CN104562212A - Cone shell venom tube cDNA library as well construction method and application thereof - Google Patents
Cone shell venom tube cDNA library as well construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a cone shell venom tube cDNA library as well a construction method and application thereof. The construction method of the cone shell venom tube cDNA library comprises the following steps: conducting reverse transcription on total RNA of the cone shell venom tube into cDNA; conducting enzyme digestion for recycling fragments of 400-1400bp in cDNA to acquire target fragments, wherein the cone shell venom tube cDNA library is composed of the target fragments. By adopting the constitution method, a cone shell venom tube full-length cDNA library aiming at the target fragments in specific length can be efficiently constructed, the full-length gene sequence of cone shell toxin peptide in the cone shell venom tube can be further determined on the basis of the cDNA library, and the probability of acquiring conotoxin can be effectively improved.
Description
Technical field
The present invention relates to the method building cone shell venom duct cDNA library, particularly, relate to cone shell venom duct cDNA library and construction process thereof and purposes.More specifically, relate to cone shell venom duct cDNA library and construction process thereof, determine the method for conotoxin peptide full-length gene order in cone shell venom duct.
Background technology
Conotoxin has high genetic diversity, its chemical structure is novel, be rich in disulfide linkage, and bioactive functions is strong, the targets such as ligand-gated ion channel, voltage gated ion channel and G-protein associated receptor can be acted on by highly selective, and can distinguish various ionic channel hypotype and subunit thereof, become the important tool of pharmacology and Neuroscience Research and the new source of new drug development, cause the extensive concern of international bio and pharmacy circle.Although conotoxin is of a great variety, but up to now, the conotoxin peptide found and study is less than the thousandth of appreciable amt amount, and this shows that cone shell venom is a potentiality unlimited " natural marine medicine treasure-house ", waits the deep research of the mankind, development and utilization.In recent years, study conotoxin, be developed to new type nerve polypeptide drug and become international popular research direction.
Traditional conotoxin drug screening is separating natural conotoxin from the venom duct of nature live body cone shell mostly, then investigation and application is directly used in or in this, as pharmaceutical chemical lead compound, design further again, process, synthesize, screen effective function medicament.But this method has certain blindness, the screening cycle is long.
Therefore, the research in the screening of conotoxin still needs deeply.
Summary of the invention
The present invention is intended at least to solve one of technical problem existed in prior art.For this reason, one object of the present invention is a kind of method proposing high flux screening conotoxin full-length gene.
According to an aspect of the present invention, the invention provides a kind of method building cone shell venom duct cDNA library.According to embodiments of the invention, the method comprises the following steps: be cDNA by the total serum IgE reverse transcription of described cone shell venom duct; And enzyme cuts back to close the fragment of 400-1400bp in described cDNA, to obtain object fragment, described object fragment forms described cone shell venom duct cDNA library.Thus, utilize method of the present invention efficiently can build cone shell venom duct cDNA library for special length object fragment, determine conotoxin peptide full-length gene order in cone shell venom duct based on this cDNA library further, effectively can improve the probability obtaining conotoxin.
According to embodiments of the invention, Trizol LS method is utilized to extract the total serum IgE of described cone shell venom duct.Thus, extract that the total serum IgE quality of cone shell venom duct obtained is higher, integrity good, do not degrade, and simple to operate, convenient and swift.
According to embodiments of the invention, Creator SMART cDNA test kit is utilized to carry out described reverse transcription, CDSIII/3primer primer in wherein said Creator SMART cDNA test kit is replaced by CDS-3M adapter, and the sequence of described CDS-3Madapter is: 5 '-AAGCAGTGGTATCAACGCAGAGTGGCCGAGGCGGCCd (T) 20VN-3 '.Thereby, it is possible to effectively obtain the reverse transcription product cDNA of RNA sequence.
According to embodiments of the invention, carrying out before described enzyme cuts back to close, comprising further: described cDNA is carried out homogenization process.Thus, reduce the abundance of the cDNA that high copy exists in library, improve the probability finding stochastic sequence and rare gene, thus more how low-abundance conotoxin cDNA can be obtained, and then the greatest differences effectively overcome on gene transcription level is to library screening with analyze the obstacle brought.
According to embodiments of the invention, Trimmer-Director test kit is utilized to carry out described homogenization process.Thereby, it is possible to effectively realize the homogenization of full-length cDNA, and homogenization effect is better.
According to embodiments of the invention, utilize Sfi I Enzyme to carry out described enzyme and cut.Thereby, it is possible to efficiently carrying out enzyme to cDNA cuts process, obtain the object fragment of length-specific, improve digesting efficiency.
According to embodiments of the invention, QIAquick Gel Extraction test kit is utilized to carry out described recovery.Thus, the rate of recovery of object fragment is higher, and simple to operate, convenient and swift.
According to embodiments of the invention, comprise further and purifying is carried out to each step products.Thus, the quality of each step products can be improved, be beneficial to the carrying out of subsequent step.
According to embodiments of the invention, comprise further and described object fragment is connected to carrier and is converted in intestinal bacteria.Thereby, it is possible to effectively set up cone shell venom duct cDNA library.
According to a further aspect in the invention, present invention also offers a kind of cone shell venom duct cDNA library.According to embodiments of the invention, this cone shell venom duct cDNA library is built by the method for phase foregoing structure cone shell venom duct cDNA library to obtain.Thus, determine conotoxin peptide full-length gene order in cone shell venom duct based on cone shell venom duct cDNA library of the present invention, effectively can improve the probability obtaining conotoxin.
And then, in accordance with a further aspect of the present invention, present invention also offers a kind of method determining conotoxin peptide full-length gene order in cone shell venom duct.According to embodiments of the invention, the method comprises the following steps: according to the method for foregoing structure cone shell venom duct cDNA library, build the cDNA library of described cone shell venom duct; Described cDNA library is checked order, to obtain sequencing result; And based on described sequencing result, determine conotoxin peptide full-length gene order in described cone shell venom duct.Thus, utilize method of the present invention greatly can improve the probability obtaining conotoxin, can disposable, the cDNA sequence that obtains conotoxin polypeptide in large quantities, and the conotoxin sequence obtained is with a high credibility, can verify transcript profile sequencing data, make up transcript profile data assembling mismatch problems.
According to embodiments of the invention, utilize be selected from Hiseq2000, SOLID, 454 and at least one of single-molecule sequencing device carry out described order-checking.
According to embodiments of the invention, based on described sequencing result, determine that in described cone shell venom duct, conotoxin peptide full-length gene order comprises further: described sequencing result is carried out pre-treatment, to obtain through pretreated sequencing result; Compare described through pretreated sequencing result and conotoxin storehouse, to determine the conotoxin peptide full-length gene order of conotoxin in described cone shell venom duct.Thus, can efficiently determine conotoxin peptide full-length gene order, accuracy is higher.
It should be noted that, the present invention at least has the following advantages:
(1) the present invention is the mRNA structure cDNA library based on coding conotoxin peptide, obtains the entire open reading frame (ORF) of conotoxin, and then obtains the conotoxin precursor peptide of total length;
(2) homogenization process can obtain more how low-abundance conotoxin cDNA;
(3) cut the object segment cDNA for special length, greatly can improve the probability obtaining conotoxin;
(4) the high-flux sequence method that utilizes of the present invention can disposable, the cDNA sequence that obtains conotoxin polypeptide in large quantities;
(5) conotoxin sequence utilizing method of the present invention to obtain is with a high credibility, can verify transcript profile sequencing data, makes up transcript profile data assembling mismatch problems.
Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage will become obvious and easy understand from accompanying drawing below combining to the description of embodiment, wherein:
Fig. 1 shows the electrophoresis detection figure of conotoxin total serum IgE according to an embodiment of the invention;
Fig. 2 shows the electrophoresis detection figure after two chain cDNA synthesis according to an embodiment of the invention;
Fig. 3 shows according to an embodiment of the invention through the cDNA electrophoresis detection figure of homogenization process;
Fig. 4 shows according to an embodiment of the invention through the electrophoresis detection figure of the cDNA secondary PCR amplified production of homogenization process; And
Fig. 5 shows bacterium colony PCR electrophoresis detection figure according to an embodiment of the invention.
Embodiment
Embodiments of the invention are described below in detail.Embodiment described below is exemplary, only for explaining the present invention, and can not be interpreted as limitation of the present invention.
Cone shell venom duct cDNA library and construction process thereof
According to an aspect of the present invention, the invention provides a kind of method building cone shell venom duct cDNA library.According to embodiments of the invention, the method comprises the following steps: be cDNA by the total serum IgE reverse transcription of described cone shell venom duct; Enzyme cuts back to close the fragment of 400-1400bp in described cDNA, to obtain object fragment, described object fragment forms described cone shell venom duct cDNA library.Thus, utilize method of the present invention efficiently can build cone shell venom duct cDNA library for special length object fragment, utilize this cDNA library to determine conotoxin peptide full-length gene order in cone shell venom duct further, effectively can improve the probability obtaining conotoxin.
According to embodiments of the invention, the method extracting the total serum IgE of described cone shell venom duct is not particularly limited, as long as can obtain complete, undegradable RNA, those skilled in the art can select as the case may be flexibly.According to one embodiment of present invention, Trizol LS method is utilized to extract the total serum IgE of described cone shell venom duct.Thus, extract that the total serum IgE quality of cone shell venom duct obtained is higher, integrity good, do not degrade, and fast simple, convenient.
According to embodiments of the invention, the method for described reverse transcription is not particularly limited, and can only effectively obtain conotoxin full-length cDNA, and those skilled in the art can select according to specific experiment condition.According to one embodiment of present invention, Creator SMART cDNA test kit is utilized to carry out described reverse transcription, CDSIII/3primer primer in wherein said Creator SMART cDNA test kit is replaced by CDS-3M adapter, the sequence of described CDS-3M adapter is: 5 '-AAGCAGTGGTATCAACGCAGAGTGGCCGAGGCGGCCd (T) 20VN-3 ' (SEQ ID NO:1, wherein, this CDS-3M adapter sequence also can direct representation be: 5 '-AAGCAGTGGTATCAACGCAGAGTGGCCGAGGCGGCCTTTTTTTTTTTTTTTTTTTT VN-3 ').Thereby, it is possible to effectively obtain the reverse transcription product cDNA of RNA sequence.
According to embodiments of the invention, carrying out before described enzyme cuts back to close, needing described cDNA to carry out homogenization process.According to one embodiment of present invention, include but not limited to utilize Trimmer-Director test kit to carry out described homogenization process to the method for described cDNA being carried out homogenization process.Thus, reduce the abundance of the cDNA that high copy exists in library, improve the probability finding stochastic sequence and rare gene, thus more how low-abundance conotoxin cDNA can be obtained, and then the greatest differences effectively overcome on gene transcription level is to library screening with analyze the obstacle brought.
In order to improve the probability obtaining conotoxin, needing that enzyme is carried out to conotoxin cDNA and cutting process, to obtain the object segment cDNA of special length, and object fragment being reclaimed.According to embodiments of the invention, the method that described enzyme cuts process is not particularly limited, as long as can efficiently obtain object segment cDNA, those skilled in the art can select flexibly according to actual conditions.According to one embodiment of present invention, utilize Sfi I Enzyme to carry out described enzyme to cut.Thereby, it is possible to efficiently carrying out enzyme to cDNA cuts process, obtain the object fragment of length-specific, improve digesting efficiency.According to another embodiment of the invention, the mode reclaimed object segment cDNA includes but not limited to utilize QIAquick Gel Extraction test kit to carry out described recovery.Thus, the rate of recovery of object fragment is higher, and simple to operate, convenient and swift.
According to embodiments of the invention, comprise further and purifying is carried out to each step products.Thus, the quality of each step products can be improved, be beneficial to the carrying out of subsequent step.
According to embodiments of the invention, comprise further and described object fragment is connected to carrier and is converted in intestinal bacteria.Thereby, it is possible to effectively set up cone shell venom duct cDNA library.
According to a further aspect in the invention, present invention also offers a kind of cone shell venom duct cDNA library.According to embodiments of the invention, this cone shell venom duct cDNA library is built by the method for phase foregoing structure cone shell venom duct cDNA library to obtain.Thus, determine conotoxin peptide full-length gene order in cone shell venom duct based on cone shell venom duct cDNA library of the present invention, effectively can improve the probability obtaining conotoxin.
Determine the method for conotoxin peptide full-length gene order in cone shell venom duct
And then, in accordance with a further aspect of the present invention, present invention also offers a kind of method determining conotoxin peptide full-length gene order in cone shell venom duct.According to embodiments of the invention, the method comprises the following steps: according to the method for foregoing structure cone shell venom duct cDNA library, build the cDNA library of described cone shell venom duct; Described cDNA library is checked order, to obtain sequencing result; And based on described sequencing result, determine conotoxin peptide full-length gene order in described cone shell venom duct.Thus, utilize method of the present invention greatly can improve the probability obtaining conotoxin, can disposable, the cDNA sequence that obtains conotoxin polypeptide in large quantities, and the conotoxin sequence obtained is with a high credibility, can verify transcript profile sequencing data, make up transcript profile data assembling mismatch problems.
According to embodiments of the invention, be not particularly limited the method that described cDNA library checks order, those skilled in the art can select as the case may be flexibly.According to concrete examples more of the present invention, can utilize be selected from ABI3730xl, Hiseq2000, SOLID, 454 and at least one of single-molecule sequencing device carry out described order-checking, preferred ABI3730xl sequenator.
According to embodiments of the invention, based on described sequencing result, determine that in described cone shell venom duct, conotoxin peptide full-length gene order comprises further: described sequencing result is carried out pre-treatment, to obtain through pretreated sequencing result; Compare described through pretreated sequencing result and conotoxin storehouse, to determine the conotoxin peptide full-length gene order of conotoxin in described cone shell venom duct.Thus, can efficiently determine conotoxin peptide full-length gene order, accuracy is higher.
Below in conjunction with embodiment, the solution of the present invention is made an explanation.It will be understood to those of skill in the art that the following examples only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition in embodiment, (such as show with reference to J. Pehanorm Brooker etc. according to the technology described by the document in this area or condition, " Molecular Cloning: A Laboratory guide " that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, be and by the conventional products of commercial acquisition, such as, can be able to purchase from Illumina company.
Embodiment 1 builds cone shell venom duct cDNA library
The acquisition of 1.1 barrel-shaped cone shells
The barrel-shaped cone shell individuality that the present embodiment adopts picks up from surrounding waters, Lingshui county, Sanya, Hainan Province on May 26th, 2012 to June 7, artificial breeding in fresh and alive air transport on June 8 to BGI-Shenzhen aquarium in 2012.
1.2 barrel-shaped cone shell venom duct Total RNAs extraction
In aseptic experiment room, separate plane cone shell, take out venom duct, directly put into the mortar that liquid nitrogen is housed after removing its hetero-organization be adhered, be ground to Powdered.Then adopt the Trizol LS method of improvement to extract total serum IgE, concrete steps are as follows:
(1) by the barrel-shaped cone shell venom duct powder transfer of above-mentioned acquisition in 2.0mL Eppendorf pipe, add 1.0mLTrizol reagent, concussion mixing, leave standstill 5min;
(2) in 4 DEG C, centrifugal 5min under 12,000 × g conditions, transfer supernatant liquor, in new 2.0mL Eppendorf pipe, then adds 200 μ L chloroforms, concussion mixing, then in 4 DEG C, centrifugal 10min under 12,000 × g conditions;
(3) shift in supernatant liquor to new 2.0mL Eppendorf pipe, then add isopyknic chloroform/primary isoamyl alcohol (24:1), fully mix, in 4 DEG C, centrifugal 10min under 12,000 × g conditions;
(4) chloroform/primary isoamyl alcohol (24:1) extractive process is repeated;
(5) shift in supernatant liquor to new 2.0mL Eppendorf pipe, add equal-volume Virahol, in-20 DEG C of standing 1h, then in 4 DEG C, centrifugal 20min under the condition of 13,600rpm, abandon supernatant;
(6) add 75% ethanol of precooling, in 4 DEG C of standing 3min, then in 4 DEG C, centrifugal 3min under 13,600rpm conditions, remove supernatant, repeat rinsing once;
(7) precipitation is placed in super clean bench dry air, then adds 40 μ L Nuclease-free H
2o dissolves RNA precipitation.The barrel-shaped cone shell venom duct total serum IgE obtained is after electrophoresis and UV spectrophotometer measuring, and packing is also placed in-80 DEG C of preservations.Detected result, in table 1 and Fig. 1, extracts the OD of the total serum IgE obtained
260/ OD
280be between RNA normal ratio scope 1.8 ~ 2.2, RNA banding pattern is special.
Table 1 UV spectrophotometer measuring result
| Title | OD260/OD280 | OD260/OD230 | Concentration (ng/ μ L) | Volume (μ L) |
| Total serum IgE | 1.89 | 0.67 | 788 | 16 |
The structure of 1.3 barrel-shaped cone shell venom duct cDNA libraries
(1) synthesis of cDNA mono-chain, two chains
Adopt Creator SMART cDNA Kit(Clonetech, Cat.No634903) chain and two chains are synthesized, CDSIII/3primer primer in test kit CDS-3M adapter replaces, primer sequence is: 5 '-AAGCAGTGGTATCAACGCAGAGTGGCCGAGGCGGCCd (T) 20VN-3 ' (SEQ ID NO:1), wherein, N=A, C, G or T; V=A, G or C.Through electrophoresis detection after two chain cDNA synthesize, the results are shown in Figure 2, wherein, the 1st swimming lane is 4uL DL2000plus marker, is 5000,3000,2000,1000,750,500,250,100bp from top to bottom, and the 2nd swimming lane is the two chain cDNA PCR primer of 5uL.Can be seen by Fig. 2, the length of two chain cDNA PCR primer is uniformly distributed from 200bp-5000bp, has the gene of obviously high copy between 1-2k.
(2) homogenization process
Utilize Trimmer-Director kit(Evrogen, Cat.No.NK002) homogenization process is carried out to the cDNA of above-mentioned acquisition, add component according to table 2 and carry out DSN process, cDNA electrophoresis detection through homogenization process the results are shown in Figure 3, and wherein, the 1st swimming lane is 4uL DL2000plus marker, 5000,3000,2000 from top to bottom, 1000,750,500,250,100bp, 2-4 swimming lane is the Control of 9 circulations, 1/2 ferment treatment, 1 times of ferment treatment.Then the cDNA through homogenization process is carried out twice PCR amplification, the electrophoresis detection of secondary PCR amplified production the results are shown in Figure 4, and wherein, the 1st swimming lane is 4uL DL2000plus marker, 5000,3000,2000,1000 from top to bottom, 750,500,250,100bp; 2nd swimming lane is secondary PCR product.Then QIAquick PCR Purification Kit(QIAGENCat.No.28104 is used) by the PCR system purifying of the double-strand cDNA of synthesis.CDNA is dissolved in 15 μ L sterilized waters, gets 1 μ L and measure concentration, regulate concentration to 100ng/ μ L with sterilized water.Before hybridization, hybridization solution room temperature is placed 15-20min, guarantee in hybridization solution without precipitation.
As can be seen from Figure 3, high copy gene 1 times of ferment treatment is excessive, and 1/2 ferment treatment is suitable, therefore carries out secondary PCR with 1/2 ferment treatment.As seen from Figure 4, make a PCR more concentrated by secondary PCR.
Table 2DSN process gradient
| Component/pipe | Pipe 1(DSN1) | Pipe 2(DSN1/2) | Pipe 3(contrasts) |
| DSN enzyme liquid | 1μL | - | - |
| 1/2DSN enzyme liquid | - | 1μL | - |
| DSN preserves damping fluid | - | - | 1μL |
(3) object fragment reclaims
Double stranded cDNA purification after adopting QIAquick PCR purification kit (QIAGEN Cat.No.28104) to carry out homogenization, final volume 80 μ L ddH
2o dissolves.Cut glue recovery concrete operations as follows:
A. add following system to carry out enzyme and cut:
| cDNA(Purified cDNA) | 79μL |
| 10 × Sfi I damping fluid | 10μL |
| Sfi I enzyme | 10μL |
| 100×BSA | 1μL |
| Cumulative volume | 100μL |
B.50 DEG C temperature bath 2h, digestion products adopts 1% agarose gel electrophoresis, and 60V electrophoresis 2h, detects;
C. marker EB is dyeed, under ultraviolet, mark 400bp and 1400bp region;
D. by the 400-1400bp region of digestion products be greater than 1400bp region and cut, reclaim with QIAquick Gel ExtractionKit (QIAGEN Cat.No.28704), measure concentration.
(4) conversion is connected
A. added by following component in 0.2mL PCR pipe, mixing, 16 DEG C, connection is spent the night;
| cDNA | 4.0μL(100ng) |
| pDNR-LIB(100ng/μL) | 1.0μL |
| 10X connects damping fluid | 1.0μL |
| ATP(10mM) | 1.0μL |
| T4DNA ligase enzyme | 1.0μL |
| Deionized water | 2.0μL |
| Cumulative volume | 10.0μL |
B.70 DEG C, sex change, after 10 minutes, places 5min on ice, of short duration centrifugal, connection product is added to desalting and purifying 1h on 0.25um Millipore purification membrane;
C. get 2 μ L products, voltage 2.1KV electroporated in intestinal bacteria DH10B after, put into 37 DEG C of shaking tables, 120rpm recovery 1h, add 80% glycerine of 50% volume;
D. getting 2 μ L bacterium liquid is applied on paraxin flat board, 37 DEG C of overnight incubation.
(5) PCR colony identification
A. get 2 μ L bacterium liquid to be coated with dull and stereotyped (diameter 15cm), calculate the storage capacity number on flat board, be about 2000, calculating bacterium drop degree is accordingly 1.0 × 10
6cfu/mL, whole connection storage capacity is greater than 3 × 10
6.Bacterium drop degree (cfu/mL)=flat-plate bacterial colony number/dull and stereotyped bacteria liquid amasss; Storage capacity (cfu)=bacterium drop degree × bacterium liquid cumulative volume;
B. from library, random picking 30 clones carry out bacterium colony PCR qualification, are detected the clip size of PCR primer by 1% agarose gel electrophoresis.Electrophoresis result is shown in accompanying drawing 5, and wherein, the 1st swimming lane is 4 μ L DL2000plus marker, is 5000,3000,2000,1000,750,500,250,100bp from top to bottom; 2-31 swimming lane is 400-1400bp and 30 the Random clones bacterium colony PCR primer being greater than 1400bp two libraries.From the result of Fig. 5, Insert Fragment is much to be distributed between 0.75-3k in 750bp, meets and builds library standard.
Embodiment 2 determines conotoxin peptide full-length gene order in cone shell venom duct
The cone shell venom duct cDNA library built based on embodiment 1 below carries out following experiment:
The high-flux sequence of 2.1 barrel-shaped cone shell venom duct cDNA libraries
Random choose 10000 mono-clonals from the 400-1400bp library built, carry out high-flux sequence.
A. get the 400-1400bp library bacterium liquid built, dilute 700 times with LB liquid nutrient medium, the bacterium liquid that each flat board coating 200 μ L have diluted;
B. the LB solid plate will coated, closes lid and to be placed upside down in 37 DEG C of greenhouses after incubated overnight 16-18h, puts into Cool Room 4 DEG C colour developing and preserves;
C.96 add 900 μ L LB bacterium liquid in hole depth orifice plate, picking is full, size is identical, color is milky white single bacterium colony, is put in 96 orifice plates, and sealed membrane seals, and cultivates 14h under 220rpm, 37 DEG C of conditions;
D. in room temperature, under 4000rpm condition, centrifugal 4min, abandons supernatant, tips upside down on after thieving paper blots waste liquid, extracts plasmid.
E. reagent is added by following system, ddH
2o mends to 5 μ L.
| Template | 100-200ng |
| BigDye v3.1 | 0.5μL |
| 25 × damping fluid | 0.2μL |
| Primer | 3.2pmol |
F. sample is put in PCR instrument, and debug first 96 DEG C of 2min, and then following program is carried out in 30 circulations: 96 DEG C of 10S, 50 DEG C of 10S, 60 DEG C of 3min;
After g.PCR EP (end of program), carry out purifying to reaction product, the every hole of purifying adds 5 μ L deionized formamides, after the centrifugal 1min of 1000rpm, adopts ABI3730xl instrument to carry out high-flux sequence.
2.2 series processing and compare of analysis
A. after using phrep software to carry out basecalling operation to lower machine data, remove carrier with cross_match software, obtain raw data data;
B. polyA gone to the sequence removing carrier, filter short sequence, obtain Clean data;
C. Clean data and the local conotoxin storehouse built are compared, annotation qualification toxin sequence;
D. obtain 194 whole toxin sequences altogether, obtain whole toxin sequence 65 after deduplication, the sequencing results is in table 3.
Table 3 cone shell cDNA sequencing sequence analytical results
As can be seen from Table 3, contriver has found complete conotoxin peptide sequence 65, and wherein 6 are found before this; Superfamily 12 kinds, wherein 2 kinds is the new superfamily of Late Cambrian; Halfcystine pattern 12 kinds, wherein 3 kinds are less than 4 halfcystines, to be determined.
In the description of this specification sheets, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referred to the schematic representation of above-mentioned term.And the specific features of description, structure, material or feature can combine in an appropriate manner in any one or more embodiment or example.
Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.
Claims (10)
1. build a method for cone shell venom duct cDNA library, it is characterized in that, comprise the following steps:
Be cDNA by the total serum IgE reverse transcription of described cone shell venom duct; And
Enzyme cuts back to close the fragment of 400-1400bp in described cDNA, to obtain object fragment, described object fragment forms described cone shell venom duct cDNA library.
2. method according to claim 1, is characterized in that, utilizes Trizol LS method to extract the total serum IgE of described cone shell venom duct.
3. method according to claim 1, it is characterized in that, Creator SMART cDNA test kit is utilized to carry out described reverse transcription, CDSIII/3primer primer in wherein said Creator SMART cDNA test kit is replaced by CDS-3Madapter, and the sequence of described CDS-3M adapter is: 5 '-AAGCAGTGGTATCAACGCAGAGTGGCCGAGGCGGCCd (T) 20VN-3 '.
4. method according to claim 1, is characterized in that, is carrying out before described enzyme cuts back to close, comprising further:
Described cDNA is carried out homogenization process.
5. method according to claim 4, is characterized in that, utilizes Trimmer-Director test kit to carry out described homogenization process.
6. method according to claim 1, is characterized in that, utilizes Sfi I Enzyme to carry out described enzyme and cuts, and optionally, utilizes QIAquick Gel Extraction test kit to carry out described recovery.
7. method according to claim 1, is characterized in that, comprises further and carries out purifying to each step products, optionally, comprises further and described object fragment is connected to carrier and is converted in intestinal bacteria.
8. a cone shell venom duct cDNA library, it is built by the method described in any one of claim 1-7 to obtain.
9. determine a method for conotoxin peptide full-length gene order in cone shell venom duct, it is characterized in that, comprise the following steps:
Method according to any one of claim 1-7, builds the cDNA library of described cone shell venom duct;
Described cDNA library is checked order, to obtain sequencing result; And
Based on described sequencing result, determine conotoxin peptide full-length gene order in described cone shell venom duct.
10. method according to claim 9, is characterized in that, utilize be selected from ABI3730xl, Hiseq2000, SOLID, 454 and at least one of single-molecule sequencing device carry out described order-checking, preferred ABI3730xl sequenator,
Optionally, based on described sequencing result, determine that in described cone shell venom duct, conotoxin peptide full-length gene order comprises further:
Described sequencing result is carried out pre-treatment, to obtain through pretreated sequencing result; And
Compare described through pretreated sequencing result and conotoxin storehouse, to determine the conotoxin peptide full-length gene order of conotoxin in described cone shell venom duct.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6197554B1 (en) * | 1998-11-20 | 2001-03-06 | Shi-Lung Lin | Method for generating full-length cDNA library from single cells |
| US20020198145A1 (en) * | 1999-06-10 | 2002-12-26 | Cognetix, Inc. | MuO-conopeptides and their use as local anesthetics |
| CN102352415A (en) * | 2011-10-31 | 2012-02-15 | 云南大学 | Preparation method of gene chip responding Xanthomonas oryzae, gene chip and application of gene chip |
-
2013
- 2013-10-21 CN CN201310497170.0A patent/CN104562212A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6197554B1 (en) * | 1998-11-20 | 2001-03-06 | Shi-Lung Lin | Method for generating full-length cDNA library from single cells |
| US20020198145A1 (en) * | 1999-06-10 | 2002-12-26 | Cognetix, Inc. | MuO-conopeptides and their use as local anesthetics |
| CN102352415A (en) * | 2011-10-31 | 2012-02-15 | 云南大学 | Preparation method of gene chip responding Xanthomonas oryzae, gene chip and application of gene chip |
Non-Patent Citations (5)
| Title |
|---|
| 冯建成等: "海南产桶形芋螺毒管cDNA文库构建", 《中国海洋药物杂志》 * |
| 李宝珠等: "利用SMART技术构建疣缟芋螺毒管cDNA噬菌体文库及芋螺毒素新基因的克隆", 《中国生物化学与分子生物学报》 * |
| 李晨等: "大豆种子不同发育时期全长均一化cDNA文库的构建", 《中国农业科学》 * |
| 边万平: "除虫菊花均一化cDNA文库的构建及EST序列分析", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
| 陈琴: "海南产桶形芋螺与勇士芋螺毒管cDNA文库的构建", 《中国学位论文全文数据库》 * |
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