CN103172707A - Diagnosis marker Talin 1 segment for human immunodeficiency virus infection and application thereof - Google Patents
Diagnosis marker Talin 1 segment for human immunodeficiency virus infection and application thereof Download PDFInfo
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- CN103172707A CN103172707A CN2011104398245A CN201110439824A CN103172707A CN 103172707 A CN103172707 A CN 103172707A CN 2011104398245 A CN2011104398245 A CN 2011104398245A CN 201110439824 A CN201110439824 A CN 201110439824A CN 103172707 A CN103172707 A CN 103172707A
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Abstract
The invention belongs to the field of biological medicine and relates to a diagnosis marker for human immunodeficiency virus infection. The invention initially provides a Talin-1 segment which can serve as a diagnosis marker for human immunodeficiency virus infection and a kit thereof. The Talin 1 segment is shown in SEQ ID NO 1 or SEQ ID NO 2. The invention also provides a corresponding kit and an application thereof. When the diagnosis marker or the kit is used for screening human immunodeficiency virus infection sufferers, a method is simple and easy to understand, operation is easy, cost is low, a diagnosis result can be provided more quickly, an infection sufferer can be helped to know pathogenic condition as early as possible, and a therapeutic schedule can be planned as early as possible and the pathogenic condition can be controlled as early as possible; and the diagnosis marker and the kit are especially applicable to detection on samples with low human immunodeficiency viral load. The diagnosis marker provided by the invention can be combined with other diagnosis methods or diagnosis markers, so that accuracy rate and sensibility of diagnosis are further increased.
Description
Technical field
The invention belongs to biomedicine field, relate to a kind of AIDS viral infection diagnostic flag.In particular to a kind of AIDS diagnosis mark and test kit thereof take Talin 1 fragment as core.The present invention also provides using method and the application of corresponding reagent box.
Background technology
Acquired immune deficiency syndrome (AIDS), namely acquired immune deficiency syndrome (AIDS) (translate again: acquired immunity defective syndrome), the transliteration of english abbreviation AIDS (Acquired Immune Deficiency Syndrome).Be divided into amphitypy: HIV-1 type and HIV-2 type are that the human injection has infected the transmissible disease that " human immunodeficiency virus " (HIV-human immunodeficiency virus) (claiming again hiv virus) causes.Acquired immune deficiency syndrome (AIDS) is called as " pestilence in century after history ", is also referred to as " super cancer " and " century killer ".
Acquired immune deficiency syndrome (AIDS) is kind of a zoonosis, causes by infecting " HIV " virus.HIV is a kind of virus that can attack the human immune system.As target of attack, considerable damage T4 Lymphoid tissue produces high fatefulue interior exhaustion most important T4 Lymphoid tissue in the human immune system for it.This virus infects in the region throughout one's life, destroys people's immunologic balance, makes human body become the carrier of various diseases.
Patients infected hiv is counted from initial infection, pass through the several years, even reaches 10 years or just can develop into AIDS patients after longer latent period.AIDS patients because of resistivity extremely decline multi-infection can appear, as zoster, Fungi Infection of Oral, pulmonary tuberculosis, the enteritis that the specific disease pathogenic microorganism causes, pneumonia, encephalitis, candidiasis, the severe infections that the multiple pathogens such as lung sac worm cause etc., malignant tumour usually occurs in the later stage, until consume because of long-term, and cachexia and death.
Acquired immune deficiency syndrome (AIDS) is seriously threatening the mankind's existence, has caused the great attention of the World Health Organization and national governments.Acquired immune deficiency syndrome (AIDS) propagation worldwide is more and swifter and more violent, and the mankind's health and the development of society in serious threat, have become the fourth-largest killer who threatens health of people.UNAIDS's declaration on May 30th, 2006 is since confirming acquired immune deficiency syndrome (AIDS) first in June, 1981, and between 25 years, whole world accumulative total has 6,500 ten thousand people's aids infections poison, and wherein 2,500,000 people are dead.
Although the numerous medical research personnel in the whole world have paid huge effort, not yet develop so far the specific medicament of radical cure acquired immune deficiency syndrome (AIDS), not can be used for the effective vaccine that prevents yet.At present, this case fatality rate is almost listed in the Class B Notifiable disease by China up to 100% " super cancer ", and is listed in one of border monitoring of hygiene transmissible disease.So we call it " super incurable disease ".
Hiv virus belongs to RNA viruses, and because RNA is strand, it is stable like that not as the DNA double chain, so the mutation frequency of hiv virus is high, giving develops vaccine brings huge difficulty.And hiv virus is retrovirus, so-called retrovirus is that it is after invading host cell, take oneself single stranded RNA as template, according to the base complementrity pair principle, under the effect of reversed transcriptive enzyme, synthetic cDNA, new synthetic cDNA inserts in host's core DNA, copies, transcribes, translates and reach amplification purpose virus with host DNA.
The methods for the treatment of of acquired immune deficiency syndrome (AIDS) has: nutrition treatment, stem cell bone marrow transplantation, fruit treatment, anti-HIV agent, immunoregulation druge, operative treatment, vaccinetherapy etc.But, there is no at present the effective vaccine that prevents AIDS.
The acquired immune deficiency syndrome (AIDS) detection principle refers to adopt laboratory method to carry out hiv virus, antibody of AIDS virus and related immune index to blood of human body, other body fluid, histoorgan, blood derivatives etc. and detects.
The diagnosis basis of acquired immune deficiency syndrome (AIDS) is aided with following any one person mainly take virus antibody positive as main, can be experiment and makes a definite diagnosis AIDS patients.
(1) at no distant date (3-6 month) loses weight more than 10%, and persistent fever reached 38 ℃ more than one month.
(2) at no distant date (3-6 month) loses weight more than 10%, continues diarrhoea (reach 3-5 every day) more than one month.
(3) pneumocystis carinii pneumonia (PCR).
(4) kaposi's sarcoma KS.
(5) significantly mould or other conditions are pathogenic infects.
The latent period average out to 8 year to 9 year of hiv virus in human body developing into before AIDS patients, and patient's appearance looks normally, and they can be without any a lot of years of symptom ground live and work.HIV itself can't cause any disease, but after immunity system is destroyed by HIV, and human body is lost and copied the chance of immunocyte because resistivity is too low, and the disease that infects other causes the various diseases multiplicity of infection and death.How the patients infected hiv of making a definite diagnosis of morning is one of important topic of this area.
Summary of the invention
The diagnostic flag that the problem to be solved in the present invention provides a kind of hiv virus carrying capacity to detect, the assisted diagnosis patients infected hiv is especially for latent period, patient that virus load is lower.
Another problem that the present invention will solve provides a kind of diagnostic kit of AIDS viral infection.
The present invention is based on following inventive concept: studies show that, the low fragment of the molecular weight of talin 1 protein raises in HIV the infected, and the purpose fragment is positioned at the C end, illustrates that the fragmentation of Talin-1 and the infection of HIV are closely related.By protein group in the mononuclearcell that detects HIV the infected and healthy volunteer, find that in HIV the infected's body, approximately the increase of the Talin-1 fragment expression amount of 38Kda size is remarkable, and the expression amount of this Talin-1 fragment and virus load negative correlation.This negative correlation prompting, the Talin-1 of newly-generated fragmentation can be used as the clinical monitoring index at a novel HIV (human immunodeficiency virus) infection initial stage.Further complete on this basis the present invention.
The invention provides a kind of diagnostic flag of AIDS viral infection, described diagnostic flag is Talin 1 fragment; Described Talin 1 fragment is as shown in SEQ ID NO 1 or SEQ ID NO 2.
Described diagnostic flag can be comprised of the polypeptide shown in the polypeptide shown in SEQ ID NO 1 and SEQ ID NO 2.These two polypeptide chains independently unit of respectively doing for oneself, the independently polypeptide chain of both can respectively having done for oneself also can be subordinated to the same polypeptide chain, namely is connected in sequentially on the same polypeptide chain one of midfeather or several amino-acid residues.
Described diagnostic flag can be also the DNA encoding sequence of Talin 1 fragment; Described Talin 1 fragment is as shown in SEQ ID NO 1 or SEQ ID NO 2.
Described diagnostic flag can be comprised of the DNA encoding sequence of polypeptide shown in the DNA encoding sequence of polypeptide shown in SEQ ID NO 1 and SEQ ID NO 2.These two DNA chains independently unit of respectively doing for oneself both can be in independently DNA chain separately, also can be subordinated to same DNA chain, namely was connected in sequentially on the same polypeptide chain one of both midfeather or several Nucleotide.
The present invention also provides the application of above-mentioned diagnostic flag, in brief, sample and above-mentioned any one diagnostic flag is compared, and detects in sample whether contain above-mentioned diagnostic flag.
Preferably, can further detect the quantity that contains described diagnostic flag in sample.
In one embodiment of the invention, the method that detects Talin 1 fragment is as follows: collect peripheral blood, the separation and Culture peripheral blood mononuclear cell is got protein sample, utilizes the immunoblotting assay method to detect the wherein content of Talin 1 fragment.
The invention provides a kind of test kit of diagnosis of aids, this test kit contains above-mentioned any one diagnostic flag.
Described test kit also contains molecular weight marker reagent.In addition, can also contain damping fluid, the albumen of small molecular weight or nucleic acid marking, internal reference reagent or specification sheets, etc.
An aspect of of the present present invention provides a kind of Talin 1 fragment polypeptide of separation, and it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with aminoacid sequence of SEQ ID NO:1h or 2.Preferably, this polypeptide is the polypeptide with aminoacid sequence of SEQ ID NO:1 or 2.
Another aspect of the present invention provides a kind of polynucleotide encoding sequence of Talin 1 fragment polypeptide of separation, and it comprises: polynucleotide encoding sequence and degenerate sequence thereof with aminoacid sequence of SEQ ID NO:1 or 2.This degenerate sequence refers to, the sequence that has one or more codons to be encoded to produce after the degenerate codon of same amino acid replaces.Due to the degeneracy of codon, so be low to moderate approximately 70% degenerate sequence also can encode out SEQ ID NO:1 or 2 described aminoacid sequences with the nucleotide coding sequence homology of SEQ ID NO:1 or 2.
In practice, can be according to this polypeptide of aminoacid sequence synthetic of Talin 1 fragment polypeptide.Also the encoding sequence of Talin 1 fragment polypeptide of the present invention can be connected with carrier, be used for amplification or express Talin 1 fragment polypeptide.
In another aspect of this invention, provide a kind of generation to have the method for Talin 1 fragment polypeptide, the method comprises:
(a) will the encode nucleotide sequence of Talin 1 fragment polypeptide operationally is connected in expression regulation sequence, forms Talin 1 fragment expression of polypeptides carrier, and described Talin 1 fragment polypeptide is identical with the aminoacid sequence of SEQ ID NO:1 or 2;
(b) change the expression vector in step (a) over to host cell, form the reconstitution cell of Talin 1 fragment polypeptide;
(c) be fit to express under the condition of Talin 1 fragment polypeptide the reconstitution cell in culturing step (b);
(d) isolate the polypeptide with Talin 1 fragment polypeptide active.
In another aspect of this invention, provide a kind of method that detects AIDS viral infection, the method comprises: collect person's to be detected blood sample or body fluid, detect the wherein content of Talin 1 fragment polypeptide.
In one embodiment, provide sample to be tested whether to have the method for Talin 1 fragment polypeptide, it comprises step: the specific antibody of testing sample with anti-Talin 1 fragment polypeptide contacted; Detect whether formed immunocomplex, form immunocomplex and just represent that this sample to be tested contains Talin 1 fragment polypeptide.
In practice, can also be by detecting the nucleic acid coding sequence that whether has Talin 1 polypeptide in sample, it comprises step: with the Auele Specific Primer of Talin 1 fragment polypeptid coding sequence, with the increase mRNA of this sample of RT-PCR method; Whether have estimate Talin 1 fragment polypeptide amplified production, exist amplified production just to represent that HIV (human immunodeficiency virus) infection has occured this sample if detecting.
In another embodiment, the method is the transcript that whether has Talin 1 fragment polypeptide in sample by detecting, and it comprises step: with the specific probe of Talin 1 fragment polypeptide-nucleic acid encoding sequence, with the cDNA sample hybridization of this sample; Whether have hybridization product, exist the hybridization product just to represent that HIV (human immunodeficiency virus) infection has occured this sample if detecting.
In another aspect of this invention, also provide a kind of test kit that detects AIDS viral infection, it contains: the primer pair of (1) specific amplification Talin 1 fragment nucleic acid coding sequence, the required reagent of reverse transcription mRNA that (2) are optional.The another kind of test kit that detects AIDS viral infection contains the specific antibody for Talin 1 fragment polypeptide.The another kind of test kit that detects AIDS viral infection, containing can be specifically and the probe of the mRNA hybridization of Talin 1 fragment polypeptide.
In the present invention, can select various carrier known in the art, as commercially available carrier.Such as, select commercially available carrier, then the nucleotide sequence with code book invention polypeptide operationally is connected in expression regulation sequence, can form protein expression vector.
As used herein, " operationally being connected in " refers to a kind of like this situation, and namely some part of linear DNA sequence can affect the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjoining, means in reading frame adjacent for the secretion leader sequence.
In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is eukaryotic cell, as Chinese hamster ovary celI, COS cell etc.
On the other hand, the present invention comprises that also Talin 1 fragment polypeptide is had specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " refers to that antibody capable is incorporated into Talin 1 fragment polypeptide.Preferably, refer to that those can be combined with Talin 1 fragment polypeptide but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.In the present invention antibody comprise those can in conjunction with and suppress the molecule of Talin 1 fragment polypeptide, comprise that also those do not affect the antibody of Talin 1 fragment polypeptide function.The present invention also comprise those can with the antibody of modifying or being combined without the Talin 1 fragment polypeptide of modified forms.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab)
2Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered scFv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the Talin 1 fragment polypeptide of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expression Talin 1 fragment polypeptide or its cell with antigenic fragment can be used to immune animal and produce antibody.Antibody of the present invention can be also monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology prepare (see the people such as Kohler,
Nature256; 495,1975; The people such as Kohler,
Eur.J.Immunol.6:511,1976; The people such as Kohler,
Eur.J.Immunol.6:292,1976; The people such as Hammerling,
In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block Talin 1 fragment polypeptide function and the antibody that does not affect Talin 1 fragment polypeptide function.Each antibody-like of the present invention can utilize fragment or the functional zone of Talin 1 fragment polypeptide, obtains by the routine immunization technology.These fragments or functional zone can utilize the recombination method preparation or utilize Peptide synthesizer synthetic.The antibody of being combined with the unmodified form of Talin 1 fragment polypeptide can come immune animal and produce with the gene product of producing in prokaryotic cell prokaryocyte (for example E.Coli); The antibody of being combined with the posttranslational modification form (as albumen or the polypeptide of glycosylation or phosphorylation) can come immune animal and obtain with the gene product that produces in eukaryotic cell (for example yeast or insect cell).
The present invention provides Talin-1 fragment and the test kit thereof that can be used as the AIDS viral infection diagnostic flag first.Use this diagnostic flag or test kit screening AIDS viral infection patient, method is simple, and is easy to operate, with low cost, easy-to-understand, and diagnostic result can be provided faster, helps infected patient to understand as early as possible the state of an illness, formulates treatment plan, controls the state of an illness.Be particularly suited for detecting the low sample of hiv virus virus load.Diagnostic flag of the present invention can be united use with other diagnostic methods or diagnostic flag, with accuracy rate and the susceptibility of further raising diagnosis.
Description of drawings
Fig. 1 is the electrophoresis partial enlarged drawing of Talin-1 fragmentation in HIV patient.Wherein, N1, N2, and N3 represents three repeated experiments of human normal plasma, H1, H2 and H3 represent the AIDS patient's sample without the HAART treatment.TLN1 base Talin-1 of the present invention in figure.
Fig. 2 is the 38KDa frag info of Mass Spectrometric Identification talin-1.
Fig. 3 is the expression of the 38KDa fragment of immunoblotting assay talin 1 in 11 healthy volunteers and 12 AIDS patients' PBMC.
Fig. 4 is near amount and the virus load negative correlation figure of the Talin-1 fragment 38KDa.
Fig. 5 is MRM detection by quantitative Talin-1C end peptide section.Wherein 1-4 is low virus load sample, and 5-7 is normal specimen, and 8-10 is high virus load sample.
Fig. 6 is that after HIV pseudovirus infection TZM-bl cell, fragmentation and the apoptosis in corresponding stage of Talin-1 detects.
Fig. 7 is that the relevant apoptosis of virus load detects.Cell model in 3.2 is carried out the immunoblot experiment of caspase usually, and result shows: in whole pseudovirus stimulating course, obvious apoptosis does not all occur in cell.
Embodiment
Embodiment 12DE-MS finds the Talin-1 fragmentation first in HIV the infected
The technological line of employing 2DE-MS detects without protein group in HIV the infected of HAART treatment and healthy volunteer's mononuclearcell, find talin 1, vinculin, the low fragment of the molecular weight of the protein such as filamin A raises (seeing Fig. 1) in HIV the infected, and the purpose fragment is positioned at the C end.TLN1 base Talin-1 of the present invention in figure.
This results suggest: in the HIV the infected who does not treat through HAART, fragmentation has occured at its C end in Talin-1, and has obtained good checking in clinical sample.
The dependency of embodiment 2 information biology checking Talin 1 and interacting protein and HIV protein
Adopt STRING 8.0 softwares (http://string.embl.de) and HIV and the interactional database of host cell (http://www.ncbi.nlm.nih.gov/RefSeq/HIVInteractions.) to analyze the interaction of talin 1 with HIV protein and host cell proteins matter, find that talin 1 has participated in the pol of HIV and the network of vif gene regulating.This result with Fig. 1 is consistent: the several protein in this network all have the fragment (seeing Fig. 1) of up-regulated in HIV the infected.Show: the fragmentation of Talin-1 is not to be a kind of independently fortuitous phenomena, but is positioned among the interactive network of a complexity.Therefore its fragmentation might be very the active reaction that a kind of host takes in the face of the HIV invasion, thus the further infection of impact virus.
3.1.Western Blotting detects the relation of Talin-1 fragment and HIV virus load
Collection through HAART treatment after 6 months virus load lower than 50 or higher than 70 patient EDTA anticoagulated whole blood, separate PBMC, equal protein matter (50 μ g) loading is carried out immunoblot experiment with the primary antibodie of anti-talin-1.
Result shows: amount and the virus load negative correlation (Fig. 4) of near the Talin-1 fragment 38KDa.
3.2MRM test the fragment of quantitative talin 1
A. nonredundancy peptide section screening
According to the peptide section of Mass Spectrometric Identification, tentative prediction 38KDa fragment is the C-end fragment of talin 1.Then analyze and finally selected the unique peptide conduct of VGAIPANALDDGQWSQGLISAAR (SEQ ID NO 1) and these two Talin-1 of LAQAAQSSVATITR (SEQ ID NO 2) Talin-1 to be carried out the foundation of mass spectrum detection by quantitative.
B. the quantitative analysis of polypeptide
Adopt Dai Anna to rise liquid chromatography Ultra3000 series connection Brooker ion trap mass spectrometry HCT and carry out the Talin-1MRM detection by quantitative.Get the aids patient PBMC sample of 4 HIV virus loads＜50 aids patients, 3 Healthy Peoples and 3 HIV virus load＞50, extract total cellular proteins, after measuring protein content, respectively get 50ug and carry out one dimension SDS-PAGE electrophoresis, electrophoresis carries out coomassie brilliant blue staining after finishing.Cut the band of 38kDa, decolour dewatered freeze-dried after, carry out reductive alkylation with DTT and IAA, add approximately 20 μ l pancreatin (0.02 μ g/ μ l) in every pipe.37 ℃ of enzymolysis spend the night (16～18h).Enzyme is cut after the peptide section extracts, and carries out the MRM detection by quantitative by Dai Anna upgrade liquid chromatogram Ultimate 3000 series connection heavy body ion trap mass spectrometries (LC-MS/MS).Peptide section sample first passes through C18 pre-column (300 μ m id x5mm, 5 μ m) desalination, and flow velocity is 20 μ L/min, and moving phase is 2%ACN and 0.1%FA.Then the flow velocity with 300nL/min separates through the reverse post of C-18 (75 μ m internal diameter x 15cm length, 3 μ m), and the gradient of chromatogram is 4-48%, and the gradient time is 60min.The peptide section of separating by the C18 chromatographic column enters the HCT mass spectrum and carries out real-time ionization analyzing and testing by receiving the stream nozzle needle.Choose the detection by quantitative that Talin-1C end peptide section m/z770.8 (sequence is VGAIPANALDDGQWSQGLISAAR) and m/z 708.9 (sequence is LAQAAQSSVATITR) carry out MRM, corresponding daughter ion is respectively y4 (m/z 404.2) and y5 (m/z 561.3), when setting chromatogram gradient runs to 20-28min, mass spectrum MRM detects ion pair 770.8/404.2, and during 30.5-42min, MRM detects 708.9/561.3.
The results are shown in Figure 5.
Result shows, 4 samples of HIV virus load＜50 all can detect two Talin-1 peptide sections, only have 1 in 3 normal specimen and Talin-1 peptide section can be detected, and also only have 1 sample two talin-1 peptide sections can be detected simultaneously in 3 samples of virus load＞50.
This presentation of results Talin-1 fragmentation and virus load be negative correlation.
MRM shows in conjunction with Western Blotting result, and the fragmentation of Talin-1 and HIV virus load present strong negative correlation, for the invention provides strong evidence.Because if the fragmentation of Talin-1 is the degraded of skimble-skamble simple protein to be caused or promotes viral propagation in host, so, its fragmentation should be proportionate with virus load.
And be just this negative correlation hint, the Talin-1 of newly-generated fragmentation is likely to possess the functional peptide fragment that suppresses the HIV effect, can be used as the clinical monitoring index at a novel HIV (human immunodeficiency virus) infection initial stage.
At first packing obtains functional HIV pseudovirus in the 293T cell, obtains pseudovirus by titer determination, attacks malicious TZM-bl clone with active HIV pseudovirus, respectively after hatching altogether 0.5,1,2,4,8, collected the equivalent cell in 12,24,48 and 72 hours, cracking obtains total protein of cell, carries out immediately the Western Blotting of talin 1.Experimental result shows that the expression of 38KDa fragment begins to increase from 2h, keeps substantially constant in 2 to 48 hours, and after 72 hours, expression amount reduces (Fig. 6) relatively.
The fragmentation of this results suggest: Talin1 may occur at the poisoning intrusion cell stage.In addition, whole course of infection does not detect apoptosis and occurs, and shows that the Talin-1 fragmentation is not due to apoptosis induction cuts.
Be by due to the no apoptosis that causes due to virus infection in order to understand fragmentation, to the clinical sample of using in embodiment 3, carry out apoptosis degree and detect, according in caspase 35 and the power of two western blot hybridization bands of 17KDa judge.
WB analyzes the relation of Caspase-3 cutting and HIV-1 carrying capacity, result shows, although at virus load during lower than 50 copy/ml, still the cutting of Caspase-3 can be detected, but in higher virus copy content sample, more Caspase-3 cutting can be detected, point out low Talin-1 fragmentation in high virus load sample be not due to the albumen that increasing apoptosis causes more extensively degrade cause (Fig. 7).
Cell model in embodiment 4 is carried out the immunoblot experiment of caspase usually, and result shows: in whole pseudovirus stimulating course, obvious apoptosis (seeing Fig. 6) does not all occur in cell.
Think there is no obvious dependency between the fragmentation of Talin-1 and apoptosis according to above-mentioned experimental result.
SEQUENCE LISTING
＜110〉Shanghai City public health clinical center
＜120〉diagnostic flag Talin 1 fragment and the application thereof of AIDS viral infection
<130> 201191
<160> 2
<170> PatentIn version 3.1
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<211> 23
<212> PRT
＜213〉synthetic
<400> 1
Val Gly Ala Ile Pro Ala Asn Ala Leu Asp Asp Gly Gln Trp Ser Gln
1 5 10 15
Gly Leu Ile Ser Ala Ala Arg
20
<210> 2
<211> 14
<212> PRT
＜213〉synthetic
<400> 2
Leu Ala Gln Ala Ala Gln Ser Ser Val Ala Thr Ile Thr Arg
1 5 10
Claims (10)
1. the diagnostic flag of an AIDS viral infection, is characterized in that, described diagnostic flag is Talin 1 fragment; Described Talin 1 fragment is as shown in SEQ ID NO 1 or SEQ ID NO 2.
2. diagnostic flag as claimed in claim 1, is characterized in that, described diagnostic flag is comprised of the polypeptide shown in the polypeptide shown in SEQ ID NO 1 and SEQ ID NO 2.
3. the diagnostic flag of an AIDS viral infection, is characterized in that, described diagnostic flag is the DNA encoding sequence of Talin 1 fragment; Described Talin 1 fragment is as shown in SEQ ID NO 1 or SEQ ID NO 2.
4. diagnostic flag as claimed in claim 3, is characterized in that, described diagnostic flag is comprised of the DNA encoding sequence of aminoacid sequence shown in the DNA encoding sequence of aminoacid sequence shown in SEQ ID NO 1 and SEQ ID NO 2.
5. the application of any one diagnostic flag in claim 1-4 is characterized in that, any one diagnostic flag in sample and claim 1-4 is compared, and detects in sample whether contain any one diagnostic flag in claim 1-4.
6. the application of any one diagnostic flag in claim 1-4 is characterized in that, detects the quantity that contains described diagnostic flag in sample.
7. the application method of the described diagnostic flag of claim 1, is characterized in that, the method in turn includes the following steps:
(1) collect peripheral blood sample to be measured;
(2) separate, cultivate peripheral blood mononuclear cell;
(3) get protein sample;
(4) utilize the immunoblotting assay method to detect the wherein content of Talin 1 fragment.
8. the test kit of a diagnosis of aids virus infection, is characterized in that, this test kit contains in claim 1-4 any one diagnostic flag.
9. test kit as claimed in claim 8, is characterized in that, described test kit also contains molecular weight marker reagent.
10. test kit as claimed in claim 8, is characterized in that, described test kit also contains the antibody of Talin 1 fragment.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040265295A1 (en) * | 2003-06-25 | 2004-12-30 | Anderson Richard A. | Methods of preventing or treating cell-migration mediated conditions or diseases |
| WO2009075883A2 (en) * | 2007-12-12 | 2009-06-18 | University Of Georgia Research Foundation, Inc. | Glycoprotein cancer biomarker |
| WO2009118205A2 (en) * | 2008-03-28 | 2009-10-01 | Fraunhofer Gesellschaft zur Förderung der angewandten Forschung e.V. | Biomarkers for monitoring or predicting the treatment of cancer |
| CN102174109A (en) * | 2011-01-21 | 2011-09-07 | 中华人民共和国吉林出入境检验检疫局 | Acquired immune deficiency syndrome virus and treponema pallidum fusion protein as well as preparation method and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040265295A1 (en) * | 2003-06-25 | 2004-12-30 | Anderson Richard A. | Methods of preventing or treating cell-migration mediated conditions or diseases |
| WO2009075883A2 (en) * | 2007-12-12 | 2009-06-18 | University Of Georgia Research Foundation, Inc. | Glycoprotein cancer biomarker |
| WO2009118205A2 (en) * | 2008-03-28 | 2009-10-01 | Fraunhofer Gesellschaft zur Förderung der angewandten Forschung e.V. | Biomarkers for monitoring or predicting the treatment of cancer |
| CN102174109A (en) * | 2011-01-21 | 2011-09-07 | 中华人民共和国吉林出入境检验检疫局 | Acquired immune deficiency syndrome virus and treponema pallidum fusion protein as well as preparation method and application thereof |
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