CN1253584C - Method for evaluating curative effect of medication to cure HIV through detecting change of TCR gene of human T cell receptor before and after treatment - Google Patents
Method for evaluating curative effect of medication to cure HIV through detecting change of TCR gene of human T cell receptor before and after treatment Download PDFInfo
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- CN1253584C CN1253584C CN 02131070 CN02131070A CN1253584C CN 1253584 C CN1253584 C CN 1253584C CN 02131070 CN02131070 CN 02131070 CN 02131070 A CN02131070 A CN 02131070A CN 1253584 C CN1253584 C CN 1253584C
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Abstract
The present invention provides a method for evaluating the curative effect of drugs by that designed specific primers are used for completing PCR reaction and amplifying the fore-and-aft variation of HIV virus infected patient's T cell receptor TCR gene after receiving drug treatment. The technology provided by the present invention fills the technical empty, and has the advantages of high sensitivity and good accuracy.
Description
Technical field:
The present invention relates to the evaluation method of curative effect of medication, be meant especially by detecting the variation of human T cell receptor tcr gene before and after treatment and estimate the method for the curative effect of treatment HIV medicine.
Background technology:
Human immunodeficiency virus (HIV) is a kind of T of having a liking for cellularity virus, and the target cell of infection is the CD4T lymphocyte, thereby causes acquired immune deficiency syndrome (AIDS) (AIDS).HIV infects can cause the minimizing of cd4 t cell quantity and the imbalance of immunologic function, also can cause the disorder on lymphocytic height activation of CD8T and the function.Effectively pharmacological agent can not only be controlled the quantity and the function of duplicating and can effectively improve or recover the T cell of virus.Detecting T cell quantity and functional status is the strongest biology and the immunology foundation of identifying curative effect of medication.
The method that generally adopts is to detect the change of T cell quantity with fluidic cell numeration instrument at present.This kind method is to adopt the fluorescent mark monoclonal antibody to carry out the change situation of tracing observation cell quantity.Yet observe the immunological status of patient body's inner cell, only the quantitative analysis by this cell phenotype is far from being enough.Therefore the slight change of adopting molecular biology and molecular immune to learn a skill to observe the T cell interior has become an important detection means.
The T lymphocyte is to come recognition protein antigenic specific by TXi Baoshouti (TCR).The polypeptide chain of forming TXi Baoshouti comprises the different dimerization γ δ of α β heterodimer.Wherein the β chain of TXi Baoshouti is because of its exist (ND or NDN) that contains the insertion of the N region sequence that non-template relies on and D district, is that TCR resets the critical area that diversity can uniqueness.Therefore frequently has clinical value to detect the expression of TCR β chain gene or amino acid.TCR β chain is by variable region gene (V gene), diversity region gene (D gene), and land gene (J gene) and constant region gene (C gene) coding form.Virus causes an optionally change of V β gene family usually.The semi-quantitative analysis method of having set up is to adopt the quantitatively relative percentage of each gene family genetic expression of the terminal primer of isotopic labeling C district gene.The sensitivity of this quantivative approach and specificity are all lower, and need a large amount of T cells (1 * 10
7Cell).Forming all by V-D-J, complementation decision family district (CDR3) of TCR is the cog region of antigen-specific.This district is that TCR produces diversity and specific key area.Therefore can utilize the TXi Baoshouti gene of rearrangement, as the sign of T cell clone.Research data shows at present, and the CDR3 section is reset or claimed to change and almost occurs in the cell-mediated immunological disease of all T-.Detecting this district's gene and amino acid whose variation has broad prospects on the evaluation curative effect of medication.
Summary of the invention:
The object of the present invention is to provide a kind of method of effectively estimating the curative effect of treatment HIV medicine by the variation of detection human T cell receptor tcr gene before and after treatment.
Above-mentioned purpose of the present invention can realize by the following technical solutions.
1.T the collection of cell, separation
Blood sample separates with Ficoll-Hypaque layering liquid after gathering, and obtains peripheral blood mononuclear cell (PBMC), has the magnetic bead of CD4 or CD8 antibody further to isolate CD4 or cd8 t cell with coupling again.
2.cDNA synthetic
The separation of lymphocytic mRNA and the method for purification are to adopt the technology (Chen that has announced, 1994 proc such as Z.W, Matl.Acai.Sci.Usa.91:7501-7505), with mRNA with 1 μ g hexanucleotides (Promega arbitrarily, Wisconsin, USA Madison state), 5 μ l supertranscriptaseII (GibcoBRL, USA New York Long Island) obtain cDNA 37 ℃ of hybrid reaction post-heating to 70 ℃ terminations.
3.TCR genome primer design:
At first design 5 ' end primer.Then at C gene regions design 3 ' end primer.This primer is used for the usefulness of the first step amplification.
In order to measure more accurately when HIV virus during with the TCR interaction of molecules, the variation of its CDR3 district nucleic acid or aminoacid sequence, we have designed the CDR3 section atlas analysis that one group of inboard primer is used for the TCR molecule.Extremely designing 22 inboard V β gene primers and an inboard C β gene primer near CDR3 section V gene regions.C β gene primer adopts highly sensitive fluorescence (6-FAM) mark.We insert seven non-template bases (GTTTCTT) at 5 of C district gene primer ' end first.
4.PCR reaction conditions
For making the PCR reaction have the specificity of height, not only need the primer of high special, and also should strict control on primer concentration.Our used primer concentration is 1/10th to 1/20th of a conventional amount used, promptly only needs 2.5pmol in 50 μ l reaction volumes.In order to prevent the base mispairing of the reactant that the TaqDNA enzyme causes, we reduce to 15 circulations (being generally 35-40 circulation) with the cycle index of PCR.
5.CDR3 the analysis that gene changes
Through fluorescently-labeled PCR product together with the dimension mark thing with after methane amide mixes 94 ℃ of sex change 2 minutes, sample is placed the sequencing gel that contains 6% acrylamide, operation is 6 hours on the dna sequencing machine, the data that obtain are analyzed with the ABIPRISMGeneScan analysis software and quantized, get corresponding results.
The present invention has following advantage:
1. the present invention has filled up the blank of technical field, is the curative effect of estimating treatment HIV medicine first with this method.
2. Gao Du susceptibility, method provided by the invention can be with the demand of T cell by about 1 * 10
7Reduce to 1 * 10
5
3. Gao Du specificity, the used primer concentration of method provided by the invention is 1/10th to 1/20th of a conventional amount used.
4. Gao Du repeatability, method provided by the invention not only have the repeatability of height in same sample, and all present the location of consistent nucleic acid at the sample of same patient's different time collection.
Description of drawings:
Fig. 1 is the gene mapping that V β 1 gene is showed after effective pharmacological agent.
Fig. 2 is the gene mapping that V β 2 genes are showed after effective pharmacological agent.
Fig. 3 is the gene mapping that V β 7 genes are showed after effective pharmacological agent.
Gene mapping when a represents not carry HIV
B represents the gene mapping before HIV virus infection person treats
C represents the gene mapping after the HIV virus infection person treatment
Embodiment:
Elaborate the specific embodiment of the present invention below in conjunction with embodiment:
After the blood sample of accepting the patient of AIDS-treating medicine treatment and healthy volunteer gathered respectively, separate with Ficoll-Hypaque layering liquid, after horizontal was centrifugal, mononuclearcell was intensive in the interface of plasma layer and layering liquid, draw this confluent monolayer cells and pass through washing the high heart resuspendedly, obtain PBMC.There is the magnetic bead of CD4 or CD8 antibody further to isolate CD4 or cd8 t cell with coupling again.
The separation of the mRNA of T cell and the method for purification are to adopt the technology (Chen that has announced, 1994 proc such as Z.W, Matl.Acai.Sci.Usa.91:7501-7505), with mRNA with 1 μ g hexanucleotides (Promega arbitrarily, Wisconsin, USA Madison state), the total volume of 5 μ l supertranscriptaseII (Gibco BRL, USA New York Long Island) is controlled at 20 μ l, mix 1 hour post-heating to 70 ℃ 10 minutes termination reaction at 37 ℃, obtain cDNA.
Design of primers is shown in subordinate list 1.
The PCR parameter designing is as follows:
First set reaction: 95 ℃ of sex change, 45 seconds; 60 ℃ of renaturation, 45 seconds; Extend 72 ℃, 45 seconds, 34 circulations, 72 ℃ 10 minutes.
| 5 ' primer (10pmol), 3 ' primer (10pmol) dNTPs (10mM) | 1μl 1μl 1μl |
| 10x reaction buffer tri-distilled water cDNA Taq archaeal dna polymerase | 2.5μl 18.75μl 0.5μl 0.25μl |
Reaction for the second time: 95 ℃ of sex change, 30 seconds; 60 ℃ of renaturation, 30 seconds; Extend 72 ℃, 30 seconds, 15 circulations, 72 ℃ 10 minutes.
| 5 ' primer, (10pmol) 3 ' primer, (10pmol) dNTPs, (10mM) 10x reaction buffer tri-distilled water PCR product first time Taq archaeal dna polymerase | 0.5μl 0.5μl 1μl 2.5μl 19.75μl 0.5μl 0.25μl |
10x reaction buffer wherein
*Component as follows,
| Tris-HCl(pH8.3) MgCl 2The KCl gelatin | 300mM 50mM 25mM 0.01％ |
After reaction is finished, mix the back with methane amide 94 ℃ of sex change 2 minutes with fluorescently-labeled PCR product together with the dimension mark thing, sample is placed the sequencing gel that contains 6% acrylamide, operation is 6 hours on the dna sequencing machine, the data that obtain are analyzed by using the ABIPRISMGeneScan analysis software, got corresponding results.
We compare mensuration to the patient and the 20 routine normal peoples of 50 routine HIV virus infectiones to adopt this technology, and the result shows that the patient that HIV infects compares its tcr gene collection of illustrative plates with the normal people have significant difference (P＜0.001).Fig. 1 Fig. 2 Fig. 3 is the gene mapping that 3 representative V β gene families are showed after effective pharmacological agent.Gene mapping is a cloning propagation before the treatment as seen from the figure, has only a main peak, then returns to normal distribution fully after the active drug treatment.This shows that this technology is being very successful aspect the evaluation curative effect of medication.
Table 1 human TCR V β and C β primer
| V β out PCR primer | V β in PCR primer |
| Vβ1out 5′GGA GTC ACA CAA ACC CCA AAG CAC CTG 3′ Vβ2out 5′GTT ATC TGT AAG AGT GGA ACC 3′ Vβ3out 5′CTC GTA GAT GTG AAA GTA ACC CAG AG 3′ Vβ4out 5′GAT AGC CAA GTC ACC ATG ATG TTC 3′ Vβ5.1out 5′GTC GTT CTC AGG GCG CCA GTT CTC 3′ Vβ6out 5′GAG TCT CCC AGA ACC CCA GAC ACA AG 3′ Vβ7out 5′GGT CAT GGG AAT GAC AAA TAA GAA G 3′ Vβ8out 5′CGA TAG ATG ATT CAG GGA TGC CCG AG 3′ Vβ9out 5′GAC AAG TCC ATT AAA TGT GAA C 3′ Vβ11out 5′CTG ACA TCT ACC AGA CCC CAA GAT AC 3′ Vβ12out 5′GAT ACT GAC AAA GGA GAA GTC 3′ Vβ13out 5′CAA GAC CCA GGC ATG GGG CTG AAG 3′ Vβ14out 5′GGG AGA TGT TCC TGA AGG GTA C 3′ Vβ15out 5′CTC GAC AGG CAC AGG CTAAATTC 3′ Vβ16out 5′GATTCTT AGC TGA AAG GAC TGG AGG 3′ Vβ17out 5′GAA GGG TAC AGC GTC TCT CGG GAG AAG 3′ Vβ18out 5′GCC GGC GTC ATG CAG AAC CCA AGA C 3′ Vβ20out 5′CTC TCA GCC TCC AGA CCC CAG 3′ Vβ21out 5′GCC TGT GGC TTT TTG GTG CAA TC 3′ Vβ22out 5′CAC ACA GAT GGG ACA GGA AG 3′ Vβ23out 5′CCC TGA TCG ATT CTC AGC TCA AC 3′ Vβ24out 5′CGG CCG AAC ACT TCT TTC TG 3′ Cβout 5′CAG GCC TCG GCG CTG ACG ATC TGG GTG AC 3′ | Vβ1in 5′CTA AAC CTG AGC TCT CTG G 3′ Vβ2in 5′GCA GCT TCT ACA TCT GCA G 3′ Vβ3in 5′GAT TCT GGA GTC CGC CAG 3′ Vβ4in 5′CAG CAC CAT ATA TCT CTG C 3′ Vβ5.1in 5′GAC TCG GCC CTT TAT CTT TG 3′ Vβ6in 5′GGA GAT CCA GCG CAC AGA G 3′ Vβ7in 5′CCT GAA TGC CCC AAC AGC TC 3′ Vβ8in 5′CCA GCC CTC AGA ACC CAG G 3′ Vβ9in 5′CTG GAG CTT GGT GAC TCT GTG 3′ Vβ11in 5′CCT GGA GTC TGC CAG GCC 3′ Vβ12in 5′CTC ACT CTG GAG TCG CTA C 3′ Vβ13in 5′GAG GAT TTC CCG CTC AGG C 3′ Vβ14in 5′GAG AAG AGG AAT TTC AAT TTC C 3′ Vβ15in 5′GAG TCT GCC ATC CCC AAC 3′ Vβ16in 5′CAG AAC TGG AGG ATT CTG G 3′ Vβ17in 5′CGG CCC AAA AGA ACC CGA CAG C 3′ Vβ18in 5′GTG CGA GGA GAT TCG GCA G 3′ Vβ20in 5′CAG GAC CGG CAG TTC ATC TTC 3′ Vβ21in 5′CCT GCA GAG CTT GGG GAC 3′ Vβ22in 5′CAC TCT GAA GAT CCG GTC C 3′ Vβ23in 5′GAG CTC CTT GGA GCT GGG G 3′ Vβ24in 5′CAT CCG CTC ACC AGG CCT GGG 3′ 3′Cβin * 5′ GTT TCT TGATCTCT GCT TCT GAT GGC TCA AAC 3′ *6-FAM labelling |
Claims (1)
1. estimate the method for treatment HIV curative effect of medication by detecting the variation of human T cell receptor tcr gene before and after treatment, the steps include:
A. blood sample obtains peripheral blood mononuclear cell after gathering separation, further isolates CD4 or cd8 t cell again;
B. separate and the mRNA of the T cell of purifying out after, by the synthetic cDNA that obtains of mRNA;
C. extremely designing 22 inboard V β gene primers and an inboard C β gene primer near CDR3 section V gene regions, 5 of C β gene primer ' end inserts seven non-template bases G TTTCTT, C β gene primer adopts highly sensitive fluorescent mark, and described V β gene primer and C β gene primer are as follows:
V β out PCR primer:
Vβ1out 5′GGA GTC ACA CAA ACC CCA AAG CAC CTG 3′，
Vβ2out 5′GTT ATC TGT AAG AGT GGA ACC 3′，
Vβ3out 5′CTC GTA GAT GTG AAA GTA ACC CAG AG 3′，
Vβ4out 5′GAT AGC CAA GTC ACC ATG ATG TTC 3′，
Vβ5.1out 5′GTC GAT TCT CAG GGC GCC AGT TCT C 3′，
Vβ6out 5′GAG TCT CCC AGA ACC CCA GAC ACA AG 3′，
Vβ7out 5′GGT CAT GGG AAT GAC AAA TAA GAA G 3′，
Vβ8out 5′CGA TAG ATG ATT CAG GGA TGC CCG AG 3′，
Vβ9out 5′GAC AAG TCC ATT AAA TGT GAA C 3′，
Vβ11out 5′CTG ACA TCT ACC AGA CCC CAA GAT AC 3′，
Vβ12out 5′GAT ACT GAC AAA GGA GAA GTC 3′，
Vβ13out 5′CAA GAC CCA GGC ATG GGG CTG AAG 3′，
Vβ14out 5′GGG AGA TGT TCC TGA AGG GTA C 3′，
Vβ15out 5′CTC GAC AGG CAC AGG CTA AAT TC 3′，
Vβ16out 5′GAT TCT TAG CTG AAA GGA CTG GAG G 3′，
Vβ17out 5′GAA GGG TAC AGC GTC TCT CGG GAG AAG 3′，
Vβ18out 5′GCC GGC GTC ATG CAG AAC CCA AGA C3′，
Vβ20out 5′CTC TCA GCC TCC AGA CCC CAG3′，
Vβ21out 5′GCC TGT GGC TTT TTG GTG CAA TC 3′，
Vβ22out 5′CAC ACA GAT GGG ACA GGA AG 3′，
Vβ23out 5′CCC TGA TCG ATT CTC AGC TCA AC 3′，
Vβ24out 5′CGG CCG AAC ACT TCT TTC TG 3′；
V β in PCR primer:
Vβ1in 5′CTA AAC CTG AGC TCT CTG G 3′，
Vβ2in 5′GCA GCT TCT ACA TCT GCA G 3′，
Vβ3in 5′GAT TCT GGA GTC CGC CAG 3′，
Vβ4in 5′CAG CAG CAT ATA TCT CTG C 3′，
Vβ5.1in 5′GAC TCG GCC CTT TAT CTT TG 3′，
Vβ6in 5′GGA GAT CCA GCG CAC AGA G 3′，
Vβ7in 5′CCT GAA TGC CCC AAC AGC TC 3′，
Vβ8in 5′CCA GCC CTC AGA ACC CAG G 3′，
Vβ9in 5′CTG GAG CTT GGT GAC TCT GTG 3′，
Vβ11in 5′CCT GGA GTC TGC CAG GCC 3′，
Vβ12in 5′CTC ACT CTG GAG TCG CTA C 3′，
Vβ13in 5′GAG GAT TTC CCG CTC AGG C 3′，
Vβ14in 5′GAG AAG AGG AAT TTC AAT TTC C 3′，
Vβ15in 5′GAG TCT GCC ATC CCC AAC 3′，
Vβ16in 5′CAG AAC TGG AGG ATT CTG G 3′，
Vβ17in 5′CGG CCC AAA AGA ACC CGA CAG C 3′，
Vβ18in 5′GTG CGA GGA GAT TCG GCA G 3′，
Vβ20in 5′CAG GAC CGG CAG TTC ATC TTC 3′，
Vβ21in 5′CCT GCA GAG CTT GGG GAC 3′，
Vβ22in 5′CAC TCT GAA GAT CCG GTC C 3′，
Vβ23in 5′GAG CTC CTT GGA GCT GGG G 3′，
Vβ24in 5′CAT CCG CTC ACC AGG CCT GGG 3′；
Cβout 5′CAG GCC TCG GCG CTG ACG ATC TGG GTG AC 3′，
3′Cβin
*5′GTT TCT TG ATC TCT GCT TCT GAT GGC TCA AAC 3′，
*6-FAM labelling；
D. react the cDNA that increases with the primer of design with by mRNA synthetic cDNA by twice PCR, the condition of described twice PCR reaction is:
First set reaction: 95 ℃ of sex change, 45 seconds; 60 ℃ of renaturation, 45 seconds; Extend 72 ℃, 45 seconds, 34 circulations, 72 ℃ 10 minutes; Reaction system is:
5 ' primer 10pmol, 1 μ l,
3 ' primer 10pmol, 1 μ l,
dNTPs 10mM 1μl，
10 * reaction buffer, 2.5 μ l,
Tri-distilled water 18.75 μ l,
cDNA 0.5μl，
Taq archaeal dna polymerase 0.25 μ l;
Reaction for the second time: 95 ℃ of sex change, 30 seconds; 60 ℃ of renaturation, 30 seconds; Extend 72 ℃, 30 seconds, 15 circulations, 72 ℃ 10 minutes; Reaction system is:
5 ' primer 10pmol, 0.5 μ l,
3 ' primer 10pmol, 0.5 μ l,
dNTPs 10mM 1μl，
10 * reaction buffer, 2.5 μ l,
Tri-distilled water 19.75 μ l,
PCR product 0.5 μ l for the first time,
Taq archaeal dna polymerase 0.25 μ l;
Wherein the component of 10 * reaction buffer is as follows:
Tris-HCl pH8.3 300mM，
MgCl
2 50mM，
KCl 25mM，
Gelatin 0.01%;
E. will be through twice PCR reaction amplification and by fluorescently-labeled TXi Baoshouti gene, mix back 94 ℃ of sex change with methane amide, denatured products is placed the sequencing gel that contains 6% acrylamide, on the dna sequencing machine, move, the data that obtain are analyzed with the ABIPRISMGeneScan analysis software and are quantized, by the variation of CDR3 gene mapping before and after relatively HIV virus infection person treats, draw the evaluation result of curative effect of medication.
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