CN102206255B - Sequences of mutants of Tat protein of human immunodeficiency virus type I and use thereof - Google Patents
Sequences of mutants of Tat protein of human immunodeficiency virus type I and use thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biomedical engineering. Human immunodeficiency virus (HIV)-1 trans-activator of transcription (Tat) is an important regulatory protein in the early stage of HIV-1 infection. The invention aims to screen sequences of HIV-1 Tat protein mutants which are relatively stable in molecular conformation and can arouse strong immunoreactions. The amino acid sequences of the HIV-1 Tat protein mutants provided by the invention are represented by SEQ ID No.2, SEQ ID No.4 and SEQ ID No.6. The rabbit antiserums of the three Tat mutant proteins all can undergo specific reactions of the full-length Tat protein of HIV-1, the reactivity is high, and thus, the three Tat mutant proteins can be used for preparing components of anti-HIV infection polypeptide medicines or used as candidate antigens for preparing HIV Tat vaccines.
Description
Technical field
The present invention relates to bio-pharmaceutical engineer technology domain, be specifically related to human immunodeficiency virus I type Tat protein mutant sequence, and the application in preparation HIV immunogen, the application in preparation prevention and therapeutic HIV vaccine, the application in preparation HIV detection kit.
Background technology
Human immunodeficiency virus (Human immunodeficiency virus, HIV), mainly through propagation such as blood, property contact and the vertical approach of mother and baby, can cause acquired immune deficiency syndrome (AIDS) (AIDS), i.e. acquired immune deficiency syndrome (AIDS).According to new statistics in the end of the year 2009 of UNAIDS, current global HIV the infected approximately 6,000 ten thousand, 2,500 ten thousand people dies from AIDS-related diseases.The existing HIV the infected of China and AIDS patients approximately more than 70 ten thousand, be wherein transmission through sex more than 70%, 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 to control propagation and the infection of HIV.The HIV that infects the mankind 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 Major Epidemic in African west area.
It is that HIV-1 infects the early stage important regulation protein produced 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 transcriptional activation activity completely; 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.Research shows, in cells infected, but the initial sum that Tat trans-activation HIV genome is transcribed extend, thereby start and promote copying of virus; Also there is 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.Neurotoxicity of HIV-1Tatprotein:involvement of D1 dopamine 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, among changing dynamically fast.Sequence and the constitutional features of above-mentioned HIV-1 Tat molecule itself, make natural Tat remain 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.
Summary of the invention
The object of the invention is to prepare conformation relatively stable and can induce the stronger immunoreactive novel HIV Tat immunogen of generation.
The present invention by the natural Tat of human immunodeficiency virus I type (HIV-1) HXB2 strain (referring to document: Opi S, P é loponese JM Jr, Esquieu D, Campbell G, de Mareuil J, Walburger A, Solomiac M, Gr é goire C, Bouveret E, Yirrell DL, Loret EP.Tat HIV-1 primary andtertiary structures critical to immune response against non-homologous variants.JBiol Chem.2002, 277 (39): 35915-35919.) the different sections of molecule carry out Reasonable, get Tat1-37, Tat1-61 and Tat22-101 section build respectively 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, and identify through SDS-PAGE gel electrophoresis analysis and Western b1ot, three kinds of conformations of screening acquisition are relatively stable and can induce the stronger immunoreactive novel HIV-1Tat immunogen of generation, the HIV-1Tat immunogen that the present invention prepares has no report at home and abroad.
The invention provides following three-type-person's para-immunity defective virus I type Tat protein mutant sequence:
One, Tat mutant antigen 1 (Tat1-37): 37 amino acid, consist of, its aminoacid sequence is H
2n-MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVC-COOH (as shown in SEQ ID NO:2);
The nucleotides sequence of coding Tat1-37 mutant is classified as:
5 '-ATGGAACCGGTTGACCCGCGTCTGGAACCGTGGAAACACCCGGGTTCTCAGCCGAA AACCGCTTGCACCAACTGCTACTGCAAAAAATGCTGCTTCCACTGCCAGGTTTGC-3 ' (as shown in SEQ ID NO:1).
Two, Tat mutant antigen 2 (Tat1-61): 61 amino acid, consist of, its aminoacid sequence is H
2n-MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFITKALGISYGRKKRRQ RRRAHQN-COOH (as shown in SEQ ID NO:4);
The nucleotides sequence of coding Tat1-61 mutant is classified as:
5 '-ATGGAACCGGTTGACCCGCGTCTGGAACCGTGGAAACACCCGGGTTCTCAGCCGAA AACCGCTTGCACCAACTGCTACTGCAAAAAATGCTGCTTCCACTGCCAGGTTTGCT TCATCACCAAAGCTCTGGGTATCTCTTACGGTCGTAAAAAACGTCGTCAGCGTCGT CGTGCT CACCAGAAC-3 ' (as shown in SEQ ID NO:3).
Three, Tat mutant antigen 3 (Tat22-101): 80 amino acid, consist of, its aminoacid sequence is H2N-CTNCYCKKCCFHCQVCFITKALGISYGRKKRRQRRRAHQNSQTHQASLSKQP ASQPRGDPTGPKESKKKVERETETDPVD-COOH (as shown in SEQ ID NO:6);
The nucleotides sequence of coding Tat22-101 mutant is classified as:
5 '-CCGAAAGAATCTAAAAAAAAAGTTGAACGTGAAACCGAAACCGACCCGGTTGACTC TGGGTATCTCTTACGGTCGTAAAAAACGTCGTCAGCGTCGTCGTGCTCACCAGAAC TCTCAGACCCACCAGGCTTCTCTGTCTAAACAGCCGGCTTCTCAGCCGCGTGGTGA CCCGACCGGTTGCACCAACTGCTACTGCAAAAAATGCTGCTTCCACTGCCAGGTTT GCTTCATCACCAAAGC-3 ' (as shown in SEQID NO:5).
The present invention also provides above-mentioned human immunodeficiency virus I type Tat protein mutant sequence and the application of encoding gene in preparation HIV immunogen thereof.
The present invention also provides above-mentioned human immunodeficiency virus I type Tat protein mutant sequence and the application of encoding gene in preparation prevention and therapeutic HIV vaccine thereof.
The present invention also provides above-mentioned human immunodeficiency virus I type Tat protein mutant sequence and the application of encoding gene in preparation HIV detection kit thereof.
With natural HIV-1Tat antigen, compare, the expression amount of above three kinds of HIV-1Tat fused antigens is high, conformation is relatively stable, and experimental result shows that above three kinds of anti-Tat mutant protein rabbit anti-serums (anti-Tat1-37-PET, anti-Tat1-61-PET and anti-Tat22-101-PET) all are specific reaction with HIV-1 total length Tat albumen; Especially Tat22-101 can induce the stronger immune response for HIB-1Tat C end antigen of generation.
Immunogenic preparation method is as follows for HIB-1Tat mutant of the present invention:
(1) with the pMD18-T-tat1-101 plasmid (referring to document: Chen Lu, Pan Wei, Cao Jie, Lu Haimei, He Jun, Jiang Shaohua, Tang Ping, Li Cunmei, Liao Wenting, Deng Songhua; Partitioned representation and the Analysis of Immunogenicity of HIV-1HXB2 strain Tat albumen in intestinal bacteria; Medical University Of Anhui's journal, 2008; 43 (3): 239-243) be template, DNA fragmentation (tat1-37, tat1-61 and tat22-101) with 3 goal gene of PCR method amplification, through 1.5% agarose gel electrophoresis, detect, the goal gene clip size of pcr amplification conforms to theoretical value as a result, is respectively 111bp, 183bp and 240bp.
(2) reclaim test kit with glue and reclaim the PCR product, these 3 kinds of purpose fragment tat1-37, tat1-61 are connected with cloning vector pMD18-T (T carrier) respectively with tat22-101, connect product and transform intestinal bacteria (E.coli) DH5 α, get mono-clonal and transform bacterium colony after PCR is accredited as the positive, then the evaluation of checking order.
(3) the positive colony plasmid that extracting is checked order correct, take its DNA fragmentation increased respectively containing 3 kinds of goal gene by PCR method as template, reclaim test kit with glue and reclaim the PCR product, respectively through BamH I and two kinds of digestion with restriction enzyme of Hind III and reclaim the purpose fragment, is connected respectively with equally again connection product Transformed E .coli DH5 α with the expression vector pET32a after Hind III enzyme is cut through BamH I.Go forward side by side performing PCR, enzyme of the plasmid that the extracting mono-clonal transforms bacterium colony cut and identified and sequencing, and the recombinant plasmid that checks order correct is called after pET32a-Tat1-37, pET32a-Tat1-61 and pET32a-Tat22-101 respectively.
(4) with above-mentioned 3 kinds of recombinant plasmids difference Transformed E .coli BL21 (DE3), the IPTG abduction delivering, through SDS-PAGE, detect and Western blot test, result shows that the molecular size range of 3 kinds of Tat mutant fusion proteins (Tat1-37-PET, Tat1-61-PET and Tat22-101-PET) of expression conforms to theoretical value, be respectively 22 kilodaltons (KDa), 24.9KDa and 26.9KDa, respectively above-mentioned 3 kinds of Tat albumen are carried out to purifying by affinity chromatography, show that through Glyko BandScan 5.0 software analysis purity is respectively 94.4%, 86.6%, 86.6%.
(5) get above-mentioned 3 kinds of Tat mutant proteins respectively through the subcutaneous injection new zealand white rabbit, 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 immunity in two weeks 1 time, for the last time within immune two weeks, by arteria carotis communis, measure greatly blood, get supernatant in-40 ℃ of preservations after centrifugal.
(6) get above-mentioned 3 kinds of immunize rabbit antiserum(antisera) (anti-Tat1-37-PET, anti-Tat1-61-PET and anti-Tat22-101-PET) detect respectively the HIV-1 total length Tat albumen (Tat1-101-PEP of itself and amalgamation and expression, *-PEP is the fusion rotein that another carrier pPEPTIDE2 expresses), the reactivity of Tat C-terminal amalgamation and expression antigen (Tat49-101-PEP and Tat60-101-PEP), through enzyme linked immunosorbent assay (ELISA), detect, these 3 kinds of rabbit anti-serums all are specific reaction with HIV-1 total length Tat albumen, wherein anti-Tat22-101-PET serum is stronger with the reactivity of total length Tat albumen with the reactivity ratio of above-mentioned two kinds of Tat C end antigen.
Analyze and Western blot evaluation through SDS-PAGE, show that three kinds of HIV-1Tat mutant proteins of the present invention (Tat1-37-PET, Tat1-61-PET and Tat22-101-PET) have all obtained good representation.Through the ELISA verification experimental verification, the rabbit anti-serum that shows three kinds of Tat mutant proteins of anti-this invention all is specific reaction with HIV-1 total length Tat albumen, wherein anti-Tat22-101-PET serum is stronger with the reactivity of total length Tat albumen with the reactivity ratio of two kinds of Tat C end antigen Tat49-101-PET and Tat60-101-PET, prompting Tat22-101 can be used as a kind of new Tat immunogen, can induce and produce the antibody for Tat C end that higher titre is arranged, 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.
The accompanying drawing explanation
Fig. 1 is the PCR product agarose electrophoresis figure of external synthetic three kinds of goal gene fragments
M, DL2000Marker (the DNA fragmentation size is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom); 1, tat1-37 (111bp); 2, tat1-61 (183bp); 2.tat22-101 (240bp).(annotate: the band above 1,2,3 swimming lanes is the purpose band, the dimer formed between primer when following band is pcr amplification).
Fig. 2 is the PCR qualification result of three kinds of recombinant expression plasmids
A, PCR identifies recombinant plasmid pET32a-Tat1-37; M, DL2000Marker; 1-7, the PCR product of positive monoclonal bacterium colony plasmid; 8, negative control (the empty bacterium contrast of DH5 α).B, PCR identifies recombinant plasmid pET32a-Tat1-61; M, DL2000Marker; 1-8, the PCR product of positive monoclonal bacterium colony plasmid.9. negative control (the empty bacterium contrast of DH5 α.C, PCR identifies recombinant plasmid pET32a-Tat22-101; M, DL2000Marker; 1-5, the PCR product of positive monoclonal bacterium colony plasmid; 6, negative control (the empty bacterium contrast of DH5 α).
Fig. 3 is that the enzyme of three kinds of recombinant expression plasmids is cut qualification result
A, enzyme is cut and is identified recombinant plasmid pET32a-Tat1-37; M, DL2000Marker; 1, Hind III and Bam H I enzyme are cut after product; 2, enzyme is not cut pET32a-Tat1-37; B, enzyme is cut and is identified recombinant plasmid pET32a-Tat1-61; M, DL2000Marker; 1, Hind III and Bam H I enzyme are cut after product; 2, enzyme is not cut pET32a-Tat1-37; C, enzyme is cut and is identified recombinant plasmid pET32a-Tat22-101; M, DL2000Marker; 1, Hind III and Bam H I enzyme are cut after product; 2, enzyme is not cut pET32a-Tat22-101.In figure, arrow means DNA fragmentation identical with expection goal gene size in the double digestion after product.
Fig. 4 is the SDS-PAGE analytical results that three kinds of Tat mutant fusion proteins are expressed
M, albumen Marker; 1, Tat1-37-PET fusion rotein is the cell pyrolysis liquid supernatant after the IPTG abduction delivering; The cell pyrolysis liquid supernatant that 2, Tat1-37-PET fusion rotein is induced without IPTG; 3, Tat1-61-PET fusion rotein is the cell pyrolysis liquid supernatant after IPTG induces; The cell pyrolysis liquid supernatant that 4, Tat1-61-PET fusion rotein is induced without IPTG; 5, Tat22-101-PET fusion rotein is the cell pyrolysis liquid supernatant after IPTG induces; The cell pyrolysis liquid supernatant that 6, Tat22-101-PET fusion rotein is induced without IPTG; 7, negative control (the empty bacterium lysate of BL21 (DE3) supernatant).
Fig. 5 is that after three kinds of Tat mutant fusion protein purifying, SDS-PAGE analyzes and Western blot qualification result
A, SDS-PAGE analytical results after three kinds of Tat mutant fusion protein purifying.M, albumen Marker;
1, Tat1-37-PET fusion rotein after purifying; 2, Tat1-61-PET fusion rotein after purifying; 3, Tat22-101-PET fusion rotein after purifying.B, Western blot qualification result after three kinds of Tat mutant fusion protein purifying.1, Tat1-101-PET fusion rotein (positive control); 2, Tat1-37-PET fusion rotein after purifying; 3, Tat1-61-PET fusion rotein after purifying; 4, Tat22-101-PET fusion rotein after purifying; 5, pET32a carrier proteins (negative control).
Fig. 6 is the antibody titers measurement result that three kinds of rabbit anti-serums react with HIV-1 total length Tat fusion rotein (Tat1-101-PEP).
Tetra-kinds of anti-Tat rabbit anti-serums of Fig. 7 are held respectively the reactivity of antigens with two kinds of HIV-1Tat C
A, anti-Tat rabbit anti-serum (anti-Tat1-37-PET, anti-Tat1-61-PET, anti-Tat22-101-PET and anti-Tat1-101-PET) is held respectively the reactivity of antigen Tat49-101-PEP with Tat C; B, anti-Tat rabbit anti-serum (anti-Tat1-37-PET, anti-Tat1-61-PET, anti-Tat22-101-PET and anti-Tat1-101-PET) is held respectively the reactivity of antigen Tat60-101-PEP with Tat C.
Embodiment
Below in conjunction with embodiments of the invention and accompanying drawing, enforcement of the present invention is elaborated; following examples are implemented take technical solution of the present invention under prerequisite; provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1: the Analysis of Immunogenicity of structure, prokaryotic expression and fusion rotein by recombinant plasmid obtains can be with HIV-1 total length Tat albumen the Tat protein mutant sequence of three kinds of brachymemmas of specific reaction
1, goal gene is external synthetic
DNA fragmentation by tri-peptide sections of the external composite coding HIV-1Tat of PCR method.The PCR reaction system comprises: template pMD18-T-tat1-101 plasmid 10ng; Take respectively primer 37-U and 37-D, 61-U and 61-D and 101-U and 101-D respectively gets 2 μ l (concentration is 0.01mM) (primer sequence is in Table 1) as the upstream and downstream primer; Taq enzyme 1.5U, dNTP 100 μ mol, Mg
2+3mmol, reaction volume 50 μ l.The pcr amplification condition is: 94 ℃ of 5min; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 40s, 30 circulations; 72 ℃ of 10min.Through 1.5% agarose gel electrophoresis, the PCR product is detected, 3 of pcr amplification purpose fragments (tat1-37, tat1-61 and tat22-101) size conforms to theoretical value as a result, is respectively 111bp, 183bp and 240bp (seeing Fig. 1).
Primer title and the sequence thereof of the external synthetic goal gene of table 1
2, goal gene is cloned into the pMD18-T carrier
After reclaiming respectively the PCR product of purifying tat1-37, tat1-61 and tat22-101 with glue recovery test kit 3S DNA Gel Purification Kit 3.1, these 3 kinds of target DNA fragments are connected to 16h with the pMD18-T carrier ratio of 1: 1 in molar ratio in 16 ℃ respectively, get intestinal bacteria (E.coli) the DH5 α competent cell that 200ng adds 200 μ l to prepare by conventional Calcium Chloride Method and transformed.Concrete conversion condition is: put ice-water bath 25min, 42 ℃ of 90s, ice-water bath 1-2min, add the LB of 800 μ l to cultivate based on after 37 ℃, 150rpm shaking culture 1h, be coated with LB dull and stereotyped (containing Amp 100 μ g/ml and Kana 35 μ g/ml), put 37 ℃ of incubator overnight incubation.To transforming, the mono-clonal formed carries out PCR and enzyme is cut evaluation.The PCR reaction system is: the bacterium liquid 2 μ l after the mono-clonal of take amplification are template, take 37-U and 37-D, 61-U and 61-D and 101-U and 101-D respectively as each 2 μ l (concentration is 0.01mM) (primer sequence is in Table 1) of upstream and downstream primer, Taq enzyme 1.5U, dNTP 100 μ mol, Mg
2+3mmol, reaction volume 50 μ l.The pcr amplification condition is: 94 ℃ of 5min; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 40s, 30 circulations; 72 ℃ of 10min.Enzyme is cut the reaction system of identifying: clone's plasmid vector 15 μ l, and each 1.5 μ l of Hind III and BamH I, 10 * Kbuffer, 3 μ l, aseptic double-distilled water 9 μ l, reaction cumulative volume 30 μ l, cut 2h in 37 ℃ of enzymes, through 1.5% agarose gel electrophoresis, detects.The mono-clonal plasmid of the qualification result positive is cut in extraction through PCR and enzyme, entrust BGI-Shenzhen to carry out sequencing, sequencing result DNASTAR software analysis, recombinant plasmid difference called after pMD18-T-Tat1-37, pMD18-T-Tat1-61 and pMD18-T-Tat22-101 that sequence is correct.
3, the structure of recombinant expression vector
Take pMD18-T-Tat1-37, pMD18-T-Tat1-61 and pMD18-T-Tat22-101 respectively as template, take T carrier universal primer RV-M and M13-47 as the upstream and downstream primer, amplification is containing the DNA fragmentation of 3 kinds of goal gene respectively, after reclaiming test kit 3S DNA Gel Purification Kit 3.1 recovery DNA fragmentations with glue, with BamH I and two kinds of restriction enzymes of Hind III, carry out double digestion to obtain the goal gene containing sticky end.The endonuclease reaction system comprises: DNA fragmentation 25 μ l (6 μ g), each 2.5 μ l (25U) of BamH I and Hind III, 10 * K Buffer, 5 μ l, 1mg/ml BSA 5 μ l, aseptic double-distilled water 10 μ l, reaction cumulative volume 50 μ l.Goal gene after enzyme is cut with BamH I, with the expression vector pET-32a of Hind III double digestion, be connected equally.Condition of contact: goal gene is connected with 1: 1 ratio of mol ratio with the pET-32a carrier, by 1/10 of cumulative volume 20 μ l, adds efficient connecting fluid 2 μ l to connect 16h in 16 ℃.Get and connect product 10 μ l transformed competence colibacillus cell E.coli DH5 α.Extract mono-clonal and transform that the bacterium colony plasmid carries out PCR and enzyme is cut evaluation, the PCR reaction system comprise and amplification condition the same.Enzyme is cut the reaction system of evaluation: recombinant expression plasmid 15 μ l, and each 1.5 μ l of Hind III and Bam HI, 10 * K buffer, 3 μ l, aseptic double-distilled water 9 μ l, reaction cumulative volume 30 μ l, cut 2h in 37 ℃ of enzymes, through 1.5% agarose gel electrophoresis, detects.To through PCR evaluation (seeing Fig. 2) and enzyme, cutting evaluation (seeing Fig. 3) the mono-clonal plasmid of the positive as a result, entrust BGI-Shenzhen to carry out its sequencing.Sequencing result DNASTAR software analysis, the recombinant plasmid that checks order correct is called after pET32a-Tat1-37, pET32a-Tat1-61 and pET32a-Tat22-101 respectively.
4, the expression of Tat mutant fusion protein and purifying
Conventional Calcium Chloride Method prepares e. coli bl21-TRXB (DE3) competence bacterium; Getting respectively above-mentioned 3 kinds of recombinant plasmid pET32a-Tat1-37, pET32a-Tat1-61 and each 200ng of pET32a-Tat22-101 adds in 200 μ l competence bacterium BL21-TRXB (DE3), ice-water bath 30min, 42 ℃, 90s, the LB that adds again 800 μ l after 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 700 μ l bacterium liquid that spends the night, add 300 μ l glycerine and prepare glycerine and preserve bacterial classification in-20 ℃ of preservations, the another inoculum size by 1: 10 is transferred to (containing 100 μ g/ml Amp and 35 μ g/ml Kana) in fresh 3ml LB substratum by the bacterium liquid of incubated overnight, in 37 ℃, 220rpm shaking culture 3h to OD
600it is 1.0 o'clock, add the IPTG 3 μ l of 1M to final concentration 1mM, inducing culture 4h, in the centrifugal 3min of 10000rpm, abandon supernatant, the precipitation add 300 μ l 0.01M phosphate buffered saline buffers (PBS) resuspended after, getting the resuspended liquid of 20 μ l PBS adds 2 * SDS sampling liquid, 20 μ l to mix, make the SDS sample, after collecting empty bacterium BL21-TRXB (DE3) nutrient solution of inducing without IPTG simultaneously and making by the same method the SDS sample, carry out the SDS-PAGE electrophoresis detection: preparation 12%SDS polyacrylamide gel, by 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, then be placed in methyl alcohol-glacial acetic acid solution decolouring 3-4 hour.Result visible respectively containing 3 kinds of recombinant plasmid pET32a-Tat1-37, pET32a-Tat1-61 and pET32a-Tat22-101 be cloned in the about 22KDa of relative molecular mass, 24.9KDa and all there is purpose fusion rotein band at the 26.9KDa place, empty bacterium BL21-TRXB (DE3) has no this band (seeing Fig. 4).Through Glyko BandScan5.0 software analysis, the expression amount of 3 kinds of target proteins accounts for the bacterial protein ratio and is respectively 54.8%, 36.7% and 42.1%.Subsequently the positive bacteria clone is carried out to a large amount of abduction deliverings and purifying: will be seeded to (containing 100 μ g/ml Amp and 35 μ g/ml Kana) in 50ml LB substratum according to 1: 10 inoculum size containing pET32a-Tat1-31/BL21, pET32a-Tat1-61/BL21 and pET32a-Tat22-101/BL21 glycerine bacterial classification respectively, after 37 ℃, 170rpm shaking culture spend the night, be forwarded to (containing 100 μ g/ml Amp and 35 μ g/mlKana) in fresh 450ml LB, in 37 ℃, 220rpm shaking culture 3.5h to OD
600be 1.0, add afterwards 1M IPTG 500 μ l to final concentration be 1mM, then collect bacterium liquid after 37 ℃, 220rpm shaking culture 4h, in 4 ℃, the centrifugal 10min of 5000rpm, abandon supernatant, precipitation adds 30ml 8M urea (pH 8.0) cracking, in 4 ℃, spends the night.Next day, split product, in 4 ℃, the centrifugal 30min of 10000rpm, is got supernatant to another centrifuge tube, after getting wherein 20 μ l supernatant liquors and adding 2 * SDS sampling liquid, 20 μ l and mix and make the SDS sample, through the 12%SDS-PAGE electrophoresis detection, analyzes, all the other urea lysates are in 11000rpm, 4 ℃ centrifugal 20 minutes, get supernatant and cross metal chelate affinity chromatography medium post (Ni-NTA, purchased from German QIAGEN company), the Ni-NTA post is with 8M urea (pH 8.0) balance, urea lysate supernatant upper prop, 8M urea (pH 7.0) is washed post, with 8M urea (pH 4.5) wash-out, collect elution peak, with 0.1M Tris, neutralize, with PBS, dialyse, analyze purification effect through 12%SDS-PAGE, result is presented at the about 22KDa of relative molecular weight, 24.9KDa and the visible clear purpose fusion rotein band in 26.9KDa place, after Glyko BandScan5.0 software analysis shows 3 kinds of recombinant protein purifications, purity is respectively 94.4%, 86.6%, 86.6% (seeing Fig. 5 A).
5, the Western blot of Tat mutant fusion protein detects
Take out the SDS-PAGE running gel and do suitably to prune, be soaked in transfering buffering liquid and prepare transferring film; 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 gel, then stacks 6 filter paper, carry out albumen current stabilization transferring film 50min under the 300mA condition; Transferring film adds appropriate confining liquid (containing 0.01mol/L PBS, 10% skim-milk, 0.1%Tween-20,0.2% Sodium Mercurothiolate) and spends the night in 4 ℃ of sealings after finishing; With PBST washing lotion washing 5 times, add the anti-Tat monoclonal antibody of mouse (dilution in 1: 1000) (preparation of microbiology teaching and research room of The 2nd Army Medical College), hatch 2h for 37 ℃, rinsing liquid is washed 5 times, sheep anti-mouse igg-the HRP (dilution in 1: 1000) that adds horseradish peroxidase (HRP) mark, hatch 45min for 37 ℃, rinsing liquid washing 5 times, add substrate diaminobenzidine (Diaminobenzidine, DAB) colour developing 5-10 minute, the color development stopping reaction appears using immediately distilled water flushing after brown band.The visible 3 kinds of Tat mutant fusion proteins of result (Tat1-37-PET, Tat1-61-PET and Tat22-101-PET) and total length Tat fusion rotein Tat1-101-PET all are positive with the anti-Tat monoclonal antibody of mouse, and the pET32a carrier proteins is without this reaction (being shown in Fig. 5 B).
6, animal immune experiment
Initial immunity is got after purifying Tat1-37-PET, Tat1-61-PET and each 300 μ l (protein content 0.2mg) of Tat22-101-PET fusion rotein respectively with after the Freund's complete adjuvant equal-volume mixes, in four limbs and back injecting immune Healthy female new zealand rabbit (purchased from the The 2nd Army Medical College Experimental Animal Center), every group of 3 experimental rabbits; After 2 weeks, the 2nd immunity mixes with 0.1mg protein sample equal-volume with Freund's incomplete adjuvant, injection rabbit four limbs and back.Every 2 weeks booster immunizations 1 time, booster immunization is 2 times altogether, and method and dosage are all with the 2nd time.With the immunity before rabbit anteserum as negative control.For the last time within immune two weeks, by arteria carotis communis, measure greatly blood, only, the centrifugal 10min of 10000rpm, get upper serum by 3ml/ packing to about 60ml/, frozen standby in-40 ℃.
7, the ELISA of immunize rabbit serum detects
Use respectively 50mM carbonate buffer solution (pH 9.6) by the coated 96 hole enzyme-linked reaction plates of dilution in 1: 200 the HIV-1 total length Tat fusion rotein (Tat1-101-PEP) of purifying and 2 kinds of Tat C-terminal fusion roteins (Tat49-101-PEP and Tat60-101-PEP), every kind of antigen coated 3 multiple holes of row, 0.5 μ g/ hole, hatch 2h for 37 ℃, abandon coating buffer, add 180 μ l 10% skim-milks in 37 ℃ of sealing 2h, with after PBST washing lotion washing 4 times, the anti-total length Tat serum of rabbit (anti-Tat1-101-PET) and 3 kinds of anti-Tat mutant protein rabbit anti-serum (the anti-Tat1-37-PET that add respectively 1: 2 doubling dilution, anti-Tat1-61-PET and anti-Tat22-101-PET) each 100 μ l, last 1 hole does not add, hatch 45min for 37 ℃, with PBST washing 5 times, the goat anti-rabbit igg (dilution in 1: 1000) 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 by microplate reader (Bio Rad)
450).With rabbit anteserum before immunity as negative control, with higher than negative control A
450be worth the numerical value of 3 times as positive judging criterion.Visible these the 3 kinds anti-Tat mutant protein rabbit anti-serums (anti-Tat1-37-PET, anti-Tat1-61-PET and anti-Tat22-101-PET) of result all are specific reaction (seeing Fig. 6) with HIV-1 total length Tat albumen, and wherein the reactivity of anti-Tat22-101-PET serum and above-mentioned two kinds of Tat C end antigen compares and the reactivity of total length Tat albumen (seeing Fig. 7) more by force.
Claims (8)
1. human immunodeficiency virus I type Tat protein mutant, its aminoacid sequence is as shown in SEQ ID NO:6.
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. the encode nucleotide sequence of human immunodeficiency virus I type Tat protein mutant as claimed in claim 1, as shown in SEQ ID NO:5.
6. the nucleotides sequence of coding human immunodeficiency virus I type Tat protein mutant according to claim 5 is listed in the application in preparation HIV immunogen.
7. 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 therapeutic HIV vaccine.
8. the nucleotides sequence of coding human immunodeficiency virus I type Tat protein mutant according to claim 5 is listed in the application in preparation HIV detection kit.
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Non-Patent Citations (6)
| Title |
|---|
| Barbara E. et al..Tat protein of HIV-1 stimulates growth of cells derived from Kaposi s sarcoma lesions of AIDS patients.《nature》.1990,第345卷84-86. |
| E. Moreau 等.Important B-cell epitopes for neutralization of human immunodeficiency virus type 1 Tat in serum samples of humans and different animal species immunized with Tat protein or peptides.《Journal of General Virology》.2004,第85卷2893–2901. |
| Important B-cell epitopes for neutralization of human immunodeficiency virus type 1 Tat in serum samples of humans and different animal species immunized with Tat protein or peptides;E. Moreau 等;《Journal of General Virology》;20041231;第85卷;2893–2901 * |
| tat protein [Human immunodeficiency virus 1];Wei,M.等;《NCBI database》;20040830;GenBank: AAT09645.1 * |
| Tat protein of HIV-1 stimulates growth of cells derived from Kaposi s sarcoma lesions of AIDS patients;Barbara E. et al.;《nature》;19900503;第345卷;84-86 * |
| Wei,M.等.tat protein [Human immunodeficiency virus 1].《NCBI database》.2004,GenBank: AAT09645.1. |
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