CN108676792B - Camel source-based PPRV-resistant cDNA library and preparation method and application thereof - Google Patents
Camel source-based PPRV-resistant cDNA library and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a camel-source-based PPRV-resistant cDNA library, a preparation method and application thereof, and belongs to the technical field of biology. The PPRV attenuated vaccine and the concentrated and purified attenuated vaccine are utilized to prepare the antigen immune camel to obtain high-quality immune serum and whole blood; reverse transcription of total mRNA extracted from peripheral blood lymphocytes is carried out by utilizing primers with SfiIA and SfiIB enzyme cutting sites at two ends respectively, a first chain of full-length cDNA is synthesized, PCR amplification is carried out, SfiI single enzyme cutting is carried out, and then a specific vector pGADT7-SfiI is directionally inserted, so that a camel-source anti-peste des petits ruminants virus antibody cDNA library is obtained. And (3) identification: library capacity>About 2.0X 106cfu/mL, about 400bp to 2Kbp of insert fragment, about 1Kbp of average length, good diversity and about 100 percent of recombination rate; functional analysis showed that: an anti-peste des petits ruminants virus VHH antibody having a neutralizing activity exists in the constructed cDNA library. The invention provides a platform for screening the anti-Peste des petits ruminants virus VHH antibody and provides a new technical means for early treatment and diagnosis of Peste des petits ruminants.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a camel-source PPRV-resistant cDNA library, and a preparation method and application thereof.
Background
Peste des petits ruminants (PPR), also known as Peste des petits pseudoplague, pneumoenteritis, stomatitis pneumoenteritis complex, are acute, febrile, highly contagious diseases of Peste des petits ruminants caused by Peste des petits ruminants virus (PPRV). PPR was first reported in West Africa in 1942 and subsequently prevalent in most African countries between the sub-Saharan desert and the equator, and later its distribution expanded to the middle east, Iran, the south Asian subcontinent, Turkey and parts of the middle Asia, where PPR first appeared in 1987 in the southern region of India, introduced in 1993-1995 into the Arabian peninsula, the middle east and the south Asian subcontinent and became local endemic. In 2007, PPR is firstly introduced into Tibet of China, spreads to Xinjiang in 2013, the epidemic spread is fast, the span is large, the risk is high, only two months, the epidemic occurs in Gansu, inner Mongolia, Ningxia, Hunan, Liaoning, Anhui and Chongqing 7 provinces, the epidemic has spread nearly 22 provinces and 261 counties in 2014, the general spreads from West to east (Zhanglian, Wangwang, Liujiasheng, etc.. serology and molecular epidemiology of the small animal plague in Yunnan red river [ J ] animal medicine progress, 2018,39 (1): 79-84.) causes threat to animal production and animal disease prevention and control in China, and international trade and economic development in China are seriously influenced. To prevent and control the disease, the pathogenesis of the virus needs to be determined by starting from a functional antibody of the virus, and a single domain antibody (VHH) is the minimum structural unit which has stable structure, complete function and can be combined with antigen at present, and becomes an important target molecule for current molecular image research due to the characteristics of low immunogenicity, easy expression, high affinity, heat resistance and the like. Therefore, how to successfully obtain functional VHH antibodies specifically aiming at the peste des petits ruminants virus is the key for solving the problem, and the construction of a VHH antibody cDNA library aiming at the peste des petits ruminants virus is a breakthrough.
A cDNA library (cDNA library) is a collection of clones formed by ligating cDNA fragments, which are all reverse-transcribed mRNA of an organism at a certain development period, with a vector. Each clone contained only one mRNA message, and the sum of a sufficient number of clones contained the full mRNA message of the cell. cDNA libraries are convenient for cloning and mass amplification, from which the desired gene of interest can be screened and used for expression. The cDNA library has special advantages in the aspects of researching the expression state of genome in a specific cell and identifying the function of an expressed gene, so that the cDNA library has wider application value in the research of life phenomena such as ontogenesis, cell differentiation, cell cycle regulation, cell senescence, death regulation and the like, and is the most commonly used gene library in research work. Yishuanghui and the like construct a hog cholera lapinized virus vaccine strain E2 antigen-to-VHH antibody cDNA library, and successfully screen a hog cholera virus E2 protein-to-VHH antibody from the library, so that hog cholera virus tracing is performed to reveal the pathogenesis of the hog cholera virus, a hog cholera virus antibody detection method is established, and plum soldiers and the like successfully construct a hybridoma cell cDNA library secreting anti-influenza virus B (flu B) antibody, thereby providing an effective tool for further screening target genes, manufacturing gene chips and the like. Schlempe chekiangensis et al constructed a cDNA library of the host target gene of Marek's disease virus miR-M4-5 p. Researchers use the single domain antibody screened from the constructed cDNA library to be fused with lactobacillus surface protein and express the single domain antibody on the surface of lactobacillus, so that the incidence rate of dysentery caused by rotavirus can be obviously reduced by feeding mice. The capacity and diversity of the cDNA antibody library largely influence the probability of panning out specific antibodies from it as well as the affinity of the antibodies. Since the first cDNA library constructed in 1976, the method is continuously improved, and the cDNA library constructed by the method of SfiIA, SfiIB enzyme cutting site SMART (switching mechanism at 5' end of the RNA transcript) and CDS can represent the abundance of mRNA in the original sample, and the integrity of biological genetic information is maintained. The method utilizes the endogenous terminal transferase activity of reverse transcriptase, can be completed in one step by only one tube without additional cDNA extraction, purification, precipitation or additional enzyme reaction, can obtain a high-quality and high-yield cDNA library by only 25ng of mRNA or 50ng of Total RNA, and can be applied to direct gene amplification, cDNA library construction, full-length cDNA (RACE) with a known sequence, amplification of cDNA probes for chip detection and the like.
In conclusion, the antibody library is established to screen the antibodies with PPRV specificity, good biological activity, strong antigen binding capacity and potential neutralization function, especially the novel antibodies such as nano antibodies, and the method has extremely important significance for establishing a sensitive clinical diagnosis method of PPRV and researching the infection mechanism of viruses.
Disclosure of Invention
The invention aims to provide a camel-source PPRV-resistant cDNA library which has the advantages of small relative molecular mass, strong stability, good solubility, good antigen binding performance, easy expression and the like.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a preparation method of a camel-derived PPRV-resistant cDNA library specifically comprises the following steps:
step A: obtaining immune serum through Peste des petits ruminants immune camel procedure;
and B: detecting immune serum according to the titer of a serum antibody, collecting immune camel whole blood after camel immunity is qualified, adding an anticoagulant into the immune camel whole blood, shaking up, diluting, adding the immune camel whole blood into camel lymphocyte separation liquid to obtain lymphocytes for separating camel peripheral blood, extracting Total RNA from the lymphocytes, and separating and purifying mRNA;
and C: reverse transcription of total mRNA extracted from peripheral blood lymphocytes is carried out by utilizing primers with SfiIA and SfiIB enzyme cutting sites at two ends respectively, a first chain of full-length cDNA is synthesized, amplification is carried out, SfiI single enzyme cutting is carried out, and then the target vector is directionally inserted into a specific vector pGADT7-SfiI vector, so that a camel-source PPRV-resistant cDNA library is obtained.
In the step A, the Peste des petits ruminants immune camel program comprises the following steps: camel left foreleg deltoid intradermal injection 2.5x105cfu BCG vaccine, right side neck subcutaneous injection sheep 3 PPRV attenuated vaccine for first immunity, 2w blood sampling and immune concentration, purified cell culture attenuated vaccine, 2 months later for three immunity, the way is two immunity.
Based on the immunization program, after the Peste des petits ruminants are immunized, the camel serum antibody titer is as follows: blank control OD450=0.055, negative control OD450=0.059, positive control OD450=2.340, nonimmune serum OD450=0.065, the primary immune serum titer is 1:40, the secondary immune serum titer is 1:2560, and the post-tertiary immune serum titer is 1: 16000.
After the camel is qualified in immunization, 3w of camel whole blood anticoagulation is collected after the third immunization, camel peripheral blood lymphocytes are separated, the concentration of Total RNA is 5162ng/uL, the R value is 1.97, the concentration of purified mRNA poly (A) is about 251ng/uL, and the R value is 1.98.
In the step C, the specific process of synthesizing the full-length cDNA is as follows: the following components are sequentially added into a PCR amplification tube: uniformly mixing mRNA, GGCCATTACGGCC SMART primers with enzyme cutting sites and GGCCGCCTCGGCC CDS primers with enzyme cutting sites by clicking, placing in a metal bath at 72 ℃, incubating for 2min, immediately placing on ice, cooling for 2min, and collecting by clicking; sequentially adding 5X First strand Buffer, DTT, dNTP Mix and MMLV reverse transcriptase, clicking and uniformly mixing, placing in a 42 ℃ metal bath, carrying out reverse transcription for 1h, cooling at room temperature, and adding 1uL of an RNase inhibitor to obtain a product, namely the full-length cDNA.
In the step C, selecting Advantage2 PCR Kit for double-chain amplification: 1uL full-length cDNA 100ng/uL, 40uL ddH20, 5uL 10X Advantage2 PCR buffer, SMART primer 1uL, CDS primer 1uL, 50X dNTP Mix1uL, 50X Advantage2 polymerase Mix1 uL; the amplification program comprises the steps of 1min at 95 ℃, 30s at 95 ℃, 3min at 68 ℃, 5s increase of the extension time of each cycle of 25-30cycles, and 3min at 68 ℃ to obtain the double-stranded cDNA.
In the step C, after the cDNA and the pGADT7-SfiI vector are subjected to enzyme digestion treatment by using a restriction enzyme SfiI, the cDNA subjected to enzyme digestion is subjected to column chromatography treatment by using CHROMA SPIN-1000-TE, short fragments are removed, and the cDNA and the pGADT7-SfiI vector are subjected to PCI/CI purification treatment, ethanol refining and ddH2O elution, and finally, the post-column cDNA was ligated to GADT7-SfiI vector using DNA ligation Kit at 12 ℃ to obtain a primary cDNA library by purifying and refining the ligation solution.
The invention utilizes the primary cDNA library to convert the electric transfer to the electric transfer with the impact condition of 1.8KV, 200 omega and 25 muF, and the electric transfer is performedThe cDNA library was obtained by coating 10 cDNA tags 24.5cm in length2Large plate, calculated library capacity greater than 2.0 x106cfu/mL, the distribution range of the insert length is about 400 bp-2 Kbp, the average length is more than 1Kbp, the diversity is good, and the recombination rate is about 100%.
Validation of the anti-PPRV VHH antibody cDNA library of the invention: culturing attenuated cell vaccine, concentrating, purifying and immunoblotting reaction with cDNA library.
The invention also provides application of the cDNA library of the anti-PPRV VHH antibody in screening the anti-peste des petits ruminants virus VHH antibody.
Compared with the prior art, the invention has the beneficial effects that:
the invention utilizes PPRV attenuated vaccine and concentrated and purified attenuated vaccine to prepare antigen immune camel, and high-quality immune serum and whole blood are obtained; reverse transcription of total mRNA extracted from peripheral blood lymphocytes is carried out by utilizing primers with SfiIA and SfiIB enzyme cutting sites at two ends respectively, a first chain of full-length cDNA is synthesized, PCR amplification is carried out, SfiI single enzyme cutting is carried out, and then a specific vector pGADT7-SfiI is directionally inserted, so that a camel-source anti-peste des petits ruminants virus antibody cDNA library is obtained. And (3) identification: library capacity>About 2.0X 106cfu/mL, about 400bp to 2Kbp of insert fragment, about 1Kbp of average length, good diversity and about 100 percent of recombination rate; functional analysis showed that: an anti-peste des petits ruminants virus VHH antibody having a neutralizing activity exists in the constructed cDNA library. The invention provides a platform for screening the anti-Peste des petits ruminants virus VHH antibody and provides a new technical means for early treatment and diagnosis of Peste des petits ruminants.
For obtaining high-quality antibody cDNA library, antigen preparation, immunization dose and immunization program are very critical links, and are also the basis for successfully constructing the cDNA antibody library, and after some immunization, antibodies are not produced or the antibody titer is very low, the invention adopts camel left foreleg deltoid muscle intradermal injection of 2.5x105cfu BCG vaccine, right neck subcutaneous injection sheep 3 PPRV attenuated vaccine for first immunity; 2w post-blood sampling and immunization for self-preparation of antigen: inoculating the attenuated vaccine in the market to cells, culturing in a large amount, collecting, concentrating, purifying, and adding an equivalent amount of adjuvant for emulsification to obtain the attenuated vaccine; after 2 months, three-free method is carried outThe formula is as follows. The results obtained camel serum antibody titers were: blank control OD450=0.055, negative control OD450=0.059, positive control OD450=2.340, nonimmune serum OD450The titer of the primary immune serum is 1:40, the titer of the secondary immune serum is 1:2560, and the titer of the serum after the tertiary immune is 1:16000, which shows that the invention successfully solves the early key link involved in the construction of the cDNA antibody library and obtains high-quality immune serum and whole blood.
The invention successfully constructs a cDNA library, and the acquisition of mRNA is a crucial factor. Firstly, the mRNA must be intact and not degraded, the more mRNA species, the more complete the cDNA library is constructed, and secondly, it should be noted that the mRNA cannot be contaminated with DNA. RNA is a highly degradable nucleic acid molecule, present in the cytoplasm and nucleus. Mammals can contain on average 5-10ug of RNA per million cells, with 80% -85% rRNA, 10% -16% tRNA, and only 1% -5% mRNA. The mRNA molecules are various in types and are heterogeneous in molecular weight, but most of the mRNA molecules have 20-250 polyadenylic acid poly (A) at the 3' end, the mRNA with a poly (A) tail can form a stable RNA-DNA hybrid chain with a short chain oligo (dT) connected on a cellulose medium, the mRNA molecules are purified from the Total RNA, the concentration of Total RNA extracted is 5162ng/uL, the R value is 1.97, the concentration of purified mRNA poly (A) is about 251ng/uL, and the R value is 1.98.
Eukaryotic mRNA has a process similar to that of "wearing shoes and caps" during processing, so that mRNA has a fragment of Poly A at its 3' end, which is the basis for preparing cDNA library by reverse transcriptase. However, since the 5' -end sequences of cDNAs are different, it has been a problem how to obtain full-length cDNAs, how to amplify cDNA libraries obtained by reverse transcription from a trace amount of mRNA, and how to obtain full-length cDNAs (i.e., RACEs) using known fragment sequences. The cDNA library constructed by using SMART (switching mechanism at 5' end of the RNA transcript) with SfiIA and SfiIB enzyme cutting sites and a CDS method can represent the abundance of mRNA in the original sample and keep the integrity of biological genetic information. The capacity of the constructed anti-PPRV VHH antibody cDNA library is more than about 2.0X 106cfu/mL, 16 random samples from the plateThe clones were subjected to colony PCR, and the results of electrophoresis showed that the inserts of the library were all different and distributed between 400 to 2kbp (FIG. 8), and the average insert was about 1kbp, indicating that the diversity of the constructed library was good. The recombination rate of the library was 100% as seen from the colony PCR results. The constructed library is amplified, cloned and sequenced, a proper sequence is further expressed, and after an Elisa reaction is carried out on an expressed protein and a prepared antigen, a specific strip, the size and an expected value are found to be generated, so that the constructed library contains an active anti-PPRV VHH antibody.
Drawings
FIG. 1 is a healthy Vero/s/v cell;
FIG. 2 shows Vero/s/v cells inoculated with PPRV attenuated seedlings;
FIG. 3 shows lymphocyte layers in the case of lymphocyte separation;
FIG. 4 shows all lymphocytes collected;
FIG. 5 shows the result of RNA12g/L agarose denaturing gel electrophoresis;
m2 is 250bp DNA Ladder; 1 is Total RNA;
FIG. 6 shows the result of LD-synthesized ds cDNA 1.0% agarose gel electrophoresis;
m2 is 250bp DNA Ladder; 1 is the Smear band result of the cDNA amplified by LD;
FIG. 7 shows the result of 1.0% agarose gel electrophoresis of cDNA after removal of short fragments;
m2 is 250bp DNA Ladder; 1 short fragment removal of cDNA;
FIG. 8 shows the result of 1.0% agarose gel electrophoresis of the library amplification assay fragment;
m2 is 250bp DNA Ladder; 1 is the detection result of 1-16 insertion fragments;
FIG. 9 is a plate diagram of the identification of the amplification required for small transformations of the library;
FIG. 10 is a graph showing the identification of the anti-PPRV activity of the library.
Detailed Description
The invention will be further explained and explained in detail with reference to the drawings, in which:
a preparation method of a camel-source PPRV-resistant cDNA library comprises the following specific steps:
i, immune camel of Peste des petits ruminants: camel left foreleg deltoid intradermal injection 2.5x105cfu BCG vaccine, right side neck subcutaneous injection sheep 3 PPRV attenuated vaccine for first immunity, during the period, culturing PPRV cell attenuated vaccine in large quantity, collecting, concentrating, purifying, preparing concentrated antigen, 2w blood sampling and immunizing the concentrated antigen emulsified by equivalent adjuvant, 2 months later, performing third immunity, and the way of two immunity.
Detecting immune serum, and collecting 200mL blood separated serum for later use after camel immunity is qualified; preparing a high-pressure anticoagulant, adding 35mL of anticoagulant into every 200mL of whole blood, separating camel peripheral blood lymphocytes by using camel peripheral blood lymphocyte separation liquid, extracting Total RNA (ribonucleic acid), and purifying mRNA poly (A).
And III, reverse transcription and extension are carried out by using CDS primers with SfiIB restriction sites, SMART primers with SfiIA restriction sites and reverse transcriptase to synthesize a first chain of the full-length cDNA, a complementary chain is synthesized by amplification, and amplification is continued.
And IV, amplifying the product in the SfiI single enzyme digestion III and a specific vector GADT7-SfiI vector to obtain cDNA with two sticky ends different, directionally inserting the cDNA into the specific vector GADT7-SfiI vector after enzyme digestion through T4 ligase, and electrically transforming the product to HST08 to obtain a primary cDNA library.
V. when the cDNA library was harvested, 10 cDNA library pieces were spread at 24.5cm2And (4) large-plate, calculating the library capacity of the obtained library, and picking part of clones to identify diversity and recombination rate.
And VI, verifying that the obtained cDNA library has a VHH antibody with neutralizing activity by using the culture concentrated antigen.
Examples
Experimental materials and reagents: MEM powder and 1:250 Trypsin were purchased from GIBCO company, and cell culture solution and pancreatin cell digestive juice were prepared in the laboratory; newborn bovine serum was purchased from Lanzhou bright-bright Biotechnology Inc.; 3-month-old female Bactrian camels were purchased from Jinchang farmers (no Bucky disease detected); camel peripheral blood lymphocyte isolate was purchased from the tertiary ocean of tianjin; RNAlater was purchased from Qiagen; SMART cDNA Library Construction Kit, Advantage2 PCR Kit, CHROMA SPIN-1000-TE and enzymes and related vectors were purchased from Clontech; the DNA ligation Kit, TaKaRa MiniBEST DNA Fragment Purification Kit, the electrotransformation Escherichia coli competent cell HST08 and PCR-related reagents were purchased from TaKaRa; PPRV antibody ELISA detection kit was purchased from IDEXX; other biochemical reagents are all made in China and analyzed to be pure.
High quality immune serum and whole blood
1.1. Preparation of concentrated antigen by culturing PPRV attenuated seedling
Selecting three bottles of healthy Vero/s/v cells (see patent application number 201611208506.7) (shown in figure 1) constructed in the laboratory, inoculating two bottles of Xinjiang Tiankang PPRV attenuated seedlings, taking one bottle as negative control, and after 5 days, repeatedly freezing and thawing in a refrigerator, and recording as F1 generation; collecting for 3.5 days until F9 generation, inoculating 5 bottles (shown in figure 2) for F10 generation, repeatedly freezing and thawing, collecting, centrifuging, concentrating, purifying, packaging 1.5mL, and storing in refrigerator at-70 deg.C.
1.2 PPRV attenuated vaccine immune camel
Selecting healthy female camels of 3 months old, collecting blood, detecting negative of disease distribution antibody, collecting 100mL blood, separating, preparing negative serum, injecting 2.5x10 into left anterior 1 leg deltoid muscle skin5cfu BCG vaccine, right side neck subcutaneous injection sheep 3 PPRV attenuated vaccine for first immunity, 2w blood sampling, the antigen prepared above with equal amount of adjuvant emulsification for second immunity, 2 months after three immunity, the way is two immunity. And (5) detecting the effect after immunization.
1.3. As a result:
after the Peste des petits ruminants are immunized according to the immunization program, the camel serum antibody titer is as follows: blank control OD450=0.055, negative control OD450=0.059, positive control OD450=2.340, nonimmune serum OD450=0.065, the titer of the primary immune serum is 1:40, the titer of the secondary immune serum is 1:2560, and the titer of the post-tertiary immune serum is 1:16000, therefore, the titer of the serum antibody is better, which indicates that the immunity is qualified.
Separating peripheral blood lymphocytes, extracting Total RNA, separating and purifying mRNA
2.1. Blood sampling
And adding 35mL of anticoagulant into a 500mL saline bottle, collecting 200mL of immune qualified camel whole blood, shaking up, and adding the whole blood diluent in equal amount to obtain the anticoagulation blood.
2.2. Isolation of peripheral blood lymphocytes
Adding 10mL of camel lymphocyte separation liquid into a 30mL glass centrifuge tube, slowly adding diluted anticoagulation liquid into the glass centrifuge tube, slightly putting the glass centrifuge tube into a group of 16 tubes, controlling the temperature to be 18-22 ℃, performing centrifugal force at 1800rpm for 20min, and performing manual braking after the centrifugation is finished; carefully collecting lymphocyte layer (shown in figure 3) in a new collection tube with Pasteur pipette, adding cell washing solution at a ratio of 1:5, centrifuging at about 20 deg.C at 1000rpm for 10 min; discarding the supernatant, repeating twice, removing the red blood cells to obtain the precipitate as the separated lymphocytes (as shown in FIG. 4), suspending the separated lymphocytes in RNA sample preservation solution RNAlater, and preserving at-70 deg.C for later use.
2.3. Extraction of lymphocyte TotalRNA and mRNA separation and purification
Exogenous RNA enzyme pollution is removed as much as possible, the activity of endogenous RNA enzyme is inhibited, and an 'RNA enzyme-free environment' is created. The combination of guanidinium isothiocyanate, beta-mercaptoethanol and N-lauryl sarcosine (SLS) serving as a detergent serving as a strongest RNase inhibitor is used for inhibiting RNA degradation and enhancing the dissociation of a nuclear protein complex, so that the separation of RNA and protein improves the yield of RNA: adding 1mLTRIzol into 400uL of lymphocyte sample preservation solution, and operating according to the steps; then 200uL of chloroform was added; after centrifugation, the supernatant was carefully collected and an equal amount of isopropanol was added; washing the precipitate with 75% anhydrous ethanol, dissolving the dried precipitate with 25uL of RNAse-free water to obtain the Total RNA, and determining the Total RNA concentration by ND 2000.
Heating Total RNA solution at 65 ℃ for 15min, rapidly cooling to 0 ℃, and performing denaturation to destroy the primary structure of RNA, fully expose polyA tail and improve the recovery rate of polyA; simultaneously dissociating the mRNA from the rRNA, and separating and purifying by oligo (dT) -cellulose column chromatography: suspending 0.2g of oligo (dT) -cellulose in 0.1N NaOH, transferring to 2mL glass wool sealed internal syringe, washing with RNAse-free water and 0.01M Tris-HCl, 0.5M KCl (pH 7.5) 1x high salt to pH 7.5; uniformly mixing the denatured total RNA with equal volume of 0.02MTris-HCl and 1MKCl (pH 7.5) 2x high salt, loading onto a column, covering, placing a rotary shaking table for 10min, adding 1x high salt for 1mL, collecting effluent, denaturing again, loading onto the column, washing with 4mL high salt for twice, discarding effluent, washing with 3mL low salt for 1min, centrifuging for 500g, collecting centrifuge, denaturing, adding 0.01MTris-HCl, loading onto a second oligo (dT) -cellulose column with 1MKCl (pH 7.5), eluting with high salt and low salt according to the first column, 400uL of 0.01MTris-HCl (pH 7.5) to obtain mRNA, adding 2.5 times of cold ethanol, 0.1 volume of 3M sodium acetate, pH5.2, standing at 20 ℃ for 2h, centrifuging at 4 ℃ for 20min to precipitate mRNA, washing with 75% ethanol, drying, dissolving mRNA with 2uL of water to obtain purified mRNA, and detecting.
2.4. Results
As can be seen from FIGS. 3 and 4, the lymphocyte layer and the lymphocytes separated and collected by the present invention are clear and meet the requirements, the concentration of the extracted peripheral blood lymphocyte Total RNA measured by ND2000 is 5162ng/uL, the R value is 1.97, the concentration of the mRNA poly (A) obtained after the purification by oligo (dT) -cellulose column chromatography is 251ng/uL, and the R value is 1.98. Three distinct bands, 28S, 18S and 5S, were visualized by denaturing agarose gel electrophoresis at 12g/L (FIG. 5), 28S: 18S being approximately 2: 1. The purity of RNA and mRNA is high, and the requirement of library construction is met.
full-Length cDNA Synthesis, double-stranded cDNA Synthesis and purification
The primers used in constructing the primary cDNA library are SMART and CDS with SfiIA and SfiIB restriction enzyme cutting sites at two ends respectively, and the used vector is as follows: pGADT7-SfiI vector, with SfiI cleavage sites. The construction of the primary cDNA library is as follows:
3.1. synthesis of full Length cDNA
The following components are sequentially added into a 200uL PCR amplification tube: 3uL of mRNA, 1uL of primer with enzyme cutting site GGCCATTACGGCC SMART and 1uL of primer with enzyme cutting site GGCCGCCTCGGCC CDS are clicked and mixed evenly, placed in a metal bath at 72 ℃, incubated for 2min, immediately placed on ice, cooled for 2min and clicked for collection. Sequentially adding 5X First strand Buffer 2uL, 20mM DTT1uL, 10mM dNTP Mix1uL and 200u/uL MMLV reverse transcriptase 1uL, clicking, mixing uniformly, placing in a 42 ℃ metal bath, carrying out reverse transcription for 1h, cooling at room temperature, adding 2u/uL RNase inhibitor 1uL to obtain a product which is full-length cDNA, and storing at-20 ℃ for later use.
3.2. Amplification of
Advantage2 is a multifunctional mixed polymerase, 3 times as high as wild type Taq in fidelity, and is suitable for amplifying various types of DNA templates including cDNA. Therefore, Advantage2 PCR Kit is selected for double-strand amplification: 1uL of the above full-length cDNA (100 ng/uL), 40uL ddH20, 5uL 10X Advantage2 PCR buffer, SMART primer 1uL, CDS primer 1uL, 50X dNTP Mix1uL, 50X Advantage2 polymerase Mix1 uL. Amplification program 95 ℃ 1min, 95 ℃ 30s, 68 ℃ 3min, 25-30cycles (extension time increase 5s per cycle), 68 ℃ 3 min. The double-stranded cDNA of 8uL was identified by electrophoresis on a 1.0% agarose gel.
3.3. Purification of
The amplified cDNA was purified and recovered using TaKaRa MiniBEST DNA Fragment Purification Kit according to the instructions.
3.4. Results
mRNA is synthesized by reverse transcription, LD amplification is carried out, and recovery and purification are carried out to obtain a No. 1 band in figure 6, which is the sum of fragments, and Smear band appears in electrophoresis result, which indicates that the obtained cDNA result is reliable.
Construction, quality evaluation and identification of cDNA library:
4.1. cDNA library construction
Firstly, carrying out enzyme digestion treatment on cDNA and pGADT7-SfiI vectors by using a restriction enzyme SfiI; as too many fragments below 400bp can seriously affect the quality of subsequent connection and library and need to remove short fragments, CHROMA SPIN-1000-TE is used for carrying out column processing on the cDNA after enzyme digestion to remove short fragments, and after PCI/CI purification processing, ethanol refining and ddH2Dissolving out O; and finally, connecting pGADT7-SfiI vector with a proper amount of post-column cDNA by using a DNA ligation Kit at 12 ℃, and purifying and refining the obtained connecting solution to obtain a primary cDNA library.
4.2. Evaluation of library quality
Taking a small amount of primary library connecting liquid, and electrically transforming competent cells HST 08; coating an Amp resistant LB plate with a proper amount of transformation liquid, and culturing at 37 ℃ overnight; taking the cotransformation product bacterial liquid by the number of colonies growing on 1/10 plates, diluting according to 1/10, 1/100 and 1/1000, respectively coating 100uL of diluent to 100mm plates, carrying out inverted culture at 30 ℃ for 3-5d until clones appear, counting the growing single colonies according to the formula: plate clone number/plating volume (mL) X dilution = plaque forming unit/mL.
4.3. Library amplification identification
Randomly picking 16 single colonies, performing colony PCR with pGADT-Rec universal sequencing primer, and analyzing the size of the library insert and recombination rate. 8uL of the PCR product was analyzed by electrophoresis on a 1.0% agarose gel.
4.4. Million clone amplification and plasmid extraction of primary cDNA library
From the data of the library capacity assay, the volume of the required linker was calculated from the amount of 100 million clones, and the cells were electroporated into competent cells HST08, plated with 10 large LB plates of 24.5X 24.5cm, and cultured overnight at 37 ℃. Monitoring the number of amplified clones obtained from the actual transformation; the amplified colonies were recovered and plasmid extraction was performed using the NucleoBond Xtra Midi EF kit. 100ng of the amplified library plasmid was subjected to 1.0% agarose gel electrophoresis.
4.5. Amplifying the constructed library, cloning, sequencing, further expressing a proper sequence, carrying out Elisa reaction on the expressed protein and the prepared antigen, and detecting the reactivity of the expressed protein and the prepared antigen.
4.6. Results
When constructing the library, the result after removing the short fragment is shown in FIG. 7, which shows that the constructed library has no redundant sequence; the library volume calculated according to the formula was > about 2.0 x106cfu/mL. FIG. 8 shows the electrophoresis result that the coverage of the cDNA library is 400-2000bp, the average insert is about 1000bp, the diversity of the constructed library is good, and the recombination rate of the library is 100% according to the colony PCR result. Identify libraries and collect library plates as shown in FIG. 9. After amplification, cloning, sequencing, further expression of appropriate sequences, and Elisa reaction of the expressed protein and the prepared antigen, a binding band is found to be generated, which indicates that the constructed library contains an active anti-PPRV VHH antibody, and the result is shown in FIG. 10.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Claims (7)
1. A camel-derived PPRV-resistant cDNA library, which is characterized in that: anti-peste des petits ruminants virus VHH antibodies with neutralizing activity are present in the cDNA library.
2. The camel-derived anti-PPRV cDNA library of claim 1, wherein: library capacity>2.0ｘ106cfu/mL, length distribution range of 400 bp-2 Kbp, average length of more than 1Kbp, and recombination rate of 100%.
3. A method for preparing a camel-derived anti-PPRV cDNA library according to claim 1 or 2, wherein: the method specifically comprises the following steps:
step A: obtaining immune serum through Peste des petits ruminants immune camel procedure;
and B: detecting immune serum according to the titer of a serum antibody, collecting immune camel whole blood after camel immunity is qualified, adding an anticoagulant into the immune camel whole blood, shaking up, diluting, adding the immune camel whole blood into camel lymphocyte separation liquid to obtain lymphocytes for separating camel peripheral blood, extracting Total RNA from the lymphocytes, and separating and purifying mRNA;
and C: reverse transcribing total mRNA extracted from peripheral blood lymphocytes by using primers with enzyme cutting sites SfiIA and SfiIB at two ends respectively, synthesizing a full-length cDNA first chain, amplifying, directionally inserting the single enzyme cutting SfiI into a specific vector pGADT7-SfiI vector to obtain a camel source PPRV-resistant cDNA library;
in the step C, the specific process of synthesizing the full-length cDNA is as follows: the following components are sequentially added into a PCR amplification tube: uniformly mixing mRNA, GGCCATTACGGCC SMART primers with enzyme cutting sites and GGCCGCCTCGGCC CDS primers with enzyme cutting sites by clicking, placing in a metal bath at 72 ℃, incubating for 2min, immediately placing on ice, cooling for 2min, and collecting by clicking; sequentially adding 5X First strand Buffer, DTT, dNTP Mix and MMLV reverse transcriptase, clicking and uniformly mixing, placing in a 42 ℃ metal bath, carrying out reverse transcription for 1h, cooling at room temperature, and adding an RNase inhibitor to obtain a product, namely a full-length cDNA;
in the step C, selecting Advantage2 PCR Kit for double-chain amplification: 1uL full-length cDNA 100ng/uL, 40uL ddH2O, 5uL 10X Advantage2 PCR buffer, SMART primer 1uL, CDS primer 1uL, 50X dNTP Mix1uL, 50X Advantage2 polymerase Mix1 uL; the amplification program comprises the steps of 1min at 95 ℃, 30s at 95 ℃, 3min at 68 ℃, 5s increase of the extension time of each cycle of 25-30cycles, and 3min at 68 ℃ to obtain double-stranded cDNA;
in step C, the cDNA and pGADT7-SfiI vector are subjected to enzyme digestion treatment by using restriction enzyme SfiI, the digested cDNA is subjected to column chromatography treatment by using CHROMA SPIN-1000-TE, short fragments are removed, and PCI/CI purification treatment, ethanol refining and ddH treatment are carried out2O elution, and finally, the post-column cDNA was ligated to GADT7-SfiI vector using DNA ligation Kit at 12 ℃ to obtain a primary cDNA library by purifying and refining the ligation solution.
4. The method for preparing the camel-derived anti-PPRV cDNA library according to claim 3, which is characterized in that: in the step A, the Peste des petits ruminants immune camel program comprises the following steps: camel left foreleg deltoid intradermal injection 2.5x105cfu BCG vaccine, right side neck subcutaneous injection sheep 3 PPRV attenuated vaccine for first immunity, 2w blood sampling and immune concentration, purified cell culture attenuated vaccine, 2 months later for three immunity, the way is two immunity.
5. The method for preparing the camel-derived anti-PPRV cDNA library according to claim 4, which is characterized in that: in step B, the serum antibody titer after immunization is: blank control OD450=0.055, negative control OD450=0.059, positive control OD450=2.340, nonimmune serum OD450=0.065, the titer of the primary immune serum is 1:40, the titer of the secondary immune serum is 1:2560, and the titer of the tertiary immune serum is 1: 16000; collecting third immune camel whole blood anticoagulation, separating peripheral blood lymphocyte, extracting Total RNA with concentration of 5162ng/uL and R value of 1.97, and purifying mRNA poly (A) with concentration of 251ng/uL and R value of 1.98.
6. Use of a camel-derived anti-PPRV cDNA library according to claim 1 or 2 for screening anti-PPRV VHH antibodies.
7. The camel-derived anti-PPRV cDNA library according to claim 6, characterized in that: the anti-PPRV VHH antibody cDNA library was validated using the following method: culturing attenuated cell vaccine, concentrating, purifying and immunoblotting reaction with cDNA library.
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