CN103073625A - Hydrophobic-region-deletion HIV (Human Immunodeficiency Virus) type I Tat protein mutant sequence and applications thereof - Google Patents

Abstract

The invention relates to the technical field of biomedical engineering, in particular to a hydrophobic-region-deletion HIV (Human Immunodeficiency Virus) type I Tat protein mutant sequence and applications thereof. An HIV type I Tat protein mutant is a hydrophobic-region-deletion Tat delta (31-45) mutant, and the amino acid sequence of the mutant is as shown in SEQ ID NO:1. The invention also provides the applications of the Tat protein mutant in preparation of HIV immunogens, preventative and therapeutic HIV vaccines and HIV detection kits. The Tat delta (31-45) mutant protein and gold-labeling particles thereof are conducive to the stabilization of protein conformation, the enhancement of immunogenicity and the show of protein N-terminal and the 46th-61th amino acid epitopes and are expected to be as candidate antigen of HIV vaccine development and HIV infection detection diagnosis.

Description

A kind of human immunodeficiency virus I type Tat protein mutant sequence and application thereof that lacks hydrophobic region

Technical field

The present invention relates to bio-pharmaceutical engineer technology domain, be specifically related to a kind of human immunodeficiency virus I type (HIV-1) Tat protein mutant sequence that lacks hydrophobic region, and the application in preparation HIV immunogen, in preparation prevention and therapeutic HIV vaccine, in preparation HIV detection kit.

Background technology

Human immunodeficiency virus (Human immunodeficiency virus, HIV) can cause acquired immune deficiency syndrome (AIDS) (AIDS), i.e. acquired immune deficiency syndrome (AIDS) mainly through propagation such as blood, property contact and the vertical approach of mother and baby.According to UNAIDS's statistics in 2012, global HIV the infected approximately 3,400 ten thousand, the existing HIV the infected of China and AIDS patients approximately 380,000, and acquired immune deficiency syndrome (AIDS) has become the great public health problem that China and the whole world face.Adopt at present highly active antiretroviral therapy (HAART) though can delay the development of the acquired immune deficiency syndrome (AIDS) state of an illness, can not remove virus fully; Behavior intervention also only slows down HIV transmission of infection speed and can't stop that it is popular.Therefore, in the urgent need to developing new effective medicine with propagation and the infection of control HIV.Infecting human HIV has HIV-1 and HIV-2 amphitypy, and both nucleotide sequences differ and can surpass 40%.That Major Epidemic is HIV-1 all over the world, HIV-2 then Major Epidemic in African west area.

It is that HIV-1 infects the early stage important regulation protein that produces that HIV-1 transcribes trans-activating factor (trans-activator of transcription, Tat).According to the difference of HIV-1 strain, Tat albumen is comprised of 86-101 amino-acid residue, and by two exons codings independently: the 1st exons coding 1-72 amino acids (aa) has completely transcriptional activation activity; Exon 2 coding Tat C-terminal district (73-101aa).Test confirm Tat copy, spread at HIV-1 and cause a disease in play an important role.Studies show that, in cells infected, but the initial sum that Tat trans-activation HIV genome is transcribed prolongation, thus start and promote copying of virus; Also has the outer activity of various born of the same parents and secrete and be released into extracellular Tat, as participate in the pathogenic courses such as immunosuppression, nervous system injury and the formation of Kaposi sarcoma, be called as " morbid poison " (referring to document: Silvers JM, Aksenova MV, Aksenov MY, Mactutus CF, Booze RM.Neurotoxicityof HIV-1Tat protein:involvement of D1dopamine receptor.Neurotoxicology.2007; 28 (6): 1184-1190.).The above-mentioned biological characteristics of Tat and the effect of causing a disease have made one of its candidate antigens that becomes the preventative and therapeutic vaccine of HIV.

The HIV-1Tat molecule is natively unfolded protein, belongs to the molecule of random coil type.Although the Tat sequence has high conservative between the HIV-1 different subtype, its shortage is stablized secondary structure and higher structure, makes its molecular conformation extremely unstable, is among the fast dynamic variation.Natural Tat remains the deficiencies such as low at the induce protective immunity antibody titers, that cross reactivity is poor as the candidate antigens of HIV vaccine.

Therefore by molecular biology method, the natural Tat molecular structure of HIV-1 being transformed, is to obtain the immunogenic effective way of the metastable novel Tat of molecular conformation, has potential HIV vaccine development and is worth.The applicant has applied for Chinese invention patent on March 25th, 2011, and relate to three-type-person's para-immunity defective virus I type Tat protein mutant sequence: Tat mutant antigen 1 (Tat1-37) is comprised of 37 amino acid; Tat mutant antigen 2(Tat1-61) formed by 61 amino acid; Tat mutant antigen 3(Tat22-101) formed (referring to Chinese patent application CN201110074185.7 by 80 amino acid, denomination of invention is " human immunodeficiency virus I type Tat protein mutant sequence and application thereof ", application publication number is CN102206255A, and Shen Qing Publication day is on October 5th, 2011).

Summary of the invention

The object of the invention is to further in depth the natural Tat molecular structure of HIV-1 be transformed, seek the stable new Tat protein mutant sequence of molecular conformation, and the application of this Tat protein mutant sequence in preparation HIV immunogen, in preparation prevention and therapeutic HIV vaccine, in preparation HIV detection kit is provided.

The applicant is by carrying out the hydrophobicity analysis discovery to Tat albumen, Tat 31-45 amino acids has stronger hydrophobicity, and rest part all has stronger wetting ability (Fig. 1).In view of the hydrophobicity of Tat31-45 peptide section has larger impact for the Tat protein expression, applicant's imagination is passed through molecular biology method, the natural Tat molecular structure of HIV-1 is transformed, clip hydrophobic section in the Tat albumen, be expected to improve Tat protein expression amount and obtain the metastable novel Tat immunogen of molecular conformation, have potential HIV vaccine development and HIV and infect the detection diagnostic value.

A first aspect of the present invention has provided a kind of human immunodeficiency virus I type Tat protein mutant sequence (following Tat Δ (31-45) or Tat Δ (31-45) mutant or Tat Δ (31-45) sequence of also claiming) that lacks hydrophobic region:

Tat Δ (31-45) mutant is comprised of 86 amino acid, and its aminoacid sequence is:

H ₂N—MEPVDPRLEPWKHPGSQPKTACTNCYCKKCSYGRKKRRQRRRAHQNSQTHQASLSKQPASQPRGDPTGPKESKKKVERETETDPVD—COOH（SEQ?ID?NO:1）；

The nucleotides sequence of coding Tat Δ (31-45) mutant is classified as:

5′-ATGGAACCGGTTGACCCGCGTCTGGAACCGTGGAAACACCCGGGTTCTCAGCCGAAAACCGCTTGCACCAACTGCTACTGCAAAAAATGCTCTTACGGTCGTAAAAAACGTCGTCAGCGTCGTCGTGCTCACCAGAACTCTCAGACCCACCAGGCTTCTCTGTCTAAACAGCCGGCTTCTCAGCCGCGTGGTGACCCGACCGGTCCGAAAGAATCTAAAAAAAAAGTTGAACGTGAAACCGAAACCGACCCGGTTGAC-3′（SEQ?ID?NO:2）。

A second aspect of the present invention has provided human immunodeficiency virus I type Tat protein mutant sequence and the application of encoding gene in preparation HIV immunogen thereof of above-mentioned disappearance hydrophobic region.

Further, the present invention also provides above-mentioned a kind of human immunodeficiency virus I type Tat protein mutant sequence and the application of encoding gene in preparation prevention and therapeutic HIV vaccine thereof that lacks hydrophobic region.

Further, the present invention also provides above-mentioned a kind of human immunodeficiency virus I type Tat protein mutant sequence and the application of encoding gene in preparation HIV detection kit thereof that lacks hydrophobic region.

A third aspect of the present invention is to prepare that a kind of prokaryotic expression amount that does not contain Tat hydrophobic region (Tat 31-45 amino acids) is high, conformation is relatively stable and can induce the stronger immunoreactive novel HIV-1Tat immunogen of generation.

The present invention obtains the natural Tat(of human immunodeficiency virus I type (HIV-1) HXB2 strain referring to document by the splicing PCR method: Opi S, P é loponese JM Jr, Esquieu D, Campbell G, de MareuilJ, Walburger A, Solomiac M, Gr é goire C, Bouveret E, Yirrell DL, Loret EP.TatHIV-1primary and tertiary structures critical to immune response againstnon-homologous variants.J Biol Chem.2002; 277 (39): 35915-35919.) Tat Δ (31-45) dna fragmentation of the high hydrophobic section 31-45 amino acids disappearance of molecule, make up its prokaryotic expression plasmid, through transforming intestinal bacteria (E.coli) BL21 (DE3), with sec.-propyl-β-D-sulfo-galactopyranoside (isopropyl β-D-1-thiogalactopyranoside, IPTG) abduction delivering, after SDS-PAGE gel electrophoresis analysis and Western blot evaluation correctly, a kind of conformation of formation of being combined with the colloid gold particle of straight average diameter 75 ± 15nm is relatively stable and can induce the stronger immunoreactive novel HIV-1Tat Δ of generation (31-45) immunogen mixture.

Tat Δ of the present invention (31-45) mutant is compared with the Tat22-101 albumen of natural total length HIV-1Tat antigen and Tat N-terminal disappearance, the prokaryotic expression amount of HIV-1Tat Δ (31-45) mutant protein significantly increases, conformation is more stable, and experimental result shows that anti-HIV-1 Tat Δ (31-45) mouse resisting anteserum and HIV-1 total length Tat albumen are specific reaction; And can induce the stronger immune response for HIV-1Tat N end of generation.In addition, compare with HIV-1Tat Δ (31-45) mutant protein, the golden standard type immunocomplex that HIV-1Tat Δ (31-45) association colloid gold forms has stronger immunogenicity, and the immune response of the golden standard type mouse resisting anteserum of anti-Tat Δ (31-45) and Tat38-61 obviously strengthens.

Concrete technical scheme of the present invention is as follows:

(1) with the synthetic tat1-101-HCVC122-191 dna fragmentation of the full gene of method of Overlap PCR, glue reclaims test kit and reclaims the PCR product, through Ase I and two kinds of digestion with restriction enzyme of BamH I and reclaim the purpose fragment, is connected connection product Transformed E .coli Top10 competent cell through Ase I with pPEPTIDE carrier (Fig. 2) after BamH I enzyme is connected again with equally.The extracting mono-clonal transforms the plasmid of bacterium colony and carries out Sequence Identification, the recombinant plasmid called after pPEP-Tat1-101-HCVC (122-191) that checks order correct.

(2) take pPEP-Tat1-101-HCVC (122-191) plasmid as template, with two dna fragmentation tat1-30 of PCR method amplification and tat46-101-HCVC (122-191), detect through 1.5% agarose gel electrophoresis, the dna fragmentation size conforms to theoretical value, is respectively 90bp, 381bp.Take above-mentioned two fragments as template, with Overlap pcr amplification tat △ (31-45)-HCVC (122-191).Detect through 1.5% agarose gel electrophoresis, the pcr amplified dna clip size conforms to theoretical value as a result, is 471bp.Take tat (△ 31-45)-HCVC (122-191) as template, through the pcr amplification goal gene, detect through 1.5% agarose gel electrophoresis, the pcr amplified dna clip size conforms to theoretical value as a result, is 258bp again.

(3) reclaim test kit with glue and reclaim the PCR product, respectively through Nco I and two kinds of digestion with restriction enzyme of BamH I and reclaim the purpose fragment, is connected connection product Transformed E .coli Top10 competent cell through the Nco I with expression vector pET32a after BamH I enzyme is connected again with equally.The extracting mono-clonal transforms the plasmid of bacterium colony and carries out enzyme and cut evaluation and Sequence Identification, the recombinant plasmid called after pET32a-Tat △ (31-45) that checks order correct.

(4) with above-mentioned recombinant plasmid transformed E.coli BL21 (DE3), the IPTG abduction delivering, detect and Western blot test through SDS-PAGE, the result shows that the molecular size range of the mutant fusion protein Tat △ (31-45) of expression-PET conforms to theoretical value, be 27.6 kilodaltons (KDa), accounting for the bacterial protein ratio through the expression amount of GlykoBandScan5.0 software analysis target protein is 55.0%.By affinity chromatography above-mentioned albumen is carried out purifying, show that through Glyko BandScan5.0 software analysis purity is 90.0%.

(5) with above-mentioned affinity chromatography purifying Tat △ (31-45)-PET protein labeling average diameter size is the colloid gold particle of 75nm, form Tat △ (31-45)-PET albumen gold standard type immunocomplex.Experimental results show that through NaCl coagulation under the condition of different pH Tat △ (31-45)-PET albumen gold standard type immunocomplex can keep stable under the physiological pH condition.

(6) with the Tat △ (31-45) of above preparation-PET albumen and Tat △ (31-45)-PET albumen gold standard type immunocomplex respectively through the subcutaneous injection C57BL mouse, the first immunisation Freund's complete adjuvant, second and third, full freund's adjuvant toos many or too much for use for four times, interval two weeks immunity 1 time, extract eyeballs after last two weeks of immunity and get blood, get supernatant in-40 ℃ of preservations after centrifugal.

(7) get above-mentioned two kinds of immune mouse antiserum(antisera)s (anti-Tat △ (31-45)-PET, anti-Tat △ (31-45)-PET gold standard type immunocomplex) and detect respectively reactivity with HIV-1 total length Tat albumen (using the pPEPTIDE2 vector expression), Tat N-terminal amalgamation and expression antigen Tat1-21-PEP, Tat38-61-PEP, Tat C-terminal amalgamation and expression antigen Tat60-101-PEP.Detect through enzyme linked immunosorbent assay (ELISA), these 2 kinds of mouse resisting anteserums all are specific reaction with HIV-1 total length Tat albumen, behind the mark Radioactive colloidal gold immunogenicity of Tat △ (31-45)-PET albumen are had enhancement.

Analyze and Western blot evaluation through SDS-PAGE, show that HIV-1Tat mutant protein Tat △ of the present invention (31-45)-PET has obtained than high expression level.Through the ELISA verification experimental verification, the mouse resisting anteserum and the HIV-1 total length Tat albumen that show the Tat △ (31-45) of anti-this invention-PET albumen and Tat △ (31-45)-PET gold standard type immunocomplex are specific reaction, wherein anti-Tat △ (31-45)-PET mice serum and HIV-1Tat N hold, the reactivity of Tat C end and Tat38-61 is than stronger with the reactivity of total length Tat albumen, prompting Tat △ (31-45) can be used as a kind of new Tat immunogen, can induce to produce holding for Tat N of higher titre arranged, the antibody of Tat C end and Tat38-61, be expected to reach the effect that blocking-up wild-type Tat protein biology is learned function, can infect the moiety of polypeptide drugs or the candidate antigens of conduct preparation HIV Tat vaccine in order to prepare anti-HIV.In addition, the reactivity of anti-Tat △ (31-45) albumen gold standard type immunocomplex antiserum(antisera) and HIV-1 total length Tat albumen is better than anti-Tat △ (31-45) serum, and hold with HIV-1Tat N, the reactivity of Tat C end and Tat38-61 is than stronger with the reactivity of total length Tat albumen, show that Tat △ (31-45) mutant protein and the golden standard type particle thereof that lack hydrophobic region all help the stable of this protein conformation, immunogenic enhancing and this albumen n end, the displaying of C end and 38-61 amino acids epi-position is expected to infect the candidate antigens that detects diagnosis as HIV vaccine development and HIV.

Description of drawings

Fig. 1 is with DNAssist Version2.2 software analysis Tat albumen hydrophobicity result

Wherein: ordinate zou represents hydrophilic and hydrophobic, and is hydrophobic on the occasion of representative, and the negative value representative is hydrophilic.31-45 amino acids (elliptic region) has stronger hydrophobicity, and all the other zones all have stronger wetting ability.

Fig. 2 is pPEPTIDE expression vector collection of illustrative plates

Wherein: its N end merges 163 amino acid of high efficient expression and 50 amino acid of His label, contains the insertion that Ase I and BamH I cloning site are used for the purpose fragment.This carrier is applicable to small molecular weight polypeptide or protein expression.

Fig. 3 is the agarose electrophoresis figure of pcr amplification Tat △ (31-45) dna fragmentation

Wherein: M, DL2000Marker(from top to bottom dna fragmentation size are followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp); 1, tat1-31; 2, tat (45-101)-HCVC (122-191); 3, tat △ (31-45)-HCVC (122-191); 4, Tat △ (31-45).

Fig. 4 is that the enzyme of recombinant expression plasmid pET32a-Tat △ (31-45) is cut qualification result

Wherein: M, DL5000 Marker(from top to bottom dna fragmentation size are followed successively by 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp); 1, empty carrier pET32a; 2, pET32a is through Nco I and BamH I double digestion after product; 3, enzyme is not cut tat △ (31-45); 4, tat △ (31-45) is through Nco I and BamH I double digestion after product; 5, enzyme is not cut pET32a-Tat △ (31-45); 6, pET32a-Tat △ (31-45) is through Nco I and BamH I double digestion after product.

Fig. 5 is SDS-PAGE analytical results behind Tat △ (31-45) mutant fusion protein prokaryotic expression product and the purifying thereof

Wherein: M, albumen Marker; 1, pET32a-Tat △ (31-45) is without IPTG abduction delivering product (cell pyrolysis liquid supernatant); 2, pET32a-Tat △ (31-45) is through IPTG abduction delivering product; 3, BL21 (DE3) empty bacterium lysate supernatant (negative control); 4, Tat △ (31-45) purpose fusion rotein (27.6kDa) behind the purifying.

Fig. 6 is Tat △ (31-45) mutant fusion protein Western blot qualification result

Wherein: M, albumen dye Marker in advance; 1, Tat △ (31-45)-PET fusion rotein behind the purifying; 2, total length Tat-PET fusion rotein (positive control); 3, pET32a carrier proteins (negative control).

Fig. 7 detects the colloid gold particle size of preparation and equal results once with transmission electron microscope

Wherein: the Radioactive colloidal gold median size is 75.0 ± 15nm, does not have agglomerating gathering situation, and is all once good.

Fig. 8 is the antibody titers measurement result of four kinds of mouse resisting anteserums (anti-Tat-PEP, anti-Tat △ (31-45)-PET, anti-Tat △ (31-45)-PET gold standard type mixture and normal mouse serum) and HIV-1 total length Tat fusion rotein (Tat1-101-PEP) reaction

Fig. 9 be four kinds of mouse resisting anteserums (anti-Tat-PEP, anti-Tat △ (31-45)-PET, anti-Tat △ (31-45)-PET gold standard type mixture and normal mouse serum) respectively with the reactive result of 3 kinds of HIV-1Tat mutant antigens (Tat1-21-PEP, Tat38-61-PEP and Tat60-101-PEP)

Wherein: A, anti-Tat mouse resisting anteserum (anti-Tat-PEP, anti-Tat △ (31-45)-PET, anti-Tat △ (31-45)-PET gold standard type mixture) is held respectively antigen Tat1-21-PEP with Tat N reactivity; B, anti-Tat mouse resisting anteserum (anti-Tat-PEP, anti-Tat △ (31-45)-PET, anti-Tat △ (31-45)-PET gold standard type mixture) respectively with the reactivity of Tat38-61-PEP; C, anti-Tat mouse resisting anteserum (anti-Tat-PEP, anti-Tat △ (31-45)-PET, anti-Tat △ (31-45)-PET gold standard type mixture) is held respectively antigen Tat60-101-PEP with TatC reactivity.

Embodiment

Now in conjunction with the embodiments and accompanying drawing, the invention will be further described, but enforcement of the present invention is not limited in this.

Embodiment 1: structure, prokaryotic expression by recombinant plasmid obtain a kind of Tat albumen that lacks hydrophobic region of energy

1.1, goal gene external synthetic

With the synthetic tat1-101-HCVC122-191 dna fragmentation of the full gene of method of Overlap PCR, glue reclaims test kit and reclaims the PCR product, through Ase I and two kinds of digestion with restriction enzyme of BamH I and reclaim the purpose fragment, again with equally through Ase I and pPEPTIDE(after BamH I enzyme is connected available from TAKARA company) carrier (Fig. 2) is connected connection product Transformed E .coli Top10 competent cell.The extracting mono-clonal transforms the plasmid of bacterium colony and carries out Sequence Identification, the recombinant plasmid called after pPEP-Tat1-101-HCVC (122-191) that checks order correct.

Dna fragmentation with the external composite coding Tat1-31 of PCR method and Tat46-101-HCVC (122-191) peptide section.The PCR reaction system comprises: template pPEP-Tat1-101-HCVC (122-191) plasmid 10ng; Respectively get 2 μ l(concentration as 0.01mM take primer Tat-U and Tat31-D, Tat45-U and T7-Terminator as the upstream and downstream primer respectively) (primer sequence sees Table 1); Taq enzyme 1.5U, dNTP100 μ mol, Mg ²⁺3mmol, reaction volume 50 μ l.The pcr amplification condition is: 94 ℃ of 5min; 94 ℃ of 30s, 57 ℃ of 30s, 72 ℃ of 40s, 30 circulations; 72 ℃ of 10min.Through 1.5% agarose gel electrophoresis the PCR product is detected, 2 of pcr amplification purpose fragments (tat1-31 and tat46-101-HCVC (122-191)) size conforms to theoretical value as a result, is respectively 90bp, 381bp(Fig. 3 the 1st, the 2nd swimming lane).

Above two PCR product stostes are template Overlap pcr amplification tat1-101 (△ 31-45)-HCVC (122-191) dna fragmentation.The PCR reaction system comprises: template is each 2.5ul of previous step PCR stoste; Primer Tat-U and T7-Terminator are that respectively to get 2 μ l(concentration be 0.01mM to the upstream and downstream primer) (primer sequence sees Table 1); Taq enzyme 1.5U, dNTP100 μ mol, Mg ²⁺3mmol, reaction volume 50 μ l.The pcr amplification condition is: 94 ℃ of 5min; 94 ℃ of 30s, 57 ℃ of 30s, 72 ℃ of 40s, 30 circulations; 72 ℃ of 10min.Through 1.5% agarose gel electrophoresis Overlap PCR product is detected, the purpose fragment tat1-101 (△ 31-45) of Overlap pcr amplification-HCVC (122-191) size conforms to theoretical value as a result, is 471bp(Fig. 3 the 3rd swimming lane).

Again take tat1-101 (△ 31-45)-HCVC (122-191) as template, pcr amplification goal gene Tat1-101 (△ 31-45), template is upper Overlap PCR product 2.5ul; Respectively get 2 μ l(concentration as 0.01mM take primer Tat-U and Tat-D as the upstream and downstream primer respectively) (primer sequence sees Table 1); Taq enzyme 1.5U, dNTP100 μ mol, Mg ²⁺3mmol, reaction volume 50 μ l.The pcr amplification condition is: 94 ℃ of 5min; 94 ℃ of 30s, 57 ℃ of 30s, 72 ℃ of 40s, 30 circulations; 72 ℃ of 10min.Detect through 1.5% agarose gel electrophoresis, pcr amplification tat1-101 (△ 31-45) dna fragmentation size conforms to theoretical value as a result, is 258bp(Fig. 3 the 4th swimming lane).

Primer title and the sequence thereof of the external synthetic goal gene of table 1

1.2, the structure of recombinant expression vector

Reclaim test kit 3S DNA Gel Purification Kit3.1(available from Shanghai TIANGEN company with glue) reclaim dna fragmentation after, carry out double digestion contains sticky end with acquisition goal gene with Nco I and two kinds of restriction enzymes of BamH I.The endonuclease reaction system comprises: dna fragmentation 25 μ l(6 μ g), each 2.5 μ l(25U of Nco I and BamH I), 10 * K Buffer5 μ l, 1mg/ml BSA5 μ l, aseptic double-distilled water 10 μ l, reaction cumulative volume 50 μ l.Goal gene after enzyme cut be connected expression vector pET-32a(available from TAKARA company with the Nco I with BamH I double digestion equally) be connected.Condition of contact: goal gene is connected with mol ratio 1:1 ratio with the pET-32a carrier, presses 1/10 of cumulative volume 20 μ l and adds efficient connecting fluid 2 μ l in 16 ℃ of connection 16h.Get and connect product 10 μ l transformed competence colibacillus cell E.coli Top10(available from the biological company limited of outstanding Lee in Shanghai).Extracting mono-clonal transforms the bacterium colony plasmid and carries out the enzyme evaluation of cutting and check order.Enzyme is cut the reaction system of evaluation: recombinant expression plasmid 15 μ l, and each 1.5 μ l of Nco I and BamH I, 10 * K buffer3 μ l, aseptic double-distilled water 9 μ l, reaction cumulative volume 30 μ l cut 2h in 37 ℃ of enzymes, detect (Fig. 4) through 1.5% agarose gel electrophoresis.To cut the mono-clonal plasmid of the qualification result positive through enzyme, entrust the outstanding Lee's bio tech ltd in Shanghai to carry out sequencing.Sequencing result DNASTAR software analysis, the recombinant plasmid that checks order correct is called after pET32a-Tat (△ 31-45) respectively.

1.3, expression and the purifying of Tat (△ 31-45) fusion rotein

Conventional Calcium Chloride Method prepares e. coli bl21-TRXB (DE3) competence bacterium; Getting above-mentioned recombinant plasmid pET32a-Tat (△ 31-45) 200ng adds among the 200 μ l competence bacterium BL21-TRXB (DE3) (available from the biological company limited of outstanding Lee in Shanghai), ice-water bath 30min, 42 ℃, 90s, the LB that adds again 800 μ l behind the ice-water bath 1-2min cultivates based on 37 ℃, 150rpm and is coated with LB culture medium flat plate (containing 100 μ g/ml Amp and 35 μ g/ml Kana) after cultivating 1h, in 37 ℃ of overnight incubation.Picking mono-clonal conversion next day bacterium colony is seeded to 3ml LB liquid nutrient medium (containing 100 μ g/ml Amp and 35 μ g/ml Kana), after 37 ℃, 150rpm shaking culture spend the night, get the 500 μ l bacterium liquid that spends the night, add 500 μ l glycerine and prepare glycerine and preserve bacterial classification in-20 ℃ of preservations; By the inoculum size of 1:10 the bacterium liquid of incubated overnight is transferred in the fresh 3ml LB substratum in addition and (contains 100 μ g/ml Amp and 35 μ g/ml Kana), in 37 ℃, 220rpm shaking culture 3h to OD ₆₀₀It is 1.0 o'clock, add the IPTG3 μ l of 1M to final concentration 1mM, inducing culture 4h, in the centrifugal 3min of 10000rpm, abandon supernatant, the precipitation add 300 μ l0.01M phosphate buffered saline buffers (PBS) resuspended after, get the resuspended liquid of 20 μ l PBS and add 2 * SDS sampling liquid, 20 μ l mixings, make the SDS sample, after collecting simultaneously the bacterium liquid of inducing without IPTG and empty bacterium BL21-TRXB (DE3) nutrient solution and making by the same method the SDS sample, carry out the SDS-PAGE electrophoresis detection: preparation 12%SDS polyacrylamide gel, with above-mentioned SDS sample and protein standard substance boiling water bath 5 minutes, application of sample, initial press 5V/cm, after entering separation gel, tetrabromophenol sulfonphthalein is increased to 12V/cm, until tetrabromophenol sulfonphthalein reaches the separation gel bottom.Electrophoresis finishes, and takes out gel and spends the night with coomassie brilliant blue staining, places methyl alcohol-glacial acetic acid solution to decolour 3-4 hour again.The result as seen contain recombinant plasmid pET32a-Tat (△ 31-45) be cloned in relative molecular mass approximately 27.6 kilodaltons (KDa) locate to express purpose fusion rotein band, bacterium liquid and empty bacterium BL21-TRXB (DE3) nutrient solution of inducing without IPTG have no this band.Through Glyko BandScan5.0 software analysis, the protein expression amount accounts for the bacterial protein ratio and is respectively 55%.Subsequently positive bacteria clone is carried out a large amount of abduction deliverings and purifying: will contain pET32a-Tat (△ 31-45)/BL21 glycerine bacterial classification and be seeded in the 50ml LB substratum according to the 1:10 inoculum size and (contain 100 μ g/ml Amp and 35 μ g/ml Kana), after 37 ℃, 170rpm shaking culture spend the night, be forwarded among the fresh 450ml LB and (contain 100 μ g/ml Amp and 35 μ g/ml Kana), in 37 ℃, 220rpm shaking culture 3.5h to OD ₆₀₀Be 1.0, add afterwards 1M IPTG500 μ l to final concentration be 1mM, behind 37 ℃, 220rpm shaking culture 4h, collect again bacterium liquid, in 4 ℃, the centrifugal 10min of 5000rpm, abandon supernatant, precipitation adds 30ml8M urea (pH8.0) cracking, spends the night in 4 ℃.Next day, split product is got supernatant to another centrifuge tube in 4 ℃, the centrifugal 30min of 10000rpm, after getting wherein 20 μ l supernatant liquors and adding 2 * SDS sampling liquid, 20 μ l mixings and make the SDS sample, analyzes through the 12%SDS-PAGE electrophoresis detection; Get supernatant and cross metal chelate affinity chromatography medium post (Ni-NTA, available from German QIAGEN company), the Ni-NTA post is with 8M urea (pH8.0) balance, urea lysate supernatant upper prop, 8M urea (pH7.0) is washed post, with 8M urea (pH4.5) wash-out, collect elution peak, neutralize with 0.1M Tris, dialyse with PBS, analyze purification effect through 12%SDS-PAGE, the result is presented at the approximately visible clear purpose fusion rotein band in 27.6KDa place of relative molecular weight, and purity is 90.0%(Fig. 5 after the GlykoBandScan5.0 software analysis shows recombinant protein purification).

1.4, the Western blot of Tat (△ 31-45) fusion rotein detects

Take out the SDS-PAGE running gel and do suitably to prune, be soaked in and prepare transferring film in the transfering buffering liquid; Cut 12 and gel filter paper of the same size and 1 nitrocellulose filter, the operation of band gloves, use the immersion of transferring film damping fluid after 15 minutes filter paper and nitrocellulose filter, 6 filter paper are neatly stacked, gel (near negative pole) placed on it, nitrocellulose filter (near anodal) is placed on the gel, stack again 6 filter paper, under the 300mA condition, carry out albumen current stabilization transferring film 50min; Adding an amount of confining liquid (containing 0.01mol/L PBS, 10% skim-milk, 0.1%Tween-20,0.2% Sodium Mercurothiolate) after transferring film finishes spends the night in 4 ℃ of sealings; With PBST washing lotion washing 5 times, add the anti-Tat monoclonal antibody of mouse (available from abcam company) (1:1000 dilution), hatch 2h for 37 ℃, rinsing liquid is washed 5 times, add the sheep anti-mouse igg-HRP(of horseradish peroxidase (HRP) mark available from abcam company) (1:1000 dilution), hatch 45min for 37 ℃, rinsing liquid washing 5 times, add substrate diaminobenzidine (Diaminobenzidine, DAB) colour developing is 5-10 minute, the color development stopping reaction appears using immediately distilled water flushing behind the brown band.The visible Tat of result (△ 31-45)-PET fusion rotein and total length Tat fusion rotein all are positive with the anti-Tat monoclonal antibody of mouse, and the pET32a carrier proteins is then without this reaction (Fig. 6).

Embodiment 2: obtain Tat albumen gold standard type immunocomplex by preparation and mark, and it is carried out quality evalution.

2.1, the preparation of Radioactive colloidal gold

0.01% chlorauric acid solution 100ml is heated to boiling, and 200rpm stirs the lower 1% trisodium citrate 0.7ml that accurately adds, and golden yellow chlorauric acid solution becomes red-purple.Continue heated and stirred 15min, constant volume is to 100ml after the cooling.

2.2, Electron microscopy colloid gold particle size and all once

Transmission electron microscope detects colloid gold particle size and all once, finds that the Radioactive colloidal gold average particulate diameter is 75nm, all once better (Fig. 7).

2.3, Tat △ (31-45)-PET protein labeling colloidal gold solution

Get behind the purifying Tat (△ 31-45)-PET albumen to 0.005Mol/L pH 9.0NaCl solution 4 ℃ of dialysis measure protein concentration after 9 hours.Regulate colloidal gold solution pH value to 9.6 with 0.1Mol/L K2CO3 or 0.1Mol/L HCl, stir to add Tat (△ 31-45)-PET albumen after the dialysis until solution no longer by the 10%NaCl coagulation, the centrifugal 10min of 1200rpm, cleer and peaceful precipitation in the separation, behind the protein concentration, add the resuspended precipitation of an amount of supernatant and be made into the Tat that protein adsorption quantity is 0.1mg/ml (△ 31-45)-PET albumen gold standard type solution for standby in the mensuration supernatant.

2.4, NaCl coagulation experimental identification Tat △ (31-45) albumen gold standard type stability

Get above golden standard type solution 2 pipes, every pipe 50ul, each leaves standstill 2h after adding 5ul10%NaCl solution, observes the coagulation situation.Found that not the colloidal gold solution in conjunction with Tat △ (31-45)-PET albumen after adding 10%NaCl solution coagulation occurs, solution colour becomes pewter by burgundy, and the colloidal gold solution that combines capacity Tat △ (31-45)-PET albumen keeps stable after adding 10%NaCl solution, solution colour does not change, and still keeps burgundy.

2.5, the NaCl coagulation identifies Tat △ (31-45) albumen gold standard type stability under the condition of different pH

Get above golden standard type solution 7 pipes, every pipe 50ul, each adds behind the 5ul10%NaCl solution 0.1Mol/LK2CO3 or 0.1Mol/L HCl, and to regulate its pH be 2.0,3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0,12.0,13.0.Leave standstill 2h, observe the coagulation situation.Found that Tat (△ 31-45)-PET albumen gold standard type all can keep stable, coagulation does not occur in the pH5.0-11.0 scope.When the pH value less than 5.0 or greater than 11.0 the time, add 10%NaCl solution after, solution colour becomes pewter by burgundy, shows Tat △ (31-45)-PET albumen gold standard type mixture generation coagulation.

Embodiment 3: detect the immunogenic change of Tat mutant and colloid gold label to the impact of its epitope display by animal immune experiment

3.1, animal immune experiment

Initial immunity gets that total length Tat-PET, Tat (△ 31-45)-PET fusion rotein and Tat (△ 31-45)-each 100 μ l(protein content of PET albumen gold standard type are 0.01mg behind the purifying) respectively with after the Freund's complete adjuvant equal-volume mixes, in sole and abdominal part hypodermic immune health female six all C57BL mouses in age (The 2nd Army Medical College Experimental Animal Center), every group of 5 experiment mices; Carry out one time afterwards booster immunization per 2 weeks, the immunity Freund's incomplete adjuvant, booster immunization is 3 times altogether, and method and dosage are all identical with initial immunity.Set up simultaneously Radioactive colloidal gold and physiological saline control group.Extract eyeballs after last two weeks of immunity and get blood, approximately 1.2ml/ only, the centrifugal 10min of 10000rpm gets upper serum frozen for subsequent use in-40 ℃.

3.2, the ELISA of immune serum detects

HIV-1 total length Tat fusion rotein (Tat1-101-PEP) and 3 kinds of Tat mutant fusion protein (Tat1-21-PEP with purifying, Tat38-61-PEP and 60-101-PET) use respectively 50mM carbonate buffer solution (pH9.6) to press the coated 96 hole enzyme-linked reaction plates of 1:200 dilution, every kind of multiple hole of antigen coated 5 rows, 0.5 μ g/ hole, hatch 2h for 37 ℃, abandon coating buffer, add 180 μ l10% skim-milks in 37 ℃ of sealing 2h, after PBST washing lotion washing 4 times, the anti-Tat1-101-PET serum of mouse that adds respectively the 1:2 doubling dilution, anti-Tat (△ 31-45)-PET serum, anti-Tat (△ 31-45)-PET albumen gold standard type serum, each 100 μ l of colloidal gold solution group mice serum and physiological saline group mice serum, last 1 hole does not add, hatch 45min for 37 ℃, with PBST washing 5 times, the goat anti-rabbit igg (1:1000 dilution) that adds the HRP mark, 100 μ l/ holes, hatch 45min for 37 ℃, PBST washing 5 times, through 3,3 ', 5,5 '-tetramethyl benzidine (3,3 ', 5,5 '-tetramethylbenzidine, TMB) colour developing, add 2mol/L sulfuric acid termination reaction, measure absorbance (A in 450nm wavelength place with microplate reader (Bio Rad) ₄₅₀).With physiological saline group mice serum as negative control, to be higher than negative control A ₄₅₀Be worth 3 times numerical value as positive judging criterion.

Anti-Tat (△ 31-45) albumen mouse resisting anteserum and anti-Tat (△ 31-45) albumen gold standard type mouse resisting anteserum all are specific reaction with HIV-1 total length Tat albumen, can obviously strengthen the immunogenicity (Fig. 8) of Tat (△ 31-45) albumen behind the mark Radioactive colloidal gold.

Compare with anti-total length Tat mouse resisting anteserum, Tat (△ 31-45) and golden standard type immune mouse antiserum(antisera) thereof all hold with Tat N and the reactivity of Tat38-61 antigen strengthens (Fig. 9 A, 9B); The reactivity stronger (Fig. 9 B) of immune mouse antiserum(antisera) and Tat38-61 antigen behind Tat (△ 31-45) the mark Radioactive colloidal gold; Anti-total length Tat, anti-Tat (△ 31-45) and the golden standard type three's mouse resisting anteserum of anti-Tat (△ 31-45) and Tat60-101 reactivity are all apparently higher than control group, but no significant difference (Fig. 9 C) between the three.

Above demonstration and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that describes in above-described embodiment and the specification sheets just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.

Claims (10)

1. human immunodeficiency virus I type Tat protein mutant, its aminoacid sequence is shown in SEQ ID NO:1.

2. the application of human immunodeficiency virus I type Tat protein mutant according to claim 1 in preparation HIV immunogen.

3. the application of human immunodeficiency virus I type Tat protein mutant according to claim 1 in preparation prevention and therapeutic HIV vaccine.

4. the application of human immunodeficiency virus I type Tat protein mutant according to claim 1 in preparation HIV detection kit.

5. encode the nucleotide sequence of human immunodeficiency virus I type Tat protein mutant as claimed in claim 1 shown in SEQ ID NO:2.

6. the nucleotides sequence of coding human immunodeficiency virus I type Tat protein mutant according to claim 5 is listed in the application in the preparation HIV immunogen.

7. the nucleotides sequence of coding human immunodeficiency virus I type Tat protein mutant according to claim 6 is listed in the application in the preparation HIV immunogen, it is characterized in that, it is the nucleotide sequence of human immunodeficiency virus I type Tat protein mutant of to encode, make up its recombined pronucleus expression plasmid, through transforming intestinal bacteria, behind sec.-propyl-β-D-sulfo-galactopyranoside abduction delivering, be combined with the colloid gold particle of diameter 75 ± 15nm and form a kind of immunogen mixture.

8. the nucleotides sequence of coding human immunodeficiency virus I type Tat protein mutant according to claim 7 is listed in the application in the preparation HIV immunogen, it is characterized in that, the natural Tat molecular structure of HIV-1 is transformed, clip in the Tat albumen hydrophobic section 31-45 amino acid rear clone to prokaryotic expression plasmid pET-32a, transform e. coli bl21, with sec.-propyl-β-D-sulfo-galactopyranoside abduction delivering, behind ni-sepharose purification, be combined with the colloid gold particle of diameter 75 ± 15nm and form immunocomplex.

9. the nucleotides sequence of coding human immunodeficiency virus I type Tat protein mutant according to claim 5 is listed in the application in preparation prevention and the therapeutic HIV vaccine.

10. the nucleotides sequence of coding human immunodeficiency virus I type Tat protein mutant according to claim 5 is listed in the application in the preparation HIV detection kit.

Images