-env5 and the counterpart v-env5NY in vaccine strains thereof can bring out the immunne response of special body fluid of LAV/HTLV III and cell participation in the macaque and these the two kinds inferior mankind's of chimpanzee primate; (b) recombinant virus can not suppress the immunne response that the T cell participates in external or the body.

6.6 the neurotoxicity of the recombinant chou that is made of the V-NY strain of vaccinia virus lowers, and some parts of 25 to 50 microlitres are contained 5 * 10 ⁷Pfu virus is injected in 5 mouse brains, inoculates back 10 days and calculates its mortality ratio.

The result of table 7 shows that the vaccine virus (v-NY bacterial strain) that the tissue culture of being purified by plaque obtains and kind of the v-env5NY that derives thereof compare with the WR bacterial strain and the kind of deriving thereof with v-env7NY with v-env2NY, and their neurotoxicity reduces.Because the bacterial strain of New York board of health is used as antismallpox vaccine, therefore being based upon the recombinant chou that obtains on this bacterial strain basis is more suitable for developing the vaccine that human body is used.

Table 7

The mouse death rate of inoculation cowpox recombinant chou in the brain

Inoculate dead mouse number/acceptance injection mouse number

v-NY 0/5

v-env5NY 0/5

v-env2NY 0/5

v-env7NY 0/5

Antismallpox vaccine (Wyeth) 3/5

v-env5 5/5

v-env2 5/5

v-env7 5/5

Salt solution 0/5

7. embodiment: baculovirus gag recombinant chou

In following each embodiment, various plasmid vectors to be made contain mosaic gene, these mosaic genes comprise and are positioned at the LAV/HTLV III gag coded sequence that AcNPV transcribes the control sequence below.

These mosaic genes that comprise Ac NPV polyhedron promoter region LAV/HTLV III gag coded sequence are inserted in the AcNPV genome by reorganization in the body.This class recombinant virus is differentiated and is purified, and from infected tissue's culturing cell the viral starting material of preparation.Playing that immunoreactive, relevant with LAV/HTLV III gag protein has been proved to be is in external generation by recombinant chou AcNPV.Be proved to be by the cell of recombinant virus and can demonstrate positive immunofluorescence.At last, from the cell lysates that infects with recombinant chou AcNPV, when handling, then in measuring, ELISA is positive as if the serum of using the AIDS patient.Each trifle is stated in the detailed description of each step of this embodiment of the present invention as follows.

7.1 general method

7.1.1 cell and virus

Spodotera frugiperda cell, clone sf9 obtains from U.S.'s typical case's culture collection product (ATCC No CRL1711), and make it at Grace's Antheraea substratum (Gibco, KC Biologicals) breeding in, substratum contains the yeastolate(Difco of 3.3 grams per liters) and the lactalbumin hydrolysate (Difco) of 3.3 grams per liters, 10% N of embryo's serum, and 0.06 grams per liter penicillin and 0.1 grams per liter Streptomycin sulphate (TNM-FH substratum).Cell is grown in 28 ℃ of temperature.Autographa califonica nucleopolyhedrosis virus system obtains (Department of Genetics and Entomology from Lois doctor Miller, UniversityofGeorgia, Athens, GA30602), and at the enterprising line space spot of the sf9 cell that contains 1% agarose and 0.8XTNM-FH purify.The virus starting material dilute with TNM-FH.

7.1.2DNA preparation, restriction and improvement

Restriction enzyme is to buy from the Bethesda research laboratory, and the condition of restriction digestion is to advise by manufacturers.With the Klenow fragment of e. coli dna polymerase with concentration be 200 units per ml put into contain 50mM Tris-HCl(pH7.4), 6mM MgCl ₂Among the solution of 10mM2-mercaptoethanol, 37 ℃ of temperature, 30 minutes time.

The viral DNA that is used for transfection is to utilize following Miller, L.K., and Miller D.W and Safer, the method for P. makes from non-closed virus (NOV) starting material of wild-type virus.Contain 25% sucrose, 5mM NaCl and 10mMEDTA by bedding and padding liquid, 90,5 ℃ of centrifugations of 000xg temperature made the NOV particle slabbing of supernatant liquor measure in 1 hour.Supernatant liquor is removed, make again virus be suspended in 0.5 milliliter the dissolving damping fluid in (10m MTris, pH7.6,10mM EDTA, 0.25%SDS).After virus particle suspends once again, add Proteinase K, making ultimate density is 500 mcg/ml, and overnight incubation (about 16 hours), temperature is 37 ℃, and stirs frequently.Use the phenol of equal volume then: chloroform: primary isoamyl alcohol (25: 24: 1) ' the solution extraction DNA that forms.DNA is that 5 the 3M sodium-acetate and the cold ethanol of 2 volumes are precipitated out under-20 ℃ by the pH that adds 1/10 volume then.After in flakes, DNA 70% washing with alcohol, drying, and before being used for transfection or restriction enzyme analysis, be suspended in TE(10mMTris, 1mM EDTA, pH7.5) among.

Plasmid DNA is to use following Summers, M.D and Smith, and the best preparation method of G.E prepares: one milliliter of Luria liquid nutrient medium (LB) that is added with suitable antibiotic is inoculated with the single bacterium colony bacterial cell in the culture dish of taking from firm preparation.After 37 ℃ are down cultivated 12 to 18 hours, again 1 milliliter of culture is transferred in the 5000-1000 ml flasks that fills 200 milliliters of LB that are added with antibiotic, then put it into and shake in the thermostatted (37 ℃) spend the night (12-18 hour).200 milliliters of cultures are moved in the centrifugal engine room, carried out centrifugation 10 minutes with 2500 rev/mins.After shifting out supernatant liquid, (8% sucrose, 0.5%Triton X-100,50mMEDTA(pH8.0), 10mMTris-Cl pH8.0), and moves in 125 ml flasks again to allow each cell piller at room temperature be suspended in 30 ° of milliliter STET damping fluids again.Adding 3 ml concns in every bottle is the N,O-Diacetylmuramidase (prepared fresh in the STET damping fluid) of 10 mg/ml, and the turn bottle makes it to mix, and at room temperature cultivates 5 minutes.Next every bottle is placed directly in turn gently on the flame, begins to solidify and change into white until cell.As soon as bubble formation just moves into flask in the boiling water bath, placed for 45 seconds, and then this bottle is put into ice-water bath cooling 2 minutes or longer.After this above-mentioned viscid mixture is slowly poured in 50 milliliters of round bottom centrifugal bottles, one with the SW28 rotor (Beckman, centrifugation is 15 minutes in preparative centrifugation machine CA), rotating speed is 16000 rev/mins.In the interior 50 milliliters of active polypropylene centrifuge tubes of the careful impouring of supernatant liquor of each pipe, Virahol or 2 volume ethanol of also adding 1 volume in the pipe, DNA is in-80 ℃ of precipitations 20 minutes (or-20 ℃ down precipitation 2 hours or longer), under 2500 * g condition centrifugal 15 minutes or longer then.After removing ethanol, in piller, add 2.5 milliliters of extraction damping fluids and (contain 12.1 gram Tris in every liter of damping fluid, 33.6 gram Na ₂EDTA 2H ₂O and 14.9 gram KCl, pH is 7.5), and with its rotation, with loosening bottle floor cells lysate.The RNase A(that then adds 10 microlitre concentration and be 10 mg/ml is dissolved among the 0.1XTE, seethes with excitement 10 minutes to carry out pre-treatment), cultivated 30 minutes down at 37 ℃.In each pipe, add the Proteinase K that is about 200 micrograms again, and cultivated 30 minutes down, at this moment add 250 microlitre 10%Sarkosyl, continue to cultivate 3 hours or the longer time at 45 ℃-50 ℃.In order to prepare the cesium chloride gradient, adding concentration in each pipe is the ethidium bromide of 10 mg/ml, is 80 microlitres in the consumption of every milliliter of dna solution.And then whenever adding 1.04 gram cesium chloride/milliliter dna solutions in by all means, turn makes its dissolving gently immediately.After in rotor, carrying out centrifugation, promptly carry out centrifugation to reach balance in 70.1Ti fixed angles rotor, rotating speed is 55.000 rev/mins, 16 hours time, produce two well-separated visible DNA bands thus, the band of its middle and upper part comprises straight line and nicked DNA.And the bottom band contains the cyclic DNA of covalent linkage sealing.Take out the band (with the cyclic DNA of covalent linkage sealing) of bottom, put into 15 milliliters of polyethylene centrifuge tubes, add water again and make each pipe volume reach 6 milliliters.Extract ethidium bromide with the equal-volume primary isoamyl alcohol from DNA, top pink liquid phase is taken out and is outwelled.Extraction process need repeat; Become colourlessly until the top liquid phase, and bottom (DNA) liquid phase is limpid colourless.At this moment, should leave 5 milliliters of dna solutions (, then should add water to 5 milliliters) in each pipe, add 2 volume dehydrated alcohols then if less than this milliliter number.Its placement under-20 ℃ is spent the night, or placed 10 minutes, the DNA precipitation is separated out at-80 ℃.Then slabbing 20 minutes under 2,500 * g.After removing all ethanol, add 50 microlitre 0.1XTE to each piller again, and make it under 65 ℃, to suspend once more 10 minutes or the longer time.DNA can preserve down at 4 ℃.

Plasmid DNA is also pressed described method preparations such as Maniatis: [(MolecularCloning, Cold Spring Harbor Laboratory(PP.8696)].Dna ligase is to buy from New England Biolabs, adds 50mMTris-HCl(pH7.8 during use), 10mM MgCl ₂, 20mM DTT, 1mMATP, making DNA concentration is 10,000 units per ml.DNA analysis in agar gel is to carry out (1982, MolecularCloning, Cold Spring Harbor Laboratory) by the method that following Maniati S etc. is provided.

7.2 contain the plasmid vector structure of the AcNPV promotor of the coded sequence that is attached to LAV/HTLV III gag gene (pA-gag1)

Following each trifle is described the structure of the plasmid vector that contains LAV/HTLV III gag genes encoding order, and wherein said gene is positioned at Ac NPV transcribes after the sequence of control, but is positioned at before the polyhedron DNA.These plasmid recombinant carriers are used for later on LAV/HTLV III gag coded sequence is inserted the genome of baculovirus by reorganization in the body.

In following each trifle, LAV/HTLV III gag coded sequence (Figure 14) is purified from pKS-5, the latter is the subclone (Wain-Hobson of λ J19, S., et.al., 1985, Cell 40:9), and among the plasmid Ac610 of below, the polyhedron promoter region of the baculovirus that is inserted among the pAC610 to be comprised, to constitute pAC-gag I (Figure 15).In this structure, the special nucleotide sequence front of LAV/HTLV III is the polyhedron promoter region, forms a mosaic gene polyhedrosis gene order.

As previously mentioned, pKS-5 be form by the fragment of one 3,148 base pair SstI to KpnI of the LAV/HTLV III DNA inset that is comprised among the λ J19, wherein λ J19 clones into SstI and the KpnI position of PUC18.It is by the SstI restricted fragment of λ J19 being cloned the into SstI position of pUC18, and obtaining pBT-1, the latter connects after with KpnI digestion again.This DNA is used for transformed into escherichia coli, and isolates plasmid pKS-5.The special DNA of LAV/HTLV III that pKS-5 comprised be arranged in LAV/HTLV III genome from the SstI position of Nucleotide 224 KpnI position (Wain-Hobson, S.et al., 1985, the Cell 40:9 to Nucleotide 3372; Also can see Figure 14).

10 microgram pKS-5 are complete with Hinc II and SstI digestion, and the fragment of generation is separated on 1% low melting glass sepharose again.1818 base pair fragments (being about 0.20 microgram) are cut off from gel.This fragment comprises the distinctive order of LAV/HTLV III from Nucleotide numbering 224 to 2042 shown in Figure 14.It comprises whole gag gene coding region.

5 microgram pAc160 are extremely complete with SmaI and SstI digestion, then fractional separation on 1% low melt sepharose; DNA band (about 0.4 microgram) is cut off (Figure 15) from gel.

Two clotting films melted 10 minutes down at 65 ℃, and the pAc610(that the specific fragment of 1818 base pair LAV/HTLV III of 3 microlitres (with Hinc II and SstI digestion) is attached to 1 microlitre digests with SmaI and SstI) on, cumulative volume 40 microlitres.Cultivated 16 hours 23 ℃ (under room temperature) then, the mixture of this connection heated 10 minutes down at 65 ℃ then, was used for transformed into escherichia coli strain HB101.Test for the plasmid DNA that has the resistance transformant to take out from penicillin toranto, from determining whether to exist the LAV/HTLV III to insert body.The structure of this plasmid pAc-gag is shown in Figure 15.

7.3 contain the structure of the recombinant baculovirus of chimeric LAV/HTLV III gag gene (Ac-gag1)

AcNPV purifies with the plaque method, breeds on the Sf19 cell.Wild-type AcNPV DNA is separated and pAc-gag1 purification (seeing the 7.1.2 joint) by previously described method.

Because the order side of the gag of pAc-gag1 is to join with the AcNPV coded sequence, therefore makes and to realize that by reorganization in the body it is possible that chimeric LAV/HTLV III gag gene is inserted AcNPV genomic constitution.PAc-gag1 plasmid DNA and the common transfection of wild-type AcNPV DNA can make the polyhedron of this plasmid recombinate in proper order and between the genomic DNA sequence homologous of AcNPV.The insertion of mosaic gene is because the result of flanking sequence reorganization.This class recombinant chou will have the mosaic gene that inserts the AcNPV polyhedrosis gene, therefore can not produce closed shape (occlusion body).Lack closed shape virus by visual inspection and can select the recombinant chou patch.

With 1 microgram AcNPV DNA, 5 microgram pAc-gag1 and 15 microgram calf thymus DNAs mix and use ethanol sedimentation.Gained throw out 70% washing with alcohol is placed on then and carries out dry air under the tissue culture ventilating hood, then makes it to be suspended in 437 microliters of water again, and adds 0.25M aluminum chloride (with the 2MCaCl of 63 microlitres ₂Be added in the aqueous dna of 437 microlitres).Making the air bulge simultaneously, dropwise add dna solution (one of per 4 second) by 500 microlitre 2XHEBS.Contain 16 milligrams of NaCl among every milliliter of 2XHEBS, 2 milligrams of D-glucose, 0.75 milligram of Repone K and 0.5 milligram of Na ₂HPO ₄12H ₂O etc., pH are 7.1).This mixture was at room temperature cultivated 30 minutes, pour one 60 millimeters tissue culture ware then into, this culture dish fills 2 milliliters of Grace's insect substratum that comprise 10% Bovine Placenta serum (FBS) and microbiotic (no Yestolate or lactalbumin hydrolysate), wherein contains 3 * 10 ⁶The Sf9 cell.Then dna solution is dropwise added in the cell.Culture dish was cultivated 4 hours down at 28 ℃.With 4 milliliters of Grace's substratum that contain FBS and antibiotic it is washed again later on, containing FBS again, cultivated for 90 seconds in the Grace's substratum of antibiotic 20% methyl-sulphoxide.

Culture dish continues to cultivate with 4 milliliters of perfect mediums down at 28 ℃ with perfect medium washing three times again.After 5 days, collect supernatant liquor, clarification is 5 minutes under 1000 * g condition.On the Sf9 cell, viral starting material are carried out titration.Recognize with visual inspection then non-closed patch to be detected the patch of non-closure, become spot again twice.These patches are expanded on the Sf9 cell, make the usefulness of viral starting material for later characterization, and Figure 16 is reorganization body structure synoptic diagram.

7.4 in the tissue culture cells that infects with recombinant baculovirus with the relevant protein expression of LAV/HTLV III gag

The recombinant chou AcNPV that carries chimeric LAV/HTLV III gag gene is proved to be able to express RNA and the protein relevant with LAV/HTLV III gag in the cells infected of tissue culture.These protein also be found can with AIDS patient's serum generation immune response.

7.4.1 be subjected to the discriminating of the special RNA of LAV/HTLV III gag in the cell that Ac-gag1 infects

(every culture dish has 10 approximately to fill the Sf9 cell with ten ⁷Cell) 100 millimeters culture dish wild-type AcNPV or its recombinant chou, Ac-gag1 infects, and infection multiplicity is 4.Infecting and took out cell in back 24 hours, is 7.2 0.15MNaCl washed twice with containing 0.01MTris-HCl pH.The method of introducing by relevant document is separated the RNA(Purchio of polyadenylation, A.F.and Fareed, G, G., 1979, J.Viro 1.29:763-769 again).Then make RNA fractional separation in 1% agarose-formaldehyde gel, (Lehrach, H., etal., 1979, Biochemistry 16:4743-4251) then forwards on the nylon membrane (Hybond) to and uses UV-irradiation.This film is mixed mutually with the pKS-5DNA of the phosphorus that contains following substance solution-35 mark, 16 hours time, 42 ℃ of temperature, described solution is made up of following: 0.9M NaCl, 50mM sodium phosphate (pH7.0), 5mM EDTA, 0.1%SDS, 4X Denhardts, 0.4mg/ml tRNA, 0.25mg/ml calf thymus DNA and 50% methane amide.The solution of forming with 0.1%SDS and 0.25XSSC, 60 ℃ of temperature with film washing 4 times, 30 minutes time, wash completely, utilize Lightening Plus intensifying screen that film is exposed on the Cronex 4x-flexible beta ray sheeting.Figure 17 shows, detects one the 2.2 thousand right broadband of alkali in the cell that recombinant chou AcNPV infects, and does not then find in the wild-type cells infected.

7.4.2 the cell expressing that utilizes immuno-precipitation to identify to infect with recombinant baculovirus with the relevant protein of LAV/HTLV III gag.

Immunoprecipitation assay described below proves that the cell that infects with recombinant baculovirus of the present invention can synthesize the protein that reacts with AIDS human serum generation specific immune.

With (each culture dish has 5 * 10 on Sf9 cell inoculation to the 60 millimeter cultivation culture dish ⁶Cell, and infect with the wild-type AcNPV among the Ac-gagl.After infection 24,48 and 72 hours, original substratum was changed with the substratum of no methionine(Met), and cell is 28 ℃ of cultivations 30 minutes down.After this substratum with contain 100 microcurie/milliliters [ ³⁵S] the no methionine(Met) substratum of methionine(Met) changes, and labeling process need carry out 2 hours under 28 ℃.

After mark finished, cell was with containing 0.01M Tris-Hcl(pH7.2) and the solution washing twice of 0.15MNaCl, centrifugal separation collection then.The cell ball that takes out in every 60 millimeters culture dish is resuspended in 1 milliliter of dissolving damping fluid, described dissolving buffered soln is composed as follows: 1%NP-40(polyoxyethylene (9) P-tert-octyl phenol), 0.5% Sodium desoxycholate, 0.1M NaCl, 0.01M Tris-HCl(pH7.4) and 1mMEDTA, cell lysates is 100, and centrifugation is 30 minutes under the 000Xg condition, to clarify.

To take from contrast crowd or AIDS patient's heat-inactivated human serum 4 microlitres, be added in the 500 ml cells lysates, to carry out immunoprecipitation.After cultivating half an hour under 4 ℃, add 80 microlitre activatory Staphylococcus aureus cell (Pansorbin cells, Cal-biochem-Behring Corp., La Jolla, CA), continue down to cultivate half an hour at 4 ℃, immunoprecipitation complex is to be that 5000 rev/mins of centrifugations were collected in 5 minutes with rotating speed in Sorvall A384 rotor under 4 ℃, then with containing 1MNaCl, 0.1%NP-40 and 0.01M Tris-Hcl, pH be 7.4 solution washing once, then use RIPA damping fluid (10mM Tris-HCl, pH7.2 again, 0.15MNaCl, 1% Sodium desoxycholate, 1% Sodium Lauryl Sulphate BP/USP, 1%Triton X-100) wash three times.Immunoprecipitate through washing is suspended in the Laemmli sample buffer of 80 microlitres once more, and ebuillition of heated was 5000 rev/mins of centrifugations 5 minutes with rotating speed in the Sorval-1A384 rotor after 1 minute.The protein that is present in the immunoprecipitation in the supernatant liquor is analyzed with electrophoretic method in the 10%SDS polyacrylamide gel.Behind the electrophoresis, gel is painted with the Coomassie blue dyes, handles (30 minutes, room temperature, 1M sodium salicylate) with sodium salicylate then, carries out photofulorography after the drying.

Someone proposes, sophisticated gag protein (24,000 dalton, P-24; 16,000 dalton, P16; 1400 dalton P-14) can obtain from a kind of 55000 bigger Dalton protein precursors processing.Figure 14 represents that molecular weight is 67,000,55,000, and 40,000,34,000,32,000 and 24,000 daltonian albumen mass-energy are precipitated by AIDS patient's seroimmunity effectively.

In order to verify any of description among Figure 18 has whether there is precursor/product relation between the immunoreactive protein, and the spy has carried out pulse-tracking test.The Sf9 cell infects according to above-described method.Infected back 24 hours, bacterium usefulness [ ³⁵S] methionine mark 5 minutes; Then wash with perfect medium.And in perfect medium, cultivated 2,4 and 8 hours.In these times, collect the cell of three different times, carry out immunoprecipitation then.The protein of immunoprecipitation, fractional separation in aforesaid 10%SDS polyacrylamide gel.Figure 19 represents a photofulorography sheet of this experiment.Data show, P67, and P55, P40, P34 and P32 be bonding mark after 5 minutes.After following the trail of in 8 hours, in P67 and P55, mark reduces, and mark increases in P34, and is constant relatively in the radio-labeling amount maintenance of P40.

7.4.3 differentiate and the relevant protein of LAV/HTLV III gag with ELISA

Ten 100 milliliters of culture dish, every fills 10 ⁷The Sf9 cell infects with Ac-gagl.In infection back 24 hours, collecting cell was used 0.01M Tris-HCl(pH7.2 with containing) and the solution washing twice of 0.15MNaCl, freeze thaw is three times then.Then in ice bath, destroyed cell 5 minutes with 2 milliliters of solution that contain the PBS of 0.5%NP-40.With cell lysates centrifugation 10 minutes under 1000 * g condition, remove supernatant liquid.Add 4 milliliters of carbonate buffer solutions (50mM yellow soda ash, pH9.6), mixture centrifugal rotation 20 minutes in the Eppenderf centrifuge tube.Remove supernatant liquor, shown in the table III, dilute with carbonate buffer solution.These cell lysates are used to be coated on the 96 well droplet culture dish, and placement is spent the night, 4 ℃ of temperature (50 microlitres/well).In every well, added 300 microlitres blocking-up reagent (5% alipoidic milk power, 0.01% thiomersal(ate), the 10XPBS solution of 0.01% foam reducing composition A) in second day, cultivated 45 minutes under the room temperature.Take out blocking-up reagent, in every well, add 50 microlitre human serums again with 100 times of blocking-up reagent dilutions.Before the adding, the serum of dilution (AIDS patients serum or normal human serum) was cultivated 45 minutes under 37 ℃ of temperature earlier.

Every well was reacted 1 hour, then with containing sodium-chlor and tween 20 (polyoxyethylene glycerine-lauric acid Isosorbide Dinitrate) washing three times down at 37 ℃.Add the anti-human body horseradish peroxidase of 100 microlitre goats binding substances (with 1000 times of the normal goats serum dilutions that contains 0.01% thiomersal(ate)) in every well, and make it 37 ℃ of reactions 1 hour.Culture dish adds the substrate that 100 microlitres cushioned and the solution of chromogen reagent again in every well after washing, the substrate that wherein cushioned is hydrogen peroxide, citric acid, phosphate buffer; Chromogen reagent is the tetramethyl benzidine in the dimethyl sulfoxide (DMSO).Culture dish was at room temperature cultivated 30 minutes.3N sulfuric acid according to adding 100 microlitres in every well makes reaction terminating.At the absorptivity of 450nm place assaying reaction mixture, be 630nm with reference to specific absorption.Gained the results are shown in table 8, and wherein TR1, Y-1, CF22C and CF9 are the AIDS human serums, and 2,16,21 and 48 is normal human's serum.

In a word, the result shows shown in Figure 17-19 and the table 8, described recombinant baculovirus can: (a) the LAV/HTLV III gag gene that express to insert, (b) improve or handle this gene product, (c) produce antigen protein with the reaction of AIDS human serum generation specific immune.

Table 8

The specific absorption numerical value that ELISA measures

Aa-gagl cytolysis thing extent of dilution

Test

Antiserum(antisera) 50 ^-1100 ^-1200 ^-1400 ^-1800 ^-11000 ^-1

AIDS

TR1 2.619 2.471 2.235 2.076 1.786 1.499

2.504 2.479 2.216 2.119 1.877 1.500

Y-1 2.502 2.354 2.046 1.564 1.298 1.014

2.305 2.306 2.006 1.595 1.282 1.078

CF22C 2.486 2.317 1.924 1.393 1.009 0.811

2.319 2.241 1.838 1.331 1.068 0.848

CF9 2.303 2.110 1.339 0.855 0.636 0.512

2.209 2.012 1.305 0.850 0.659 0.455

The normal people:

2 0.221 0.204 0.312 0.199 0.228 0.222

0.229 0.187 0.176 0.227 0.208 0.210

16 0.176 0.175 0.217 0.131 0.144 0.129

0.165 0.135 0.139 0.210 0.175 0.145

21 0.193 0.190 0.216 0.189 0.237 0.165

0.205 0.176 0.170 0.199 0.197 0.224

48 0.200 0.185 0.217 0.177 0.190 0.134

0.249 0.169 0.163 0.175 0.173 0.143

8. embodiment: cowpox gag genetic recombinants

In following embodiment, the plasmid vector of formation contains the mosaic gene of the LAV/HTLV III gag genes encoding order of the below that comprises the transcripting controling area that is positioned at vaccinia virus.The mosaic gene that contains vaccinia virus 7.5k promoter region and LAV/HTLV III gag gene order is recombinated in body and is inserted in the gene of vaccinia virus.The virus of this reorganization differentiated and purify and prepare viral original seed by infected tissue culture cells.In in vitro tests, can demonstrate recombined vaccinia virus and produce protein involved with immunoreactive LAV/HTLV III gag gene.Following chapters and sections will at length be narrated each step of this embodiment of the present invention.The general technology that is adopted in the present embodiment is described in 6.1 joints.

8.1 contain the formation of the plasmid vector of the vaccinia virus promotor on a kind of coded sequence of the LAV/HTLV of being bound up on III gag gene.

Structure contains five kinds of plasmid carriers of the 7.5k promotor that is bound up on the vaccinia virus on the genomic different lengths of LAV/HTLV III with gag coded sequence.LAV/HTLV III gag coded sequence derives from people, Cell40:1985 such as λ J19(Wain-Hobson) subclone pKS-5 and the pSS-5 relevant with two kinds.Plasmid pKS-5 contains-3148 base pairs, and the SstI of the LAV/HTLV III DNA that promptly inserts on the corresponding position of pUC18 is to 224 pairs 3372 of KPnI(Nucleotide numberings) fragment.Plasmid pSS-5 contains-5107 base pairs, and the SstI of the LAV/HTLV III DNA that promptly inserts on the corresponding position of pUC18 is to 224 pairs 5221 of SalI(Nucleotide numberings) fragment.Be inserted into the plasmid pGS62 from the different lengths of pKS-5 or pSS-5 deutero-gag number order, except having a single EcoRI position that is positioned at single SmaI position below (referring to Figure 20), it is identical with pGS20 (people, 1984 such as Mackett, JVirol, 49:857-864).The particular order that is inserted into the LAV/HTLV III among the pGS62 all starts from the top of the initiator codon that is positioned at the gag gene and the ThaI(FnuD II of 76 base pairs) position (Nucleotide 257).It is 4194 through EcoRI(respectively that plasmid pv-gag1, pv-gag2, pv-gag3, pv-gag4 and pv-gag5 contain from the ThaI position), KpnI(is 3372), EcoRV(is 2523), BclI(is 1975) or the special order of LAV/HTLV III of Bgl II (being 1642) position.With reference to Figure 20 the structure of every kind of plasmid being done following narration can be more convenient.

The structure of pv-gag1: by restriction enzyme ThaI and EcoRI complete digestion 5 microgram plasmid pSS-5DNA.Separating obtained fragment on the 1%LMP sepharose.Isolate the 3.9Kbp fragment that contains LAV/HTLV III gag coded sequence and be coupled on the above-mentioned pGS62 with SmaI and EcoRI digestion.Connect mixture with so that intestinal bacteria HB101(ECcli HB101) transform and select anti-penbritin to clone.The regenerated situation is measured on existence by 3.9Kbp inset in the individual clone's of restriction analysis the plasmid and the EcoRI conjugation position.The constituting body of gained contains the complete coded sequence of gag and proteolytic enzyme (Prt) gene, and the open reading frame of most of Pol gene, up to the EcoRI position of Nucleotide 4194.

The formation of pv-gag2: by restriction enzyme ThaI and SmaI complete digestion 5 microgram plasmid pKS-5.The SmaI position of pKS-5 system contiguous KpnI position, this position be the LAV/HTLV III derive and the order inserted and pUC18 plasmid multiple clone site between joint.Separating obtained fragment on the 1%LMP sepharose.Separate the fragment of the 3.2Kbp obtain containing LAV/HTLV III gag order and be coupled to above-mentioned with SmaI digest in advance and the pGS62DNA that handles with CIAP on.Connect the clone of mixture in order to transformed into escherichia coli HB101 and the anti-penbritin of selection.Whether the plasmid that the individuality clone is obtained exists the 3.1Kbp fragment to measure, and is determined the orientation of inset by restriction analysis.Sign indicating number is read in the opening that the constituting body of pv-gag2 gained contains complete gag gene, prt gene and a part of pol gene, up to the KpnI position of Nucleotide 3372.

The formation of pv-gag3: produce the 2.3Kbp fragment except by ThaI and EcoRV pKS-5 being digested, be used for being inserted into outside the pGS62 in the SmaI position, its structure is similar to pv-gag2.The final structure of pv-gag3 contains complete gag and prt gene and sub-fraction and is incorporated into the pol gene of the vaccinia virus TK gene of 3' part.

The formation of pv-gag4: by SstI and BclI complete digestion 5 microgram plasmid pSS-5DNA.The purification gained contains the fragment of LAV/HTLV III gag order.Make this fragment be attached to above-mentioned prior people such as plasmid vector PIC19R(Marsh with SstI and BamHI cutting, 1984, Gene32:481-485) go up formation one intermediate plasmid.Gag is inserted in the BamI position of PIC19R in proper order, causes the BclI site in the LAV/HTLV III order and puts EcoRI position in this multidigit point is cloned.By restriction enzyme ThaI and EcoRI digestion this in the middle of structure body and produce the fragment that 1.72kbp can be easy to be inserted into SmaI and EcoRI position among the plasmid pGS62.The final formation thing of pv-gag4 contains a complete LAV/HTLV III gag gene and a part and is incorporated into prt gene on the vaccinia virus TK gene of 3' part.

The formation system of plasmid pv-gag5 utilizes the two step strategies identical with pv-gag4.Digest 5 microgram pSS-5 with restriction enzyme Sst I and Bgl II.Separate the 1.42kbp fragment that contains LAV/HTLV III gag order, and on the 1%LMP sepharose, separate and purify, be inserted on plasmid PIC19RSst I and the Bgl II position.To be attached in proper order at the gag on the Bgl II position and (a) be produced have and the open reading frame of the in-phase terminator codon of gag and (b) Bgl II site and place the EcoRI position of the vicinity on the multiple clone site plasmid.Then, with ThaI and this middle constituting body of EcoRI digestion.Purification contains the 1.39Kbp fragment of LAV/HTLV III gag order, and is linked on the plasmid pGS62 on SmaI and EcoRI position.The final plasmid pv-gag5 that forms contains great majority rather than all is attached at the gag(on the translation termination order in the framework up to Bgl II position) coded sequence.

8.2 contain the formation of the recombined vaccinia virus of chimeric LAV/HTLV III gag gene

According to the same approach that makes up v-env5 and v-env2 (referring to 6.3 joints), chimeric LAV/HTLV III gag gene is inserted in the vaccinia virus genome in plasmid vector pv-gag1, pv-gag2, pv-gag3, pv-gag4 and pv-gag5.In some following embodiment, all recombinant viruses constitute by plaque purifying and New York City Board of Health strain (V-NY is referring to the 6.1.1 joint).Select TK ^-Recombinant chou and develop into be applicable to later evaluation and viral starting material before with plaque purifying three times.To in pv-gag1, pv-gag2, pv-gag3, pv-gag4 and pv-gag5, be referred to as v-gag1NY, v-gag2NY, v-gag3NY, v-gag4NY and v-gag5NY respectively by the deutero-recombinant virus.

8.3 expression at LAV/HTLV III gag protein involved in the tissue culture cells of recombinant chou vaccinia virus infection

The protein involved of LAV/HTLV III gag during cell infection during the recombinant chou vaccinia virus that has an above-mentioned chimeric LAV/HTLV III gag gene can expression tissue be cultivated.Some recombinant chous in these recombinant chous can make precursor gag protein p55 be processed into sophisticated protein p25 and p18.Have found that these protein are to be immune response with the monoclonal antibody of AIDS patients serum and/or anti-gag protein p25 or p18.

Under 10moi, by parental form vaccinia virus (v-NY) or use its recombinant derivative: v-gag1NY, v-gag2NY, v-gag3NY, v-gag4NY or v-gag5NY in 60 millimeters culture dish, to make to merge the BSC-40 cell and infected.Infection was carried out 12 hours, and harvested cell washs once centrifugal again collection with PBS then.Make infected cell pellet resuspending in 0.3 milliliter of Laemmli sample buffer, and seethe with excitement and made its dissolving in 4 minutes.Getting 20 microlitre cytolysates electrophoresis on the 15%SDS polyacrylamide gel, to separate whole protein component.The LAV/HTLV III virus particle of purifying and the cytolytic aliquots containig of simulated infection are as contrast.

Make and contain the thing electrotransfer to nitrocellulose membrane in the gel.At first make film and AIDS patients serum reaction or with the composition react of the monoclonal antibody of anti-gag protein p25 and p18.After thoroughly washing, make again film and the anti-human immunoglobulin's antibody response of alkaline phosphatase (AP-combines) bonded goat (when using the patients serum) or with the anti-rat immune globulin antibody response of AP bonded goat, (when using the monoclonal antibody of mouse).Described in first and second kind antibody response condition and wash conditions such as the 6.4.1 joint.For the second time after the antibody response, with being added with 0.1M NaCl and 5mM MgCl ₂0.1M Tris(PH9.5) do washing at last.By containing 0.1M Tris(PH9.5), 0.1MNa Cl, 5mM Mg Cl ₂0.33mg/ml antigen bonded antibody on detection of the solution reaction of phosphoric acid bromine chloro-indole (bromo-chloroindolgl phosphate) and 0.17mg/ml nitrogen blue tetrazole and the film.Make after the reaction of film and chromogen, rinse in water dries in the air and takes a picture.

Shown in Figure 21 is this analytical results.V-gag1NY and v-gag2NY recombinant chou contain complete gag and prt gene.A family of the LAV/HTLV III protein involved of the common migration of main gag protein p55, p40, p25 and p18 of these recombinant chous expression and LAV/HTLV III.These protein are the immunoreactive products of mouse monoclonal antibody (Figure 21 A) with AIDS patients serum (Figure 21 B) and anti-p25 and p18.The v-gag3NY recombinant chou contains gag and prt gene, but the open sign indicating number of reading of its pol is bound up on C-terminal part homophase with cowpox TK gene.V-gag3NY can also express initial gag gene product p55.As if yet the working (machining) efficiency of p55 is low than v-gag1NY and v-gag2NY, because p55 is than p25 and p18 much less in the cytolysate that infects with v-gag3NY.Recombinant chou v-gag4NY contains complete gag gene, but has only part prt gene.It is similar to p55 that it has synthesized a kind of size, the proteins associated matter of LAV/HTLV III.Recombinant chou v-gag5NY only contains part gag gene, express the protein (Figure 21 c) of the brachymemma of a kind of 43kD, as if though v-gag4NY and v-gag5NY can not handle their initial gag protein involveds effectively, the monoclonal antibody of their product and anti-p25 and p18 has immune response.In a word, although their working ability differences, five kinds of recombinant chous can both be expressed has immunoreactive protein, and it is the major antigen determinant that contains LAV/HTLV III core protein.

9. embodiment: baculovirus shell recombinant chou

In following enforcement, the plasmid vector of formation contains the mosaic gene that comprises the LAV/HTLV III env coded sequence of transcribing the control sequence downstream that is positioned at AcNPV.The mosaic gene that contains AcNPV polyhedron promoter region and LAV/HTLV III env coded sequence is inserted in the genome of AcNPV through re-constituted in vivo.Identify and the purification recombinant virus, prepare viral original seed by the supernatant liquor of infected cell.Showed already that recombinant baculovirus can produce the protein involved with immune response LAV/HTLV III env in vitro tests.To at length narrate each step of this embodiment of the present invention in the following trifle.The general technology that is adopted in the present embodiment is described in 7.1.

9.1 contain the formation of the plasmid vector of the AcNPV promoter region on the coded sequence that is bound up on LAV/HTLV III env gene

Purification LAV/HTLV III env coded sequence from pv-env5 (seeing joint 6.3), it is used to constitute the recombined vaccinia virus v-env5 that had shown expression env protein involved already.Env coding region in pv-env-5 and near 96 base pair 5' positions and near the untranslated order in 223 base pair 3' positions the most together, the side, two ends is the BamHI site.This plasmid (it also contains an inner BamHI site) is through producing the 2.9kbp fragment of the particular order that only contains the LAV/HTLV III with the part digestion of restriction enzyme BamHI.This fragment (about 0.5 microgram) is separated on the 1%LMP sepharose and is purified, and is bound up on the 0.5 microgram plasmid vector pAc610, and pAc610 system handles with the BamHI linearizing with CIAP in advance.The mixture that connects can be in order to Transformed E coli bacterial strain Mc1000, and selects the bacterium colony of anti-penbritin.Whether the plasmid DNA test that single transformant is come has LAV/HTLV III env to exist in proper order, that is, with the orientation of BamHI digestion generation 2.3kbp and 0.54kbp fragment and test inset.It is purified and represent with pAc-env-5 that LAV/HTLV III env is inserted in plasmid in the tram in proper order.Its structure is shown in Figure 22.

9.2 contain the structure of the baculovirus recombinant chou of chimeric LAV/HTLV III env gene

As described in 7.3 joints, chimeric LAV/HTLV III env gene is inserted in the AcNPV genome by recombination in the body.Same for another example joint is described, makes AcNPV DNA(1mg), pAc-env5DNA(5mg) and calf thymus DNA (15mg) transfection in the Sf9 cell.After under 28 ℃, hatching 5 days, collect supernatant liquor and centrifugal clarification 10 minutes under 1000 * g with 4 milliliters of perfect mediums.Titration virus original seed on the Sf9 cell, and at first identify recombinant virus with the plaque hybridization assay method of following described M, D, Summers and G, E, Smith:

On 60 * 15 millimeters culture plate, in the TNM-FH of serum-free substratum, the cell density on every flat board is 2.5 * 10 with the Sf9 cell inoculation ⁶After the cell attachment, remove substratum, on every ground flat board, add the virus inoculation thing of one milliliter of dilution.After cultivating 1 hour under 27 ℃, remove all inoculums on the every flat board, 4 milliliters of LMP agar over lay are added on the edge of every flat board at leisure.After coating solidified, flat board was cultivated 4-6 days in wet environment, perhaps until form plaque well.Make dull and stereotyped dried overnight or longer then, in the environment that this moment, the chien shih plate cannot not be placed on wetly.Reaching enough when dry, remove agar over lay, the exsiccant nitrocellulose membrane is placed on the cell stayed in the flat board above.With solution A (0.05M Tris-HCl, pH7.4,0.15M NaCl) make the l Water Paper of 47 millimeters girths of What-man3MM saturated and be placed on the Nitrocellulose film, wait the seal after, nitrocellulose membrane is removed carefully, and it is placed on on the saturated Whatman filter paper of solution B (0.5NNaOH, 1.5M NaCl), cell faces up.After 2-3 minute, on paper handkerchief, make nitrocellulose membrane, transfer to then and use on the saturated Whatman filter paper of solution C (1.0M Tris-HCl, PH7.4,1.5M NaCl) at air drying.After 2-3 minute, make nitrocellulose membrane dry on air on the paper handkerchief, immersing in the Petri dish of having filled solution D (0.3M NaCl, 0.03M Trisodium Citrate) already at leisure again and wash.Thereafter, take out on paper handkerchief dry, 80 ℃ of bakings 2 hours down in a vacuum again.Make film and 32P mark pRS-3 hybridization as probe.The picking recombinant virus and again titration on the Sf9 cell.With the naked eye differentiate unencapsulated plaque.These are used for preparing the viral original seed that following research is used through purification plaque more than three times.

9.3 the expression of the LAV/HTLV III env of protein involved in the tissue culture cells that infects by recombinant baculovirus Ac-envs

Showing already, is the protein involved of can be in infected tissue cultivates representing LAV/HTLV III env during cell with the reorganization AcNPV of chimeric LAV/HTLV III env gene.And point out that these protein are to be immune response with the AIDS patients serum and with the monoclonal anti physical efficiency that limits LAV/HTLV III shell glycoprotein gp110 and gp41.

Make the Sf9 cell of on 100 millimeters culture dish, inoculating (1 * 10 under the 5moi with wild-type AcNPV or its recombinant chou Ac-env-5 ⁷) infect.After infecting 28 hours, under the situation that does not have the serum supply, substratum is by the substratum replacement of no methionine(Met) and cultivated 30 minutes.After finishing, with containing 100uCi/ml[ ³⁵S] 2 milliliters of no methionine(Met) substratum of methionine(Met) replace this substratum, enable 28 ℃ of following marks 2 hours.Mark phase finishes, and with PBS washed cell twice, described in the 6.4.2 joint, dissolves resuspending in the damping fluid at 1 milliliter then.Described in 6.4.2 joint, also can be equally carry out immunoprecipitation with the monoclonal antibody of the mouse of anti-gp110 or gp41 with the AIDS patients serum.

This analytical results points out mainly to have synthesized like the represented recombinant virus Ac-env5 of Figure 23 the protein of a kind of relevant 150kD of LAV/HTLV III shell.Because it with by the common migration of cowpox reorganization v-env5 synthetic gp150 and with the monoclonal antibody immunity reaction of AIDS patients serum and anti-gp110 and gp41, so this protein is represented glycosylation precursor coat protein gp150.This molecule processing is inadequate, because can not detect gp110 or gp41 in the phase at two hour-symbols.Yet, because the most of antigenic determinants of gp110 and gp41 all are on the precursor, so this protein has potential value as diagnostic reagent with as immunogen.

10. microorganism deposits

Following intestinal bacteria (Ecoli) bacterial strain that carries listed plasmid had left the farming research cultivation collection unit (NRRL) of the U.S., Illinois (IL), peoria (Peorial) already in, and the access numbering is as follows:

Ecoli bacterial strain plasmid access numbering

K12 MC1000 PV-env1 NRRL B-18003

K12 MC1000 PV-env2 NRRL B-18004

K12 MC1000 PV-env5 NRRL B-18005

K12 MC1000 PV-env7 NRRL B-18110

K12 HB101 PV-gag1 NRRL B-18111

K12 HB101 PAC-gag1 NRRL B-18105

K12 NF1829 PAC-env5 NRRL B-18109

Following recombinant virus had left Maryland, USA (MD), Roc already in

The American type culture collection of Wei Er (Rockville), deposit number is as follows:

The recombinant virus deposit number

v-env2 ATCC VR2114

v-env5 ATCC VR2113

Following recombinant virus leaves Chinese typical culture collection center (CCTCC) on February 10th, 1987 with the host cell that carries the Recombinant HIV sequence, and deposit number is as follows:

Vaccinia virus V-env 5NY CCTCC No.V87001

Vaccinia virus V-gag 1NY CCTCC No.V87002

Baculovirus Ac-env5 CCTCC No.V87003

Baculovirus Ac-gag1 CCTCC No.V87004

E.coli Pv-env5 CCTCC No.M87007

E.coli Pac-gag1 CCTCC No.M87008

E.coli Pv-gag1 CCTCC No.M87009

E.coli Pac-env5 CCTCC No.M87010

Claims (11)

1, the method for preparing the live-virus vaccine preparation, the animal or the insect viruses that comprise the preparation reorganization, or the recombinant virus of microorganism, these viruses instruct on immunology capsid glycoprotein or relevant peptide or the protein expression of gag protein gene product with HIV, in being subjected to the patient of immunization, recombinant virus is that tool is infective, but does not cause disease, and only produce immune response

It is characterized in that the method that wherein prepares recombinant virus may further comprise the steps:

(a) with recombinant virus infection animal or insect cell or microorganism, the genome of this recombinant virus comprises the nucleotide sequence of the coding HIV capsid glycoprotein gene product of the nucleotide sequence from Nucleotide ordinal number 5767 to 8349 as shown below in fact;

Or its coding and described capsid glycoprotein gene product relevant immunogenic peptide or arbitrary part of proteic this nucleotide sequence on immunology;

Or nucleotide sequence or its arbitrary part that indivedual Nucleotide change can appear on the degeneracy basis;

Or comprise as shown below in fact, the nucleotide sequence of the coding HIVgag protein gene product of Nucleotide ordinal number 340 to 1835;

Or its coding and described gag protein gene product relevant immunogenic peptide or arbitrary part of proteinic this Nucleotide on immunology;

Or this nucleotide sequence or its arbitrary part that indivedual Nucleotide change can appear on the degeneracy basis;

This nucleotide sequence be in that regulatory gene is expressed, comprise under the control of exogenous second nucleotide sequence of transcribing and translate control signal,

The result has in fact as the peptide of figure below aminoacid sequence listed, amino-acid residue ordinal number from 1 to 861 or protein peptide or protein that form, relevant with HIV capsid glycoprotein, and with described HIV capsid glycoprotein gene product relevant immunogen part in immunity.

Or comprise the peptide of the aminoacid sequence that has in fact the amino-acid residue ordinal number of listing as figure below from 1 to 500 or the relevant immunogenicity part of gag protein gene product that albumen is formed;

Or with described gag protein gene product immunology on relevant arbitrary immunogenicity part, in the host of virus infection, express and

(b) cell that infects by breeding is bred recombinant virus.

2, by the described method of claim 1, it is characterized in that recombinant virus comprises poxvirus.

3, by the described method of claim 2, it is characterized in that wherein poxvirus comprises vaccinia virus.

4, by the described method of claim 3, it is characterized in that wherein vaccinia virus comprises and was preserved in CCTCC on February 10th, 1987, preserving number is the recombined vaccinia virus V-env5NY of No.V87001.

5, by the described method of claim 3, it is characterized in that wherein vaccinia virus comprises and was preserved in CCTCC on February 10th, 1987, preserving number is the recombined vaccinia virus V-gag1NY of No.V87002.

6, by the described method of claim 1, it is characterized in that wherein recombinant virus comprises baculovirus.

7, by the described method of claim 6, it is characterized in that wherein recombinant virus comprises nucleopolyhedrosis virus.

8, by the described method of claim 6, it is characterized in that wherein recombinant virus comprises and was preserved in CCTCC on February 10th, 1987, preserving number is the recombinant baculovirus Ac-env5 of No.V87003.

9, by the described method of claim 6, it is characterized in that wherein recombinant virus comprises and was preserved in CCTCC on February 10th, 1987, preserving number is the recombinant baculovirus Ac-gag1 of No.V87004.

10, by the described method of claim 1, it is characterized in that wherein recombinant virus comprises phage.

11, prepare the method for multivalence live-virus vaccine preparation, it is characterized in that:

(a) animal of preparation reorganization or the recombinant virus of insect viruses or microorganism, these viruses instruct peptide or proteic expression relevant with the capsid glycoprotein gene product of HIV on immunology, and recombinant virus has infectivity, but disease does not take place and only produce immune response in being subjected to the patient of vaccine inoculation, the method for preparing recombinant virus comprises following each step:

(ⅰ) with recombinant virus infection animal or insect cell or microorganism, the genome of recombinant virus comprises that essence is as shown below, the nucleotide sequence of the coding HIV capsid glycoprotein gene product of Nucleotide ordinal number from 5767 to 8349;

Or its coding and described capsid glycoprotein gene product relevant immunogenic peptide or arbitrary part of proteic this nucleotide sequence on immunology;

Or this nucleotide sequence or its arbitrary part that indivedual Nucleotide change can appear on the degeneracy basis;

The gene product of this capsid glycoprotein is to be in second nucleotide sequence that regulatory gene is expressed, comprise under the exogenous control of transcribing and translating control signal, it is listed that the result has the figure below of being essentially, the peptide of the aminoacid sequence of amino-acid residue ordinal number from 1 to 861 or albumen are formed, peptide or the albumen relevant with HIV capsid glycoprotein;

Or the arbitrary immunogenicity part relevant with described capsid glycoprotein gene product, have among this viral host in infection and to express; With

(ⅱ) breed recombinant virus by the breeding cells infected.

(b) animal of preparation reorganization or the recombinant virus of insect viruses or microorganism, these viruses instruct peptide or proteic expression relevant with the gag protein gene product of HIV on immunology, and recombinant virus has infectivity, but disease does not take place and only produce immune response in being subjected to the patient of vaccine inoculation, the method for preparing recombinant virus comprises following each step:

(ⅰ) with recombinant virus infection animal or insect cell or microorganism, the genome of recombinant virus comprises that essence is as shown below, from the coding HIVgag protein gene product of the nucleotide sequence of Nucleotide ordinal number 340 to 1835

Or its coding and described gag protein gene product relevant immunogenic peptide or arbitrary part of proteic this nucleotide sequence on immunology;

Or this nucleotide sequence or its arbitrary part that indivedual Nucleotide change can appear on the degeneracy basis;

This gag protein gene product is to be in second nucleotide sequence that regulatory gene is expressed, comprise under the exogenous control of transcribing and translating control signal, the result has listed as figure below in fact, the peptide of the aminoacid sequence of amino-acid residue ordinal number from 1 to 500 or albumen are formed, peptide or the albumen relevant with the gag protein gene product;

Or the arbitrary immunogenicity part relevant with described gag protein gene product, have among this viral host in infection and to express; With

(ⅱ) breed recombinant virus by the breeding cells infected.

(c) recombinant virus of (a) step with (b) step mixed with pharmaceutical carrier.

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