CN105085616A - Amino acid sequence and application thereof - Google Patents
Amino acid sequence and application thereof Download PDFInfo
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- CN105085616A CN105085616A CN201410222933.5A CN201410222933A CN105085616A CN 105085616 A CN105085616 A CN 105085616A CN 201410222933 A CN201410222933 A CN 201410222933A CN 105085616 A CN105085616 A CN 105085616A
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
An amino acid sequence is designed and synthesized in the invention. The amino acid sequence comprises a sequence represented by SEQ ID No.1. The amino acid sequence can be used for determining the clinic boundary of the quantity of protective CMV specific CD8+T cells in peripheral blood of a patient, and monitoring and assessing infected CMV reactivation and pathogenic possibility in bodies.
Description
Technical field
The present invention relates to a kind of aminoacid sequence, particularly relating to the application of a kind of aminoacid sequence for detecting cytomegalovirus and described aminoacid sequence.
Background technology
Human cytomegalic inclusion disease virus (humancytomegalovirus, HCMV) belongs to spore exanthema virus section, is one virus maximum in nerpes vinrus hominis's group, can causes HCMV infections.HCMV is general susceptible in crowd, be grown up to the infection rate of HCMV up to more than 90%, usual non-evident sympton, hide in host cell, for inapparent infection, and when immunologic hypofunction or defect, as gestation, repeatedly transfuse blood, transplant receptor's (HCMV virus infection is also the one of the main reasons of organ transplantation failure), AIDS patient etc., normal in exciting infection (apparent infection), virus can attack multiple organ and system, cause serious disease, severe patient can lethally be died.Therefore, early monitoring HCMV hides, reactivate infection state, most important for immuuoeorapromised host.
But because HCMV virus is at the ubiquity of people's In vivo infection, usually hide does not cause manifest symptom in host cell, brings very large difficulty to the diagnosis of HCMV reactivate infection.Current HCMV Infect And Diagnose method mainly contains:
1) separation and Culture
Carry out Isolation and culture with clinical samples, be that current diagnosis HCMV infects the most reliable method, be considered to the gold standard detecting HCMV Infection Status.The method will detect sample (blood, urine, saliva, ascites, bleeding of the umbilicus, cerebrospinal fluid, lavage of trachea liquid etc.) and be inoculated in inoblast, occur that cytopathy is the positive.But because HCMV virus in-vitro multiplication is slow, first separation at least needs within one month, just to make detection cell occur pathology, experimental period is long, easily pollute, and cannot distinguish with other virus infectiones, be often negative findings for the sample virus purification after pharmacological agent, thus clinical application is limited, is now mainly used in scientific research.
2) virus antigen detects
Main application is pp65 antigenemia detection method at present, when HCMV reactivate infection, PP65 antigen was expressed in peripheral blood lymphocyte, monocyte, polymorphonuclear leukocyte and vascular endothelial cell in 6 ~ 24 hours, it is the early diagnosis diagnosis marker of HCMV Active infection, but owing to there is false positive, false negative result, also there is dispute in the method at present.
3) Serologic detection antibody
Detection of specific antibody mainly detects the anti-cytomegalovirus IgM and IgG antibody that stimulate body to produce after HCMV infects.Anti-HCMVIgG antibody many parts of Virus monitory can be used for HCMV clinical diagnosis, serum IgG antibody positive transformants is shown to be primary infection, and monitor IgG titre continuously and sharply raise and show to there is HCMV Active infection, but HCMV is grown up, infection rate is high, IgG most people is positive, and Clinical significance of detecting is little; The anti-HCMV specific IgM antibodies of serum detects, and because its antibody lifetime is short, it is lower to examine out positive rate, and the people such as Bendiksen studies discovery in addition, and IgM level and virus replication have nothing to do, and therefore IgM can not hide, excite the good index of Infect And Diagnose.
4) nucleic acid hybridization, in situ hybridization detect
Order-checking at present for HCMV completes, and greatly facilitates the application of nucleic acid hybridization technique in HCMV diagnosis.Adopt the method directly can find inclusion body in infected tissue, more traditional immunohistochemical method is more sensitive, special.But due to the singularity in methodology, complete the impact of whole process by factors, comprise: the sample disposal time, the selection of fixing agent, the specificity etc. of cell strain of monoclonal antibody, the method is higher to blood slide examiners technical requirements in addition, method is difficult to realize high throughput testing, contamination resistance is not strong, and high cost, is difficult to popularize at present.
5) PCR method
PCR method detects HCMV, and sensitivity is 0.15fg, susceptibility and specific degree higher, the quantification to Viral diagnosis can be realized, at detection viral activity, evaluate in Anti-viral Treatment etc. and have great importance.
From current PCR detected result, alone one couple of PCR primers can cause false negative, major cause may be in the difference of HCMV virus strain in clinical samples or viral genome minimum variation and viral copy number too low caused by.In addition, design of primers sequence is for the different sites of HCMV same gene, and PCR result also has notable difference, and method also needs stdn, does not also have the PCR diagnostic kit of commercial HCMV in the market.
Summary of the invention
A kind of aminoacid sequence of design and synthesis of the present invention, can be used for measuring the clinical boundary in patient's peripheral blood with protectiveness CMV special CD8+T cell quantity, the CMV reactivation of monitoring and evaluation machine In vivo infection and pathogenic possibility.
Should be understood that, hereinafter, aminoacid sequence has identical implication with polypeptide or peptide sequence in the present invention.
First aspect of the present invention provides a kind of aminoacid sequence, and described aminoacid sequence comprises sequence shown in SEQIDNo.1.
SEQIDNo.1：LysMetMetTyrMetCysTyrArgAsnIle。
Hereinafter, SEQIDNo.1 also can be abbreviated as: KMMYMCYRNI.
In foregoing of the present invention, described aminoacid sequence be preferably in sequence shown in SEQIDNo.1 and polymer thereof any one or a few.
Wherein, described polymer can be preferably dimer, tripolymer, the tetramer or more aggressiveness.
The present invention second aspect is to provide the application of a kind of aminoacid sequence in preparation detection cytomegalovirus reagent, and described aminoacid sequence comprises sequence shown in SEQIDNo.1.
In foregoing of the present invention, described aminoacid sequence be preferably in sequence shown in SEQIDNo.1 and polymer thereof any one or a few.
Wherein, described polymer can be preferably dimer, tripolymer, the tetramer or more aggressiveness.
Third aspect of the present invention is to provide a kind of test kit detecting cytomegalovirus, and described test kit comprises a kind of aminoacid sequence, and described aminoacid sequence comprises sequence shown in SEQIDNo.1.
In foregoing of the present invention, described aminoacid sequence be preferably in sequence shown in SEQIDNo.1 and polymer thereof any one or a few.
Wherein, described polymer can be preferably dimer, tripolymer, the tetramer or more aggressiveness.
In a kind of preferred embodiment of third aspect of the present invention, described test kit can also comprise fluorescent mark.
Wherein, described fluorescent mark can be any one or a few in fluorescein, radionuclide, enzyme, chemiluminescent substance, metallic particles.
The example of described fluorescein comprise in FITC, RB200, TRITC any one or a few.
The example of described radionuclide comprises
3h,
14c,
32p,
125i,
131any one or a few in I.
The example of described enzyme comprise in HRP, AP any one or a few.
More preferably, described fluorescent mark is fluorescein.
In a kind of preferred embodiment of third aspect of the present invention, described test kit can also comprise in HLA gene epitopes polypeptide and polymer thereof any one or a few.
In a kind of preferred embodiment of third aspect of the present invention, described test kit can also comprise damping fluid.
Wherein, described damping fluid at least comprises any one or a few in PBS damping fluid, FACS damping fluid.
In a kind of preferred embodiment of third aspect of the present invention, described test kit also comprises anti-CD8 antibody and/or fluorescently-labeled anti-CD8 antibody.
The invention provides a kind of amino acid and the polymer thereof that can identify the MHCclassI molecule/CMV specific polypeptides of CMVTCR; in order to detect the quantity of the special CD8+T cell of CMV in patient's peripheral blood; thus in order to measure the clinical boundary in Chinese patient's peripheral blood with protectiveness CMV special CD8+T cell quantity, the CMV reactivation of monitoring and evaluation machine In vivo infection and pathogenic possibility.
Accompanying drawing explanation
Fig. 1 is SEQIDNo.1 sequence polymer of the present invention and peripheral blood mononuclear cell Dual culture after 48 hours, and the cell proliferation of reacting positive increases.
Fig. 2 is the result that SEQIDNo.1 sequence polymer of the present invention detects the special CD8+T cell of CMV in peripheral blood.
Embodiment
Research finds, CMV specific C D8+T cell plays a part very crucial in suppression CMV reactivation, in health adult's body, the antiviral immunity of virus reactivation and CMV special CD8+T cell controls to be in a kind of equilibrium state, when this specific T-cells quantity reduces or function reduces, this balance is broken, and virus just reactivation again, human body just may be fallen ill and be occurred clinical symptom.In cmv infection crowd, the CMV hidden again reactivation is topmost a kind of mode of infection, so the Cellular Immune Status of the anti-CMV of body is the important indicator whether monitoring CMV can cause a disease.
Based on this, a kind of aminoacid sequence of design and synthesis of the present invention, can be used for measuring the clinical boundary in patient's peripheral blood with protectiveness CMV special CD8+T cell quantity, the CMV reactivation of monitoring and evaluation machine In vivo infection and pathogenic possibility.
Embodiment 1, verifies that T cell is to the reactivity of SEQIDNo.1 sequence on a cellular level
Gather cmv infection person peripheric venous blood, separating peripheral blood mononuclear cells.
Give polypeptide to stimulate, after SEQIDNo.1 peptide sequence and co-culture of cells 48h, detect cell proliferation change situation.Meanwhile, with TK-10, YK-10, NA-10, SK-10-1, YV-10, KV-10, AR-10, CK-10, MF-10, QF-10, LL-10 in contrast.The results are shown in Figure 1.
As can be seen from Figure 1, SEQIDNo.1 sequence of the present invention (KI-10 sequence) and co-culture of cells are after 48 hours, and reacting positive cell proliferation is obvious.
Embodiment 2, detects the quantity of the special CD8+T cell of CMV in peripheral blood
By SEQIDNo.1 sequence (KI-10) and the synthesizing fluorescently labeled polymer of HLA genotype, measure the quantity of the special CD8+T cell of CMV with stream type cell analyzer.Step is as follows:
1) 100 μ L whole bloods are added in 12x75mm test tube.
2) mix after adding 10 μ L polymers, lucifuge incubated at room 10min.
3) add appropriate fluorescently-labeled anti-CD8 antibody (as DakocloneDK25) to mix afterwards.Lucifuge 2-8 DEG C, hatches 10min.
4) 2mLEasyLyse is added
tMworkingsolution (DakocodeS2364), hatches 10min.
5) 2mL0.01mol/LPBS is added, and centrifugal 5 minutes (300xg), supernatant discarded.
6) heavy cell is suspended from FACS damping fluid, as 0.4mLPBS, and upper machine analysis.The results are shown in Figure 2.
As can be seen from Figure 2, the special CD8+T cell proportion of CMV in the peripheral blood mononuclear cell of reacting positive of the present invention is increased.
Can find out; SEQIDNo.1 sequence of the present invention and polymer thereof effectively can detect the special CD8+T cell of CMV in peripheral blood mononuclear cell, thus provide a kind of high sensitive, high efficiency reagent and method for having the clinical boundary of protectiveness CMV special CD8+T cell quantity, the CMV reactivation of monitoring and evaluation machine In vivo infection and pathogenic possibility in the Chinese patient's peripheral blood of mensuration.
Be described in detail specific embodiments of the invention above, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and substituting also all among category of the present invention.Therefore, equalization conversion done without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.
Claims (10)
1. an aminoacid sequence, is characterized in that, described aminoacid sequence comprises sequence shown in SEQIDNo.1: SEQIDNo.1:LysMetMetTyrMetCysTyrArgAsnIle.
2. aminoacid sequence according to claim 1, is characterized in that, described aminoacid sequence is any one or a few in sequence shown in SEQIDNo.1 and polymer thereof.
3. aminoacid sequence detects the application in cytomegalovirus reagent in preparation, and it is characterized in that, described aminoacid sequence comprises sequence shown in SEQIDNo.1.
4. application according to claim 4, is characterized in that, described aminoacid sequence is any one or a few in sequence shown in SEQIDNo.1 and polymer thereof.
5. detect a test kit for cytomegalovirus, it is characterized in that, described test kit comprises a kind of aminoacid sequence, and described aminoacid sequence comprises sequence shown in SEQIDNo.1.
6. test kit according to claim 5, is characterized in that, described test kit also comprises fluorescent mark.
7. the test kit according to claim 5 or 6, is characterized in that, described test kit also comprise in HLA gene epitopes polypeptide and polymer thereof any one or a few.
8. test kit according to claim 5, is characterized in that, described aminoacid sequence is any one or a few in sequence shown in SEQIDNo.1 and polymer thereof.
9. test kit according to claim 5, is characterized in that, described test kit also comprises damping fluid, described damping fluid at least comprise in PBS damping fluid, FACS damping fluid any one or a few.
10. test kit according to claim 5, is characterized in that, described test kit also comprises anti-CD8 antibody and/or fluorescently-labeled anti-CD8 antibody.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410222933.5A CN105085616A (en) | 2014-05-23 | 2014-05-23 | Amino acid sequence and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410222933.5A CN105085616A (en) | 2014-05-23 | 2014-05-23 | Amino acid sequence and application thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1476480A (en) * | 2000-09-26 | 2004-02-18 | ·��ά��֢�о�Ժ | Isolated HLA-C molecule-binding peptides and uses thereof |
| CN1893971A (en) * | 2003-10-17 | 2007-01-10 | 贝勒医学院 | A method for increasing CD8<+> cytotoxic T cell reponses and for treating multiple sclerosis |
| WO2010037397A1 (en) * | 2008-10-01 | 2010-04-08 | Dako Denmark A/S | Mhc multimers in cmv immune monitoring |
-
2014
- 2014-05-23 CN CN201410222933.5A patent/CN105085616A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1476480A (en) * | 2000-09-26 | 2004-02-18 | ·��ά��֢�о�Ժ | Isolated HLA-C molecule-binding peptides and uses thereof |
| CN1893971A (en) * | 2003-10-17 | 2007-01-10 | 贝勒医学院 | A method for increasing CD8<+> cytotoxic T cell reponses and for treating multiple sclerosis |
| WO2010037397A1 (en) * | 2008-10-01 | 2010-04-08 | Dako Denmark A/S | Mhc multimers in cmv immune monitoring |
Non-Patent Citations (5)
| Title |
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| 何贤辉等: "加载HCMV 抗原肽的HLA_A＊0201单体及其四聚体制备和鉴定", 《生物工程学报》 * |
| 李丰耀等: "负载HCMV pp65 抗原肽的HLA-A *1101-GPI 四聚体的制备", 《现代免疫学》 * |
| 查庆兵等: "HLA-A2+供者外周血hCMV特异性CTL的定量及其表型分析", 《细胞与分子免疫学杂志》 * |
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