CN105887208A - Anti-goatpox virus bactrian camel VHH heavy-chain single-domain antibody cDNA library and preparation method thereof - Google Patents
Anti-goatpox virus bactrian camel VHH heavy-chain single-domain antibody cDNA library and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an anti-goatpox virus bactrian camel VHH heavy-chain single-domain antibody cDNA library and a preparation method thereof and belongs to the technical field of biology. The single-domain antibody cDNA library contains an antigen dose, an immune procedure and a serum antibody titer of a goatpox attenuated vaccine immune camel. The preparation method includes the steps of camel lymphocyte separation, total RNA extraction and mRNA separation and purification. CDS III and SMART primers are subjected to reverse transcription to synthesize a full-length cDNA; two amplification primers are designed, a homologous recombination method is adopted to co-transfect the amplification primers and pGADT7 to yeast cells Y187; a VHH gene antibody cDNA library of camel anti-goatpox viruses is established; the capacity of the library is calculated, and cDNA library amplification is performed for quality evaluation. The anti-goatpox virus bactrian camel VHH heavy-chain single-domain antibody cDNA library has the advantages of being small in relative molecular mass, good in stability, good in solubility, good in antigen combining performance and easy to express.
Description
Technical field
The present invention relates to a kind of anti-capripox virus two-humped camel VHH single domain heavy chain antibody cDNA library and preparation method thereof, belong to
In biological technical field.
Background technology
Sheep pox (Capripox) be caused by capripox virus (Capripoxvirus, CPV) can infect goat, sheep and
Ox cause goatpox (variolacaprin, Goatpox), sheep pox (variolacaprin, Sheeppox) and the lump of ox
The one of carbuncle skin disease (Lumpy skin disease) is acute, hot, contagious disease.Sheep pox is in all animal acne diseases
The most serious one, because of itself and sheep infective warts (being commonly called as sore mouth virus), to there is the similitude in clinical symptoms, serology anti-
The intercrossing answered, and taking place frequently of visceratonia sheep pox cause bigger difficulty to making a definite diagnosis of this disease.Single domain antibody (VHH) is current
Can obtain have Stability Analysis of Structures, fully functional, can the minimum structural unit of conjugated antigen, due to its immunogenicity table low, easy
Reach, affinity is high, the feature such as heat-resisting becomes the important target molecules of current molecular image research.Sheep pox to be set up quickly is examined in early days
Disconnected technology, specifies the pathogenesis of capripox virus, need to start with from the functional antibodies of virus, is resisted by virus novel nano VHH
It is the key solving this problem that viral function antibody is screened in body library, and building VHH cDNA library is the base solving this problem
Plinth.
VHH single domain antibody can specific recognition virus and the special construction albumen of other pathogenic microorganisms, assist the anti-sense of host
Dye immune defense is widely used.There is researcher by this single domain antibody and Lactobacillus surface protein fusion, can be expressed in
Lactobacillus surface, can significantly reduce, with this feeding mouse, the incidence of dysentery caused by rotavirus.Due to most parasites
Can escape host immune defenses by all kinds of glycoprotein that polypide surface covers, single domain antibody can also be used for examining of parasitic infection
After breaking and treating little molecular specific single domain antibody coupling fluorescent dye, recognizable parasite surface glycoprotein also penetrates into worm
Body, detects the parasitic infection in blood with this, if coupling beta lactamase, can be toxicity medicine by pro-drug conversion such as cynnematins
Thing kills parasite.The research such as Kim shows, SPIO nano particle (SPIONs) and ScFv are formed abf-
SPIONs, has latent effect to treatment carcinomebryonic antigen (CEA).Shanta etc. point out, application is for amyloid precusor protein
(APP) activity of ScFv suppression Alzheimer's cell beta-secretase, solves mAb difficulty over the course for the treatment of.
Respiratory syncytial virus (RSV) (the Respiratory that Hultberg A etc. are connected by 9 glycine serine residues
Syncytial Virus, RSV) bispecific nano antibody RSV-D3/E4 (9GS) microneutralization RSV virus ability be RSV-
17 times of E4/D3 (9GS), IC50 is 6nmol/L.
The capacity of antibody library and diversity largely affect therefrom elutriation and go out probability and the antibody of specific antibody
Affinity.Since first cDNA library built for 1976, its method is constantly improved, wherein SMART
The cDNA library that (switching mechanism at 5 ' end of the RNA transcript) method builds can generation
In the original sample of table, the abundance of mRNA, saves the integrality of biological heredity information.And the method has only to few to 25ng's
The Total RNA of mRNA or 50ng can be obtained by the cDNA storehouse of high-quality, high yield, it is often more important that the cDNA energy obtained
Enough represent the abundance of mRNA in original sample, can apply to direct amplification gene, construction cDNA library, known array fish complete
Long cDNA (RACE), and the amplification etc. of the cDNA probe for chip detection.Yeast-two hybrid technique can be from structure simultaneously
Host cell cDNA library filters out the albumen interacted with known destination protein, therefore builds Yeast libraries and seem particularly
Important.
In sum, building anti-capripox virus yeast cDNA antibody library, screening has capripox virus type specificity, biology
Learning the antibody that activity is good, conjugated antigen ability strong, have potential neutralization function, especially the novel antibodies of single domain antibody etc, right
In the methods for clinical diagnosis of the capripox virus setting up sensitivity, the infection mechanism of research virus is extremely important.
Summary of the invention
In view of this, the one of the present invention anti-capripox virus two-humped camel VHH single domain heavy chain antibody cDNA library and preparation thereof
Method, it is provided that one possesses the advantages such as relative molecular mass is little, stability is strong, solubility is good, antigen-binding good, the easy expression of energy
The two-humped camel VHH single domain heavy chain antibody yeast cDNA library of anti-capripox virus and preparation method thereof.
The present invention is by the techniques below means above-mentioned technical problem of solution:
One anti-capripox virus two-humped camel VHH single domain heavy chain antibody cDNA library of the present invention and preparation method thereof, a kind of
Anti-capripox virus two-humped camel VHH single domain heavy chain antibody cDNA library, it includes the antigen dose of sheep pox Attenuate vaccine immunity camel, exempts from
Epidemic disease program and serum antibody titer.
A kind of anti-capripox virus two-humped camel VHH single domain heavy chain antibody cDNA library, described goatpox Attenuate vaccine immunity camel
Dosage is: head exempts from 6 parts, two exempts from 12 parts, three exempts from 8 parts, four exempts from 8 parts；Described immune programme for children is: BCG vaccine sensitization 1 week
After, Attenuated Goat Pox Virus Vaccine head exempts from, within the 15th day two, exempt from, within the 30th day three, exempt from, within the 60th day four, exempt from；Described serum antibody titer is: empty
White OD450=0.049, negative control OD450=0.054, positive control OD450=2.250, four exempt from rear serum OD450=3.055.
A kind of preparation method of anti-capripox virus two-humped camel VHH single domain heavy chain antibody cDNA library, it comprises the following steps:
(1) camel separation of lymphocytes, Total RNAs extraction and mRNA separate, purify；
(2) CDS III and SMART primer reverse transcription synthesis full-length cDNA；
(3) design two set amplimer, uses the method for homologous recombination by thin with pGADT7 cotransfection yeast for amplified production
Born of the same parents Y187；
(4) the VHH gene cDNA library of the anti-sheep of virus of camel is built；
(5) library storage capacity is calculated；
(6) amplification cDNA library carries out quality evaluation.
Separating Total RNA concentration in lymphocyte in described step (1) is 4329ng/ul, and purity OD260/OD280 is
1.98。
In described step (3), first round amplimer is:
SEQ ID NO.1:5'-GTCCTGGCTGCTTCTACAAGG-3 ',
SEQ ID NO.2:5'-GGTACGTGGTTGAACTGTTCC-3 '.
Second takes turns amplimer is:
SEQ ID NO.3:5'-TTCCACCCAAGCAGTGGTATCAAGTGGGAGTCTGGGGGAGG-3 ',
SEQ ID NO.4:5'-GTATCGATGCCCACCCTCTAGCCGAGGCGGCCGACATGGAGACGGTGA CCTGGG
T-3’。
Building yeast cDNA library storage capacity in described step (4) is 2.0x106cfu/ml。
The present invention relates to the separation of lymphocyte: in peripheral blood, mononuclearcell includes lymphocyte and monocyte etc.,
Its volume, form are different from other cell with density, according to this principle, utilize lymphocyte separation medium, through density gradient
The centrifugal cell making certain density is distributed by corresponding density gradient, thus is separated by various haemocytes, the pouring that will be separated to
Bar cell is resuspended in RNAlater, and this RNAlater is the Preservation of lymphocytes agent preventing RNA from degrading, it is to avoid RNA is due to for a long time
Preservation or improper use cause degraded thus do not reach requirement for construction data base.
The present invention relates to the preparation of whole VHH cDNA, under general case, mRNA with oligo (dT) as primer, commonly
Carrying out reverse transcription under reverse transcriptase effect, the cDNA length of its synthesis is limited, does not often contain the abundance of mRNA, so
The library obtained does not possesses broad spectrum activity.The present invention using oligo (dT) nucleotide oligonucleotide of a kind of transformation as primer (CDS III),
The first chain is synthesized, when synthesizing the reaction of cDNA due to thing under MMLV (MMLV) reverse transcriptase catalytic action
The SMART of 3 ' end strips Oligo (dG) first added (RNA5 ' end transcribe switching mechanism) primer, when synthesis cDNA arrives mRNA
5 ' end time encounter eukaryotic mrna distinctive " cap sequence ", can cDNA end in synthesis add continuously during the most methylated G
Upper several (dC), form the extension of cDNA after several C pairing prominent with synthesis cDNA end for the Oligo (dG) of SMART primer
Template, reverse transcriptase can automatic conversion module, continue to extend cDNA strand until primer using SMART primer as extending template
End, the cDNA so obtained is the full-length cDNA of high-quality, high yield, it is often more important that the cDNA obtained can represent original
The abundance of the mRNA in sample, can apply to direct construction cDNA library, amplification gene, known array fishing full-length cDNA
(RACE) amplification etc. of the cDNA probe with for chip detection.
The yeast cDNA library that the present invention builds has that relative molecular weight is little, tissue penetration is strong, internal removing is fast, be prone to
Escherichia coli fermentation produces and carries out the advantages such as genetic engineering improvement, therefore has bigger than the monoclonal antibody of complete length
Advantage and development prospect.It addition, the cDNA library that the present invention is directed to capripox virus structure is that one possesses type specificity, biology
Activity is good, conjugated antigen ability strong, have the novel anti-of the antibody library of potential neutralization function, especially single domain antibody etc
Body, for setting up the methods for clinical diagnosis of sensitive capripox virus, the infection mechanism of research virus has extremely important effect.
The invention have the advantage that the invention provides one and possess that relative molecular mass is little, stability is strong, solubility is good,
Antigen-binding can the advantages such as good, easy expression anti-capripox virus two-humped camel VHH single domain heavy chain antibody yeast cDNA library and
Its preparation method.
Accompanying drawing explanation
The invention will be further described with embodiment below in conjunction with the accompanying drawings.
Fig. 1 is total serum IgE integrality electrophoresis detection figure, wherein DNA molecular amount marker:Lambda EcoT14I digest；
250bp DNA Ladder marker；
Fig. 2 is pGADT7-cDNA Library plasmid the result figure；
Fig. 3 is that pGADT7-cDNA library inserts expands the result figure, wherein DNA molecular amount marker:Lambda
EcoT14I digest；250bp DNA Ladder marker.
Detailed description of the invention
Below with reference to accompanying drawing, the present invention is described in detail.
Embodiment 1
Sheep pox Attenuate vaccine immunity camel is tested
Choosing two monthly age mother camels, negative serum is prepared in first blood sampling, then intracutaneous injection BCG vaccine outside foreleg deltoid muscle
Carrying out sensitization and observe, after 1w, inside camel opposite side root of the tail and inside stock, intracutaneous branch carries out immunity: first immunisation
Dosage is the immunity amount of 6 part sheep；Within 15th day, carrying out two to exempt from, dosage is 12 parts；Within 30th day, carrying out three to exempt from, dosage is 8 parts；
Within 60th day, carrying out four to exempt from, dosage is 8 parts；Within 70th day, gather camel blood in jugular vein, separate serum, sheep pox antibody test reagent
Box (Q&D company of the U.S.) detection serum antibody titer.
Testing result is: blank OD450=0.049；Negative control OD450=0.054；Positive control OD450=2.250；The
Camel serum OD after four immunity450=3.055.Visible, serum antibody titer is relatively good.
Embodiment 2
Separate lymphocyte, prepare total serum IgE and purify mRNA
1. prepare anti-coagulants: 480mg/100ml；Sodium citrate 1.32g/100ml, glucose 1.47g/100ml, high pressure goes out
After bacterium standby, anti-coagulants consumption is 17.5%.
2. separate lymphocyte
After camel the 4th immunity 1 week, jugular vein gathered camel anticoagulation 100ml, and equivalent adds whole blood dilution, mixing；
First add lymphocyte separation medium with the ratio of 5:9, the slowest afterwards after add the anticoagulation diluted soon, horizontal centrifuge 1800rpm,
18-22℃,20min；Careful collection buffy coat, in new collecting pipe, dilutes it with 5 times amount cell washing solution, horizontal centrifugal
Machine 1000rpm, 18-22 DEG C, 10min；Abandoning supernatant, precipitation dilutes with cell washing solution again, horizontal centrifuge 1000rpm, 18-22
DEG C, 10min is centrifuged；Abandoning supernatant, precipitation is the lymphocyte being separated to, is suspended in RNAlater, puts-70 DEG C of preservations
Standby.
3. cell total rna results
Drawing above-mentioned lymphocyte liquid 400ul, add 1mlTRizol, mixing, 4 DEG C stand 5min；Add 200ul trichlorine
Methane, mixing, room temperature stands 3min, 12000r/min, 4 DEG C, centrifugal 15min；Draw 750ul supernatant, add equivalent isopropanol, room
Gentle and quiet put 10min, 10000r/min, 4 DEG C, centrifugal 10min；Gently abandon supernatant, precipitate and add 75% ethanol, 8000r/min, 4 DEG C, from
Heart 5min；Precipitation is put fume hood 10min and is allowed to dry；20ul is the total serum IgE of results without the water-soluble precipitation of RNase,
It is 4329ng/ul that ND2000 records Total RNA concentration, and purity OD260/OD280 is 1.98.Its electrophoresis detection is shown in Fig. 1
4.mRNA purifies
In cell Total RNA, mRNA utilizes Oligotex mRNA Mini Kit isolation and purification method to carry out separating pure
Change, above-mentioned total serum IgE is diluted to 500ul volume, adds 500 μ LOBB buffer solutions, mixing, hatching 3min in 70 DEG C of water-baths, 25 DEG C
Stand 10min, 12000r/min, 4 DEG C, 2min；Supernatant is abandoned in suction, the 400 μ L resuspended precipitations of OW1 buffer solution, full dose upper prop,
12000r/min, 4 DEG C, 1min；Abandoning filtrate, post washed by 400 μ L OW2 buffer solutions, 12000r/min, 4 DEG C, 1min；Post is placed in newly
1.5mL without RNase centrifuge tube, inhale 50 μ LOEB buffer solutions to post, room temperature stands 1min, 12000r/min, 4 DEG C, 1min,
Gained is the mRNA purified, and its concentration is 101.3ng/ul, and purity OD260/OD280 is 1.96；28S, 18S, 5S 3 bands are clear
Clear, 28S:18S is about 2:1, illustrates that RNA and mRNA purity is higher, meets the needs of library construction.The sex change fine jade of integrity verification
Sepharose electrophoresis such as Fig. 1.
Embodiment 3
Full-length cDNA synthesis, cDNA synthesis double-strand and purifying
1. full-length cDNA synthesis
Expand at the PCR of 0.2ml and pipe be sequentially added into following component:
Click on mixing, centrifuge tube is positioned over 72 DEG C of metal baths, hatches 2min, put on ice immediately, cool down 2min, click on and receive
Collection, is sequentially added into following component:
Clicking on mixing, centrifuge tube is positioned over 42 DEG C of metal baths, reverse transcription 1h, room temperature cools down, and adds 1ulRNA enzyme level
Agent (2u/ul), the product obtained is full-length cDNA, puts-20 DEG C and saves backup.
2.cDNA synthesis double-strand and purifying
Expand in the PCR first round of 0.2ml and pipe be sequentially added into following component:
Above-mentioned amplification program: 95 DEG C of 1min；95 DEG C of 15s, 68 DEG C of 5min, 20 circulations；68℃5min.By amplified production electricity
Swimming, gel-purified reclaims 900bp product, and-20 DEG C save backup.
Take turns to expand at the PCR second of 0.2ml and pipe be sequentially added into following component:
Above-mentioned amplification program: 95 DEG C of 1min；95 DEG C of 15s, 68 DEG C of 5min, 20 circulations；68℃5min.Amplified production electricity
Swimming, gel
Reclaim kit purified pool 400bp product from gel, put-20 DEG C and save backup.
Embodiment 4
The structure of cDNA library, quality evaluation and qualification:
1.cDNA library construction
PEG/LiAc method is used to prepare yeast Y187 competent cell.Every 600uL competent cell converts 20ul double-strand
CDNA and 6ul pGADT7-Rec, is spread evenly across the bacterium solution of conversion on the SD/-Leu flat board with 150mm of diameter 100mm,
It is inverted for 30 DEG C and cultivates 3-5d until clone occurs.Again flat board is placed in 4 DEG C of refrigerators, places 3-4h.5mL is added to every piece of flat board
Frozen stock solution (the YPDA fluid nutrient medium containing 25% glycerine), and add sterile glass beads, horizontal direction is shaken repeatedly, collects afterwards
Bacterium solution, products therefrom is yeast cDNA primary libraries, plasmid identification result such as Fig. 2.
2. the quality evaluation in library
Take cotransformation product bacterium solution, by 1/10,1/100 and 1/1000 dilution, be coated with 100ul dilution extremely respectively
100mmSD/-Leu flat board, is inverted for 30 DEG C and cultivates 3-5d until clone occurs, the single colony counting to growth, according to formula: plank
Clone's number/Each FLIPR (ml) X extension rate=bacterial plaque forms unit/ml.Calculating library storage capacity is 2.0x106cfu/ml。
3. amplified library is identified
Random 16 single bacterium colonies of picking, carry out bacterium colony PCR with pGADT-Rec universal sequencing primer thing, analyze library and insert sheet
The size of section and recombination fraction.PCR response parameter: 94 DEG C of denaturations 5min；94 DEG C of sex change 1min, 56 DEG C of annealing 1min, 72 DEG C
Extending 2min, totally 30 circulations, last 72 DEG C extend 10min.Take 7ul PCR primer to carry out 1.2% agarose gel electrophoresis and divide
Analysis.The coverage of Fig. 3 electrophoresis result display cDNA library is 400-2000bp, and average Insert Fragment is about 1000bp.By bacterium
The PCR result that falls is appreciated that the recombination fraction in library is 100%.
Finally illustrating, above example is only in order to illustrate technical scheme and unrestricted, although with reference to relatively
The present invention has been described in detail by good embodiment, it will be understood by those within the art that, can be to the skill of the present invention
Art scheme is modified or equivalent, and without deviating from objective and the scope of technical solution of the present invention, it all should be contained at this
In the middle of the right of invention.
Claims (6)
1. an anti-capripox virus two-humped camel VHH single domain heavy chain antibody cDNA library, it is characterised in that: include that sheep pox Attenuate vaccine is exempted from
The antigen dose of epidemic disease camel, immune programme for children and serum antibody titer.
One the most according to claim 1 anti-capripox virus two-humped camel VHH single domain heavy chain antibody cDNA library, its feature exists
In: described goatpox Attenuate vaccine immunity camel dosage is: head exempts from 6 parts, two exempts from 12 parts, three exempts from 8 parts, four exempts from 8 parts；Institute
Stating immune programme for children is: BCG vaccine sensitization is after 1 week, and Attenuated Goat Pox Virus Vaccine head exempts from, within the 15th day two, exempt from, within the 30th day three, exempt from, the 60th day
Four exempt from；Described serum antibody titer is: blank OD450=0.049, negative control OD450=0.054, positive control OD450=
2.250, four exempt from rear serum OD450=3.055.
The preparation side of a kind of anti-capripox virus two-humped camel VHH single domain heavy chain antibody cDNA library the most according to claim 1
Method, it is characterised in that comprise the following steps:
(1) camel separation of lymphocytes, Total RNAs extraction and mRNA separate, purify；
(2) CDS III and SMART primer reverse transcription synthesis full-length cDNA；
(3) design two set amplimer, uses the method for homologous recombination by amplified production and pGADT7 cotransfection yeast cells
Y187；
(4) the VHH gene cDNA library of the anti-sheep of virus of camel is built；
(5) library storage capacity is calculated；
(6) amplification cDNA library carries out quality evaluation.
The preparation side of a kind of anti-capripox virus two-humped camel VHH single domain heavy chain antibody cDNA library the most according to claim 3
Method, it is characterised in that: separating Total RNA concentration in lymphocyte in described step (1) is 4329ng/ul, purity OD260/
OD280 is 1.98.
One the most according to claim 3 anti-capripox virus two-humped camel VHH single domain heavy chain antibody cDNA library and preparation thereof
Method, it is characterised in that: in described step (3), first round amplimer is:
SEQ ID NO.1:5'-GTCCTGGCTGCTTCTACAAGG-3 ',
SEQ ID NO.2:5'-GGTACGTGGTTGAACTGTTCC-3 '.
Second takes turns amplimer is:
SEQ ID NO.3:
5'-TTCCACCCAAGCAGTGGTATCAAGTGGGAGTCTGGGGGAGG-3 ',
SEQ ID NO.4:
5'-GTATCGATGCCCACCCTCTAGCCGAGGCGGCCGACATGGAGACGGTGACCTGGGT-3’。
One the most according to claim 3 anti-capripox virus two-humped camel VHH single domain heavy chain antibody cDNA library and preparation thereof
Method, it is characterised in that: building yeast cDNA library storage capacity in described step (4) is 2.0x106cfu/ml。
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108676792A (en) * | 2018-05-23 | 2018-10-19 | 中国农业科学院兰州兽医研究所 | One kind is based on anti-PPRV cDNA libraries in hunchbacked source and its preparation method and application |
| CN108912225A (en) * | 2018-07-27 | 2018-11-30 | 中国农业科学院兰州兽医研究所 | One kind being based on the anti-goat capripoxvirus VHH-4-1 of two-humped camel and purposes |
| CN108912226A (en) * | 2018-07-27 | 2018-11-30 | 中国农业科学院兰州兽医研究所 | One kind being based on the anti-goat capripoxvirus VHH-4-2 of two-humped camel and purposes |
| CN110139952A (en) * | 2016-12-09 | 2019-08-16 | 深圳华大生命科学研究院 | The primer combination and application of Camelidae antibodies variable region immune group library building |
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| CN110139952B (en) * | 2016-12-09 | 2022-11-25 | 深圳华大生命科学研究院 | Primer combination for constructing camelidae antibody variable region immune repertoire and application |
| CN108676792A (en) * | 2018-05-23 | 2018-10-19 | 中国农业科学院兰州兽医研究所 | One kind is based on anti-PPRV cDNA libraries in hunchbacked source and its preparation method and application |
| CN108676792B (en) * | 2018-05-23 | 2020-12-25 | 中国农业科学院兰州兽医研究所 | Camel source-based PPRV-resistant cDNA library and preparation method and application thereof |
| CN108912225A (en) * | 2018-07-27 | 2018-11-30 | 中国农业科学院兰州兽医研究所 | One kind being based on the anti-goat capripoxvirus VHH-4-1 of two-humped camel and purposes |
| CN108912226A (en) * | 2018-07-27 | 2018-11-30 | 中国农业科学院兰州兽医研究所 | One kind being based on the anti-goat capripoxvirus VHH-4-2 of two-humped camel and purposes |
| CN108912225B (en) * | 2018-07-27 | 2020-06-09 | 中国农业科学院兰州兽医研究所 | Bactrian camel-based anti-goatpox virus VHH-4-1 and application |
| CN108912226B (en) * | 2018-07-27 | 2020-06-09 | 中国农业科学院兰州兽医研究所 | Bactrian camel-based anti-goatpox virus VHH-4-2 and application |
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