CN109851664A

CN109851664A - A kind of protein based on the reversed epitope design of antibody and its in the application prepared in AIDS virus resisting vaccine

Info

Publication number: CN109851664A
Application number: CN201711240643.3A
Authority: CN
Inventors: 张林琦; 汪桦; 史宣玲
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2017-11-30
Filing date: 2017-11-30
Publication date: 2019-06-07

Abstract

The invention discloses a kind of protein based on the reversed epitope design of antibody and its in the application prepared in AIDS virus resisting vaccine.The present invention provides a histone matter: protein shown in sequence 1；Protein shown in sequence 3；Protein shown in sequence 5；The protein being successively made of following element from N-terminal to C-terminal: function section, tripolymer folded domain；The protein being successively made of following element from N-terminal to C-terminal: gp67 signal peptide, function section, link peptide, tripolymer folded domain, His₆Label.Protein provided by the invention has good epitope conformation and combines activity, shows targeting immunogenicity well.Using protein provided by the invention as immunogen immune biology, biology can be promoted to generate the antibody that there is pervasive neutralization activity to a variety of AIDS viruses.The present invention has great application and popularization value to the treatment of AIDS.

Description

A kind of protein based on the reversed epitope design of antibody and its preparing anti-AIDS disease Application in malicious vaccine

Technical field

The present invention relates to a kind of protein based on the reversed epitope design of antibody and its preparing AIDS virus resisting vaccine In application.

Background technique

Since self-discovery, AIDS virus (Human immunodeficiency virus, HIV) has added up to cause the whole world About 39,000,000 people are dead.To prevent and treat HIV infection, it is annual caused by global economy burden be up to about 200 hundred million beauty at present Member.HIV infection has become serious major global public health problem.

According to the World Health Organization (World Health Organization, WHO) and The Joint Programme on AIDS (The Joint United Nations Programme on HIV/AIDS, UNAIDS) recent statistics, by December, 2016, It is about 1,800,000 that the number of the infected of HIV, which is about newly-increased HIV infection number in 36,700,000,2016, in global range, and death toll is about It is 1,000,000.

Summary of the invention

The object of the present invention is to provide a kind of protein based on the reversed epitope design of antibody and its preparing anti-AIDS Application in viral vaccine.

It is any one following of (a1) into (a13) the present invention provides a histone matter:

(a1) protein that the amino acid sequence shown in sequence 1 in sequence table forms；

(a2) protein that the amino acid sequence shown in sequence 3 in sequence table forms；

(a3) protein that the amino acid sequence shown in sequence 5 in sequence table forms；

(a4) fusion protein containing (a1) or (a2) or (a3)；

(a5) protein being successively made of following element from N-terminal to C-terminal: function section, tripolymer folded domain；Institute Stating function section is (a1) or (a2) or (a3)；

(a6) protein being successively made of following element from N-terminal to C-terminal: function section, link peptide, tripolymer fold knot Structure domain；The function section is (a1) or (a2) or (a3)；

(a7) protein being successively made of following element from N-terminal to C-terminal: gp67 signal peptide, function section, link peptide, Tripolymer folded domain；The function section is (a1) or (a2) or (a3)；

(a8) protein being successively made of following element from N-terminal to C-terminal: function section, link peptide, tripolymer fold knot Structure domain, His₆Label；The function section is (a1) or (a2) or (a3)；

(a9) protein being successively made of following element from N-terminal to C-terminal: gp67 signal peptide, function section, link peptide, Tripolymer folded domain, His₆Label；The function section is (a1) or (a2) or (a3)；

(a10) fusion (a11) that connection label obtains in the end of (a1) or (a2) or (a3) or (a5) or (a6) exists (a1) or the obtained fusion protein of the N-terminal connection signal peptide of (a2) or (a3) or (a5) or (a6)；

(a12) in the N-terminal connection signal Peptide C end of (a1) or (a2) or (a3) or (a5) or (a6), connection label is obtained Fusion protein；

(a13) any one protein by (a1) into (a12) by one or several amino acid residues substitution and/ Or deletion and/or addition and with protein with the same function as derived from it.

Protein shown in sequence 3 in protein shown in the sequence 5 of sequence table with sequence table.3 institute of sequence of sequence table Show protein shown in the sequence 1 in protein with sequence table.

Gp67 signal peptide is specific as shown in the sequence 7 of sequence table.Link peptide is specific as shown in the sequence 9 of sequence table.Trimerization Folding structure domain is specific as shown in the sequence 11 of sequence table.His₆Label is specific as shown in the sequence 13 of sequence table.

The gene of coding any description above protein also belongs to protection scope of the present invention.

The gene is specially following (b1) to (b10) any described DNA molecular:

(b1) code area DNA molecular as shown in sequence 2 in sequence table；

(b2) code area DNA molecular as shown in sequence 4 in sequence table；

(b3) code area DNA molecular as shown in sequence 6 in sequence table；

(b4) DNA molecular that code area is successively made of following element from upstream to downstream: the gene of encoding function section, The coded sequence of tripolymer folded domain；

(b5) DNA molecular that code area is successively made of following element from upstream to downstream: the gene of encoding function section, The coded sequence of the coded sequence of link peptide, tripolymer folded domain；

(b6) DNA molecular that code area is successively made of following element from upstream to downstream: the code sequence of gp67 signal peptide The coded sequence of column, the gene of encoding function section, the coded sequence of link peptide, tripolymer folded domain；

(b7) DNA molecular that code area is successively made of following element from upstream to downstream: the gene of encoding function section, The coded sequence of link peptide, the coded sequence of tripolymer folded domain, His₆The coded sequence of label；

(b8) DNA molecular that code area is successively made of following element from upstream to downstream: the code sequence of gp67 signal peptide Column, the gene of encoding function section, the coded sequence of link peptide, the coded sequence of tripolymer folded domain, His₆Label Coded sequence；

(b9) hybridize under strict conditions with (b1) to (b8) any described DNA molecular and the DNA of code for said proteins Molecule；

(b10) there is 95% or more homology and code for said proteins with (b1) to (b8) any described DNA molecular DNA molecular.

The sequence 2 of gene such as sequence table or the sequence 4 of sequence table of the encoding function section or 6 institute of the sequence of sequence table Show.

The coded sequence of gp67 signal peptide is specific as shown in the sequence 8 of sequence table.The coded sequence of link peptide is specifically such as sequence Shown in the sequence 10 of list.The coded sequence of tripolymer folded domain is specific as shown in the sequence 12 of sequence table.His₆Label Coded sequence it is specific as shown in the sequence 14 of sequence table.

Recombinant expression carrier, expression cassette, transgenic cell line containing any description above gene or recombinant bacterium tool belong to Protection scope of the present invention.

The recombinant plasmid is concretely by the multiple cloning sites of any description above gene insertion pFastBac1 carrier The recombinant plasmid that (such as between BamHI and NotI restriction enzyme site) obtains.

The present invention also protects the application of any description above protein, is at least one of following (c1) to (c8):

(c1) AIDS virus resisting vaccine is prepared；

(c2) drug of preparation treatment AIDS；

(c3) preparation inhibits the drug of AIDS virus；

(c4) preparation inhibition AIDS virus enters the drug of cell；

(c5) AIDS virus resisting；

(c6) AIDS is treated；

(c7) inhibit AIDS virus；

(c8) AIDS virus is inhibited to enter cell.

The tripolymer that the present invention also protects any description above protein to be formed.

The trimer preparation method is as follows: by any description above channel genes mammalian cell, cultivating cell And supernatant is collected, wherein containing the tripolymer.

The present invention also protects the application of the tripolymer, is at least one of following (c1) to (c8):

(c1) AIDS virus resisting vaccine is prepared；

(c2) drug of preparation treatment AIDS；

(c3) preparation inhibits the drug of AIDS virus；

(c4) preparation inhibition AIDS virus enters the drug of cell；

(c5) AIDS virus resisting；

(c6) AIDS is treated；

(c7) inhibit AIDS virus；

(c8) AIDS virus is inhibited to enter cell.

The present invention also protects a kind of composition, is made of component first, component second and component third；

The component first is the tripolymer that albumen first is formed；The albumen first be (e1) or (e2) or (e3) or (e4) or (e5)；The component second is the tripolymer that albumen second is formed；The albumen second is (f1) or (f2) or (f3) or (f4) or (f5)； The component third is the tripolymer that albumen third is formed；The albumen third is (g1) or (g2) or (g3) or (g4) or (g5)；

(e1) protein being successively made of following element from N-terminal to C-terminal: function section, tripolymer folded domain；Institute Stating function section is the protein that the amino acid sequence shown in sequence 1 in sequence table forms；

(e2) protein being successively made of following element from N-terminal to C-terminal: function section, link peptide, tripolymer fold knot Structure domain；The function section is the protein that the amino acid sequence shown in sequence 1 in sequence table forms；

(e3) protein being successively made of following element from N-terminal to C-terminal: gp67 signal peptide, function section, link peptide, Tripolymer folded domain；The function section is the protein that the amino acid sequence shown in sequence 1 in sequence table forms；

(e4) protein being successively made of following element from N-terminal to C-terminal: function section, link peptide, tripolymer fold knot Structure domain, His₆Label；The function section is the protein that the amino acid sequence shown in sequence 1 in sequence table forms；

(e5) protein being successively made of following element from N-terminal to C-terminal: gp67 signal peptide, function section, link peptide, Tripolymer folded domain, His₆Label；Function section amino acid sequence shown in sequence 1 in sequence table forms Protein.

(f1) protein being successively made of following element from N-terminal to C-terminal: function section, tripolymer folded domain；Institute Stating function section is the protein that the amino acid sequence shown in sequence 3 in sequence table forms；

(f2) protein being successively made of following element from N-terminal to C-terminal: function section, link peptide, tripolymer fold knot Structure domain；The function section is the protein that the amino acid sequence shown in sequence 3 in sequence table forms；

(f3) protein being successively made of following element from N-terminal to C-terminal: gp67 signal peptide, function section, link peptide, Tripolymer folded domain；The function section is the protein that the amino acid sequence shown in sequence 3 in sequence table forms；

(f4) protein being successively made of following element from N-terminal to C-terminal: function section, link peptide, tripolymer fold knot Structure domain, His₆Label；The function section is the protein that the amino acid sequence shown in sequence 3 in sequence table forms；

(f5) protein being successively made of following element from N-terminal to C-terminal: gp67 signal peptide, function section, link peptide, Tripolymer folded domain, His₆Label；Function section amino acid sequence shown in sequence 3 in sequence table forms Protein.

(g1) protein being successively made of following element from N-terminal to C-terminal: function section, tripolymer folded domain；Institute Stating function section is the protein that the amino acid sequence shown in sequence 5 in sequence table forms；

(g2) protein being successively made of following element from N-terminal to C-terminal: function section, link peptide, tripolymer fold knot Structure domain；The function section is the protein that the amino acid sequence shown in sequence 5 in sequence table forms；

(g3) protein being successively made of following element from N-terminal to C-terminal: gp67 signal peptide, function section, link peptide, Tripolymer folded domain；The function section is the protein that the amino acid sequence shown in sequence 5 in sequence table forms；

(g4) protein being successively made of following element from N-terminal to C-terminal: function section, link peptide, tripolymer fold knot Structure domain, His₆Label；The function section is the protein that the amino acid sequence shown in sequence 5 in sequence table forms；

(g5) protein being successively made of following element from N-terminal to C-terminal: gp67 signal peptide, function section, link peptide, Tripolymer folded domain, His₆Label；Function section amino acid sequence shown in sequence 5 in sequence table forms Protein.

In the composition, the quality proportioning of component first, component second and component third are as follows: 0.5-2:0.5-2:0.5-2.

In the composition, the quality proportioning of component first, component second and component third are as follows: 1:1:1.

The function of the composition is following (d1), (d2), (d3) or (d4):

(d1) AIDS virus resisting；

(d2) AIDS is treated；

(d3) inhibit AIDS virus；

(d4) AIDS virus is inhibited to enter cell.

The present invention also protects the application of the composition, is at least one of following (c1) to (c8):

(c1) AIDS virus resisting vaccine is prepared；

(c2) drug of preparation treatment AIDS；

(c3) preparation inhibits the drug of AIDS virus；

(c4) preparation inhibition AIDS virus enters the drug of cell；

(c5) AIDS virus resisting；

(c6) AIDS is treated；

(c7) inhibit AIDS virus；

(c8) AIDS virus is inhibited to enter cell.

The present invention also protects any description above gene preparing the application in vaccine；The function of the vaccine is as follows (d1), (d2), (d3) or (d4):

(d1) AIDS virus resisting；

(d2) AIDS is treated；

(d3) inhibit AIDS virus；

(d4) AIDS virus is inhibited to enter cell.

The present invention also protects a kind of vaccine, and active constituent is any description above protein or any description above trimerization Body or any description above composition；The function of the vaccine is following (d1), (d2), (d3) or (d4):

(d1) AIDS virus resisting；

(d2) AIDS is treated；

(d3) inhibit AIDS virus；

(d4) AIDS virus is inhibited to enter cell.

Any description above AIDS virus concretely HIV subtype B.

Any description above AIDS virus concretely Chinese epidemic strain.

Any description above AIDS virus concretely CNE14, CNE4, CNE10, CNE11 or CNE57.

Protein provided by the invention has good epitope conformation and combines activity, shows targeting immunogene well Property.Using protein provided by the invention as immunogen immune biology, biology can be promoted to generate to a variety of AIDS viruses Antibody with pervasive neutralization activity.The present invention has great application and popularization value to the treatment of AIDS.

Detailed description of the invention

Fig. 1 is to carry out the chromatogram that molecular-exclusion chromatography is purified with Buffer 1.

Fig. 2 is the electrophoretogram that six kinds of solution carry out reproducibility PAGE gel electrophoresis.

Fig. 3 is the testing result of serum titer in embodiment 4.

Specific embodiment

Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even Mean value.Unless otherwise specified, PBS buffer solution used in embodiment is the PBS buffer solution of pH7.2,0.01M.

PFastBac1 carrier: Invitrogen company, article No. 10359-016.Escherichia coli DH10 Bac: Invitrogen company, article No. 10359-016.Sf9 cell: Invitrogen company, article No. 11496-015.Dunkin Hartley Female guinea pigs: company, tonneau China, article No. 601 are tieed up in Beijing.293T cell: ATCC, article No. CRL-3216. PcDNA3.1 (+) carrier: Invitrogen company, article No. V790-20.Skeleton plasmid pNL4-3R-E-luciferase:NIH AIDS Reagent company, article No. 3418.TZM-bl cell: NIH AIDS Reagent company, article No. 8129.Insect is thin Born of the same parents' culture medium (Insect-XpressTM Media): Lonza Wokingham Ltd., article No. 12-730F.

The discovery of embodiment 1, V61 albumen and its encoding gene

C-terminal by way of random cutting is connect with the antigen fragment library generated after recombination T-A with Aga2 albumen Fusion is shown under corresponding inductive condition on saccharomyces cerevisiae surface.Table is neutralized using full length antibody and as other of control Position is screened in yeast display library, is obtained its corresponding positive yeast monoclonal and is carried out the dyeing of yeast fluidic cell and tests Card, sifts out epitope antigen segment.Wherein three epitope antigen segments are respectively epitope antigen segment V61, epitope antigen segment V51 With epitope antigen segment V17.

Epitope antigen segment V61, also known as V61 albumen, (by 227 amino acid residue groups as shown in the sequence 1 of sequence table At).The expection molecular weight of V61 albumen is 30-35KDa.The nucleic acid of coding V61 albumen is named as V61 gene, the coding in cDNA Area is as shown in the sequence 2 of sequence table.

Epitope antigen segment V51, also known as V51 albumen, (by 254 amino acid residue groups as shown in the sequence 3 of sequence table At).The expection molecular weight of V51 albumen is 30-35KDa.The nucleic acid of coding V51 albumen is named as V51 gene, the coding in cDNA Area is as shown in the sequence 4 of sequence table.

Epitope antigen segment V17, also known as V17 albumen, (by 261 amino acid residue groups as shown in the sequence 5 of sequence table At).The expection molecular weight of V17 albumen is 30-35KDa.The nucleic acid of coding V17 albumen is named as V17 gene, the coding in cDNA Area is as shown in the sequence 6 of sequence table.

The expression and purifying of embodiment 2, albumen

One, construction recombination plasmid

Specific DNA molecular first-I is inserted between BamHI the and NotI restriction enzyme site of pFastBac1 carrier, is recombinated Plasmid first-I.It in specific DNA molecular first-I, is successively made of from upstream to downstream following element: the code sequence of gp67 signal peptide It arranges, V61 gene, His shown in the sequence 2 of sequence table₆The coded sequence of label, terminator codon (TAA).By recombinant plasmid First-I, which imports cell and cultivates, can express differential protein first-I.In differential protein first-I, from N-terminal to C-terminal successively by following member Part composition: gp67 signal peptide, sequence table sequence 1 shown in V61 albumen, His₆Label.Differential protein first-I is in culture supernatant In in the form of monomer exist.

Specific DNA molecular first-II is inserted between BamHI the and NotI restriction enzyme site of pFastBac1 carrier, is recombinated Plasmid first-II.It in specific DNA molecular first-II, is successively made of from upstream to downstream following element: the coding of gp67 signal peptide V61 gene shown in sequence, the sequence of sequence table 2, the coded sequence of link peptide, tripolymer folded domain coded sequence, His₆The coded sequence of label, terminator codon (TAA).Recombinant plasmid first-II, which is imported cell and cultivated, can express specifically Albumen first-II.In differential protein first-II, be successively made of following element from N-terminal to C-terminal: gp67 signal peptide, sequence table sequence V61 albumen, link peptide shown in column 1, tripolymer folded domain, His₆Label.Differential protein first-II in culture supernatant with The form of tripolymer exists.

Specific DNA molecular second-I is inserted between BamHI the and NotI restriction enzyme site of pFastBac1 carrier, is recombinated Plasmid second-I.It in specific DNA molecular second-I, is successively made of from upstream to downstream following element: the code sequence of gp67 signal peptide It arranges, V51 gene, His shown in the sequence 4 of sequence table₆The coded sequence of label, terminator codon (TAA).By recombinant plasmid Second-I, which imports cell and cultivates, can express differential protein second-I.In differential protein second-I, from N-terminal to C-terminal successively by following member Part composition: gp67 signal peptide, sequence table sequence 3 shown in V51 albumen, His₆Label.Differential protein second-I is in culture supernatant In in the form of monomer exist.

Specific DNA molecular second-II is inserted between BamHI the and NotI restriction enzyme site of pFastBac1 carrier, is recombinated Plasmid second-II.It in specific DNA molecular second-II, is successively made of from upstream to downstream following element: the coding of gp67 signal peptide V51 gene shown in sequence, the sequence of sequence table 4, the coded sequence of link peptide, tripolymer folded domain coded sequence, His₆The coded sequence of label, terminator codon (TAA).Recombinant plasmid second-II, which is imported cell and cultivated, can express specifically Albumen second-II.In differential protein second-II, be successively made of following element from N-terminal to C-terminal: gp67 signal peptide, sequence table sequence V51 albumen, link peptide shown in column 3, tripolymer folded domain, His₆Label.Differential protein second-II in culture supernatant with The form of tripolymer exists.

Specific DNA molecular propyl- I is inserted between BamHI the and NotI restriction enzyme site of pFastBac1 carrier, is recombinated Plasmid propyl- I.It in specific DNA molecular propyl- I, is successively made of from upstream to downstream following element: the code sequence of gp67 signal peptide It arranges, V17 gene, His shown in the sequence 6 of sequence table₆The coded sequence of label, terminator codon (TAA).By recombinant plasmid Propyl- I, which imports cell and cultivates, can express differential protein propyl- I.In differential protein propyl- I, from N-terminal to C-terminal successively by following member Part composition: gp67 signal peptide, sequence table sequence 5 shown in V17 albumen, His₆Label.Differential protein propyl- I is in culture supernatant In in the form of monomer exist.

Specific DNA molecular propyl- II is inserted between BamHI the and NotI restriction enzyme site of pFastBac1 carrier, is recombinated Plasmid propyl- II.It in specific DNA molecular propyl- II, is successively made of from upstream to downstream following element: the coding of gp67 signal peptide V17 gene shown in sequence, the sequence of sequence table 6, the coded sequence of link peptide, tripolymer folded domain coded sequence, His₆The coded sequence of label, terminator codon (TAA).Recombinant plasmid propyl- II, which is imported cell and cultivated, can express specifically Albumen propyl- II.In differential protein propyl- II, be successively made of following element from N-terminal to C-terminal: gp67 signal peptide, sequence table sequence V17 albumen, link peptide shown in column 5, tripolymer folded domain, His₆Label.Differential protein propyl- II in culture supernatant with The form of tripolymer exists.

In above six recombinant plasmids, the coded sequence of the gp67 signal peptide (coded sequence as shown in the sequence of sequence table 8 Polypeptide shown in the sequence 7 of table), the coded sequence of the link peptide (sequence 9 of polynucleotide as shown in the sequence of sequence table 10 Shown in polypeptide), the coded sequence of the tripolymer folded domain (sequence of polynucleotide as shown in the sequence of sequence table 12 Polypeptide shown in 11), His₆The coded sequence of label is as shown in the sequence of sequence table 14 (shown in the sequence 13 of polynucleotide Polypeptide).

Two, the preparation of Bacmid is recombinated

1, recombinant plasmid is added in the Escherichia coli DH10 Bac competent cell just melted, places 30min on ice； Then 42 DEG C of heat shock 75s put back to 2min on ice；Then 500 μ l LB liquid mediums are added, in 37 DEG C of recovery 5h；Then it draws 10 μ l are coated on containing 50 μ g/ml kanamycins, 7 μ g/ml gentamicins, 10 μ g/ml tetracyclines, 40 μ g/ml IPTG and 100 μ g/ The LB solid medium tablets of ml X-gal are protected from light culture three days to growing clearly blue hickie.

2, picking white single colonie is inoculated in 5mL containing 50 μ g/ml kanamycins, 7 μ g/ml gentamicins, 10 μ g/ml tetra- The LB liquid medium of ring element, 37 DEG C, 220rpm shaken cultivation 12 hours.

3, the cultivating system for taking step 2 to obtain, the small extraction reagent kit of the plasmid of use (QIAprep Spin Miniprep Kit, Qiagen company, article No. 27106, wherein contain P1 reagent, P2 reagent and P3 reagent) extract plasmid, specific steps according to It is secondary as follows:

(1) cultivating system 13000rpm is centrifuged 2min, collects bacterial sediment, thallus is resuspended with P1 reagent；

(2) P2 reagent is added, is slowly mixed by inversion 6-8 times；

(3) P3 reagent is added, is slowly mixed by inversion 6-8 times (visible white precipitating), 13000rpm is centrifuged 10min, takes Clear liquid；

(4) supernatant for taking 600 μ l steps (3) to obtain, the isopropanol of addition 800 μ l pre-cooling, -20 DEG C of placement 10min, so 13000rpm is centrifuged 15min afterwards, collects precipitating；

(5) precipitating that step (4) obtain being resuspended with 70% ethanol water of 500 μ l pre-cooling, 13000rpm is centrifuged 5min, Precipitating is collected, after alcohol is dried up completely, with the ddH of 65 DEG C of preheatings₂O dissolution precipitates, 13000rpm centrifugation 5min, in absorption Clearly, the solution of Bacmid, abbreviation Bacmid solution are as recombinated.

When recombinant plasmid is recombinant plasmid first-I, obtained Bacmid solution is named as Bacmid solution first-I.Recombinate matter When grain is recombinant plasmid first-II, obtained Bacmid solution is named as Bacmid solution first-II.Recombinant plasmid is recombinant plasmid When second-I, obtained Bacmid solution is named as Bacmid solution second-I.When recombinant plasmid is recombinant plasmid second-II, obtain Bacmid solution is named as Bacmid solution second-II.When recombinant plasmid is recombinant plasmid propyl- I, obtained Bacmid solution is named For Bacmid solution propyl- I.When recombinant plasmid is recombinant plasmid propyl- II, obtained Bacmid solution is named as Bacmid solution Propyl- II.

Three, the preparation and amplification of recombinant virus

1, well-grown Sf9 cell is taken, is added in 10cm culture dish, stands 10min, cell is adherent, sees under microscope It examines, guarantees that there are about 70%-80% for culture dish bottom and covered by cell.

2, Cellfectin II Reagent15 μ l is taken, is diluted with 100 μ l insect cell mediums.

3,15-20 μ lBacmid solution is taken, is diluted with 100 μ l insect cell mediums.

4, the liquid phase for obtaining step 2 is slowly added in the liquid phase that step 3 obtains, and slowly piping and druming uniformly, is stored at room temperature 30min is diluted to 2ml with insect cell medium.

5, the culture dish for taking into step 1, discards supernatant, and the liquid phase for slowly uniformly obtaining step 4 is added dropwise to culture It in ware, is inhaled after 27 DEG C of stationary culture 5h and abandons supernatant, the fresh insect cell medium of 7ml is added, 27 DEG C after being sealed with sealed membrane Stationary culture 8 days, culture solution is collected, 600g is centrifuged 6min, takes supernatant, and fetal calf serum is added and makes its volumetric concentration 2- 5%, the virus liquid of long-term preservation, as P0 for recombinant virus.

6, it takes P0 for the virus liquid of recombinant virus, is 2 according to the cell concentration that shaking flask culture is added in the volume ratio of 1:1000 ×10⁶In the Sf9 cell liquid of a cell/mL, 27 DEG C, 110rpm culture 5 days collect culture solution, 600g is centrifuged 6min, takes supernatant Liquid, as P1 are for the virus liquid of recombinant virus, and abbreviation P1 is for virus liquid.

When Bacmid solution is Bacmid solution first-I, obtained P1 is named as P1 for virus liquid first-I for virus liquid. When Bacmid solution is Bacmid solution first-II, obtained P1 is named as P1 for virus liquid first-II for virus liquid.Bacmid is molten When liquid is Bacmid solution second-I, obtained P1 is named as P1 for virus liquid second-I for virus liquid.Bacmid solution is Bacmid When solution second-II, obtained P1 is named as P1 for virus liquid second-II for virus liquid.Bacmid solution is Bacmid solution propyl- I When, obtained P1 is named as P1 for virus liquid propyl- I for virus liquid.When Bacmid solution is Bacmid solution propyl- II, obtain P1 is named as P1 for virus liquid propyl- II for virus liquid.

Four, the expression and purifying of albumen

1, take P1 for virus liquid, it is 2 × 10 that 1L cell concentration, which is added, according to the volume ratio of 1:100⁶The Sf9 of a cell/mL In cell liquid, 27 DEG C, 125rpm culture 72 hours, 4000rpm is centrifuged 15min, collects supernatant.

2, the supernatant for obtaining step 1 is filtered with double-deck 0.45 μm of the glass fibre membrane, collects filtrate.

3, with cross-flow ultrafiltration system (Masterflex PharMed BPT Tubing system, Cole-Parmer company, Article No. is 06508-24) filtrate that step 2 obtains is concentrated, while be added Buffer 1 (pH7.2 containing 150mMNaCl, 10mM HEPAS buffer) constantly dilution, make albumen displacement into Buffer 1, then 13000rpm centrifugation 30min, in collection Clear liquid.

4, affinity chromatography

(1) Ni-NTA purification media is added in the supernatant that step 3 obtains, 4 DEG C are incubated for 3 hours, 400rpm centrifugation 5min takes precipitating.

(2) precipitating obtained with 1 washing step (1) of the Buffer of 100mL imidazoles containing 20mM, to remove foreign protein.

(3) precipitating obtained with 1 washing step (2) of the Buffer of 20mL imidazoles containing 300mM collects solution.

5, the solution that step 4 obtains is concentrated using 10kD concentration tube, obtains protein concentrated solution.

6, the protein concentrated solution obtained using solvent resistant column (Superdex200, GE Healthcare) purification step 5, It is eluted with Buffer 1, flow velocity 1mL/min.

P1 for virus liquid be P1 for virus liquid first-I when, collect in step 6 retention volume be 14.5-17.5mL cross column after Solution, the as solution containing differential protein first-I existing for monomeric form, are named as V61 solution.

P1 for virus liquid be P1 for virus liquid first-II when, it is that crossing for 12-15mL is molten after column that retention volume is collected in step 6 Liquid, the as solution containing differential protein first-II existing for trimeric form, are named as V61tri solution.

P1 for virus liquid be P1 for virus liquid second-I when, collect in step 6 retention volume be 14.5-17.5mL cross column after Solution, the as solution containing differential protein second-I existing for monomeric form, are named as V51 solution.

P1 for virus liquid be P1 for virus liquid second-II when, it is that crossing for 12-15mL is molten after column that retention volume is collected in step 6 Liquid, the as solution containing differential protein second-II existing for trimeric form, are named as V51tri solution.

P1 for virus liquid be P1 for virus liquid propyl- I when, collect in step 6 retention volume be 14.5-17.5mL cross column after Solution, the as solution containing differential protein propyl- I existing for monomeric form, are named as V17 solution.

P1 for virus liquid be P1 for virus liquid propyl- II when, it is that crossing for 12-15mL is molten after column that retention volume is collected in step 6 Liquid, the as solution containing differential protein propyl- II existing for trimeric form, are named as V17tri solution.

Fig. 1 is seen with the chromatogram that Buffer 1 is eluted, and shows more single peak point.All trimer proteins are than it Corresponding monomeric protein has obvious peak point to move forward, and illustrates that it is successfully assembled by itself tripolymer folded domain Trimeric form.

7, six kinds of solution for obtaining step 6 carry out reproducibility PAGE gel electrophoresis, as a result see Fig. 2.All 6 hatching eggs Bai Jun has purer main band in about 55KDa.

The combination activity of embodiment 3, epitope antigen segment and antibody

By surface plasma resonance (Surface Plasmon Resonance, SPR), detect epitope antigen segment with The combination activity of the associated antibodies such as CD4bs.

Have chosen 7 kinds of CD4bs VRC01class antibody (VRC01,12A12, VRC-CH31,3BNC60,3BNC117, NIH45-46, VRC-PG04) and a kind of V3-glycan antibody (PGT135).Using 3C11 antibody (for the anti-of influenza virus HA Body) it is control antibodies.VRC01 antibody, light chain such as GENBANK ACCESSION NO.GU980703.1 (30-AUG-2010) institute Show, shown in heavy chain such as GENBANK ACCESSION NO.GU980702.1 (30-AUG-2010).12A12 antibody, light chain is such as Shown in GENBANK ACCESSION NO.HE584540.1 (25-JUL-2016), heavy chain such as GENBANK ACCESSION Shown in NO.HE584539.1 (25-JUL-2016).VRC-CH31 antibody, light chain such as GENBANK ACCESSION Shown in NO.JN159438.1 (29-MAR-2012), heavy chain such as GENBANK ACCESSION NO.JN159435.1 (29-MAR- 2012) shown in.3BNC60 antibody, shown in light chain such as GENBANK ACCESSION NO.HE584536.1 (25-JUL-2016), Shown in heavy chain such as GENBANK ACCESSION NO.HE584535.1 (25-JUL-2016).3BNC117 antibody, light chain is such as Shown in GENBANK ACCESSION NO.HE584538.1 (25-JUL-2016), heavy chain such as GENBANK ACCESSION Shown in NO.HE584537.1 (25-JUL-2016).NIH45-46 antibody, light chain such as GENBANK ACCESSION Shown in NO.HE584544.1 (25-JUL-2016), heavy chain such as GENBANK ACCESSION NO.HE584543.1 (25-JUL- 2016) shown in.VRC-PG04 antibody, light chain such as GENBANK ACCESSION NO.JN159466.1 (29-MAR-2012) institute Show, shown in heavy chain such as GENBANK ACCESSION NO.JN159464.1 (29-MAR-2012).PGT135 antibody, light chain is such as Shown in GENBANK ACCESSION NO.JN201920.1 (26-SEP-2011), heavy chain such as GENBANK ACCESSION Shown in NO.JN201903.1 (26-SEP-2011).3C11 antibody, light chain such as GENBANK ACCESSION NO.JF274049.1 Shown in (22-FEB-2012), shown in heavy chain such as GENBANK ACCESSION NO.JF274048.1 (22-FEB-2012).

Each albumen prepared by the segment of epitope antigen used in the present embodiment, that is, embodiment 2, the specific form that is added is real Apply V61 solution, V61tri solution, V51 solution, V51tri solution, V17 solution or the V17tri solution of the preparation of example 2.

Epitope antigen segment is fixed to CM5 chip surface by amino coupled kit (GE Healthcare), and is made It reaches 200-300 response units (RU).When carrying out the dynamic analysis of Ag-Ab combination: antibody is pressed 1- 500nM initial concentration respectively flows through chip (binding time 3min, 30 μ l/min of flow velocity；Dissociation time 5-30min, 30 μ l/ of flow velocity Min), then lived again 1min using the glycine-HCl buffer of pH2.0,10mM with 50 μ l/min flow velocitys；It is originated again with 2 times Concentration repeat above in conjunction with, dissociate, step of living again；Again with 4 times of initial concentrations repeat above in conjunction with, dissociate, step of living again；It uses again 8 times of initial concentrations repeat above in conjunction with, dissociate, step of living again；Again with 16 times of initial concentrations repeat above in conjunction with, dissociate, live again Step；Finally, being carried out using Biacore Evaluation software analytic dynamics data using 1:1Langmuir model Analysis, finally obtains binding constant (K_on, on-rate constant), dissociation constant (K_off, off-rate constant) and Equilibrium dissociation constant (K_D, equilibrium dissociation constant).Experimental temperature is 25 DEG C, buffer used For HBS-EP (10mMHepes, 150mMNaCl, 3mM EDTA, 0.005% Surfactant P20, pH7.4).

Equilibrium dissociation constant the results are shown in Table 1 (ND representative is not detected in conjunction with activity).The epitope antigen piece of monomeric form Section and the epitope antigen segment of trimeric form have higher affine activity with a variety of HIV antibodies.

Table 1

Embodiment 4, immunized guinea pigs experiment

One, it is immunized and collects serum

Experimental animal are as follows: 8-10 week old Dunkin Hartley Female guinea pigs.The V61tri prepared using embodiment 2 is molten Liquid, V51tri solution or V17tri solution.Protein content is in terms of total protein.Dilution is all made of PBS buffer solution.Immunization ways are Subcutaneous inoculation.

First group (6 experimental animals, be successively named as Seq01-Seq06): the 0th day progress initial immunity of test, test 28th day progress first time booster immunization, tests second of the booster immunization of progress in the 84th day, and the test third time of progress in the 140th day adds Strong immune, test takes a blood sample and collects serum on the 154th day；When initial immunity, by V17tri solution dilution after with complete Freund's adjuvant It is immunized after isometric mixing, single animal immune total volume is 600 μ l, and the protein content of single animal immune is 300 μ g；The When booster immunization, it is immunized after being mixed in equal volume after the dilution of V51tri solution with incomplete Freund's adjuvant, it is single only dynamic It is 600 μ l that total volume, which is immunized, in object, and the protein content of single animal immune is 300 μ g；When second of booster immunization, by V61tri solution It is immunized after being mixed in equal volume after dilution with incomplete Freund's adjuvant, single animal immune total volume is 600 μ l, single animal Immune protein content is 300 μ g；It, will be isometric with incomplete Freund's adjuvant after the dilution of V17tri solution when third time booster immunization It is immunized after mixing, single animal immune total volume is 600 μ l, and the protein content of single animal immune is 300 μ g；

Second group (6 experimental animals, be successively named as Mix01-Mix06): the 0th day progress initial immunity of test, test 28th day progress first time booster immunization, tests second of the booster immunization of progress in the 84th day, and the test third time of progress in the 140th day adds Strong immune, test takes a blood sample and collects serum on the 154th day；It is when initial immunity, V61tri solution, V51tri solution and V17tri is molten It being immunized after being mixed in equal volume after liquid mixed diluting with complete Freund's adjuvant, single animal immune total volume is 600 μ l, single The protein content of animal immune is 300 μ g (V61tri solution, V51tri solution and V17tri solution respectively provide 100 μ g albumen)；The When booster immunization, by after V61tri solution, V51tri solution and V17tri solution mixed diluting with incomplete Freund's adjuvant It is immunized after isometric mixing, single animal immune total volume is 600 μ l, and the protein content of single animal immune is 300 μ g (V61tri solution, V51tri solution and V17tri solution respectively provide 100 μ g albumen)；When second of booster immunization, by V61tri It is immunized after being mixed in equal volume after solution, V51tri solution and V17tri solution mixed diluting with incomplete Freund's adjuvant, it is single Animal immune total volume is 600 μ l, the protein content of single animal immune be 300 μ g (V61tri solution, V51tri solution and V17tri solution respectively provides 100 μ g albumen)；It is when third time booster immunization, V61tri solution, V51tri solution and V17tri is molten It is immunized after being mixed in equal volume after liquid mixed diluting with incomplete Freund's adjuvant, single animal immune total volume is 600 μ l, single The protein content of animal immune is 300 μ g (V61tri solution, V51tri solution and V17tri solution respectively provide 100 μ g albumen)；

Third group (control group) (3 experimental animals, be successively named as NC01-NC03): test is exempted from the 0th day for the first time Epidemic disease tests the 28th day progress first time booster immunization, tests second of the booster immunization of progress in the 84th day, tests the 140th day and carries out Third time booster immunization, test take a blood sample and collect serum on the 154th day；When initial immunity, by PBS buffer solution and complete Freund's adjuvant It is immunized after isometric mixing, single animal immune total volume is 600 μ l；When first time booster immunization, by PBS buffer solution with Incomplete Freund's adjuvant is immunized after mixing in equal volume, and single animal immune total volume is 600 μ l；Second of booster immunization When, it is immunized after PBS buffer solution is mixed in equal volume with incomplete Freund's adjuvant, single animal immune total volume is 600 μ l； It when third time booster immunization, is immunized after PBS buffer solution is mixed in equal volume with incomplete Freund's adjuvant, single animal immune Total volume is 600 μ l.

Two, serum titer

The serum for taking step 1 to obtain detects titre as test serum, by ELISA.When ELISA is detected, envelope antigen Peridium concentration be 2 μ g/ml.V61tri solution, V51tri solution or the V17tri that envelope antigen is prepared by embodiment 2 respectively are molten Liquid provides (concentration is in terms of total protein concentration).The titre of test serum is according to OD under greatest dilution_450nmReadings be greater than yin Property serum control determine.

As a result see Fig. 3.The test serum result that control group experimental animal obtains is feminine gender.First group of experimental animal and second The test serum that group experimental animal obtains is 10 for the titre of 3 kinds of coating antigens⁶-10⁷。

Three, neutralization activity detects

The serum for taking step 1 to obtain is as test serum.In order to verify the neutralization activity of test serum, choose several existing The pseudovirus of toxic strain is carried out in pseudovirus and is tested respectively as strain to be measured.

1, each pseudovirus is prepared

Existing strain is respectively as follows: Chinese epidemic strain: CNE14, CNE4, CNE10, CNE11 and CNE57.Using murine leukemia Control virus of the viral MMLV as existing strain.

Express the plasmid (being named as memebrane protein plasmid) and skeleton plasmid pNL4-3R-E-luciferase of overall length memebrane protein Cotransfection 293T cell can obtain having infectivity but the not pseudotype virus of replication capacity after incubation, infectious with living Virus is similar.To encode the overall length memebrane protein of each existing strain gene insertion pcDNA3.1 (+) carrier HindIII and Between XhoI restriction enzyme site, each memebrane protein plasmid is obtained.By some memebrane protein plasmid and skeleton plasmid pNL4-3R-E- Luciferase cotransfection 293T cell, 37 DEG C of stationary incubations, transfection collect cells and supernatant after 48 hours, as contain phase Answer the virus liquid of pseudovirus.

The overall length memebrane protein and its encoding gene of CNE14 such as GENBANK ACCESSION NO.HM215400.1 (07- NOV-2014 shown in).The overall length memebrane protein and its encoding gene of CNE4 such as GENBANK ACCESSION NO.HM215413.1 Shown in (07-NOV-2014).The overall length memebrane protein and its encoding gene of CNE10 such as GENBANK ACCESSION Shown in NO.HM215397.1 (07-NOV-2014).Overall length memebrane protein and its encoding gene such as GENBANK of CNE11 Shown in ACCESSION NO.HM215398.1 (07-NOV-2014).The overall length memebrane protein and its encoding gene of CNE57 is such as Shown in GENBANK ACCESSION NO.HM215420.1 (07-NOV-2014).The overall length memebrane protein and its encoding gene of MMLV As shown in GENBANK ACCESSION NO.S77017.1 (26-JUL-2016).

2, neutralization activity detects

Virus liquid to be measured is respectively the virus liquid of each pseudovirus prepared by step 1.

(1) test serum doubling dilution is obtained by each of different extension rates using the DMEM culture medium containing 10%FBS Dilution.

(2) by dilution that 100 microlitres of steps (1) obtain and 50 microlitres of virus liquids to be measured, (viral level is 100TCID50) mix, 37 DEG C stationary incubation 1 hour.Be arranged the DMEM culture medium with 100 microlitres containing 10%FBS replace it is 100 micro- Rise the blank control of dilution.

(3) after completing step (2), the cell liquid of 50 microlitres of TZM-bl cells is added (containing about 2 × 10⁴A TZM-bl is thin Born of the same parents), 37 DEG C stationary incubation 48 hours.

(4) after completing step (3), 100 μ lPBS buffers and 50 μ l cell pyrolysis liquid (Bright-Glo are added^TM Luciferase Assay System, Promega, E2650), 2min is stood, then detects luciferase with Chemiluminescence Apparatus Activity.

5 multiple holes are arranged in every kind of processing, and results are averaged.

Neutralization activity=(fluorescence intensity of the fluorescence intensity of blank control group-addition dilution experimental group)/blank pair According to fluorescence intensity × 100% of group.

The dilution of corresponding test serum is ID when neutralization activity is 50%₅₀Value.

It the results are shown in Table 2 (NA represents inactive).The test serum that first group of experimental animal and second group of experimental animal obtain Can each pseudovirus of neutralization in various degree enter cell, can not only neutralize autologous virus CNE11, can also in various degree The allosomes virus such as neutralization CNE14, CNE4, CNE10 and CNE57, wherein Mix 02 can to the neutralization ID50 value of CNE14 and CNE4 Respectively up to 592 and 3226.Neutralization activity between sequential-type immune group (first group) and hybrid immune group (second group) has no Obvious statistical difference, but hybrid immune group is slightly strong.

Table 2

NC01

NC02

NC03

Seq01

Seq02

Seq03

Seq04

Seq05

Seq06

Mix01

Mix02

Mix03

Mix04

Mix05

Mix06

CNE14

<20

131

64

48

80

133

287

476

592

132

138

22

41

CNE4

<20

27

136

28

201

158

83

47

3226

94

278

<20

120

CNE10

<20

28

<20

51

<20

50

<20

CNE11

<20

65

<20

36

34

39

55

28

<20

26

37

CNE57

<20

35

<20

36

70

<20

62

94

29

248

NA

MMLV

<20

SEQUENCE LISTING

<110>Tsinghua University

<120>a kind of protein based on the reversed epitope design of antibody and its in the application prepared in AIDS virus resisting vaccine

<130> CGGNQAYX-176150

<160> 14

<170> PatentIn version 3.5

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<211> 227

<212> PRT

<213> Artificial sequence

<400> 1

Ile Arg Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala

1 5 10 15

Glu Glu Glu Val Val Ile Arg Ser Ser Asn Phe Thr Asn Asn Ala Lys

20 25 30

Val Ile Ile Val Gln Leu Asn Glu Ser Val Ile Ile Asn Cys Thr Arg

35 40 45

Pro Asn Asn Asn Thr Arg Lys Ser Ile His Leu Gly Gln Gly Arg Ala

50 55 60

Trp Tyr Thr Thr Gly Gln Ile Ile Gly Asp Ile Arg Gln Ala His Cys

65 70 75 80

Asn Leu Ser Arg Thr Glu Trp Asn Asn Thr Leu Lys Gln Ile Ala Lys

85 90 95

Lys Leu Arg Glu Gln Phe Gly Asn Lys Thr Ile Ile Phe Asn Gln Ser

100 105 110

Ser Gly Gly Asp Pro Glu Ile Val Met His Ser Phe Asn Cys Gly Gly

115 120 125

Glu Phe Phe Tyr Cys Asn Thr Ser Gln Leu Phe Asn Ser Thr Trp Asn

130 135 140

Asn Asn Ser Thr Trp Asn Asp Thr Ser Ile Trp Asn Asp Thr Thr Gly

145 150 155 160

Asn Asp Asn Ile Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn Met

165 170 175

Trp Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Ala Gly Gln

180 185 190

Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly

195 200 205

Gly Thr Asn Glu Ser Glu Thr Thr Glu Ile Phe Arg Pro Ala Gly Gly

210 215 220

Asp Met Arg

225

<210> 2

<211> 681

<212> DNA

<213> Artificial sequence

<400> 2

attaggccag tagtatcaac ccaactgctg ttaaatggca gtttagcaga agaagaggta 60

gtaattagat ctagcaattt cacgaacaat gctaaagtca taatagtaca gctgaatgaa 120

tctgtaataa ttaattgtac aagacccaac aacaatacaa gaaaaagtat acatctagga 180

caagggcgag catggtatac aacaggacaa ataataggag atataagaca agcacattgt 240

aaccttagta gaacagaatg gaataacact ttaaagcaga tagctaaaaa attaagagaa 300

caatttggga acaaaacaat aatctttaat caatcttcag gaggggaccc agagattgta 360

atgcacagtt ttaattgtgg aggggaattt ttctactgta atacatcaca actgtttaat 420

agtacttgga ataataatag tacttggaat gatactagta tttggaatga tactacagga 480

aatgacaata tcacgctccc ttgcagaata aaacaaatta taaacatgtg gcaggaagta 540

gggaaagcaa tgtatgcccc tcccattgca ggacaaatta gatgttcatc aaatattaca 600

gggttactat taacaagaga tggtggtact aatgaaagcg agaccaccga gatcttcaga 660

cctgcaggag gagacatgag a 681

<210> 3

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<212> PRT

<213> Artificial sequence

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Ala Gly Phe Ala Ile Leu Lys Cys Asn Asp Lys Thr Phe Asn Gly Thr

1 5 10 15

Gly Pro Cys Thr Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg

20 25 30

Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu

35 40 45

Glu Val Val Ile Arg Ser Ser Asn Phe Thr Asn Asn Ala Lys Val Ile

50 55 60

Ile Val Gln Leu Asn Glu Ser Val Ile Ile Asn Cys Thr Arg Pro Asn

65 70 75 80

Asn Asn Thr Arg Lys Ser Ile His Leu Gly Gln Gly Arg Ala Trp Tyr

85 90 95

Thr Thr Gly Gln Ile Ile Gly Asp Ile Arg Gln Ala His Cys Asn Leu

100 105 110

Ser Arg Thr Glu Trp Asn Asn Thr Leu Lys Gln Ile Ala Lys Lys Leu

115 120 125

Arg Glu Gln Phe Gly Asn Lys Thr Ile Ile Phe Asn Gln Ser Ser Gly

130 135 140

Gly Asp Pro Glu Ile Val Met His Ser Phe Asn Cys Gly Gly Glu Phe

145 150 155 160

Phe Tyr Cys Asn Thr Ser Gln Leu Phe Asn Ser Thr Trp Asn Asn Asn

165 170 175

Ser Thr Trp Asn Asp Thr Ser Ile Trp Asn Asp Thr Thr Gly Asn Asp

180 185 190

Asn Ile Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln

195 200 205

Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Ala Gly Gln Ile Arg

210 215 220

Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Thr

225 230 235 240

Asn Glu Ser Glu Thr Thr Glu Ile Phe Arg Pro Ala Gly Gly

245 250

<210> 4

<211> 762

<212> DNA

<213> Artificial sequence

<400> 4

gctggttttg cgattctaaa gtgtaacgat aaaacgttca atggaacagg accatgtaca 60

aatgtcagta cagtacaatg tacacatgga attaggccag tagtatcaac ccaactgctg 120

ttaaatggca gtttagcaga agaagaggta gtaattagat ctagcaattt cacgaacaat 180

gctaaagtca taatagtaca gctgaatgaa tctgtaataa ttaattgtac aagacccaac 240

aacaatacaa gaaaaagtat acatctagga caagggcgag catggtatac aacaggacaa 300

ataataggag atataagaca agcacattgt aaccttagta gaacagaatg gaataacact 360

ttaaagcaga tagctaaaaa attaagagaa caatttggga acaaaacaat aatctttaat 420

caatcttcag gaggggaccc agagattgta atgcacagtt ttaattgtgg aggggaattt 480

ttctactgta atacatcaca actgtttaat agtacttgga ataataatag tacttggaat 540

gatactagta tttggaatga tactacagga aatgacaata tcacgctccc ttgcagaata 600

aaacaaatta taaacatgtg gcaggaagta gggaaagcaa tgtatgcccc tcccattgca 660

ggacaaatta gatgttcatc aaatattaca gggttactat taacaagaga tggtggtact 720

aatgaaagcg agaccaccga gatcttcaga cctgcaggag ga 762

<210> 5

<211> 261

<212> PRT

<213> Artificial sequence

<400> 5

Asn Gly Thr Gly Pro Cys Thr Asn Val Ser Thr Val Gln Cys Thr His

1 5 10 15

Gly Ile Arg Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu

20 25 30

Ala Glu Glu Glu Val Val Ile Arg Ser Ser Asn Phe Thr Asn Asn Ala

35 40 45

Lys Val Ile Ile Val Gln Leu Asn Glu Ser Val Ile Ile Asn Cys Thr

50 55 60

Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile His Leu Gly Gln Gly Arg

65 70 75 80

Ala Trp Tyr Thr Thr Gly Gln Ile Ile Gly Asp Ile Arg Gln Ala His

85 90 95

Cys Asn Leu Ser Arg Thr Glu Trp Asn Asn Thr Leu Lys Gln Ile Ala

100 105 110

Lys Lys Leu Arg Glu Gln Phe Gly Asn Lys Thr Ile Ile Phe Asn Gln

115 120 125

Ser Ser Gly Gly Asp Pro Glu Ile Val Met His Ser Phe Asn Cys Gly

130 135 140

Gly Glu Phe Phe Tyr Cys Asn Thr Ser Gln Leu Phe Asn Ser Thr Trp

145 150 155 160

Asn Asn Asn Ser Thr Trp Asn Asp Thr Ser Ile Trp Asn Asp Thr Thr

165 170 175

Gly Asn Asp Asn Ile Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn

180 185 190

Met Trp Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Ala Gly

195 200 205

Gln Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp

210 215 220

Gly Gly Thr Asn Glu Ser Glu Thr Thr Glu Ile Phe Arg Pro Ala Gly

225 230 235 240

Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val

245 250 255

Val Lys Ile Glu Pro

260

<210> 6

<211> 783

<212> DNA

<213> Artificial sequence

<400> 6

aatggaacag gaccatgtac aaatgtcagt acagtacaat gtacacatgg aattaggcca 60

gtagtatcaa cccaactgct gttaaatggc agtttagcag aagaagaggt agtaattaga 120

tctagcaatt tcacgaacaa tgctaaagtc ataatagtac agctgaatga atctgtaata 180

attaattgta caagacccaa caacaataca agaaaaagta tacatctagg acaagggcga 240

gcatggtata caacaggaca aataatagga gatataagac aagcacattg taaccttagt 300

agaacagaat ggaataacac tttaaagcag atagctaaaa aattaagaga acaatttggg 360

aacaaaacaa taatctttaa tcaatcttca ggaggggacc cagagattgt aatgcacagt 420

tttaattgtg gaggggaatt tttctactgt aatacatcac aactgtttaa tagtacttgg 480

aataataata gtacttggaa tgatactagt atttggaatg atactacagg aaatgacaat 540

atcacgctcc cttgcagaat aaaacaaatt ataaacatgt ggcaggaagt agggaaagca 600

atgtatgccc ctcccattgc aggacaaatt agatgttcat caaatattac agggttacta 660

ttaacaagag atggtggtac taatgaaagc gagaccaccg agatcttcag acctgcagga 720

ggagacatga gagacaattg gagaagtgaa ttatataaat ataaagtagt gaaaattgaa 780

cca 783

<210> 7

<211> 38

<212> PRT

<213> Artificial sequence

<400> 7

Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr

1 5 10 15

Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala

20 25 30

Ala His Ser Ala Phe Ala

35

<210> 8

<211> 114

<212> DNA

<213> Artificial sequence

<400> 8

atgctactag taaatcagtc acaccaaggc ttcaataagg aacacacaag caagatggta 60

agcgctattg ttttatatgt gcttttggcg gcggcggcgc attctgcctt tgcg 114

<210> 9

<211> 10

<212> PRT

<213> Artificial sequence

<400> 9

Glu Asn Leu Tyr Phe Gln Gly Ala Gly Ser

1 5 10

<210> 10

<211> 30

<212> DNA

<213> Artificial sequence

<400> 10

gaaaacctgt attttcaggg cgccggcagc 30

<210> 11

<211> 26

<212> PRT

<213> Artificial sequence

<400> 11

Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys

1 5 10 15

Asp Gly Glu Trp Val Leu Leu Ser Thr Phe

20 25

<210> 12

<211> 78

<212> DNA

<213> Artificial sequence

<400> 12

ggctatattc cggaagcgcc gcgcgatggc caggcgtatg tgcgcaaaga tggcgaatgg 60

gtgctgctga gcaccttt 78

<210> 13

<211> 6

<212> PRT

<213> Artificial sequence

<400> 13

His His His His His His

1 5

<210> 14

<211> 18

<212> DNA

<213> Artificial sequence

<400> 14

catcaccatc accatcac 18

Claims

1. a kind of protein is any one following of (a1) into (a13):

(a4) fusion protein containing (a1) or (a2) or (a3)；

(a5) protein being successively made of following element from N-terminal to C-terminal: function section, tripolymer folded domain；The function Energy section is (a1) or (a2) or (a3)；

(a6) protein being successively made of following element from N-terminal to C-terminal: function section, link peptide, trimerization folding structure Domain；The function section is (a1) or (a2) or (a3)；

(a7) protein being successively made of following element from N-terminal to C-terminal: gp67 signal peptide, function section, link peptide, trimerization Folding structure domain；The function section is (a1) or (a2) or (a3)；

(a8) protein being successively made of following element from N-terminal to C-terminal: function section, link peptide, trimerization folding structure Domain, His₆Label；The function section is (a1) or (a2) or (a3)；

(a9) protein being successively made of following element from N-terminal to C-terminal: gp67 signal peptide, function section, link peptide, trimerization Folding structure domain, His₆Label；The function section is (a1) or (a2) or (a3)；

(a10) fusion protein that connection label obtains in the end of (a1) or (a2) or (a3) or (a5) or (a6)；

(a11) fusion protein obtained in the N-terminal connection signal peptide of (a1) or (a2) or (a3) or (a5) or (a6)；

(a12) in melting of obtaining of the N-terminal connection signal Peptide C end of (a1) or (a2) or (a3) or (a5) or (a6) connection label Hop protein；

(a13) any one protein by (a1) into (a12) passes through the substitution of one or several amino acid residues and/or lacks Lose and/or addition and with protein with the same function as derived from it.

2. encoding the gene of protein described in claim 1.

3. gene as claimed in claim 2, it is characterised in that: the gene is following (b1) to (b10) any described DNA Molecule:

(b1) code area DNA molecular as shown in sequence 2 in sequence table；

(b2) code area DNA molecular as shown in sequence 4 in sequence table；

(b3) code area DNA molecular as shown in sequence 6 in sequence table；

(b4) DNA molecular that code area is successively made of following element from upstream to downstream: gene, the trimerization of encoding function section The coded sequence in folding structure domain；The sequence 2 of gene such as sequence table or the sequence 4 of sequence table of the encoding function section or Shown in the sequence 6 of sequence table；

(b5) DNA molecular that code area is successively made of following element from upstream to downstream: the gene of encoding function section, connection The coded sequence of the coded sequence of peptide, tripolymer folded domain；The sequence 2 of the gene such as sequence table of the encoding function section Sequence table sequence 4 or sequence table sequence 6 shown in；

(b6) DNA molecular that code area is successively made of following element from upstream to downstream: the coded sequence of gp67 signal peptide is compiled Code the gene of function section, the coded sequence of link peptide, tripolymer folded domain coded sequence；The encoding function section Gene as shown in the sequence 2 of sequence table or the sequence 4 of sequence table or the sequence 6 of sequence table；

(b7) DNA molecular that code area is successively made of following element from upstream to downstream: the gene of encoding function section, connection The coded sequence of peptide, the coded sequence of tripolymer folded domain, His₆The coded sequence of label；The encoding function section Gene is as shown in the sequence 2 of sequence table or the sequence 4 of sequence table or the sequence 6 of sequence table；

(b8) DNA molecular that code area is successively made of following element from upstream to downstream: the coded sequence of gp67 signal peptide is compiled Code the gene of function section, the coded sequence of link peptide, tripolymer folded domain coded sequence, His₆The code sequence of label Column；The gene of the encoding function section is as shown in the sequence 2 of sequence table or the sequence 4 of sequence table or the sequence 6 of sequence table；

(b9) hybridize under strict conditions with (b1) to (b8) any described DNA molecular and the DNA molecular of code for said proteins；

(b10) there are the DNA of 95% or more homology and code for said proteins points with (b1) to (b8) any described DNA molecular Son.

4. recombinant expression carrier, expression cassette, transgenic cell line or recombinant bacterium containing gene described in Claims 2 or 3.

5. the application of protein described in claim 1 is at least one of following (c1) to (c8):

(c1) AIDS virus resisting vaccine is prepared；

(c2) drug of preparation treatment AIDS；

(c3) preparation inhibits the drug of AIDS virus；

(c4) preparation inhibition AIDS virus enters the drug of cell；

(c5) AIDS virus resisting；

(c6) AIDS is treated；

(c7) inhibit AIDS virus；

(c8) AIDS virus is inhibited to enter cell.

6. the tripolymer that protein described in claim 1 is formed.

7. a kind of composition is made of component first, component second and component third；

The component first is the tripolymer that albumen first is formed；The albumen first is (e1) or (e2) or (e3) or (e4) or (e5)；

The component second is the tripolymer that albumen second is formed；The albumen second is (f1) or (f2) or (f3) or (f4) or (f5)；

The component third is the tripolymer that albumen third is formed；The albumen third is (g1) or (g2) or (g3) or (g4) or (g5)；

(e1) protein being successively made of following element from N-terminal to C-terminal: function section, tripolymer folded domain；The function Energy section is the protein that the amino acid sequence shown in sequence 1 in sequence table forms；

(e2) protein being successively made of following element from N-terminal to C-terminal: function section, link peptide, trimerization folding structure Domain；The function section is the protein that the amino acid sequence shown in sequence 1 in sequence table forms；

(e3) protein being successively made of following element from N-terminal to C-terminal: gp67 signal peptide, function section, link peptide, trimerization Folding structure domain；The function section is the protein that the amino acid sequence shown in sequence 1 in sequence table forms；

(e4) protein being successively made of following element from N-terminal to C-terminal: function section, link peptide, trimerization folding structure Domain, His₆Label；The function section is the protein that the amino acid sequence shown in sequence 1 in sequence table forms；

(e5) protein being successively made of following element from N-terminal to C-terminal: gp67 signal peptide, function section, link peptide, trimerization Folding structure domain, His₆Label；The function section is the albumen that the amino acid sequence shown in sequence 1 in sequence table forms Matter；

(f1) protein being successively made of following element from N-terminal to C-terminal: function section, tripolymer folded domain；The function Energy section is the protein that the amino acid sequence shown in sequence 3 in sequence table forms；

(f2) protein being successively made of following element from N-terminal to C-terminal: function section, link peptide, trimerization folding structure Domain；The function section is the protein that the amino acid sequence shown in sequence 3 in sequence table forms；

(f3) protein being successively made of following element from N-terminal to C-terminal: gp67 signal peptide, function section, link peptide, trimerization Folding structure domain；The function section is the protein that the amino acid sequence shown in sequence 3 in sequence table forms；

(f4) protein being successively made of following element from N-terminal to C-terminal: function section, link peptide, trimerization folding structure Domain, His₆Label；The function section is the protein that the amino acid sequence shown in sequence 3 in sequence table forms；

(f5) protein being successively made of following element from N-terminal to C-terminal: gp67 signal peptide, function section, link peptide, trimerization Folding structure domain, His₆Label；The function section is the albumen that the amino acid sequence shown in sequence 3 in sequence table forms Matter；

(g1) protein being successively made of following element from N-terminal to C-terminal: function section, tripolymer folded domain；The function Energy section is the protein that the amino acid sequence shown in sequence 5 in sequence table forms；

(g2) protein being successively made of following element from N-terminal to C-terminal: function section, link peptide, trimerization folding structure Domain；The function section is the protein that the amino acid sequence shown in sequence 5 in sequence table forms；

(g3) protein being successively made of following element from N-terminal to C-terminal: gp67 signal peptide, function section, link peptide, trimerization Folding structure domain；The function section is the protein that the amino acid sequence shown in sequence 5 in sequence table forms；

(g4) protein being successively made of following element from N-terminal to C-terminal: function section, link peptide, trimerization folding structure Domain, His₆Label；The function section is the protein that the amino acid sequence shown in sequence 5 in sequence table forms；

(g5) protein being successively made of following element from N-terminal to C-terminal: gp67 signal peptide, function section, link peptide, trimerization Folding structure domain, His₆Label；The function section is the albumen that the amino acid sequence shown in sequence 5 in sequence table forms Matter.

8. the application of composition described in tripolymer or claim 7 described in claim 6, for following (c1) into (c8) at least It is a kind of:

(c1) AIDS virus resisting vaccine is prepared；

(c2) drug of preparation treatment AIDS；

(c3) preparation inhibits the drug of AIDS virus；

(c4) preparation inhibition AIDS virus enters the drug of cell；

(c5) AIDS virus resisting；

(c6) AIDS is treated；

(c7) inhibit AIDS virus；

(c8) AIDS virus is inhibited to enter cell.

9. gene described in Claims 2 or 3 is preparing the application in vaccine；The function of the vaccine is following (d1), (d2), (d3) or (d4):

(d1) AIDS virus resisting；

(d2) AIDS is treated；

(d3) inhibit AIDS virus；

(d4) AIDS virus is inhibited to enter cell.

10. a kind of vaccine, active constituent is that tripolymer described in protein described in claim 1 or claim 6 or right are wanted Seek 7 compositions；The function of the vaccine is following (d1), (d2), (d3) or (d4):

(d1) AIDS virus resisting；

(d2) AIDS is treated；

(d3) inhibit AIDS virus；

(d4) AIDS virus is inhibited to enter cell.