CN108676792A - One kind is based on anti-PPRV cDNA libraries in hunchbacked source and its preparation method and application - Google Patents
One kind is based on anti-PPRV cDNA libraries in hunchbacked source and its preparation method and application Download PDFInfo
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Abstract
The invention discloses one kind based on anti-PPRV cDNA libraries in hunchbacked source and its preparation method and application, belongs to biotechnology.It prepares antigen and camel is immunized using PPRV Attenuate vaccines and concentration, purifying Attenuate vaccine, obtains high quality immune serum and whole blood；Total mRNA of SfiIA, SfiIB restriction enzyme site primer reverse transcription peripheral blood lymphocytes extraction is respectively provided with using both ends, synthesize the first chain of full-length cDNA, PCR amplification, orientation is inserted into specific support pGADT7 SfiI after SfiI single endonuclease digestions, obtains based on the anti-PPR virus antibody cDNA library in hunchbacked source.It is identified：Library storage capacity>About 2.0 × 106Cfu/mL, Insert Fragment about 400bp~2kbp, average length about 1Kbp, diversity is good, recombination fraction about 100%；Functional selection shows：Existing in constructed cDNA library has the anti-PPR virus VHH antibody of neutralization activity.The it is proposed of the present invention provides platform to screen anti-PPR virus VHH antibody, and new technological means is provided for the early treatment of peste des petits ruminants and diagnosis.
Description
Technical field
The invention belongs to biotechnology, be related to it is a kind of based on anti-PPRV cDNA libraries in hunchbacked source and preparation method thereof and
Using.
Background technology
Peste des petits ruminants（Peste des petites ruminants, PPR）Also known as small ruminate the false rinderpest of beast, lung intestines
Scorching, the compound disease of stomatitis pneumoenteritis, is by PPR virus（Peste des petites ruminants virus,
PPRV）Cause that small ruminant is acute, hot, highly contagious disease.PPR in nineteen forty-two for the first time West Africa report, then
The most of African countries being popular between Sahara Desert and equator, later, distribution are expanded to the Middle East, Iran, South Asia time
The Countries in continent, Turkey and Central Asia, in Asia, PPR occurred in South India area for the first time in 1987,
The incoming Arabia Peninsula 1993-1995, the Middle East and South Asian Subcontinent some areas simultaneously become local endemic conditions disease.
2007, China Tibet incoming first PPR, the end of the year in 2013 diffused to Xinjiang, and epidemic situation propagation is fast, span is big, risk is high, only
Two months, just occurs in Gansu, Inner Mongol, Ningxia, Hunan, Liaoning, Anhui and 7, Chongqing province, end end of the year epidemic situation in 2014
Nearly 22 provinces are spread, 261 counties, (Zhang Rongping, Wang Wei, Liu Jiasheng wait Honghe Prefectures small for overall diffusion from west to east
The serology and Molecule Epidemiology Investigation [J] animal medicines for ruminating epizootic disease are in progress, and 2018,39（1）:79-84.), to China
Animal husbandry production and Field of Animal Epidemic Disease Control threaten, and seriously affect International Trade in China and economic development.Prevent and controls
The disease specifies the pathogenesis of the virus, need to start with from the functional antibodies of virus, single domain antibody（VHH）It is that can obtain at present
With stable structure, it is fully functional, in combination with antigen minimum structural unit, due to its immunogenicity it is low, it is easy expression, it is affine
The important target molecule that the features such as power is high, heat-resisting is studied as current molecular image.Therefore specificity how is successfully obtained to be directed to
The functional VHH antibody of PPR virus is the key of solving the problem, and is built and be directed to PPR virus VHH
Antibody cDNA library is breach.
CDNA library (cDNA library) refers to the mRNA that is transcribed of certain a certain developmental stage of biology all through reverse transcription
The cDNA segments of formation are connect with certain carrier and the set of clone that is formed.Each clone is containing only a kind of information of mRNA, foot
The summation of enough mesh clones contains whole mRNA information of cell.CDNA library, can be therefrom convenient for clone and large amplification
Required target gene is screened, and for expressing.The expression shape of cDNA library genome in certain specific class specific cells of research
There is special advantage, to make it in ontogeny, cell differentiation, cell cycle in terms of state and the Function Identification of expressing gene
Have in the researchs of biological phenomenas such as regulation and control, cell ageing and dead regulation and control and be more widely applied value, is in research work
The most-often used gene library arrived.Yin Shuanhui etc. builds hog cholera lapinised virus vaccine strain E2 antigens and is directed to VHH antibody cDNA libraries,
Therefrom successfully screening is directed to CSFV E 2 protein VHH antibody, on the one hand carries out swine fever virus tracer, discloses swine fever virus and causes
Anttdisease Mechanism, on the other hand establishes antibody against swine fever virus detection method, and Li Bing etc. successfully builds secretion resisiting influenza virus B（Flu B）
Antibody hybridoma cell cDNA library provides effective tool for further screening target gene, making genetic chip etc..Xue Zheng
Fly to wait the host target genes cDNA library for building Marek's disease virus miR-M4-5p.There is researcher using in construction cDNA library
The single domain antibody screened and Lactobacillus surface protein fusion, are expressed in Lactobacillus surface, can be notable with this feeding mouse
Reduce the incidence of dysentery caused by rotavirus.The capacity and diversity of cDNA antibody libraries largely influence therefrom elutriation
Go out the probability of specific antibody and the affinity of antibody.Since first cDNA library of structure in 1976, method is not
Disconnected improvement carries SfiIA, SfiIB restriction enzyme site SMART (5 ' end of the RNA of switching mechanism at
Transcript) and the cDNA library of CDS methods structure can represent the abundance of mRNA in original sample, maintain biological heredity
The integrality of information.Using the endogenous terminal transferase activity of reverse transcriptase, as long as single tube, a step can be completed, and not need volume
Outer cDNA extracting and purifyings, precipitation or additional enzyme reaction, it is only necessary to the as little as Total RNA of the mRNA of 25ng or 50ng
The libraries cDNA that can be obtained by high quality, high yield can be applied to direct amplification gene, construction cDNA library, known array fishing
Full-length cDNA（RACE）, and for chip detection cDNA probes amplification etc..
In conclusion by establishing, screening of phage antibody library is specific with PPRV, biological activity is good, in conjunction with antigenic capacity
By force, with the potential antibody for neutralizing function, the especially novel antibodies of nano antibody etc, for establishing facing for sensitive PPRV
Bed diagnostic method, the infection mechanism for studying virus are extremely important.
Invention content
Have that relative molecular mass is small, stability is strong, soluble good, antigen binding the purpose of the present invention is to provide one kind
The advantages such as performance is good, easy expression based on the anti-PPRV cDNA libraries in hunchbacked source.
To achieve the goals above, the technical solution adopted by the present invention is as follows：
A kind of preparation method based on the anti-PPRV cDNA libraries in hunchbacked source, specifically includes following steps：
Step A：Camel program adaptive immune serum is immunized by peste des petits ruminants；
Step B：Immune serum is detected according to serum antibody titer, after camel immuno-competent, acquires and camel whole blood is immunized adds anti-freezing
Agent shakes up, and camel lymphocyte separation medium is added into after dilution and obtains the lymphocyte of separation camel peripheral blood, is therefrom extracted
Total RNA, separation, purifying mRNA；
Step C：It is respectively provided with the total of SfiIA, SfiIB restriction enzyme site primer reverse transcription peripheral blood lymphocytes extraction using both ends
MRNA synthesizes the first chain of full-length cDNA, expands, and orientation is inserted into specific support pGADT7-SfiI vector after SfiI single endonuclease digestions
, obtain based on the anti-PPRV cDNA libraries in hunchbacked source.
In step A of the present invention, peste des petits ruminants is immunized camel program and is：Camel left front leg deltoid muscle intracutaneous injection
2.5x105The BCG vaccine of cfu, 3 part PPRV Attenuate vaccines progress of sheep are subcutaneously injected in right neck, and head exempts from, 2w takes a blood sample and it is dense to be immunized
The Attenuate vaccine of contracting, purifying cells culture carries out three and exempts from after 2 months, mode is exempted from two.
Based on above-mentioned immune programme, after peste des petits ruminants is immunized, camel serum antibody titer is：Blank control OD450=
0.055, negative control OD450=0.059, positive control OD450=2.340, non-immune serum OD450=0.065, one exempts from serum titer
1:40, two exempt from serum titer 1:2560, three exempt from rear serum titer 1:16000.
After camel immuno-competent, after 3w camel whole blood anticoagulations, separation camel periphery blood strangury after acquisition third time is immune
Bar cell, a concentration of 5162ng/uL of extraction Total RNA, R values are 1.97, and the concentration for the mRNA poly (A) being purified to is about
For 251ng/uL, R values are 1.98.
In the step C, synthesis full-length cDNA detailed process is：Following component is sequentially added in PCR amplification pipe：
MRNA, restriction enzyme site GGCCATTACGGCC SMART primers are carried, carry restriction enzyme site GGCCGCCTCGGCC CDS primers,
Mixing is clicked, 72 DEG C of metal baths are placed in, is incubated 2min, is set immediately on ice, cooling 2min is clicked and collected；Sequentially add 5X
First strand Buffer, DTT, dNTP Mix, MMLV reverse transcriptases click mixing, are placed in 42 DEG C of metal baths, invert
1h is recorded, RNase inhibitor 1uL is added to get being full-length cDNA to product in room temperature cooling.
In the step C, 2 PCR Kit of Advantage is selected to carry out double-stranded amplification：1uL full-length cDNAs 100ng/
UL, 40uL ddH20,5uL 10X Advantage 2 PCR buffer, SMART primer 1uL, CDS primers 1uL, 50X
dNTP Mix 1uL、50X Advantage2 polymerase Mix 1uL；Amplification program:95 DEG C of 1min, 95 DEG C of 30s,
68 DEG C of 3min, 25-30cycles each recycle extension of time and increase 5s, and 68 DEG C of 3min obtain double-strand cDNA.
In the step C, digestion processing is carried out to cDNA and pGADT7-SfiI carriers using restriction enzyme SfiI
Afterwards, column was carried out to cDNA after digestion to handle, short-movie section is removed, at PCI/CI purifications using CHROMA SPIN-1000-TE
It manages, ethyl alcohol is refined, ddH2O dissolve out, finally use DNA ligation Kit by GADT7-SfiI vector with cross column after cDNA,
12 DEG C of connections purify the connection liquid of acquisition and refine, obtain primary cDNA library.
Impact condition is the μ F of 1.8KV, 200 Ω, 25, harvest cDNA texts when the present invention is turned using the primary cDNA library electricity
10 24.5cm are coated with when library2Massive plate calculates gained library storage capacity and is more than 2.0x106Cfu/mL, Insert Fragment distribution of lengths model
About 400bp~2kbp is enclosed, average length is more than 1Kbp, and diversity is good, recombination fraction about 100%.
Verification to the anti-PPRV VHH antibody cDNA libraries of the present invention：Cell weak-toxic seedling is cultivated, is concentrated, after purification, with
CDNA library carries out immunoblotting reaction.
It is anti-in the anti-PPR virus VHH of screening that the invention also provides the cDNA libraries of the anti-PPRV VHH antibody
Purposes in body.
Compared with prior art, the beneficial effects of the present invention are：
The present invention prepares antigen and camel is immunized using PPRV Attenuate vaccines and concentration, purifying Attenuate vaccine, obtains high quality immune serum
And whole blood；It is respectively provided with the total of SfiIA, SfiIB restriction enzyme site primer reverse transcription peripheral blood lymphocytes extraction using both ends
MRNA synthesizes the first chain of full-length cDNA, and PCR amplification, orientation is inserted into specific support pGADT7-SfiI after SfiI single endonuclease digestions, obtains
Based on the anti-PPR virus antibody cDNA library in hunchbacked source.It is identified：Library storage capacity>About 2.0 × 106Cfu/mL is inserted into piece
Section about 400bp~2kbp, average length about 1Kbp, diversity is good, recombination fraction about 100%；Functional selection shows：Constructed
Existing in cDNA library has the anti-PPR virus VHH antibody of neutralization activity.The it is proposed of the present invention is that screening resists small ruminate
Epizootic disease virus VHH antibody provides platform, and new technological means is provided for the early treatment of peste des petits ruminants and diagnosis.
For obtaining high quality antibody cDNA library, antigen preparation, immunizing dose, immune programme are a very crucial rings
Section, and the successfully basis of construction cDNA antibody library, otherwise antibody or antibody titer are not generated very after some are immune
Low, the present invention uses camel left front leg deltoid muscle intracutaneous injection 2.5x105Sheep 3 is subcutaneously injected in the BCG vaccine of cfu, right neck
Part PPRV Attenuate vaccines carry out head and exempt from；It takes a blood sample and is immunized after 2w and oneself prepare antigen：Market Attenuate vaccine inoculating cell, mass propgation
Afterwards, it collects, concentrate, purifying, equivalent adjuvant emulsion is added to form；It carries out three after 2 months to exempt from, mode is exempted from two.As a result camel blood is obtained
Clear antibody titer is：Blank control OD450=0.055, negative control OD450=0.059, positive control OD450=2.340, it is not immunized
Serum OD450=0.065, one exempts from serum titer 1:40, two exempt from serum titer 1:2560, three exempt from rear serum titer 1:16000, explanation
The present invention successfully solves key link early period involved by construction cDNA antibody library, obtains high quality immune serum and whole blood.
The present invention successfully builds a cDNA library, and the acquisition of mRNA is a vital factor.First, mRNA must
Must be complete, it cannot degrade, mRNA types are more, and the libraries cDNA of structure are more complete, secondly it should be noted that mRNA cannot be by DNA dirts
Dye.RNA is a kind of nucleic acid molecules being easily degraded, and is present in cytoplasm and core.Mammal is averaged every million cells can
RNA containing 5-10ug, wherein rRNA account for 80%-85%, and tRNA accounts for 10%-16%, and mRNA only accounts for 1%-5%.MRNA molecular species is numerous
More, molecular size range is inhomogenous, but overwhelming majority mRNA molecules are held 3 ' there are 20-250 polyadenylic acid poly (A),
The present invention, which utilizes, carries poly（A）The mRNA of tail can be with the short chain oligo that is connected in cellulose media（dT）Form stabilization
RNA-DNA heterozygosis chains, are purified to mRNA molecules, a concentration of 5162ng/uL of extraction Total RNA from total RNA, and R values are
The concentration of 1.97, purifying mRNA poly (A) are about 251ng/uL, and R values are 1.98.
There are one the processes for likening " putting on shoes and wearing hates " in process by the mRNA of eukaryocyte, therefore the 3' of mRNA is last
End all carries one section of Poly A, this is the basis that cDNA library is prepared using reverse transcriptase.But the sequence at the ends 5' due to cDNA
It arranges different, how to obtain the cDNA of overall length, how to expand and cDNA library is obtained by micro mRNA reverse transcriptions, how to be utilized
Known fragment sequence obtains the cDNA of overall length（That is RACE）, be once one make us puzzlement the problem of.The present invention is utilized and is carried
SfiIA, SfiIB restriction enzyme site SMART (5 ' end of the RNA transcript of switching mechanism at)
The abundance that mRNA in original sample can be represented with the cDNA library of CDS methods structure, maintains the complete of biological heredity information
Property.The capacity of constructed anti-PPRV VHH antibody cDNA libraries is greater than about 2.0 × 106Cfu/mL, the random picking from tablet
16 clones carry out bacterium colony PCR, and electrophoresis result shows that library inserts are different, is distributed between 400~2kbp（Fig. 8）,
Average Insert Fragment is about 1kbp or so, illustrates that the diversity in constructed library is good.Library can be learnt by bacterium colony PCR results
Recombination fraction be 100%.Structure library is expanded, is cloned, is sequenced, proper sequence is further expressed, is expressed albumen and is prepared
After antigen carries out Elisa reactions, it is found that generate specific band, size and desired value, illustrate that building library contains active resist
PPRV VHH antibody, proposition of the invention provide platform to screen anti-PPRV VHH antibody, and for the early treatment of PPR and
Diagnosis provides new technological means.
Description of the drawings
Fig. 1 is health Vero/s/v cells；
Fig. 2 is the Vero/s/v cells for being inoculated with PPRV Attenuate vaccines；
Buffy coat when Fig. 3 is separation lymphocyte；
Fig. 4 is all lymphocytes being collected into；
Fig. 5 is RNA12g/L agarose denaturing gel electrophoresis results；
M2 is 250bp DNA Ladder；1 is Total RNA；
It is the 1.0% agarose gel electrophoresis results of ds cDNA of LD- synthesis in Fig. 6；
M2 is 250bp DNA Ladder；1 is the Smear slice results of cDNA after LD amplifications；
Fig. 7 is 1.0% agarose gel electrophoresis results of cDNA after short-movie section removal；
M2 is 250bp DNA Ladder；1 removes cDNA for short-movie section；
Fig. 8 is that amplified library detects 1.0% agarose gel electrophoresis result of segment；
M2 is 250bp DNA Ladder；1 is 1-16 Insert Fragment testing results；
Fig. 9 is that library converts the lithograph for needing to expand identification on a small quantity；
Figure 10 is the anti-PPRV activity identifications figure in library.
Specific implementation mode
Below in conjunction with the accompanying drawings, the present invention is described in further details and is explained：
A kind of preparation method based on the anti-PPRV cDNA libraries in hunchbacked source, is as follows：
Camel is immunized in I, peste des petits ruminants：Camel left front leg deltoid muscle intracutaneous injection 2.5x105The BCG vaccine of cfu, right neck
3 part PPRV Attenuate vaccines progress head of sheep are subcutaneously injected to exempt from, during which, mass propgation PPRV cell weak-toxic seedlings are collected, concentration, are purified,
Concentrated antigen is prepared, 2w takes a blood sample and the concentrated antigen after equivalent adjuvant emulsion is immunized, and three are carried out after 2 months and is exempted from, mode is exempted from two.
II, detects immune serum, and after camel immuno-competent, acquisition 200mL blood systems are spare from serum；It prepares and high pressure is anti-
Solidifying agent adds 35mL anti-coagulants per 200mL whole bloods, thin using camel peripheral blood lymphocytes separating liquid separation camel periphery hemolymph
Born of the same parents extract Total RNA, purifying mRNA poly (A).
III, is utilized with SfiIB restriction enzyme site CDS primers and is carried SfiIA restriction enzyme site SMART primers and reverse transcription
Enzyme, reverse transcription extend synthesis the first chain of full-length cDNA, and amplification synthesis complementary strand continues to expand.
Amplified production and specific support GADT7-SfiI vector, obtain both ends cohesive end not in IV, SfiI single endonuclease digestions III
Same cDNA directs it the specific support GADT7-SfiI vector after being inserted into digestion by T4 ligases, and product electricity turns
HST08 obtains primary cDNA library.
V, is coated with 10 24.5cm when harvesting cDNA library2Massive plate calculates gained library storage capacity, picking part clone
Identify diversity and recombination fraction.
VI, which is verified using culture concentrated antigen in gained cDNA library, has neutralization activity VHH antibody.
Embodiment
Experiment material and reagent：MEM pulvis, 1:250 Trypsin are purchased from GIBCO companies, and cell training is prepared in this laboratory
Nutrient solution and pancreatin cell dissociation buffer；Newborn bovine serum is purchased from the Lanzhou bio tech ltd Rong Ye；3 month female two-humped camels are purchased
From Jinchang peasant household（Detection is without cloth disease）；Camel peripheral blood lymphocytes separating liquid is purchased from the oceans Tianjin Hao；RNAlater is purchased from
Qiagen；SMART cDNA Library Construction Kit、 Advantage 2 PCR Kit、CHROMA SPIN-
1000-TE and enzyme and relevant carriers are purchased from Clontech；DNA ligation Kit、TaKaRa MiniBEST DNA
Fragment Purification Kit, electrotransformation competent escherichia coli cell HST08 and PCR related reagent are purchased from
TaKaRa；PPRV antibody ELISA detection kits are purchased from IDEXX；Other biochemical reagents are that domestic analysis is pure.
, high quality immune serum and whole blood acquisition
1.1. culture PPRV Attenuate vaccines prepare concentrated antigen
Choose this laboratory structure health Vero/s/v cells（It is specifically shown in number of patent application 201611208506.7）（Such as Fig. 1）Three
Bottle is inoculated with two bottles of Xinjiang Tian Kang PPRV Attenuate vaccines, and one bottle is negative control, after 5d, sets refrigerator multigelation, is denoted as F1 generation；Extremely
F9 for when, 3.5d receive, 5 bottles of F10 pickups kind（Such as Fig. 2）, multigelation, collection, centrifugation, concentration, after purification, 1.5mL packing ,-
70 DEG C of refrigerators save backup.
1.2.PPRV camel is immunized in Attenuate vaccine
Female camel of healthy 3 monthly ages is chosen, blood sampling after detecting cloth disease negative antibody, acquires 100mL blood, and separation prepares negative blood
Clearly, left front 1 crural triangle flesh intracutaneous injection 2.5x1053 part PPRV Attenuate vaccines of sheep are subcutaneously injected in the BCG vaccine of cfu, right neck
Carry out head exempt from, 2w blood samplings, the antigen of above-mentioned preparation, which adds, to carry out two after equivalent adjuvant emulsion and exempts from, and three are carried out after 2 months and is exempted from, mode is same
Two exempt from.Effect after detection is immune.
1.3. result：
After peste des petits ruminants is immunized according to above-mentioned immune programme, camel serum antibody titer is：Blank control OD450=0.055, cloudy
Property control OD450=0.059, positive control OD450=2.340, non-immune serum OD450=0.065, one exempts from serum titer 1:40, two
Exempt from serum titer 1:2560, three exempt from rear serum titer 1:16000, it is seen then that serum antibody titer is relatively good, illustrates immuno-competent.
, separation peripheral blood lymphocytes, extract Total RNA, separation, purifying mRNA
2.1. blood sampling
35mL anti-coagulants is added in 500mL saline bottles, acquires immuno-competent camel whole blood 200mL, shakes up, it is dilute that whole blood is added in equivalent
It releases liquid and obtains anticoagulation.
2.2. peripheral blood lymphocytes is detached
10mL camel lymphocyte separation mediums are added in 30mL glass centrifuge tubes, are added the anticoagulation diluted after first slow soon, 16
Guan Weiyi groups are gently placed in horizontal centrifuge, and temperature is controlled at 18-22 DEG C, centrifugal force 1800rpm, time 20min, centrifugation knot
Shu Caiyong hand brakes；Pasteur pipet careful collection buffy coat（Such as Fig. 3）In new collecting pipe, 1:5 ratio is added thin
Born of the same parents' cleaning solution, 1000rpm, centrifuge 10min by 20 DEG C or so；Supernatant is abandoned, is repeated twice, it is point to remove the precipitation after red blood cell
From to lymphocyte（Such as Fig. 4）, it is suspended in RNA sample and is preserved in liquid RNAlater, is set -70 DEG C and save backup.
2.3. the extraction of lymphocyte TotalRNA and mRNA separation, purifying
Removal exogenous rna enzyme pollution as possible inhibits endogenous RNA enzyme activity, creates " no RNA enzyme an environment.Using most strong
Inhibition is used in combination in RNase inhibitor guanidinium isothiocyanate, mono- thioglycols of β and mono- sarcosyls of detergent N (SLS)
RNA degrades, and enhances the dissociation to nucleoprotein complex, and RNA is made to improve the yield of RNA with Separation of Proteins：400uL lymphocytes
1mLTRizol is added in Sample storage liquid, according to step operation；Then 200uL chloroforms are added；Careful collection supernatant is simultaneously after centrifugation
Equivalent isopropanol is added；The washing of 75% absolute ethyl alcohol is precipitated, the precipitation after drying, without RNAse water dissolutions, is as harvested with 25uL
Total serum IgE, ND2000 measure Total RNA concentration.
65 DEG C of heating 15min of Total RNA solutions, are rapidly cooled to 0 DEG C, denaturation to destroy RNA primary structures, makes
PolyA tails fully expose, and improve the poly A rate of recovery；So that the combination of mRNA and rRNA is dissociated simultaneously, then utilizes oligomerization（dT）-
Cellulose chromatography method isolates and purifies：With 0.1N NaOH suspension 0.2g oligomerizations（dT）Cellulose is transferred to 2mL mineral wools and seals
In inner injector, with no RNAse water and 0.01MTris-HCl, 0.5MKCl（pH 7.5）1x high salt wash is to pH 7.5；Denaturation
Total serum IgE and isometric 0.02MTris-HCl, 1MKCl afterwards（pH 7.5）Rotary shaker is set in 2x mixings with high salt, upper prop, capping
1x 1mL with high salt are added in 10min, collect effluent, are denaturalized upper prop again, 4mL high salt wash twice, abandons effluent, 3mL less salts
Column is washed, 500g centrifuges 1min, collects centrifugation object, 0.01MTris-HCl, 1MKCl are added after denaturation（pH 7.5）Upper second widow
It is poly-（dT）Cellulose column, according to the first column through with high salt, less salt, 400uL 0.01MTris-HCl（pH 7.5）Elution obtains
To mRNA, the cold ethyl alcohol of 2.5 times of volumes is added, 0.1 volume 3M sodium acetates pH5.2, -20 DEG C are placed 2h, 4 DEG C of 10 000g centrifugation
20min precipitates mRNA, dry after the washing of 75% ethyl alcohol, and 2uL purifies mRNA to obtain the final product without RNAse water dissolutions, and detection is spare.
2.4. result
From Fig. 3,4 as can be seen that the present invention separation, collect buffy coat and lymphocyte it is high-visible, meet the requirements, extract
Peripheral blood lymphocytes Total RNA, through ND2000 measure a concentration of 5162ng/uL, R values be 1.97, through oligomerization（dT）-
Cellulose chromatography method after purification gained mRNA poly (A) a concentration of 251ng/uL, R values be 1.98.In the change of 12 g/L
Property agarose gel electrophoresis（Fig. 5）It can be seen that tri- clear bands of 28S, 18S, 5S, 28S:18S is about 2:1.Illustrate RNA and mRNA
Purity is higher, meets the needs of library construction.
, full-length cDNA synthesis, cDNA synthesis double-strands and purifying
The primer is the SMART and CDS that both ends are respectively provided with SfiIA, SfiIB restriction enzyme site when structure primary cDNA library,
Used carrier is：PGADT7-SfiI vector carry SfiI restriction enzyme sites.The primary cDNA library of the structure is specific as follows：
3.1. full-length cDNA is synthesized
Following component is sequentially added in 200uL PCR amplification pipes：MRNA 3uL, restriction enzyme site GGCCATTACGGCC is carried
SMART primers 1uL, restriction enzyme site GGCCGCCTCGGCC CDS primer 1uL are carried, clicks mixing, is placed in 72 DEG C of metal baths, incubates
2min is educated, is set immediately on ice, cooling 2min is clicked and collected.Sequentially add 5X First strand Buffer 2uL, 20mM
DTT1uL, 10mM dNTP Mix1uL, 200u/uL MMLV reverse transcriptase 1uL click mixing, are placed in 42 DEG C of metal baths, invert
1h is recorded, 2u/uL RNase inhibitor 1uL are added in room temperature cooling, and it is full-length cDNA to obtain product, sets -20 DEG C and saves backup.
3.2. amplification
Advantage 2 is multi-functional mixed type polymerase, and fidelity is 3 times of wild type Taq, is suitable for amplification multiple types
DNA profiling include cDNA.Therefore 2 PCR Kit of Advantage is selected to carry out double-stranded amplification：The above-mentioned full-length cDNAs of 1uL
（100ng/uL）, 40uL ddH20,2 PCR buffer, SMART primer 1uL, CDS primers of 5uL 10X Advantage
1uL、50X dNTP Mix 1uL、50X Advantage2 polymerase Mix 1uL.Amplification program:95 DEG C of 1min, 95
DEG C 30s, 68 DEG C of 3min, 25-30cycles (each cycle extension of time increases 5s), 68 DEG C of 3min.The double-strand of 8uL
CDNA is identified with 1.0% agarose gel electrophoresis.
3.3. purifying
Amplification is obtained according to operation instruction using TaKaRa MiniBEST DNA Fragment Purification Kit
CDNA carry out purifying recovery processing.
3.4. result
MRNA is synthesized by reverse transcription, and No. 1 band in Fig. 6 is obtained after carrying out LD amplifications, recovery purifying, it is the summation of segment,
There are Smear bands in electrophoresis result, illustrates to obtain cDNA credible result.
, cDNA library structure, quality evaluation and identification:
4.1. cDNA library is built
Digestion processing is carried out to cDNA and pGADT7-SfiI carriers first with restriction enzyme SfiI；Due to 400bp or less
Segment can seriously affect follow-up connection and Library Quality too much, need to be removed short-movie section, therefore use CHROMA
SPIN-1000-TE carried out column processing to cDNA after digestion, removed short-movie section, and after PCI/CI purified treatments, ethyl alcohol is refined,
ddH2O is dissolved out；Finally use DNA ligation Kit by pGADT7-SfiI vector and appropriate cDNA after crossing column, 12 DEG C of companies
It connects, carrying out purifying to the connection liquid of acquisition refines, and obtains primary cDNA library.
4.2. Library Quality is evaluated
Take a small amount of primary libraries connection liquid, Electroporation-competent cells HST08；Take appropriate conversion fluid coating Amp resistances LB flat
Plate, 37 DEG C are incubated overnight；Cotransformation product bacterium solution is taken by the bacterium colony number grown on 1/10 tablet, by 1/10,1/100 and 1/
1000 dilutions apply 100uL dilutions to 100mm tablets respectively, and 30 DEG C are inverted culture 3-5d until clone occurs, to the list of growth
Bacterium colony counts, according to formula：Plank clones number/Each FLIPR（mL）X extension rates=bacterial plaque forms unit/mL.
4.3. amplified library is identified
16 single bacterium colonies of random picking, carry out bacterium colony PCR with pGADT-Rec universal sequencing primers object, analyze library inserts
Size and recombination fraction.8uL PCR products are taken to carry out 1.0% agarose gel electrophoresis analysis.
4.4. primary million clonal expansion of cDNA library and plasmid extraction
By storage capacity detection data, the amount for taking 100 myriagrams grand calculates the connection liquid product of needs, electrotransformation to competent cell
In HST08,10 24.5x24.5cm LB massive plates are coated with, 37 DEG C are incubated overnight.The amplification clone that the practical conversion of monitoring obtains
Quantity；Recycling amplification bacterium colony, plasmid extraction is carried out using NucleoBond Xtra Midi EF kits.Take 100ng amplification texts
Library plasmid carries out 1.0% agarose gel electrophoresis.
4.5. to structure library is expanded, is cloned, is sequenced, proper sequence further express, express albumen and prepare antigen into
Row Elisa reactions, detect its reactivity.
4.6. result
When building library, result such as Fig. 7 after short-movie section removal illustrates that the library of structure does not have redundant sequence；It is calculated according to formula
Gained library storage capacity is ＞ about 2.0x106cfu/mL.Fig. 8 electrophoresis results show that the coverage area of cDNA library is 400-
2000bp, average Insert Fragment is about 1000bp, it is seen that structure library diversity is good, and library can be learnt by bacterium colony PCR results
Recombination fraction be 100%.It identifies library and collects library platings such as Fig. 9.Structure library is expanded, cloned, be sequenced, is closed
Suitable sequence further expresses, expresses albumen and prepares antigen and carry out after Elisa reacts, it is found that generates and combines band, illustrates to build
Active anti-PPRV VHH antibody is contained in library, and the results are shown in Figure 10.
Finally illustrate, the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although with reference to compared with
Good embodiment describes the invention in detail, it will be understood by those of ordinary skill in the art that, it can be to the skill of the present invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the right of invention.
Claims (10)
1. a kind of preparation method based on the anti-PPRV cDNA libraries in hunchbacked source, it is characterised in that：Specifically include following steps：
Step A：Camel program adaptive immune serum is immunized by peste des petits ruminants；
Step B：Immune serum is detected according to serum antibody titer, after camel immuno-competent, acquires and camel whole blood is immunized adds anti-freezing
Agent shakes up, and camel lymphocyte separation medium is added into after dilution and obtains the lymphocyte of separation camel peripheral blood, is therefrom extracted
Total RNA, separation, purifying mRNA；
Step C：It is respectively provided with the total of SfiIA, SfiIB restriction enzyme site primer reverse transcription peripheral blood lymphocytes extraction using both ends
MRNA synthesizes the first chain of full-length cDNA, expands, and orientation is inserted into specific support pGADT7-SfiI vector after SfiI single endonuclease digestions
, obtain based on the anti-PPRV cDNA libraries in hunchbacked source.
2. a kind of preparation method based on the anti-PPRV cDNA libraries in hunchbacked source according to claim 1, it is characterised in that：Step
In rapid A, peste des petits ruminants is immunized camel program and is：Camel left front leg deltoid muscle intracutaneous injection 2.5x105The BCG vaccine of cfu, it is right
3 part PPRV Attenuate vaccines progress head of side neck hypodermic injection sheep exempt from, 2w takes a blood sample and the weak poison of immuno-concentration, purifying cells culture
Seedling carries out three and exempts from after 2 months, mode is exempted from two.
3. a kind of preparation method based on the anti-PPRV cDNA libraries in hunchbacked source according to claim 2, it is characterised in that：Step
In rapid B, Post-immunisation serum antibody titer is：Blank control OD450=0.055, negative control OD450=0.059, positive control OD450
=2.340, non-immune serum OD450=0.065, one exempts from serum titer 1:40, two exempt from serum titer 1:2560, three exempt from rear serum effect
Valence 1:16000；Camel whole blood anticoagulation is immunized in acquisition third time, detaches peripheral blood lymphocytes, the concentration of extraction Total RNA
For 5162ng/uL, R values are 1.97, and a concentration of 251ng/uL of purifying mRNA poly (A), R values are 1.98.
4. according to a kind of preparation method based on the anti-PPRV cDNA libraries in hunchbacked source of claim 1-3 any one of them, feature
It is：In step C, synthesis full-length cDNA detailed process is：Following component is sequentially added in PCR amplification pipe：MRNA, band
There are restriction enzyme site GGCCATTACGGCC SMART primers, carry restriction enzyme site GGCCGCCTCGGCC CDS primers, clicks mixing,
72 DEG C of metal baths are placed in, 2min is incubated, are set immediately on ice, cooling 2min is clicked and collected；Sequentially add 5X First strand
Buffer, DTT, dNTP Mix, MMLV reverse transcriptase click mixing, are placed in 42 DEG C of metal baths, reverse transcription 1h, and room temperature cools down,
RNase inhibitor is added to get being full-length cDNA to product.
5. a kind of preparation method based on the anti-PPRV cDNA libraries in hunchbacked source according to claim 4, it is characterised in that：Step
In rapid C, 2 PCR Kit of Advantage is selected to carry out double-stranded amplification：1uL full-length cDNAs 100ng/uL, 40uL ddH20,
2 PCR buffer, SMART primer 1uL, CDS primers 1uL of 5uL 10X Advantage, 50X dNTP Mix 1uL, 50X
Advantage2 polymerase Mix 1uL；Amplification program:95 DEG C of 1min, 95 DEG C of 30s, 68 DEG C of 3min, 25-
30cycles each recycles extension of time and increases 5s, and 68 DEG C of 3min obtain double-strand cDNA.
6. a kind of preparation method based on the anti-PPRV cDNA libraries in hunchbacked source according to claim 1 or 5, it is characterised in that：
In step C, digestion processing is carried out to cDNA and pGADT7-SfiI carriers using restriction enzyme SfiI, uses CHROMA
SPIN-1000-TE carried out column processing to cDNA after digestion, removed short-movie section, refined through PCI/CI purified treatments, ethyl alcohol,
ddH2O dissolve out, finally use DNA ligation Kit by GADT7-SfiI vector with cross column after cDNA, 12 DEG C connection, it is right
The connection liquid purifying of acquisition is refined, obtains primary cDNA library.
7. a kind of preparation method based on the anti-PPRV cDNA libraries in hunchbacked source according to claim 6, it is characterised in that：Text
Kuku holds>2.0x106Cfu/mL, length distribution range are 400bp~2kbp, and average length is more than 1Kbp, recombination fraction 100%.
8. be prepared according to the method for claim 1 based on the anti-PPRV cDNA libraries in hunchbacked source.
9. it is anti-in screening that one kind according to claim 1,2,3,5,7 or 8 is based on the hunchbacked anti-PPRV cDNA libraries in source
Purposes in PPRV VHH antibody.
10. according to claim 9 a kind of based on the anti-PPRV cDNA libraries in hunchbacked source, it is characterised in that：The anti-PPRV
VHH antibody cDNA libraries are verified using following methods：Cell weak-toxic seedling is cultivated, is concentrated, after purification, is carried out with cDNA library
Immunoblotting reaction.
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| CN105821480A (en) * | 2016-05-03 | 2016-08-03 | 中国农业科学院兰州兽医研究所 | Anti-orf virus two-humped camel VHH heavy-chain single-domain antibody cDNA library and preparation method thereof |
| CN105887208A (en) * | 2016-05-03 | 2016-08-24 | 中国农业科学院兰州兽医研究所 | Anti-goatpox virus bactrian camel VHH heavy-chain single-domain antibody cDNA library and preparation method thereof |
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| CN105821480A (en) * | 2016-05-03 | 2016-08-03 | 中国农业科学院兰州兽医研究所 | Anti-orf virus two-humped camel VHH heavy-chain single-domain antibody cDNA library and preparation method thereof |
| CN105887208A (en) * | 2016-05-03 | 2016-08-24 | 中国农业科学院兰州兽医研究所 | Anti-goatpox virus bactrian camel VHH heavy-chain single-domain antibody cDNA library and preparation method thereof |
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| 孙圣福等: "小反刍兽疫免疫抗体检测与分析", 《山东畜牧兽医》 * |
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