CN107345242A - The processing method of TCR β chain complementary determining regions - Google Patents
The processing method of TCR β chain complementary determining regions Download PDFInfo
- Publication number
- CN107345242A CN107345242A CN201610317368.XA CN201610317368A CN107345242A CN 107345242 A CN107345242 A CN 107345242A CN 201610317368 A CN201610317368 A CN 201610317368A CN 107345242 A CN107345242 A CN 107345242A
- Authority
- CN
- China
- Prior art keywords
- dna
- processing method
- group
- tcr
- carried out
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
A kind of processing method of TCR β chain complementary determining regions, comprises the following steps：Experimental group and control group, every group of periphery blood specimen for having been detached from human body for including the different human body of several pieces are set；The lymphocyte of each periphery blood specimen is separated, and extracts the DNA in the periphery blood specimen lymphocyte respectively；Using multiple PCR technique, constructed dna library；High-flux sequence is carried out to the DNA library, data analysis is carried out to the TCR β chains complementary determining region in the experimental group and control group.The processing method of above-mentioned TCR β chain complementary determining regions, it is reasonable in design feasible, comprehensive analysis Decompensated stage hbv-liver cirrhosis graft perioperatively its internal TCR β chains complementary determining region receptoire dynamic change characterization, it can be appreciated that the immune state of patient, direction of medication usage is carried out for the monitoring of disease event after transplanting, prevention of postoperative occurs repelling, infected, and provides foundation for the pathogenesis of disease.
Description
Technical field
The present invention relates to a kind of peri-operation period liver transplant recipients information obtained as intermediate result, more particularly to a kind of processing method of TCR β chain complementary determining regions.
Background technology
Hepatic sclerosis (liver Cirrhosis, LC) it is a kind of common chronic liver disease, it is to continuingly act on liver by single or a variety of pathogenic factors, cause liver parenchyma diffusivity damage etc. further to cause a series of liver functions to be damaged, be eventually developed to portal hypertension or the final result of end-stage liver disease.It is characterized in mainly due to degeneration of liver cells necrosis, remaining liver cell nodules regeneration and fibr tissue regeneration, causes liver to be hardened etc., beyond the compensatory capacity of liver cell.Its main pathogenesis is that there occurs liver fibrosis.Liver fibrosis is due to that the collagenous fibres outside hepatic tissue inner cell are continued largely to breed, so as to constantly extrude liver cell and be gradually surrounded to form fiber every ultimately resulting in liver structure and function, there occurs the result that pathology sexually revises.It can be divided into compensatory phase (early stage) and Decompensated stage (late period) according to clinical manifestation situation difference hepatic sclerosis, and their line of demarcation unobvious and overlapping sign often be presented.Compensatory phase symptom is relatively light often to lack specificity, typically no obvious clinical manifestation, even any discomfort is not felt on body yet, mainly with lassitude and weak, become thin, based on belly flatulence, diarrhoea, epigastric discomfort and secret anguish, indices in normal range (NR) or mile abnormality can occur during row liver function test, and such case is more common in micronodular cirrhosis；Decompensated stage symptom is notable, often occur with various complication, it is mainly shown as hepatic insufficiency, multiple organ dysfunction is often accompanied by be damaged, jaundice, portal hypertension, splenomegaly, ascites, hepatic encephalopathy or UGB etc., each index is substantially abnormal during row liver function test, it is more common in major tubercle hepatic sclerosis, therefore, clinically generally by blood routine, routine urinalysis, imageological examination is combined under liver function and ascites routine and necessary condition to diagnose hepatic sclerosis, and it can be not difficult to judge decompensated liver cirrhosis from the inspection and clinical manifestation of their indices, but the diagnosis of Decompensated cirrhosis is more difficult.
And Decompensated stage hepatitis b cirrhosis is primarily due to hepatitis type B virus (hepatitis B virus, HBV) persistent infection patient and changing for the series of physiological and pathological that lesions position causes liver cell to be damaged serious, continuous apoptosis etc. and causes liver function to be caused body by heavy damage is iteratively operating on, the Clinical symptoms such as jaundice, varices of esophagus, ascites, liver cerebral disease and spleen enlargement occurs.In China, virus B hepatitis is to cause the principal element of hepatic sclerosis, next to that viral hepatitis type C, wherein chronic HBV infection turn into whole world publilc health hygienic issues.According to World Health Organization, the whole world there are about hepatic sclerosis, hepatic failure and hepatocellular carcinoma caused by 1,000,000 people die from HBV infection every year, China's resident's health and national economy are had a strong impact on, the important means for preventing the development of the patient disease state of an illness and reducing case fatality rate is exactly that accurately diagnosis and early stage are treated in time for progress.There is document report, the patient for having more than 40% chronic HBV infection can develop into serious complication, about 12% hbv-liver cirrhosis patient dies from hepatic failure, and 10% or so dies from liver cancer.Because the boundary unobvious between the diversity of clinical manifestation and the pathologic process and compensatory phase and Decompensated stage of complexity even overlapping phenomenon occur, huge challenge is proposed to disease early stage and accurately diagnosis.So that the prognosis of Decompensated stage hbv-liver cirrhosis is very poor, and patient's disease development in 5 years rapidly deteriorates mostly, and only about 14% patient has the survival rate of more than 5 years.
At present, liver transfer operation is the treatment late period unique method of irreversible hepatopath, the chance of a regeneration is provided to patient, but because the problems such as not being inconsistent for liver shortage of resources and distribution type is so that many Decompensated stage hbv-liver cirrhosis patients all over the world can not obtain or be not eligible for obtain this therapeutic modality, and uniquely the method for giving treatment to is exactly to carry out antiviral therapy to these patients.Antiviral therapy is to suppress the duplication of HBV in chronic hepatitis b cirrhosis patient and Decompensated stage hepatitis B patient body to reduce hepatic necrosis inflammatory reaction and improve its liver function situation.The raising of art is received in the development of nearly more than ten years transplantation of liver and the appearance of immunodepressant and transplanting, the annual transplanting case load in China is constantly increasing, in the liver transfer operation case load completed every year in the U.S., about 5% is all hepatitis B liver transfer operation, and this ratio has been that comparison is high in the Asian-Pacific area.Since U.S. Starzl in 1963 has carried out the 1st orthotopic liver transplantation till now, very big achievement is all achieved in terms of the quantity or the quantity and clinical efficacy transplanted of transplanting.But as the various complication such as accumulation, the rejection occurred after liver transfer operation of transplanting quantity are still unavoidable, wherein turning into the principal element for influenceing survival rates with Ia problem.Patient with operation is treated by antiviral drugs and immunodepressant after most of hepatitis B patient and row liver transfer operation, but because long-term use of this kind of medicine can cause immunity to reduce, so that patient is more vulnerable to the infection of other pathogens, it also occur that resistance, once resistance occur will lessen the curative effect, aggravation is resulted even in.
The content of the invention
Based on this, it is necessary in view of the above-mentioned problems, providing a kind of processing method of peri-operation period liver transplant recipients TCR β chain complementary determining regions.
A kind of processing method of TCR β chain complementary determining regions, comprises the following steps：
Experimental group and control group, every group of periphery blood specimen for having been detached from human body for including the different human body of several pieces are set；
The lymphocyte of each periphery blood specimen is separated, and extracts the DNA in the periphery blood specimen lymphocyte respectively；
Using multiple PCR technique, constructed dna library；
High-flux sequence is carried out to the DNA library, data analysis is carried out to the TCR β chains complementary determining region in the experimental group and control group.
In one of the embodiments, before using multiple PCR technique, the processing method also include measure DNA content and detection DNA integralities the step of.
In one of the embodiments, DNA content is determined using XRF.
In one of the embodiments, DNA integralities are detected with agarose gel electrophoresis method.
In one of the embodiments, high-flux sequence is carried out to DNA library using Illumina MiSeq platforms.
In one of the embodiments, using multiple PCR technique, constructed dna library, it is specifically included：
The amount of taking of DNA in the experimental group and control group is calculated, is weighed, and adds V areas primer and J areas primer progress multiplex PCR；
Electrophoresis detection reclaims multiple PCR products；
Add end maintenance mixed liquor and carry out end maintenance reaction, and purify；
Product after purification is connected into A bases at 3 ' ends；
Joint connection is carried out in joint coupled reaction system；
The DNA fragmentation modified by PCR reaction enrichments by joint, and row agarose gel electrophoresis are entered to PCR primer, after cutting purpose fragment, purifying recovery, obtain DNA library.
In one of the embodiments, behind constructed dna library, in addition to detect the Insert Fragment scope of the DNA library and the concentration of the DNA library is quantified.
In one of the embodiments, carrying out data analysis to the TCR β chains complementary determining region in the experimental group and control group includes：Quality Control is carried out to sequencing data and pre-processes, data is compared, sequential structure is analyzed, building immune group storehouse and variance analysis is carried out to immune group storehouse.
In one of the embodiments, carrying out Quality Control and pretreatment to sequencing data includes：Filtration treatment is carried out to the data after sequencing.
In one of the embodiments, the lymphocyte of the periphery blood specimen is isolated using Ficoll density-gradient centrifugation methods.
The processing method of above-mentioned TCR β chain complementary determining regions, it is reasonable in design feasible, comprehensive analysis Decompensated stage hbv-liver cirrhosis graft perioperatively its internal TCR β chains complementary determining region receptoire dynamic change characterization, it can be appreciated that the immune state of patient, direction of medication usage is carried out for the monitoring of disease event after transplanting, prevention of postoperative occurs repelling, infected, and provides foundation for the pathogenesis of disease.
Brief description of the drawings
Fig. 1 is the schematic flow sheet of the processing method of TCR β chain complementary determining regions in one embodiment of the invention.
Embodiment
In order to facilitate the understanding of the purposes, features and advantages of the present invention, the embodiment of the present invention is described in detail below in conjunction with the accompanying drawings.Many details are elaborated in the following description in order to fully understand the present invention.But the invention can be embodied in many other ways as described herein, those skilled in the art can make similar improvements without departing from the spirit of the invention, therefore the present invention is not limited by following public specific embodiment.
Referring to Fig. 1, one embodiment of the present of invention is, a kind of processing method of TCR β chain complementary determining regions, it comprises the following steps.
S110, experimental group and control group, every group of periphery blood specimen for having been detached from human body for including the different human body of several pieces are set, for example, the experimental group includes preoperative 1 day group, postoperative 1 day group, postoperative 7 days groups.
Such as, the periphery blood specimen that various embodiments of the present invention experimental group uses, it is respectively from the hospital's organ transplant of PLA of Guilin City the 181st and is in hospital with dialysis treatment center and carries out the patient 6 of orthotopic liver transplantation and the healthy person 6 in the same period physical examination of hospital of attached Guilin Liberation army the 181st of Nanfang Medical Univ through being diagnosed as hbv-liver cirrhosis latter stage at end.
Wherein experimental group 6, women 2, male 4,44~60 years old age, average age 48.67 ± 6.31 years old.
Healthy control group 6, wherein women 3, male 3, age is 35~50 years old, average age 44.17 ± 5.85 years old, for the healthy person of the same period physical examination of hospital of attached Guilin Liberation army the 181st of Nanfang Medical Univ, no major disease history, relevant disease without immunology, I and its lineal relative without hepatitis B medical history, without autoimmune disease.
For example, at ambient temperature, the 3~4mL of peripheric venous blood for obtaining disengaging human body is placed in 5mL EDTA anti-freezing vacuum blood collection tubes, after closeing the lid, EDTA anti-freezings vacuum blood collection tube is gently overturned after mixing for several times, be put into -80 DEG C of ultra low temperature freezers and save backup.Gather each normal healthy controls person periphery anticoagulant heparin venous blood about 5mL, each renal transplantation recipients in preoperative 1 day, it is postoperative 1 day, postoperative 7 days totally 3 time points respectively gather periphery anticoagulant heparin venous blood 5mL, 24 samples altogether, the same day isolates lymphocyte to each sample after collection.
S120, each periphery blood specimen of separation lymphocyte, and the DNA in the periphery blood specimen lymphocyte is extracted respectively.
For example, isolating the lymphocyte of the periphery blood specimen using Ficoll density-gradient centrifugation methods, by making the cell of certain density be distributed by corresponding density gradient, so as to which various haemocytes be separated, the lymphocyte of the periphery blood specimen is obtained..
And for example, step S120 comprises the following steps：
(1) 1mL peripheral bloods are drawn into 2.0mL centrifuge tube, 1mL erythrocyte cracked liquids is added and vortex oscillator mixes.
(2) it is put into centrifuge, under conditions of 25 DEG C, 10min is centrifuged with 12000rpm,.
(3) after stratification, abandoning supernatant adds 900 μ L nucleus lysate, 50 μ L 20mg/mL Proteinase K and 50 μ L 10%SDS, and mixing is placed in cracking 1h in constant-temperature mixer.
(4) cooled down rapidly after the completion of cracking, add 1mL phenol/chloroform/isoamyl alcohol, mixing of turning upside down is to without obvious boundary line.
(5) under conditions of 25 DEG C, 10min is centrifuged with 12000rpm, stand, then 900 μ L of supernatant liquid are drawn to be placed in 1.5mL centrifuge tube, and add the isopropanol and 10%3M sodium acetates of -20 DEG C of precoolings of 2/3 volume, mixing turn upside down for several times, is then positioned over -20 DEG C of refrigerator overnight.
(6) under conditions of 25 DEG C, 10min is centrifuged with 12000rpm, stood.
(7) abandoning supernatant, 750 μ L75% ethanol, washing DNA precipitations are added.
(9) under conditions of 25 DEG C, 5min is centrifuged with 12000rpm, stood, abandoning supernatant, naturally dry.
(10) collect DNA and preserved under conditions of -80 DEG C stand-by.
DNA content and integrality in S130, measure.
For example, step S130 comprises the following steps：
S131, measure DNA content, for example, determining DNA content using XRF.
Specifically, step S131 comprises the following steps：
Prepare DNA dyestuff working solutions；
For example, DNA dyestuffs are Quant-iTTMReagent, by using Quant-iTTMBuffer is diluted 200 times and obtains Quant-iTTM Working Solution。
Standard solution is prepared, and makes standard curve；
For example, Quant-iT is taken respectivelyTMStandard1 (100ng/ μ L) and Quant-iTTMAfter standard2 (100ng/ μ L) 0~10 μ L mixing, wherein Quant-iTTMStandard1 and Quant-iTTMStandard2 cumulative volume is 10 μ L, then with 190 μ L Quant-iTTMAfter Working Solution mixing, according toThe program that Fluorometer has been set carries out the making of standard curve.
DNA prepare liquids are mixed with DNA dyestuff working solutions, after being stored at room temperature 2 minutes, carry out fluoroscopic examination；
2~20 μ L DNA prepare liquids and 180~198 μ L Quant-iT are taken respectivelyTMAfter Working Solution mixing, wherein, DNA prepare liquids and Quant-iTTMWorking Solution cumulative volumes are 200 μ L, are stored at room temperature 2 minutes, DNA is fully combined with dyestuff, be subsequently placed inFluoroscopic examination is carried out in Fluorometer.
According to standard curve, the DNA content of DNA prepare liquids is calculated.
S132, measure DNA integralities, for example, determining DNA integrality using agarose gel electrophoresis method.And for example, the integrality for determining DNA prepare liquids comprises the following steps.
Specifically, 0.6g is claimed to take agarose to be dissolved in electrophoretic buffer, it is placed in micro-wave oven or boiling water bath and is heated to dissolving and shaking up completely, 0.6% Ago-Gel is made, carefully poured on electrophoresis tank level board until the temperature of Ago-Gel solution is dropped to 55 DEG C or so, and be careful not to fill to spilling, treat that Ago-Gel is solidified in backward electrophoresis tank and add appropriate electrophoretic buffer, then extract；By DNA prepare liquids after melting on ice, centrifuged after fully mixing；3 μ L DNA samples are taken to add orifice plate after being mixed with sample loading buffer, a hole in its aperture plate adds 2 μ L DNA marker, from 0.6% Ago-Gel under 120V voltages electrophoresis 50 minutes, electrophoresis to bromophenol blue migrates out suitable distance in gel, cut off electric current, gel is taken out, simultaneously scan image is checked under uviol lamp.
S140, using multiple PCR technique, constructed dna library.
For example, step S140 is specifically included：
The amount of taking of DNA in the experimental group and control group is calculated, is weighed, and adds V areas primer and J areas primer progress multiplex PCR；
For example, the DNA prepare liquids for taking DNA content to be 500ng, add after pre-designed family of V areas primer and family of J areas primer and carry out multiplex PCR with QIAGEN multiplex PCR Kit, carry out 30 circulations (cycles).What deserves to be explained is there is preferable expanding effect using slightly longer primer, for example, primer 15~20 bases of length that pre-designed family of V areas primer and family of J areas primer is more conventional；For example, pre-designed 15~20 bases of family of V areas primer and family of J areas primer extension are set；And for example, pre-designed family of V areas primer and the more conventional primer of family of J areas primer increase by 15~20 bases.
Electrophoresis detection reclaims multiple PCR products；
For example, electrophoresis detection reclaims 100~190bp multiple PCR products, and reclaimed using QIAquick Gel Extraction kit.
Add end maintenance mixed liquor and carry out end maintenance reaction, and purify；
For example, under the conditions of 20 DEG C, 10 × End Repair buffer and End Repair Mix are added, end is carried out and repairs reaction, and purified using QIAquick PCR Purification Kit.
Product after purification is connected into A bases at 3 ' ends；
For example, add adenine (A) at the end of DNA fragmentation 3 ' of filling-in end using NEBNext dA-tailing module.And for example, under conditions of 37 DEG C, NEBNext dA-tailing buffer and NEBNext dA-tailing enzyme are added, are reacted 30 minutes.And for example, the dosage of the polymerase in increase system, for example, the dosage increase by 200%~300% of more conventional polymerase；That is, the dosage of polymerase is designed as the 300%~400% of conventional amount used.
Joint connection is carried out in joint coupled reaction system；Preferably, in the reaction, according to the concentration of reaction effect Promoting Layered Buffer salt before.
Connected for example, preparing joint coupled reaction system with Adapter oligo mix and DNA ligase and carrying out joint.
The DNA fragmentation modified by PCR reaction enrichments by joint, and row agarose gel electrophoresis are entered to PCR primer, after cutting purpose fragment, purifying recovery, obtain DNA library.Effect is cut for lifting, it is preferred that melting temperature is more than 67 degrees Celsius；And for example, melting temperature is 67 degrees Celsius~70 degrees Celsius, and for example, the processing of cooling afterwards, for example, being down to room temperature.
In the present embodiment, after the product PCR amplifications after joint is connected, row agarose gel electrophoresis are entered to PCR primer, after cutting purpose fragment, glue purification recovery is carried out using QIAquick Gel Extraction kit, is dissolved in elution buffer.
In an embodiment of the present invention, in addition to detect the Insert Fragment scope of the DNA library and the concentration of the DNA library is quantified.
For example, detecting the gene library includes the concentration for the Insert Fragment scope and quantitative determination gene library for detecting the gene library.And for example, use the Insert Fragment scope in Agilent 2100Bioanalyzer (Reagents of Agilent DNA 1000) detections library, and for example, concentration is carried out to library using ABI StepOnePlus Real-Time PCR System (TaqMan Probe) to quantify.
S150, high-flux sequence is carried out to the DNA library, data analysis is carried out to the TCR β chains complementary determining region in the experimental group and control group.
High-flux sequence is carried out to the DNA library, data analysis is carried out to the TCR β chains complementary determining region in the experimental group and control group, comprised the following steps：Analysis of control group and Decompensated stage hbv-liver cirrhosis patient is preoperative 1 day, CDR3 areas diversity, the distribution of lengths feature of TCR β chains be each in postoperative 1,7 day PBLC and expression frequency, base insertion and deletion situation and the V-J pairings of each gene family；Filter out control group and Decompensated stage hbv-liver cirrhosis patient it is preoperative 1 day, postoperative 1 day, postoperative 7 days in height Clonal expansion (frequency be more than 0.5%) amino acid sequence (AA), DNA sequence dna and V-J combinations；And go to analyze that Decompensated stage hbv-liver cirrhosis patient is preoperative 1 day, postoperative 1, postoperative 7 days and NC groups TCR diversity and clonal expression difference according to Distinct (uniq) clone numbers and Simpson's coefficient.
For example, it is anticipated that upper machine data volume, is detecting qualified gene library addition 0.5mmol/L NaOH solution, is being denatured 30 minutes, makes double chain DNA fragment be denatured to form single stranded DNA.And the library after denaturation is added in FlowCell, hybridized with the joint on FlowCell, TruSeq PE Cluster Kit v3-cBot-HS reagents are then combined on cluster generating platform cBot and complete bridge-type PCR amplifications.The FlowCell prepared is finally subjected to upper machine sequencing using the sequencing systems of HiSeq 2000 and TruSeq SBS KIT-HS v3 reagents.
For example, also include after sequencing：Quality Control is carried out to sequencing data and pre-processes, the sequencing data is compared, sequential structure is analyzed, builds immune group storehouse, immune group storehouse difference analysis and statistical analysis.
For example, sequencing data progress Quality Control and pretreatment are specifically included：Under the sequential structure progress analyze data of the sequencing data after machine, filtration treatment is done to initial data first, during filtering, filters out the sequence containing joint first；Reads of the draw quality less than 15 (Illumina 0-41 quality systems) is removed；For unknown base (N bases), it is desirable to which base number can not be more than 5%；For the second-rate sequence of tail of sequence, intercepted to low quality part (quality is less than 10 base), ensure the sequence average quality after interception higher than 15, meanwhile, the length requirement of residue sequence is not less than 60 after interception.According to above filter condition, the cleandata after being filtered.After the completion of filtering, the two sequences of Pair-end (PE) data are spliced into a complete contig (contigs), in the case where reading length and being 100, splicing is divided into two steps：1st, the afterbodys of two sequences is compared, finds matching sequence, such splicing needs the overlapping of minimum 10 bases, the matching rate of overlapping region base however less than 90%；2nd, in view of the length of immune group storehouse capture region is different, that terminal sequence is tested logical in some cases can be present, in part 1 afterbody splices remaining sequence, trial head is continued to splice, and the sequence after final merging and the sequence contig of merging staple diagram can be obtained after data processing above.
For example, using high-flux sequence method (Illumina2000 genome analysises instrument), the peripheral blood TCR β chain complementary determining regions of 1 day group preoperative to experimental group, postoperative 1 day group, postoperative 7 days groups and control group totally 24 samples are sequenced.It is long (Total Reads) to obtain 17769012.83 total readings for average each sample in control group, average each sample obtains 16499515.8 total reading length in preoperative 1 day group, average each sample obtains 17158190.3 total reading length in postoperative 1 day group, and average each sample obtains 20811576.2 total reading length in postoperative 7 days groups.Then these initial data are done with filtration treatment (filter out and be less than the 15 and long Reads of reading of the unknown base (N bases) more than 5% containing joint, average quality), obtained result：The average filtration rate of control group is 4.37%, and the average filtration rate of preoperative 1 day group is 11.27%, and the average filtration rate of postoperative 1 day group is 18.70%, and the average filtration rate of postoperative 7 days groups is 11.82%.After the completion of filtering, the two sequences of Pair-end (PE) data are spliced into a complete contig, finally give the complete high-quality sequence after merging.Averagely there are 9446150.2 high-quality sequences (Total good sequences) in control group, averagely there are 9090364.7 high-quality sequences in preoperative 1 day group, averagely there are 8385257.2 high-quality sequences in postoperative 1 day group, averagely there are 12796683.8 high-quality sequences in postoperative 7 days groups.And average clone (Clones) number of control group is 79360.7, average clone (Clones) number of preoperative 1 day group is 46274.5, average clone (Clones) number of postoperative 1 day group is 28850.8, and average clone (Clones) number of postoperative 7 days groups is 80009.5.
Specifically included for example, the sequencing data is compared：φt cell receptor is corrected automatically using the milaboratory miTCR developed, corrects the sequence errors brought by reasons such as PCR, sequencings.After the completion of comparison, the expression of programming count complementary determining region clone and insertion and deletion situation.
Specifically included for example, carrying out analysis to the sequential structure of the sequencing data：The result obtained according to analysis is compared, obtain comparison and obtain the last site of V gene sequence, the initiation site of D genes, the termination site of D genes and the initiation site of J genes, the base of V-D-J insertion and deletions is found by site information and counts its distribution of lengths.The complementary determining region found according to comparison, and count the distribution of lengths of complementary determining region sequence.
And for example, to 6 (preoperative 1 day time points of liver cirrhosis due to hbv Decompensated stage patients'perioperative three, postoperative 1 day and it is postoperative 7 days) and 6 normal health person's peripheral bloods TCR β chains complementary determining region (CDR3), VD Indel and DJ Indel distribution of lengths is counted and averaged, it was found that 1 day group in the preoperative, postoperative 1 day group, in postoperative 7 days groups and control group, find TCR β CDR3 distribution of lengths between 16nt~106nt according to statistics, and it was found that 5 maximum CDR3 length of middle distribution frequency are before and after liver cirrhosis due to hbv Decompensated stage graft：It is wherein 42,39,45,36 and 48nt in 1 day group in the preoperative, is 42,45,39,48 and 36nt in 1 day after surgery group, be 42,45,39,36 and 48nt in 7 days after surgery groups, the maximum CDR3 length of 5 frequencies is 42,45,36,39,48nt in control group；DJ indel distribution of lengths is between -1~68nt, and 5 maximum DJ indel length of distribution frequency are respectively wherein in each group of liver cirrhosis due to hbv patient：It is 0,2,3,1 and 4nt in 1 day group in the preoperative, is 0,2,3,1 in postoperative 1 day group, and 4nt, be 0,2,3,1 and 4nt in postoperative 7 days groups, and in control group, 5 DJ indel length of frequency maximum are 0,3,2,1 and 4nt；VD indel distribution of lengths is -1~73nt sections, wherein 5 maximum VD indel length of distribution frequency are 0,3 in preoperative 1 day group, 2,1 and 4nt, it is 0 in postoperative 1 day group, 2,3,1 and 4nt, it is 0,3,2 in postoperative 7 days groups, 4 and 1nt, the maximum VD indel length of 5 frequencies is 1,0 in control group, 2,3 and 4nt；It can be seen from analysis result above, distribution frequency 1 day before liver cirrhosis due to hbv Decompensated stage graft, after transplanting after 1 day and transplanting in the PMNC of 7 days in TCR β CDR3, DJ inDel, VD indel distribution of lengths frequencies and control group is similar, without significant difference.
For example, the sequencing result, which is analyzed, also to be included：
1st, the structure in immune group storehouse, it is specifically included：The result obtained according to analysis is compared, the expression of each clone is counted, count DNA sequence dna, amino acid sequence respectively, V-J combines the expression of each sample obtained in the case of three kinds of different resolutions.For example, in order to weigh the diversity of sample, the distinct clone numbers of each sample are counted and calculated respectively, Simpson's coefficient and Shannon prestige receive coefficient, and three coefficients combine three kinds of different resolution ratio for DNA, amino acid, V-J respectively.For the expression each cloned in each sample, the expression each cloned according to three kinds of DNA sequence dna, amino acid sequence, V-J combinations different resolutions statistics.Filter out the DNA sequence dna of the height Clonal expansion (frequency is more than 0.5%) of each cell subsets, amino acid sequence, V-J combinations.
2nd, the variance analysis in immune group storehouse, it mainly includes：Sample Diversity and clonal expression difference two parts.Wherein, sample Diversity is intended to find out the difference of sample room entirety clonal expression pattern, and differential expression is then intended to find out the clone of significant difference.Sample Diversity first against be various property coefficient that each sample calculates, i.e. Distinct (uniq) clone numbers, Simpson and Shannon prestige receive coefficient.According to sample attribute between different groups, it is directed to DNA sequence dna, amino acid sequence, V-J and combines three kinds of resolution ratio, statistics calculates the significance of difference.Further, three aspects are combined in DNA sequence dna, amino acid sequence and V-J to be analyzed, calculates the P values of each difference pair using the corresponding verification scheme such as Poisson distribution, while P values are corrected using FDR and bonferroni.
For example, clonal expansion degree is divided into >=0.5%, 0.05~0.5%, 0.005~0.05%, 0.0005~0.005% He<0.0005% 5 grade, Clones of the definition clone's frequency more than 0.5% is height amplification property clone (highly expanded clones, HEC the clonal expansion degree), and from three kinds of DNA sequence dna, amino acid sequence, V-J combinations different resolutions statistics each cloned.The computational methods of each Clone Clonal expansion degree are：Total Total good sequences numbers of the number that the Clone occurs in the test sample/sample.In DNA sequence dna, it was found that the preoperative 1 day group (pre) of experimental group, postoperative 1 day group (post1), height amplification property clone's number (highly expanded clones, HECs) of postoperative 7 days groups (post7) and control group (NC) are respectively 49,43,41 and 14；It is corresponding in amino acid sequence, preoperative 1 day group, postoperative 1 day group, height amplification property clone's number of postoperative 7 days groups and control group is respectively 49,43,41 and 14；In V-J combinations, preoperative 1 day group, postoperative 1 day group, height amplification property clone's number of postoperative 7 days groups and control group distinguishes 292,299,304 and 232.In DNA and amino acid sequence, height amplification property clone's number in postoperative 7 days groups is all fewer than control group, preoperative 1 day group and postoperative 1 day group, and its height amplification property clone's number is most in V-J combinations.In all Clones, the ratio very little shared by height amplification property clone (HECs).By taking DNA sequence dna as an example, the clones for cloning frequency >=0.5% in NC groups has 1.11 × 10-2%, and ratio shared in all Clones HEC in pre groups is 4.99 × 10-2%, equally, there is 3.35 × 10 respectively in post1, post7-2% and 3.86 × 10-3% clones belongs to HEC, according to analysis result it is recognised that not having significant difference (p=0.180 ＞ 0.05) between this four groups.But go to analyze in terms of overall Clonal expansion degree, frequency distribution can be found to clone in tri- patient's groups of PRE, post1, post7 >=0.5%, the Clones quantity in 0.05~0.5% and 0.005~0.05% section is significantly more than NC groups, and it is substantially fewer than NC groups in 0.0005~0.005% section, this reveals that the Clonal expansion degree in pre, post1, post7 patient's group is substantially higher than control group.
For further quantitative preoperative 1 day group, postoperative 1 day group, the TCR β chain CDR3 polymorphisms of postoperative 7 days groups and control group, each Clone frequency is further counted using Simpson (Simpson) inverse exponential (1/Ds).The excursion of this index from 1 to ∞, in this range 1 show that the amplification degree of all individual Clones in sample is uneven, diversity is minimum；For ∞ then on the contrary, representing that sample Clones amplification degree is more uniform, its diversity is maximum, i.e. the amplification degree of the bigger clone for illustrating that sample is all of the value of Simpson coefficients is more uniform, and its diversity is higher.For example, combining the TCR β chain CDR3 diversity of the preoperative 1 day group of three kinds of resolution analysis, postoperative 1 day group, postoperative 7 days groups and control group from DNA sequence dna, amino acid sequence, V-J, three kinds of resolution ratio show control group：DNA level：1/Ds=1.023；Amino acid and V-J combined horizontals：1.031；The TCR β chain CDR3 diversity of preoperative 1 day group：DNA level：1/Ds≈1.007；Amino acid and V-J combined horizontals：1.017；The TCR β chain CDR3 diversity that art is organized one day after：DNA level：1/Ds≈1.005；Amino acid and V-J combined horizontals：1.014；The TCR β chain CDR3 diversity of postoperative 7 days groups：DNA level：1/Ds≈1.001；Amino acid and V-J combined horizontals：1.009), above contrast shows：Compared with control group, the diversity of preoperative 1 day group and postoperative 1 day group is relative to be suppressed, and TCR β chain CDR3 diversity is in suppression trend between 1 day after surgery group and postoperative 7 days groups.
It is it is appreciated that also relatively uniform for the high sample of diversity, its expression.According to analysis above, there are 50,43,41 and 14 HECs respectively in NC groups, pre patient's group, post1 patient's group and post7 groups, further respectively to being analyzed between Different Individual in NC groups, pre groups, post1 groups and post7 group groups either group with the presence or absence of shared HECs.As a result find, each individual has a unique group storehouse, lacks between these group of storehouse a part of overlapping.Do not have common HECs in DNA sequence dna and amino acid sequence between Different Individual simultaneously, and also without common HECs between NC groups, pre groups, post1 groups and post7 group groups；On the contrary, NC groups, pre groups are had been found that in V-J combinations, is all existed in the inconsistent shared combination of many frequencies in post1 groups and post7 patient's group group between Different Individual and between group.
The processing method of above-mentioned TCR β chain complementary determining regions, it is reasonable in design feasible, comprehensive analysis Decompensated stage hbv-liver cirrhosis graft perioperatively its internal TCR β chains complementary determining region receptoire dynamic change characterization, it can be appreciated that the immune state of patient, direction of medication usage is carried out for the monitoring of disease event after transplanting, prevention of postoperative occurs repelling, infected, and provides foundation for the pathogenesis of disease.
You need to add is that, the direct purpose of the present invention is not to obtain diagnostic result or health status, and it is the body fluid to having been detached from human body, that is peripheral blood, handled or detected to obtain the method as the information of intermediate result, or the method for handling the information, according to current medical knowledge and content disclosed in this invention, the diagnostic result of disease can not be immediately arrived in itself from the information obtained, but use the result that DNA analysis is carried out to TCR β chains in the experimental group and control group or the analysis result of the T cell antigen receptor β chain complementary determining region of the experimental group and control group, further research can be made to the acute rejection of peri-operation period liver transplant recipients as intermediate data, to be advantageous to the long-term surviving of transplant recipient and graft.
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, combination not all possible to each technical characteristic in above-described embodiment is all described, but, as long as contradiction is not present in the combination of these technical characteristics, the scope of this specification record is all considered to be.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but can not therefore be construed as limiting the scope of the patent.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
1. a kind of processing method of TCR β chain complementary determining regions, it is characterised in that comprise the following steps：
Experimental group and control group, every group of peripheral blood for having been detached from human body for including the different human body of several pieces are set
Sample；
The lymphocyte of each periphery blood specimen is separated, and extracts the periphery blood specimen lymphocyte respectively
In DNA；
Using multiple PCR technique, constructed dna library；
High-flux sequence is carried out to the DNA library, it is mutual to the TCR β chains in the experimental group and control group
Mend and determine that area carries out data analysis.
2. processing method according to claim 1, it is characterised in that before multiple PCR technique,
The processing method also includes the step of measure DNA content and detection DNA integralities.
3. processing method according to claim 2, it is characterised in that determined using XRF
DNA content.
4. processing method according to claim 2, it is characterised in that examined with agarose gel electrophoresis method
Survey DNA integralities.
5. processing method according to claim 1, it is characterised in that put down using Illumina MiSeq
Platform carries out high-flux sequence to DNA library.
6. processing method according to claim 1, it is characterised in that using multiple PCR technique, structure
DNA library is built, it is specifically included：
The amount of taking of DNA in the experimental group and control group is calculated, is weighed, and adds V areas primer and J
Area's primer carries out multiplex PCR；
Electrophoresis detection reclaims multiple PCR products；
Add end maintenance mixed liquor and carry out end maintenance reaction, and purify；
Product after purification is connected into A bases at 3 ' ends；
Joint connection is carried out in joint coupled reaction system；
The DNA fragmentation modified by PCR reaction enrichments by joint, and agarose is carried out to PCR primer
Gel electrophoresis, after cutting purpose fragment, purifying recovery, obtain DNA library.
7. processing method according to claim 6, it is characterised in that behind constructed dna library, also
Including detecting the Insert Fragment scope of the DNA library and the concentration of the DNA library being quantified.
8. processing method according to claim 1, it is characterised in that to the experimental group and control group
In TCR β chains complementary determining region carry out data analysis include：Quality Control is carried out to sequencing data and is pre-processed,
Data are compared, sequential structure is analyzed, immune group storehouse is built and immune group storehouse is carried out
Variance analysis.
9. processing method according to claim 8, it is characterised in that to sequencing data carry out Quality Control and
Pretreatment includes：Filtration treatment is carried out to the data after sequencing.
10. processing method according to claim 1, it is characterised in that using Ficoll density gradients from
Heart method isolates the lymphocyte of the periphery blood specimen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610317368.XA CN107345242A (en) | 2016-05-12 | 2016-05-12 | The processing method of TCR β chain complementary determining regions |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610317368.XA CN107345242A (en) | 2016-05-12 | 2016-05-12 | The processing method of TCR β chain complementary determining regions |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107345242A true CN107345242A (en) | 2017-11-14 |
Family
ID=60254508
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610317368.XA Pending CN107345242A (en) | 2016-05-12 | 2016-05-12 | The processing method of TCR β chain complementary determining regions |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107345242A (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011107595A1 (en) * | 2010-03-04 | 2011-09-09 | Charité Universitätsmedizin Berlin | High throughput analysis of t-cell receptor repertoires |
| CN103205420A (en) * | 2012-01-13 | 2013-07-17 | 深圳华大基因科技有限公司 | Primer composition for amplifying T cell receptor beta chain CDR3 coding sequence and application thereof |
| US20150104441A1 (en) * | 2013-10-11 | 2015-04-16 | Oslo Universitetssykehus Hf | Identification of disease-driving antigens |
| WO2015136072A1 (en) * | 2014-03-14 | 2015-09-17 | Immunocore Limited | Tcr libraries |
| CN105040111A (en) * | 2015-05-28 | 2015-11-11 | 眭维国 | Construction method of systemic lupus erythematosus map model |
| CN105087789A (en) * | 2015-08-10 | 2015-11-25 | 北京吉因加科技有限公司 | Method for detecting BCR and TCR immune repertoire in blood plasma cfDNA |
| CN105506746A (en) * | 2014-09-22 | 2016-04-20 | 深圳华大基因科技有限公司 | Method for constructing variable region sequencing library, and method for determining variable region nucleic acid sequence |
-
2016
- 2016-05-12 CN CN201610317368.XA patent/CN107345242A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011107595A1 (en) * | 2010-03-04 | 2011-09-09 | Charité Universitätsmedizin Berlin | High throughput analysis of t-cell receptor repertoires |
| CN103205420A (en) * | 2012-01-13 | 2013-07-17 | 深圳华大基因科技有限公司 | Primer composition for amplifying T cell receptor beta chain CDR3 coding sequence and application thereof |
| US20150104441A1 (en) * | 2013-10-11 | 2015-04-16 | Oslo Universitetssykehus Hf | Identification of disease-driving antigens |
| WO2015136072A1 (en) * | 2014-03-14 | 2015-09-17 | Immunocore Limited | Tcr libraries |
| CN105506746A (en) * | 2014-09-22 | 2016-04-20 | 深圳华大基因科技有限公司 | Method for constructing variable region sequencing library, and method for determining variable region nucleic acid sequence |
| CN105040111A (en) * | 2015-05-28 | 2015-11-11 | 眭维国 | Construction method of systemic lupus erythematosus map model |
| CN105087789A (en) * | 2015-08-10 | 2015-11-25 | 北京吉因加科技有限公司 | Method for detecting BCR and TCR immune repertoire in blood plasma cfDNA |
Non-Patent Citations (4)
| Title |
|---|
| PHILLIP H HORNE等: ""Recipient Immune Repertoire and Engraftment Site Influence the Immune Pathway Effecting Acute Hepatocellular Allograft Rejection"", 《CELL TRANSPLANTATION》 * |
| WEIGUO SUI等: ""Composition and variation analysis of the TCR -chain CDR3 repertoire in systemic lupus erythematosus using high-throughput sequencing"", 《MOLECULAR IMMUNOLOGY》 * |
| 中国科学技术协会: "《免疫学学科发展报告2014-2015》", 30 April 2016, 中国科学技术出版社 * |
| 吴松 等: ""深度测序揭示多种肾癌亚型的人体内和人体间TCR-β链的异质性"", 《2015中国遗传学大会论文摘要汇编》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105040111B (en) | The construction method of systemic loupus erythematosus spectrum model | |
| CN104450901B (en) | The nucleic acid markers of quick diagnosis mucocutaneous lymphnode syndrome and test kit thereof | |
| CN104372087B (en) | The diagnosing cancer of liver mark being made up of serum microRNA and diagnostic kit | |
| CN102399898B (en) | Single nucleotide polymorphism (SNP) marker related with clinically cryptogenic non-obstructive azoospermia aided diagnosis and application of SNP marker | |
| CN109576370B (en) | Biomarker and detection kit for bladder cancer diagnosis and recurrence monitoring | |
| CN107475440A (en) | A kind of application of blood plasma excretion body biomarker in breast cancer diagnosis | |
| CN105039530A (en) | Mitochondria-related seminal plasma miRNAs taken as mankind severe asthenospermia markers, and applications thereof | |
| CN106834287A (en) | A kind of SNP marker for detecting RhD negative phenotypes | |
| CN103103161A (en) | Separation and purification method for fetal nucleated red blood cells and Down syndrome screening kit | |
| CN107345242A (en) | The processing method of TCR β chain complementary determining regions | |
| CN107190071A (en) | A kind of SNP marker for being used to detect RhD variation phenotypes | |
| CN105671179B (en) | application of serum microRNA in liver cancer diagnosis and diagnosis kit | |
| CN106967808A (en) | A kind of primer sets and its application for being used to detect RhD negative blood groups | |
| CN112410414B (en) | Application of pregnant woman serum exosome miRNA marker in preparation of early diagnosis product of fetal congenital heart disease and kit | |
| CN107345240A (en) | T cell antigen receptor β chain CDR3 processing method | |
| CN107917835A (en) | A kind of blood-taking device for screening circulating tumor cell | |
| CN105132572B (en) | A kind of patau syndrome kit of noninvasive Prenatal Screening 21 | |
| CN203855580U (en) | System for IgA nephropathy detection | |
| CN103290006B (en) | A clinically unexplained NOA-related mitochondrial DNA SNP marker and applications thereof | |
| CN114015765B (en) | System, method and method establishment for IgA nephropathy detection | |
| CN115803448A (en) | Micronucleus DNA from peripheral red blood cells and uses thereof | |
| CN107345241A (en) | B cell antigen receptor H chains CDR3 processing method | |
| CN110205377A (en) | The assessment in advance of Kawasaki disease risk | |
| CN105671178B (en) | Serum microRNA diagnosing cancer of liver marker and kit | |
| Grosser et al. | A bibliometric review of peripartum cardiomyopathy compared to other cardiomyopathies using artificial intelligence and machine learning |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171114 |
|
| RJ01 | Rejection of invention patent application after publication |