CN103468697B - Aeromonas hydrophila is fit and its screening technique and application - Google Patents
Aeromonas hydrophila is fit and its screening technique and application Download PDFInfo
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Abstract
The present invention relates to Aeromonas hydrophila is fit and its screening technique and application.Offer Aeromonas hydrophila is fit and the fit screening technique of the Aeromonas hydrophila and application.The specific steps for the screening technique that the Aeromonas hydrophila is fit include：Build and synthesize the fit ssDNA libraries of Aeromonas hydrophila to be screened, and design the corresponding primer of synthesis；The appropriate ssDNA libraries are taken to be combined with Aeromonas hydrophila；Separation obtains the ssDNA combined with Aeromonas hydrophila, and PCT amplifications are carried out by template of the ssDNA, and amplified production carries out SELEX screenings until obtaining corresponding fit again.The present invention goes out the high-affinity oligonucleotide aptamers of pathogenic hydrophila gingivalis, the fit detection and analysis that can be widely applied to Aeromonas hydrophila using SELEX technology screenings.
Description
Technical field
The present invention relates to infective pathogen bacterium --- Aeromonas hydrophila common in aquaculture, more particularly to thermophilic aqueous vapor list
Born of the same parents bacterium is fit and its screening technique and application.
Background technology
Aeromonas hydrophila（Aeromonas hydrophila）In distributed in nature extensively, it is the original of a variety of aquatic animals
Hair property pathogenic bacteria, can produce a variety of virulence factors, to aquatic livestock, livestock and poultry and the mankind have pathogenic, can cause many animals
Septicemia and the mankind diarrhoea, to mankind's health care belt come threat, while also causing larger economic loss to aquaculture（1.
Kingfisher beautiful, Yu Zhouliang, Zhao Baohua, He Hong pavilions Advancement in Study on Aeromonas hydrophila [J] China Veterinary Journal, 2008,42（7）：46-
50.）.Therefore, for the Aeromonas hydrophila pollution as pollution of waterhead indicator bacteria, its inspecting force still needs to further reinforcement.
The detection of current Aeromonas hydrophila is always a stubborn problem, and traditional chemical pathological examination process is cumbersome, expends the time；
Then cost is too high for enzyme linked immunological, multiple PCR technique（2. several detection methods [J] inlands of Wang Li Fish Aeromonas Hydrophilas
Aquatic products, 2006,（2）：22-23.）.Therefore, develop a kind of easy to operate, with low cost, quickly and easily Aeromonas hydrophila is examined
Survey method, as those skilled in the art's technical barrier in the urgent need to address.
SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is to refer to
Number enrichment Fas lignand system evolution technology, which is that the one kind created by the Tuerk and Gold in the U.S. nineteen ninety is external, synthesizes the few core of screening
The chemical combination technology that thuja acid is fit（3. the Meng Qing tinkling of pieces of jade, old wound husband .SELEX technologies and its application prospect [J] Tarim University is learned
Report, 2008,20 (3):Liao's generation of 44-48. 4. is strange, Liu Wei, Wang Li .SELEX technologies application study progress [J] Gansu medicine,
2009, (02)：96-97.）.Due to fit with specificity is high, storage capacity is big, generated time is short（5. Wang Cong is gorgeous, Sun Wei, Li Jian
State, waits .SELEX technologies application study progress [J] biotechnology circulars, 2006, (S1)：232-236.）The advantages of, SELEX
Technology is widely used in the different technical field such as control and prevention of disease, drug screening, clinical diagnosis now, with the continuous wound of technology
Newly with development, gradually generate some new SELEX screening techniques, be such as oriented to SELEX technologies, compound target molecule SELEX technologies,
The research meanses of genomic SELEX innovation（6. the Meng Qing tinkling of pieces of jade, old wound husband .SELEX technologies and its application prospect [J] Tarim Basins
College journal, 2008,20 (3):44-48.）.
The content of the invention
The first object of the present invention is that offer Aeromonas hydrophila is fit.
The second object of the present invention is to provide the Aeromonas hydrophila fit screening technique.
It is fit in Aeromonas hydrophila is stated in detection that the third object of the present invention is to provide the Aeromonas hydrophila
Using.
The nucleotide sequence that the Aeromonas hydrophila is fit is as shown in the sequence in sequence table No. 1-22.
The screening technique that the Aeromonas hydrophila is fit is by index concentration Fas lignand system evolution technology（That is SELEX is sieved
Choosing）The screening fit applied to Aeromonas hydrophila, the specific steps of the SELEX screenings include：
1）Build and synthesize the fit ssDNA libraries of Aeromonas hydrophila to be screened, and design the corresponding primer of synthesis；
2）The appropriate ssDNA libraries are taken to be combined with Aeromonas hydrophila；
3）Separation obtains the ssDNA combined with Aeromonas hydrophila, and PCT amplifications, amplification production are carried out by template of the ssDNA
Thing carries out SELEX screenings until obtaining corresponding fit again.
In step 1）In, the ssDNA libraries can be：：5′-GGG AGC TCA GAA TAA ACG CTC AA-N35-
TT CGA CAT GAG GCC CGG ATC-3′。
The primer can be：
Primer I：5′-GGG AGC TCA GAA TAA ACG CTC AA-3′；
Primer II：5′-GAT CCG GGC CTC ATG TCG AA-3′；
Primer III：5 '-digoxin-GGG AGC TCA GAA TAA ACG CTC AA-3 '；
Primer IV：5 '-biotin-GAT CCG GGC CTC ATG TCG AA-3 '.
In step 3）In, the SELEX screenings carry out 12 and taken turns altogether.
The present invention goes out the high-affinity oligonucleotide aptamers of pathogenic hydrophila gingivalis, institute using SELEX technology screenings
State the exploitation of the fit Fast Detection Technique for Aeromonas hydrophila and the application in terms of aquatic products Disease management provide reference,
It can be widely applied to the detection and analysis of Aeromonas hydrophila.
Brief description of the drawings
Fig. 1 is the agarose gel electrophoresis figure of PCR primer 3%.In Fig. 1, M is 50bp DNA marker, 1,3,5,7,9,
11st, 12 the 1st, 3,5,7,9,11,12 PCR primers taken turns are represented respectively.
Fig. 2 is the fit absorbance combined with Aeromonas hydrophila.In fig. 2, abscissa is number of screening round, and ordinate is
Absorbance during A450nm.
The aptamer secondary structure figure that Fig. 3-Fig. 5 is screened for the present invention.Structure minimum free energy in Fig. 3 for-
7.60 kilocalories/rub, structure minimum free energy in Fig. 4 for 1.83 kilocalories/rub, the structure minimum free energy in Fig. 5 is -3.11 thousand
Block/rub.
Embodiment
First, the synthesis of ssDNA libraries and primer
Reference literature（7th, Liu Fengwei, research and Preliminary Applications [D] that blue small roc in-vitro screening pseudomonas aeruginosas are fit
Foochow Medical University Of Fujian, 2007.）Disclosed in method, for SELEX screen ssDNA pool and primer by Shanghai
Sheng Gong biotechnologies Services Co., Ltd synthesizes.
Library used is：5′-GGG AGC TCA GAA TAA ACG CTC AA-N35-TT CGA CAT GAG GCC
CGG ATC-3′。
The primer is respectively：
Primer I：5′-GGG AGC TCA GAA TAA ACG CTC AA-3′；
Primer II：5′-GAT CCG GGC CTC ATG TCG AA-3′；
Primer III：5 '-digoxin-GGG AGC TCA GAA TAA ACG CTC AA-3 '；
Primer IV：5 '-biotin-GAT CCG GGC CTC ATG TCG AA-3 '.
Above-mentioned primer is in post synthesis with the TE buffer solutions commonly used in molecular biology（pH8.0）Concentration is diluted to for 10 μ
Mol, -20 DEG C save backup.
2nd, prepared by experiment reagent and buffer solution
Dynabeads streptavidin magnetic bead M-280 kits, 2 × Taq PCR MasterMix, 50bp DNA
Ladder Marker, bovine serum albumin(BSA)（BSA）It is purchased from Xiamen Lu Long Bioisystech Co., Ltd, the purifying examination of PCR glue reclaims
Agent box is purchased from omega companies, and anti-digoxin alkaline phosphatase is purchased from Roche companies, and 4-NPP is Amresco public
Take charge of product.Buffer solution is with reference to bibliography (Liu Fengwei, research that blue small roc in-vitro screening pseudomonas aeruginosas are fit and preliminary
Using [D] Foochow Medical University Of Fujian, 2007.) prepare.
3rd, strain：Aeromonas hydrophila AS1.1801 is purchased from Chinese industrial Microbiological Culture Collection administrative center.
4th, SELEX is screened
Take a certain amount of ssDNA pool（First run screening consumption is 600pmol, and then often wheel reduces consumption）, add 600
μ L selection buffer solution dilution ssDNA, are denatured 5min in 95 DEG C, cool down 10min.Add 30 μ L Aeromonas hydrophila bacteria suspension
（The quantity of bacterium is about 1.5 × 108It is individual）, mixing is put centrifuges 10min at 30min, 15000r/min, 4 DEG C of shaking table room temperature combination.It
After abandon supernatant, add 600 μ L selection buffer solutions and be resuspended, such repeated washing 3 times washes away what is do not combined with Aeromonas hydrophila
SsDNA, the Aeromonas hydrophila for combining ssDNA is precipitated with 100 μ L ddH2O is resuspended, 96 DEG C of heating 5min, is taken after centrifugation
It is template clearly, entering performing PCR with primer I, IV expands, and obtains the double-stranded DNA of mark biotin（dsDNA）, it is logical using M-280 magnetic beads
The interaction separation ssDNA crossed between biotin-streptavidin, the secondary storehouse screened as next round.According to above-mentioned side
Method, SELEX screenings are carried out to secondary storehouse again, are successively carried out 12 and are taken turns SELEX screenings.
5th, fit measure and fit sequence and analysis with Aeromonas hydrophila Percentage bound is respectively taken turns
By the product of the 1st, 3,5,7,9,11,12 wheel SELEX screenings（That is the elution supernatant of the wheel）, with primer III and primer
IV enters performing PCR amplification, and each wheel expands 320 μ L.PCR amplification system is：2 × Taq PCR MasterMix 10ul, upstream is drawn
Thing (10um), anti-sense primer (10um) each 0.4ul, template 1ul, 20ul is supplied with deionized water.PCR reaction conditions are：95
DEG C pre-degeneration 3min；94 DEG C of denaturation 30s, 58 DEG C of -70 DEG C of annealing 30s, 72 DEG C of extension 20s, 24 circulations；72 DEG C of extension 3min.
Pcr amplification product is purified with glue reclaim purification kit, purified product is examined using 3% agarose electrophoresis
Survey, it can be seen that electrophoretic band is more and more finer and close, while diffusing phenomenon is fewer and fewer, band is more next with the increase of screening number of times
More neat and brightness increased.Illustrate that, with the increase of screening number of times, SPECIFIC APTAMER is enriched with, its homology may
Compare high
The dsDNA of purifying is unwind into ssDNA by PCR primer after purification with Streptavidin MagneSphere, and this ssDNA is to carry
Digoxigenin labeled, determines its content.SsDNA: Aeromonas hydrophila=300pmol: 1.5 × 10 are pressed again8Ratio, will both
30min is combined in 500 μ L selection buffer solutions, the alkali of the anti digoxin antibody mark of 100 μ L 1: 15000 dilutions is added after centrifugation
Acid phosphatase react 10min, centrifugation abandon supernatant and with 500 μ L selection buffer solution suspension Aeromonas hydrophila, repeated washing 3 times,
The alkaline phosphatase not marked with the anti digoxin antibody that ssDNA- digoxin is combined on Aeromonas hydrophila is washed away, is then added
100 μ L para-nitro-pheneye phosphates solution are developed the color, and 100 μ L 2mol/L NaOH terminating reactions are added after colour developing is good, with ELIASA in
405 nm determine absorbance, and experimental result is referring to Fig. 1.The affinity measurement result shows, with the increase of number of screening round, fits
The affinity of body and bacterium solution gradually increases, and maximum is reached when the 9th takes turns, and affinity change afterwards is little, tend towards stability, explanation
It is fit to have reached saturation.
The PCR primer of the 12nd wheel is largely expanded with primer I and primer II again, sent after purification limited in English fine horse biotechnology
It is random after company clone, clone to take 24 clone sequencings, obtain 22 satisfactory fit sequences.According to random sequence
The number of base is divided into A crowds, two groups of B groups.A groups（It is shown in Table 1）It is consistent with expected length including 13 fit sequences, B groups（See
Table 2）Comprising 9 sequences, it is shorter than expected length.Sequencing result is shown in Table 1.With the software kits of Macaw 2.05 analyze 22 it is fit
Homologous sequence, obtains 4 conserved sequences：GTTTTX（See A1-1, A1-8, A1-13, A1-17, A1-20, A1-21, A1-26,
No. A1-30 fit）；GTGGGT（See A1-1, A1-7, A1-12, A1-15, A1-22, A1-29）；GTTTX（See A1-1, A1-
5、A1-7、A1-10、A1-13、A1-14、A1-15、A1-18、A1-21、A1-22、A1-29）；XTTTT（See A1-14, A1-
26、A1-27）.Have it is fit in same conserved sequence occur multiple, there is GTTTG three times in such as A1-18；Have it is fit in then
There are multiple conserved sequences, but without find to be present in it is all it is fit in conserved sequence.
| It is fit | Numbering in sequence table | Nucleotide sequence |
| A1-3 | 1 | TGGTGGTCTGCTAGCGGTTTATGTTTTTGTCTGGT |
| A1-4 | 2 | AGGATGGATTAGTGGGATTGGTGGTTAGGGGGGG |
| A1-5 | 3 | GACCTCGTCTTCGGTGTGATCGTTTCTTGTTGTTG |
| A1-7 | 4 | GGTGTGTTGGTGGGTGTGTGTTTGGGTTCTTGGGT |
| A1-14 | 5 | TGCGGCTGTGTGGGTGGTTTGGGTTTGTTGCTTTT |
| A1-15 | 6 | GCGGTGGGTGGTAGGTTTGTGTTTGTTAATGGGTG |
| A1-17 | 7 | GTTGTCTCTGGTGTTTTGGATTGATGGCCTTGCTG |
| A1-18 | 8 | GGTGTCTGGTTTGTTGTGGTTGTTTGTGGTTTGTG |
| A1-20 | 9 | GCGGTTATGTGAAATTATGGCATGGTTTTGTGGTG |
| A1-26 | 10 | GGTCCATTGGCTGTGTTTTCTAGGTGTTATGTTTT |
| A1-27 | 11 | GGTCGTTAGACGGGGCTAGTAATATATTTCCTTTT |
| A1-29 | 12 | CTGTCGTGTGTTTGGGTGTGGGTCGTGTGGTTGGT |
| A1-31 | 13 | GTATAGATGTACTGCTGTGCATATCCTATGCAACT |
The A groups of Aeromonas hydrophilas of table 1 are fit
| It is fit | Numbering in sequence table | Nucleotide sequence |
| A1-1 | 14 | GGTGGTGGGTGTTTTGTCGGTTTGGTGTTTGGT |
| A1-8 | 15 | TGGCGGTGTTTTTGGGTGGGGTTATAGG |
| A1-10 | 16 | CTGGCTGTTCGTTTAGAATGAACCGGTCATG |
| A1-12 | 17 | GTTGTGGTGGGGTCGGAGGTTGTCTGGT |
| A1-13 | 18 | TGGTGTTTGGTTGTTTTGTTTGG |
| A1-21 | 19 | TTTGTGGCGGGGTTTGCTTGGTTTTGTGGTGG |
| A1-22 | 20 | GGGGTGGGTGGGTGTGTGTTTGGTTGGTTTG |
| A1-30 | 21 | GGGTTGGTTTGGGGGGGGCTGTTTTG |
| A1-28 | 22 | GGGTGGGTTGGTTGGTGGTTGTTTTTGTG |
The B groups of Aeromonas hydrophilas of table 2 are fit
Secondary structure analysis are carried out to 22 measured sequences with DNAMAN softwares, three can be classified as according to structure
Class：
The first kind：The fixed sequence program area base at 5 ' ends and the 6th base at 3 ' ends rise and form loop-stem structure, middle random area
Form loop-stem structure, 5 fixed base formation, one small bag structure at 3 ' ends（Fig. 3）.This structure it is fit have A1-3,
A1-5, A1-7, A1-13, A1-14, A1-20, A1-22 totally seven；
Equations of The Second Kind：3rd to the 21 bases formation loop-stem structure at 5 ' ends, 3 ' end fixed sequence program area's bases and centre are random
Area's base formation stem ring is simultaneously raised, then forms a pocket by 5 ' ends, 3 ' end fixed sequence program area's bases and middle random area again
Structure（Fig. 4）, the fit of this structure have A1-1, A1-4, A1-12, A1-15, A1-17, A1-18, A1-21, A1-27, A1-
28th, A1-29, A1-30, A1-31 totally ten two；
3rd class：5 ' ends, 3 ' end fixed sequence program area number of base form loop-stem structure with random area respectively, then by 5 '
End, 3 ' end fixed sequence program area's bases and middle random area re-form a big bag structure（Fig. 5）.The fit of this structure has
A1-8, A1-10, A1-26 tri-.
The artificial synthesized ssDNA libraries total length 78bp of applicant, middle 35bp is random nucleotide, because each site has
35 sequences just have 4 in the middle of tetra- kinds of possibility of A, G, C, T, such words35Individual to combine, i.e., its storage capacity has reached 1016It
It is many, although may be only to 10 in actual experiment14 Left and right, but this also meet screening purpose oligonucleotides the need for（8th, Li Wei
Shore, Lan little Peng, Yang Xiangyue wait utilization [J] Foochow hospital general journal of the .SELEX technologies in screening Ciclosporin A is fit,
2007,(03)：171-173）.During preceding two-wheeled is screened, the library amount of applicant's addition is relatively more, and such purpose is
Obtain how fit with Aeromonas hydrophila specific binding as far as possible.But it is secondary with the increase of number of screening round
The amount in library will be reduced accordingly, because the concentration in library is increasing.In order to increase fit specificity,
Applicant accordingly adds washing steps, while time for being combined with bacterium solution of library is also after the 30min of former wheels is reduced to
The 20min come.The optimization of PCR conditions is also particularly significant to fit screening, and each round screening, applicant will first make a ladder
PCR is spent, PCR amplifications is done again on the premise of most suitable annealing temperature is obtained, so effectively avoids non-specific amplification etc. and ask
Topic.
Applicant's experiment is Streptavidin MagneSphere to be enriched with secondary library, and it is simple to operate, need not centrifuge, only
The effect of separation is can reach under the influence of a magnetic field, while the secondary library that it is obtained is more stable, purity is higher, is difficult
The problem of there is the non-specific amplification in asymmetric PCR.In 12 wheels are screened, obtain 22 of cloning and sequencing are fit, have
13 fit consistent always with expected length.
Using DNAMAN softwares to its secondary structure analysis, as can be seen from the results, the secondary structure of these sequences is all
It is by stem ring and pocket（Spoon shape）Structure composition, this illustrates that stem ring and bag structure are probably fit and Aeromonas hydrophila knot
The architecture basics of conjunction, stem may play a part of rock-steady structure, and ring is probably its binding site.Contain spoon in some secondary structures
The pocket of shape, these pockets may also play the same effect of same ring, it is possible to be the binding site combined with acceptor.At these
In stem structure, its base is mainly based on G-C, and because G-C structure can be more than A-T, this point also illustrates that stem may be played surely
It is set for using.
The preliminary SELEX methods that establish of the invention screen the fit method of Aeromonas hydrophila, while obtaining with special
The Aeromonas hydrophila of property it is fit, and the secondary structure of fit group is tentatively studied, obtains the one of its secondary structure
A little structure compositions, this has laid certain basis for the molecular structure of research Aeromonas hydrophila later, while being also thermophilic aqueous vapor
The detection of monad provides a kind of new method.
Claims (2)
1. Aeromonas hydrophila is fit, it is characterised in that its sequence is such as
GGGAGCTCAGAATAAACGCTCAACTGGCTGTTCGTTTAGAATGAACCGGTCATGTTCGACATGAGGCCCGGAT
Shown in C.
The application that 2. Aeromonas hydrophila as claimed in claim 1 is fit in detection Aeromonas hydrophila, the application is non-
The application of medical diagnosis on disease purpose.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101418335A (en) * | 2007-10-24 | 2009-04-29 | 福建出入境检验检疫局检验检疫技术中心 | Reagent for detection of pathogenic hydrophila gingivalis and detection method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101418335A (en) * | 2007-10-24 | 2009-04-29 | 福建出入境检验检疫局检验检疫技术中心 | Reagent for detection of pathogenic hydrophila gingivalis and detection method thereof |
Non-Patent Citations (3)
| Title |
|---|
| SELEX技术在致病菌检测中应用进展;余晓峰等;《家禽科学》;20110731(第7期);摘要 * |
| 体外筛选铜绿假单胞菌适体的研究及初步应用;刘丰伟;《中国优秀硕士论文全文数据库 医药卫生科技辑》;20070115(第1期);1.2.1节-1.2.5节 * |
| 致病性嗜水气单胞菌多重PCR检测方法的建立;饶静静等;《中国水产科学》;20070930;第14卷(第5期);摘要 * |
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