CN108504654A - The yeast cDNA library and its construction method and purposes of a kind of anti-Sai Nika paddy virus VHH antibody - Google Patents

Abstract

The invention discloses a yeast cDNA library of anti-Seneca Valley virus VHH antibody and its construction method and application. The invention separates lymphocytes from the peripheral blood of camels immunized with Seneca Valley virus inactivated vaccine, and obtains lymphocytes by RT- The cDNA fragment of the anti-Seneca Valley virus VHH antibody was obtained by PCR, and the cDNA fragment of the VHH antibody was co-transformed with pGADT7‑Rec into yeast competent cells to obtain the yeast cDNA library of the anti-Seneca Valley virus VHH antibody. The results showed that the titer of the library was about 7.2×10 ⁶ cfu/ml, and the recombination rate of the library was 100%. Further preliminary functional identification of the library showed that there were VHH antibodies against Seneca Valley virus in the constructed yeast library. The present invention provides a platform for screening anti-Seneca Valley virus VHH antibodies, and provides a new method for early treatment and diagnosis of Seneca Valley.

Description

A yeast cDNA library of anti-Seneca Valley virus VHH antibody and its construction method and use

technical field

The invention belongs to the field of biotechnology and relates to a Bactrian camel VHH heavy chain antibody library against Seneca Valley virus. In addition, the present invention also relates to the construction method, library capacity identification and application of the antibody library.

Background technique

Seneca valley virus (SVV), also known as Seneca virus A (SVA), belongs to the genus Senecavirus of the Picornaviridae family (a different genus from the same family as foot-and-mouth disease virus), and is also a member of the genus sole member. The genome is single-stranded positive-sense RNA. Like other members of the picornaviridae family, SVV has a non-enveloped capsid approximately 25-30 nm in diameter with icosahedral symmetry. The SVV genome is approximately 7.2 kb in length and contains a unique open reading frame (ORF) flanked by a 5' untranslated region (MTR) (approximately 668 nucleotides) and a 3' untranslated region (approximately 68 nuclei nucleotides), where the 3' MTR is followed by a poly(A) tail. The SVV coding region encodes a 2181 amino acid precursor polyprotein that can be cleaved into a leader protein and three major polyproteins (P1, P2 and P3). The P1 genomic region encodes the structural proteins (VP4, VP2, VP3, and VP1) that make up the viral capsid, while the P2 and P3 genomic regions encode seven nonstructural proteins involved in viral replication (2A, 2B, 2C, 3A, 3B, 3C, and 3D).

Seneca valley virus (SVV) infection of pigs can cause primary vesicular disease in pigs, and the clinical symptoms are indistinguishable from foot-and-mouth disease. SVV has successively launched in the United States (2015, 2016, 2017), Canada (2007, 2011, 2015, 2016), Brazil (2014, 2015), Colombia (2016), China (2015, 2016, 2017) and Thailand (2016) and other countries caused outbreaks. Since it was introduced into China in 2015, it was distributed in 2015-2016. Since 2017, SVV has spread rapidly in Fujian, Guangdong, Guangxi, Henan (11 counties), Hebei, Shandong, Liaoning and other provinces, causing a large number of The disease occurred in the pig farm, and there were cases of mixed infection of SVV and FMDV. However, the prevalence of SVV is still unknown, and there are no commercial vaccines and diagnostic reagents available. SVV is still out of control, and the situation of SVV prevention and control is severe. And because the clinical symptoms are similar to foot-and-mouth disease, and can be mixed infection, it seriously interferes with the prevention and control of foot-and-mouth disease.

In 1993, Hamers et al. reported that half of the antibodies in camel blood naturally lacked the light chain and heavy chain constant region 1 (CH1), cloned the variable region of the heavy chain of this antibody, and constructed a single variable region consisting of only one heavy chain variable region. Domain antibody (variable domain of heavy chain of heavy 2chain antibody, VHH), has been renamed as "nanobody", which is the smallest antigen-binding fragment with complete functions. Nanobodies have been developed into highly versatile molecules with extensive biological and clinical application value since their discovery. Nanobodies have high stability and high affinity for antigen binding, and can interact with protein clefts and enzyme active sites, acting like inhibitors. Therefore, nanobodies can provide new ideas for designing small molecule enzyme inhibitors from peptidomimetic drugs. Nanobodies are easier to manufacture than mAbs due to having only heavy chains. The unique properties of nanobodies, such as stability in extreme temperature and pH environments, also have water solubility and conformational stability unmatched by conventional single domain antibodies. This monomeric domain can bind to the antigen with high specificity and high affinity, thereby neutralizing or blocking relatively hidden antigenic epitopes. With the above characteristics, nanobodies have great advantages compared with ordinary single domain antibodies. Nanobodies are currently available as the smallest unit with complete functions, stability, and antigen-binding properties. It is an important target molecule in imaging research, which provides a good source for engineered antibodies and has broad application prospects. Therefore, nanobodies are of great value in the treatment and diagnosis of diseases.

Therefore, based on the current research and utilization in the field of antibodies, it is not difficult to find that traditional antibody drugs still face challenges, and it is necessary to design and produce a new generation of more effective antibody molecules to meet clinical needs. In particular, how to realize the miniaturization of antibodies so that they can efficiently act on specific parts. For this reason, realizing the miniaturization of antibodies targeting effector molecules or other target cells in cancer cells has always been an important goal and research hotspot in antibody engineering and anti-cancer research.

The capacity and diversity of the antibody library greatly affect the probability of panning out specific antibodies and the affinity of antibodies. Since the first cDNA library was constructed in 1976, its method has been continuously improved. The cDNA library constructed by the SMART (switching mechanism at 5'end of the RNA transcript) method can represent the abundance of mRNA in the original sample and maintain the Integrity of biological genetic information. Moreover, this method only needs 25ng of mRNA or 50ng of Total RNA to obtain a high-quality, high-yield cDNA library. More importantly, the obtained cDNA can represent the abundance of mRNA in the original sample and can be applied to direct amplification. Genes, construction of cDNA libraries.

In summary, screening antibodies with SVV specificity, good biological activity, strong antigen-binding ability, and potential neutralization function through the establishment of antibody libraries, especially new antibodies such as nanobodies, is very important for the establishment of sensitive SVV clinical trials. It is of great significance to study the mechanism of virus infection as a diagnostic method.

Contents of the invention

The object of the present invention is to provide a yeast cDNA library of anti-Seneca Valley virus VHH antibody and its construction method.

In order to achieve the above object, the technical means that the present invention adopts are as follows: a kind of construction method of the yeast cDNA library of anti-Seneca Valley virus VHH antibody of the present invention, it comprises the following steps:

Step (1) Lymphocyte separation: Separation of lymphocytes from the peripheral blood of camels immunized with the Seneca Valley inactivated vaccine, and preservation for subsequent use;

Step (2) extraction of lymphocyte total mRNA: extracting the total mRNA of lymphocyte isolated in step (1);

Step (3) synthesis of whole gene cDNA: using the total mRNA of lymphocytes extracted in step (2) as a template to synthesize whole gene cDNA;

Step (4) The first round of amplification: use the whole gene cDNA synthesized in step (3) as a template, and use P1 and R1 as primers to perform the first round of amplification. The amplification program is: 95°C for 1min; 95°C for 15s, 68°C 5min at ℃, 20 cycles; 5min at 68℃, electrophoresis the amplified product, purify and collect the 600bp product, and store it at -20℃ for later use;

SEQ ID NO.1-P1:5-GTCCTGGCTGCTCTTCTACAAGG-3

SEQ ID NO.2-R1:5-GGTACGTGCTGTTGAACTGTTCC-3

Step (5) The second round of amplification: use the first round of amplification product as a template, and use P2 and R2 as primers to perform a second round of amplification. The amplification program is: 95°C for 1min; 95°C for 15s, 68°C for 5min, 20 cycles; 68°C for 5min; amplified product electrophoresis, purified and collected 400bp product, stored at -20°C for later use;

SEQ ID NO.3-P2:5-TTCCACCCAAGCAGTGGTATCAACGCAGAGTGG

GAGTCTGGGGGAGG-3

SEQ ID NO.4-R2:5-GTATCGATGCCCACCCTCTAGAGGCCGAGGCGG

CCGACATGGAGACGGTGACCTGGGT-3

Step (6) Construction and harvesting of the library: Prepare yeast competent cells, transform 20 μl of the second-round amplification product and 6 μl of pGADT7-Rec vector per 600 μL of competent cells, and evenly spread the transformed bacteria solution on the SD/-Leu plate and cultured upside down at 30°C for 3-5d until clones appeared; put the plate again in the refrigerator at 4°C for 3-4h, add YPDA liquid medium containing 25% (v/v) glycerol to each plate, and add no Bacteria glass beads were shaken repeatedly in the horizontal direction, and then the bacterial liquid was collected to obtain the yeast cDNA library of anti-Seneca Valley virus VHH antibody.

In the present invention, preferably, the lymphocyte separation is carried out by using a camel peripheral blood lymphocyte separation kit (product number: LTS1076, purchased from Tianjin Haoyang Biological Products Technology Co., Ltd.) according to the following steps:

(1) Add an equal volume of sample diluent to the anticoagulant blood of camels immunized with Seneca Valley virus inactivated vaccine, and mix to obtain the anticoagulant diluent;

(2) Measure the lymphocyte separation liquid according to the volume ratio of sample diluent and lymphocyte separation liquid of 5:9, then add anticoagulant diluent slowly and then quickly, horizontal centrifuge at 1800rpm, 18-22°C Centrifuge for 20min;

(3) Carefully collect the lymphocyte layer in a new collection tube, dilute with 5 times the amount of cell washing solution, and centrifuge at 1000rpm at 18-22°C for 10 minutes in a horizontal centrifuge; discard the supernatant, dilute the pellet with cell washing solution, and use a horizontal centrifuge Centrifuge at 1000rpm at 18-22°C for 10min; resuspend the pellet in 5ml of RNase-free water, then add 45ml of RPIM-1640 culture medium and mix well, centrifuge at 1000rpm at 18-22°C for 10min, the pellet is lymphocytes, and resuspend Store in RNALater preservation solution at -70°C for later use.

In the present invention, preferably, the extraction of the total lymphocyte mRNA is carried out according to the following method: draw 400 μl of the lymphocyte fluid separated in step (1), add 1ml TRizol, mix well, let stand at 4°C for 5 minutes; add 200 μl Chloroform, mix well, let stand at room temperature for 3min, centrifuge at 12000r/min, 4°C for 15min; absorb 750μl supernatant, add an equal amount of isopropanol, let stand at room temperature for 10min, centrifuge at 10000r/min, 4°C for 10min; Clear, add 75% ethanol to the pellet, centrifuge at 8000r/min, 4°C for 5min; put the pellet in a fume hood for 10min to dry; the pellet dissolved in 20μl RNase-free water is the harvested total mRNA.

In the present invention, preferably, the whole gene cDNA synthesis is to adopt the SMART cDNA library construction kit, and add the following components in sequence in the 0.2ml PCR amplification tube:

mRNA 3μl SMART 1μl CDSⅢ 1μl

After mixing, place the centrifuge tube in a metal bath at 72°C, incubate for 2 minutes, immediately put it on ice, cool for 2 minutes, and then add the following components in sequence:

5×First strand Bμffer 2μl DTT (20mM) 1μl dNTPMi×(10mM) 1μl MMLV reverse transcriptase (200μ/μl) 1μl

After mixing, place the centrifuge tube in a metal bath at 42°C, perform reverse transcription for 1 hour, cool at room temperature, add 1 μl RNase H, and the resulting product is whole genome cDNA, which is stored at -20°C for later use.

In the present invention, preferably, the first round of amplification is to adopt the SMART cDNA library construction kit, and the following components are sequentially added in the 0.2ml PCR amplification tube:

10×PCR buffer 10μl 10× solution 10μl 50× polymerase mix 2μl 50×dNTP 2μl

P1 primer 2μl R1 primer 2μl whole genome cDNA 2μl H ₂ O 70μl

The first round of amplification was performed under the amplification program described in step (4), the amplified product was electrophoresed, and the 600bp product was purified and collected, and stored at -20°C for future use.

In the present invention, preferably, the second round of amplification is to use the SMART cDNA library construction kit, and the following components are sequentially added to the 0.2ml PCR amplification tube:

10×PCR buffer 10μl 10× solution 10μl 50× polymerase mix 2μl 50×dNTP 2μl P2 primer 2μl R2 primer 2μl First-round amplification product 2μl H ₂ O 70μl

Carry out the second round of amplification under the amplification program described in step (5), electrophoresis the amplified product, and purify and collect the 400bp product from the gel with the gel recovery kit, and store it at -20°C for future use.

In the present invention, the kit used above for cDNA synthesis is the SMART cDNA library construction kit (product number: 634901, purchased from Treasure Biotechnology Co., Ltd.).

Furthermore, the present invention also proposes to construct the yeast cDNA library of the anti-Seneca Valley virus VHH antibody obtained according to the method described above.

In the present invention, the cDNA distribution in the library is between 400-2000bp, the titer is 5.5×10 ⁶ cfμ/ml, and the recombination rate is 100%.

Furthermore, the present invention also proposes the use of the yeast cDNA library of anti-Seneca Valley virus VHH antibody in screening anti-Seneca Valley virus VHH antibody.

The beneficial effect of the present invention compared with the prior art: the present invention identified the capacity and polymorphism of the constructed yeast cDNA library of anti-Seneca Valley virus VHH antibody, and the results showed that the titer of the library was about 7.2×10 ⁶ cfμ/ml. 16 clones were randomly selected from the SD/-Leu plate for colony PCR. The electrophoresis results showed that the library inserts were all different and distributed between 400-2000bp, as shown in Figure 4. The average insert was about 1000bp, indicating that the constructed The diversity of the library is good. From the results of colony PCR, it can be known that the recombination rate of the library is 100%. The preliminary functional identification of the yeast cDNA library of anti-Seneca Valley virus VHH antibody was carried out by hemagglutination inhibition test, which showed that there were anti-Seneca Valley virus VHH antibodies with neutralizing activity in the constructed yeast library. The present invention provides a platform for screening anti-Seneca Valley virus VHH antibodies, and provides a new technical means for the early treatment and diagnosis of Seneca Valley.

Description of drawings

Fig. 1. is the total RNA electrophoresis figure of camel peripheral blood lymphocyte;

M.DL5000 relative molecular mass standard; 1. Total RNA of camel peripheral blood lymphocytes;

Fig. 2 is the agarose gel electrophoresis picture of the second round of amplification of ds cDNA of camel peripheral blood lymphocytes;

M.DL2000 relative molecular mass standard; 1. The first round of amplified ds DNA, 600bp;

Fig. 3 is the agarose gel electrophoresis picture of the second round of amplification of ds cDNA of camel peripheral blood lymphocytes;

M.DL2000 relative molecular mass standard; 1. The second round of amplified ds DNA, 400bp;

Figure 4. is the result of monoclonal PCR amplification of camel peripheral blood lymphocyte yeast two-hybrid cDNA library;

M.DL5000 relative molecular mass standard; 1-16: PCR result of yeast colonies.

Fig. 5. is the PCR amplification result of the VHH gene of anti-Seneca Valley virus;

M: DL200 DNA molecular mass standard; 1,2: VHH PCR amplification product

Figure 6 shows the double enzyme digestion identification results of the expression vector pMAL-C2X-VHH;

M.DL5000 DNA molecular quality standard; 1,2,3: pMAL-C2X-VHH double enzyme digestion product;

Fig. 7 is the expression result of VHH gene;

M1 and M2: protein molecular mass standard; 1: pMAL-C2X whole protein; 2: pMAL-C2X supernatant; 3: pMAL-C2X precipitation; 4: pMAL-C2X-1 whole protein; 5: pMAL-C2X-1 upper clear; 6: pMAL-C2X-1 precipitation;

Fig. 8 is the result of westernblotting detection of VHH gene.

M1 and M2: protein molecular mass standard; 1: pMAL-C2X whole protein; 2: pMAL-C2X supernatant; 3: pMAL-C2X precipitation; 4: pMAL-C2X-1 whole protein; 5: pMAL-C2X-1 upper clear; 6: pMAL-C2X-1 precipitation.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description.

Example 1

Construction of Yeast cDNA Library of Anti-Seneca Valley Virus VHH Antibody

1. Materials and methods

1.1 Materials

1.1.1 Test animals, antigen, adjuvant and Seneca Valley virus antibody detection kit

1 male Bactrian camel (1 year old), 1 female Bactrian camel (6 months old); Seneca Valley virus inactivated vaccine, SVV antibody ELISA detection kit purchased from IDEXX.

1.1.2 Strains and reagents

Yeast library-related vectors were purchased from Clontech, restriction endonucleases were purchased from NEB, camel peripheral blood lymphocyte isolation kit (product number: LTS1076) were purchased from Tianjin Haoyang Biological Products Technology Co., Ltd., Trizol, nickel affinity Chromatographic resin, DNA fragment recovery kit and plasmid extraction kit were OMEGA products, SMART cDNA library construction kit (product number: 634901) and PCR-related reagents were purchased from Treasure Biotechnology Company, molecular biology reagents were from Sigma Company; other biochemical Reagents were all domestic analytically pure.

1.2 Method

1.2.1 Determination of antigen vaccination dose, immunization program and serum antibody titer

The inactivated Seneca Valley virus vaccine was inoculated subcutaneously on the neck, back and hind legs of the Bactrian camel, with at least three vaccination points for each site. Venous blood was collected before the first immunization and after each immunization, and the serum antibody titer was detected with the SVV antibody ELISA detection kit, and the venous blood was collected when the antibody blocking rate was greater than 70%. See Table 1 for specific animal immunization procedures and antigen inoculation doses:

Table 1 Bactrian camel immunization program and antigen dose

Number of immunizations First exemption Second exemption Three exemptions Dosage (ml/serving) 10 30 30 Interval time with the first exemption (days) 0 twenty one 35

1.2.2 Lymphocyte separation

After the antibody titer was determined to meet the requirements, 100ml of camel anticoagulant blood was collected from the jugular vein. Lymphocyte separation was carried out according to the operating instructions of Tianjin Haoyang TBD Camel Peripheral Blood Lymphocyte Separation Kit, specifically: add an equal amount of sample diluent to the collected anticoagulated blood and mix well; The volume ratio is 5:9, measure the lymphocyte separation solution, then add the diluted anticoagulant blood slowly and quickly, centrifuge at 1800rpm at 18-22°C for 20min; carefully collect the lymphocyte layer in a new collection Tube, diluted with 5 times the amount of cell washing solution, centrifuged at 1000rpm, 18-22°C for 10min in a horizontal centrifuge; The supernatant and the precipitate are the isolated lymphocytes. The supernatant of suspended cells is taken and counted, suspended in RNA preservation solution (RNAlater), and stored at -70°C for later use.

1.2.3 Extraction of total mRNA from lymphocytes

Aspirate 400 μl of the above lymphocyte liquid, add 1ml TRizol, mix well, and let stand at 4°C for 5 minutes; add 200 μl chloroform, mix well, let stand at room temperature for 3 minutes, 12000r/min, and centrifuge at 4°C for 15 minutes; draw 750μl supernatant, add etc. Measure isopropanol, let stand at room temperature for 10min, centrifuge at 10000r/min, 4°C for 10min; discard the supernatant, add 75% ethanol to the precipitate, and centrifuge at 8000r/min, 4°C for 5min; put the precipitate in a fume hood for 10min to dry; The water-dissolved precipitate of RNase is the harvested total mRNA.

1.2.4 Design of primers for library preparation

Primers for the first round of amplification:

SEQ ID NO.1-P1:5-GTCCTGGCTGCTCTTCTACAAGG-3

SEQ ID NO.2-R1:5-GGTACGTGCTGTTGAACTGTTCC-3

Primers for the second round of amplification:

SEQ ID NO.3-P2:5-TTCCACCCAAGCAGTGGTATCAACGCAGAGTGG

GAGTCTGGGGGAGG-3

SEQ ID NO.4-R2:5-GTATCGATGCCCACCCTCTAGAGGCCGAGGCGG

CCGACATGGAGACGGTGACCTGGGT-3

1.2.5 cDNA double-strand synthesis and purification

1.2.5.1 Whole gene cDNA synthesis

Add the following components in sequence to a 0.2ml PCR amplification tube:

Reagent material volume mRNA 3μl SMART 1μl CDSⅢ 1μl total capacity 5μl

Mix well, place the centrifuge tube in a 72°C metal bath, incubate for 2 minutes, immediately put it on ice, cool for 2 minutes, and add the following components in turn:

Reagent material volume 5×First strand Bμffer 2μl DTT (20mM) 1μl dNTPMix (10mM) 1μl MMLV reverse transcriptase (200μ/μl) 1μl total capacity 5μl

Mix well, place the centrifuge tube in a metal bath at 42°C, reverse transcribe for 1 hour, cool at room temperature, add 1 μl RNase H, the obtained product is whole genome cDNA, store at -20°C for later use.

1.2.5.2 The first round of amplification

Add the following components in sequence to a 0.2ml PCR amplification tube:

The above amplification program: 95°C for 1min; 95°C for 15s, 68°C for 5min, 20 cycles; 68°C for 5min. The amplified product was electrophoresed, and the 600bp product was purified and collected from the gel using a gel recovery kit, and stored at -20°C for future use.

1.2.5.3 The second round of amplification

Add the following components in sequence to a 0.2ml PCR amplification tube:

Reagent material (reagent) Quantity (amoμnt) 10×PCR buffer 10μl 10× solution 10μl 50× polymerase mix 2μl 50×dNTP 2μl P2 primer 2μl R2 primer 2μl First-round amplification product 2μl H ₂ O 70μl total capacity 100μl

The above amplification program: 95°C for 1min; 95°C for 15s, 68°C for 5min, 20 cycles; 68°C for 5min. The amplified product was electrophoresed, and the 400bp product was purified and collected from the gel with a gel recovery kit, and stored at -20°C for future use.

1.2.6 Library Construction and Harvesting

Yeast Y187 competent cells were prepared by PEG/LiAc method. Transform 20 μl of double-stranded cDNA and 6 μl of pGADT7-Rec per 600 μL of competent cells, evenly spread the transformed bacterial solution on SD/-Leu plates with a diameter of 100 mm and 150 mm, and incubate at 30°C for 3-5 days until clones appear. Place the plate again in a 4°C refrigerator for 3-4h. Add 5 mL of freezing solution (YPDA liquid medium containing 25% glycerol) to each plate, and add sterile glass beads, shake repeatedly in the horizontal direction, and then collect the bacterial solution.

1.2.7 Library quality assessment

Take the bacterial solution of the co-transformation product, dilute it according to 1/10, 1/100 and 1/1000, apply 100 μl of the diluted solution to a 100 mmSD/-Leu plate, and incubate it upside down at 30°C for 3-5 days until clones appear. Count the colonies and calculate the library capacity. Randomly pick 16 single colonies, carry out colony PCR with pGADT-Rec universal sequencing primers, and analyze the size of library inserts and recombination rate. PCR reaction parameters: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, extension at 72°C for 2 minutes, a total of 30 cycles, and finally extension at 72°C for 10 minutes. 7 μl of PCR product was taken for 1.2% agarose gel electrophoresis analysis.

2. Results

2.1 Extraction of total RNA and isolation and purification of camel peripheral blood lymphocytes

The extracted total RNA was determined by ultraviolet spectrophotometer, the concentration was 5077ng/μl, and the OD260/OD280 was 1.98. Denaturing agarose gel electrophoresis showed three clear bands of 28S, 18S, and 5S, and the ratio of 28S:18S was about 2:1. It shows that the purity of RNA and mRNA is high, meeting the needs of library construction (Figure 1).

2.2 Agarose gel electrophoresis of double-stranded cDNA library

The ds cDNA product amplified in the first round was electrophoresed and a 600bp band was collected. The ds cDNA product amplified in the second round was electrophoresed and a 400bp band was collected. As shown in Figure 2 and Figure 3.

2.3 Library capacity and polymorphism identification

Count single colonies on plates coated with different dilution libraries. According to the formula: library titer = number of colonies on the plate/volume of plated bacterial solution (ml) × dilution factor, the calculated pGADT7-Rec library titer is about 5.5 × 10 ⁶ cfμ/ml. 16 clones were randomly picked from the SD/-Leu plate for colony PCR. The electrophoresis results showed that the library inserts were all different, distributed between 400-2000bp, and the average insert was about 1000bp, indicating that the diversity of the constructed library was relatively high. Well, as shown in Figure 4. From the results of colony PCR, it can be known that the recombination rate of the library is 100%.

Example 2

Use of the Yeast cDNA Library of Anti-Seneca Valley Virus VHH Antibody in Screening Anti-Seneca Valley Virus VHH Antibody

1. Experimental materials and methods

1.1 Amplification of the Seneca Valley VHH gene

use The Plasmid Midi Kit OMEGA Yeast Plasmid Extraction Kit extracted the yeast library plasmid in Example 1, and designed amplification primers according to the structural characteristics of VHH, and introduced EcoRI and XholI restriction sites at the 5′ ends of the upstream and downstream primers, respectively. The primer sequences are:

VF: 5'-CCGGAATTCATGGGTATCAACGCAGAG-3';

VR: 5'-CCCTCGAGCATGGAGACGGTGACC-3', the amplification system is 50μl: 10×PCR Bμffer 5μl, dNTP 4μl, primer 1μl, Taq enzyme 1μl, ddH ₂ O 39μl, carry out PCR amplification, and the reaction condition is 94℃ pre-denaturation for 5min ; Denaturation at 94°C for 50 s, annealing at 57°C for 40 s, extension at 72°C for 1 min, a total of 30 cycles; extension at 72°C for 10 min, 5 min at 4°C. PCR products were identified by electrophoresis in 10g/L agarose gel.

1.2 Gel recovery and purification of VHH gene

According to the operation of the Bio-Gel Fragment Recovery Kit, the details are as follows: under the ultraviolet light, cut the band that conforms to the size of the VHH gene from the gel, add PC at the ratio of 0.1g gel to 100μl PC solution, and put it in a water bath at 50°C Dissolve for 10 minutes until the gel is completely dissolved; pour the dissolved gel solution into the equilibrated column, centrifuge at 12000rmp for 1min, discard the waste liquid, and recover the column; add 600μl rinse solution PW to the column, centrifuge at 12000rmp for 1min, discard the waste liquid, and Repeat the elution of the column once. 12000rmp air for 2min, room temperature for 4-5min, the purpose is to evaporate ethanol. Put the column into a 1.5mL EP tube and add 30μL of deionized water heated at 65°C. Let stand for 1-2min. Centrifuge at 12000rmp for 1min, and collect the centrifuged liquid.

1.3 Construction and identification of pMAL-C2X-VHH

EcoRI and XholI were used to double-enzyme digest the product recovered from PCR and the expression vector pMAL-C2X-1. After recovery and purification, T4 DNA ligase was used to ligate the digested VHH to pMAL-C2X-14°C overnight. The ligation product was transformed into BL21 competent cells, spread on the ampicillin-resistant LB solid plate and cultured until colonies appeared, then picked a single colony and placed it in the Kanal-resistant LB liquid medium, cultured overnight at 37°C on a shaker, The plasmids were extracted for double enzyme digestion identification and the positive plasmids were sent for sequencing verification.

1.4 Purification of recombinant target protein

Add the positive bacteria liquid to 100 mL of LB liquid medium containing Amp, and add IPTG to induce it. When the OD ₆₀₀ value does not increase any more, centrifuge at 5000 r/min for 5 min to harvest the bacterial pellet. Add 10mL 1×IB Wash Bμffer to resuspend the pellet, purify according to the instructions of the Novagen protein purification kit, elute step by step and collect the eluate of the target protein, which is the Seneca valley VHH antibody protein.

1.5 VHH activity detection

Using the Seneca Valley indirect ELISA antibody test kit, the expressed VHH was diluted according to the requirements of the operation manual, and its titer was determined.

2. Results

2.1 PCR amplification of the VHH gene of anti-Seneca Valley virus

The PCR product was detected by 10g/L agarose gel electrophoresis to obtain a clear specific band with a size of about 400bp, which was consistent with the expected fragment size (see Figure 5). This indicated that the VHH gene was successfully amplified from the constructed yeast library.

2.2 Double enzyme digestion analysis of the expression vector pMAL-C2X-VHH

The recombinant plasmid was analyzed with EcoRI and XholI restriction endonucleases, and fragments of about 400 bp and 5000 bp were obtained, which were consistent with the expected results (see Figure 6). The sequencing results proved that the VHH gene was successfully inserted into the expression vector and could be used in subsequent experiments.

2.3 Expression of VHH gene

After induction at 28°C, a specific protein band with a size of 37kDa appeared, which was in line with expectations (see Figure 7), and became more concentrated with the prolongation of induction time, and the expression level reached a peak 12 hours after induction.

2.4 Detection of the reactivity of the expressed Seneca Valley VHH antibody

The expression product was detected by Western-blotting to detect the activity of the expressed Seneca valley VHH antibody. The results of Western-blotting test showed that the expressed VHH could react with Seneca Valley virus (Fig. 8).

The above-mentioned embodiments are only exemplary and do not limit the scope of the present invention in any way. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Organization Applicant

----------------------

Street : No. 1 Xujiaping, Yanchangbao, Chengguan District, Lanzhou City

City : Lanzhou

State : Gansu

Country : China

PostalCode: 730046

Phone Number : 0931-8342982

FaxNumber : 0931-8340977

EmailAddress : xibeitian0931@163.com

<110> OrganizationName : Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences

Application Project

-------------------

<120> Title : A Yeast cDNA Library of Anti-Seneca Valley Virus VHH Antibody and Its Construction Method and Application

<130> AppFileReference : Characterization of Asia 1 sdAb from CamelsBactrianus (C. bactrianus) and Conjugation with Quantum Dots for Imaging FMDVin BHK-21 Cells

<140> CurrentAppNumber :

<141> CurrentFilingDate : ____-__-__

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Claims (10)

1. a kind of construction method of the yeast cDNA library of anti-Sai Nika paddy virus VHH antibody, it is characterised in that：Including following step Suddenly：

Step (1), separation of lymphocytes：Isolated leaching in the anticoagulation for the camel being immunized from Sai Nika paddy viral inactivation vaccines Bar cell, saves backup；

The extraction of step (2), the total mRNA of lymphocyte：Total mRNA of extraction step (1) isolated lymphocyte；

Step (3), whole gene cDNA synthesis：Using total mRNA of the lymphocyte of step (2) extraction as template, full genome is synthesized cDNA；

Step (4), first round amplification：Using the whole gene cDNA of step (3) synthesis as template, first is carried out by primer of P1, R1 Wheel amplification, by amplified production electrophoresis, purified pool 600bp products are spare；

SEQ ID NO.1-P1:5-GTCCTGGCTGCTCTTCTACAAGG-3

SEQ ID NO.2-R1:5-GGTACGTGCTGTTGAACTGTTCC-3

Step (5), the second wheel amplification：Using first round amplified production as template, first round amplification is carried out by primer of P2, R2, is expanded Increase production object electrophoresis, purified pool 400bp products are spare；

SEQ ID NO.3-P2:

5-TTCCACCCAAGCAGTGGTATCAACGCAGAGTGGGAGTCTGGGGGAGG-3

SEQ ID NO.4-R2:

5-GTATCGATGCCCACCCTCTAGAGGCCGAGGCGGCCGACATGGAGACGGTGACCTGGGT-3

Step (6), library structure and harvest：Second wheel amplified production and pGADT7-Rec carrier transformed yeast competence is thin Born of the same parents, 30 DEG C are inverted culture until clone's appearance, collects bacterium solution to get the VHH antibody yeast cDNA of anti-Sai Nika paddy virus later Library.

2. a kind of construction method of the yeast cDNA library of anti-Sai Nika paddy virus VHH antibody as described in claim 1, special Sign is：In the step (1), separation of lymphocytes uses the separating kit of camel peripheral blood lymphocytes, according to following Step carries out：

(1) it is added the Sample dilution of equal volume amounts in the anticoagulation of the immune camel of Sai Nika paddy viral inactivation vaccine, mixing, Obtain anticoagulant dilution；

(2) it is 5 according to Sample dilution and lymphocyte separation medium volume ratio:9 ratio measures lymphocyte separation medium, it Anticoagulant dilution, horizontal centrifuge 1800rpm, 18-22 DEG C of centrifugation 20min is added after elder generation is slow afterwards soon；

(3) buffy coat is collected in new collecting pipe, with 5 times of amount cell washing solution dilutions, horizontal centrifuge 1000rpm, 18- 22 DEG C of centrifugation 10min；Supernatant is abandoned, precipitation is diluted with cell washing solution again, horizontal centrifuge 1000rpm, 18-22 DEG C of centrifugation 10min；Precipitation is resuspended with 5ml without RNA enzyme water, immediately addition 45mlRPIM-1640 culture solution mixings, 1000rpm, 18-22 DEG C 10min is centrifuged, precipitation is lymphocyte, and is resuspended in RNALater and preserved in liquid, sets -70 DEG C and saves backup.

3. a kind of construction method of the yeast cDNA library of anti-Sai Nika paddy virus VHH antibody as described in claim 1, special Sign is：In the step (2), the extraction of the total mRNA of lymphocyte is to carry out in accordance with the following methods：Aspiration step (1) detaches 1mlTRizol, mixing, 4 DEG C of standing 5min are added in the 400 μ l of lymphocyte arrived；200 μ l chloroforms, mixing, room temperature is added Stand 3min, 12000r/min, 4 DEG C of centrifugation 15min；750 μ l supernatants are drawn, adds equivalent isopropanol, is stored at room temperature 10min, 10000r/min, 4 DEG C of centrifugation 10min；It is light to abandon supernatant, precipitation plus 75% ethyl alcohol, 8000r/min, 4 DEG C of centrifugation 5min；Precipitation is set Draught cupboard 10min is allowed to drying；The precipitation of 20 water dissolutions of the μ l without RNA enzyme is the total mRNA of lymphocyte of harvest.

4. a kind of construction method of the yeast cDNA library of anti-Sai Nika paddy virus VHH antibody as described in claim 1, special Sign is：The whole gene cDNA synthesis is to build kit using SMART cDNA libraries, in the PCR amplification pipe of 0.2ml Sequentially add following component：

mRNA 3μl SMART 1μl CDSⅢ 1μl

After mixing, centrifuge tube is positioned over 72 DEG C of metal baths, is incubated 2min, is set immediately on ice, then cooling 2min adds successively again Enter following component：

5×First strand Bμffer 2μl DTT 1μl dNTP Mix 1μl MMLV reverse transcriptases 1μl

After mixing, centrifuge tube is positioned over 42 DEG C of metal baths, reverse transcription 1h, 1 μ l RNA enzyme H, obtained production is added in room temperature cooling Object is full-length genome cDNA, sets -20 DEG C and saves backup.

5. a kind of construction method of the yeast cDNA library of anti-Sai Nika paddy virus VHH antibody as described in claim 1, special Sign is：The first round amplification is to build kit using SMART cDNA libraries, in the PCR amplification Guan Zhongyi of 0.2ml Secondary addition following component：

First round amplification is carried out by primer of P1, R1 in step (4), amplification program is：95℃1min；95 DEG C of 15s, 68 DEG C 5min, 20 cycles；68 DEG C of 5min, by amplified production electrophoresis, purified pool 600bp products, -20 DEG C save backup, for the Two wheel amplifications.

6. a kind of construction method of the yeast cDNA library of anti-Sai Nika paddy virus VHH antibody as described in claim 1, special Sign is：The second wheel amplification is to build kit using SMART cDNA libraries, in the PCR amplification Guan Zhongyi of 0.2ml Secondary addition following component：

10 × PCR buffer solutions 10μl 10 × lysate 10μl 50 × polymerase mixture 2μl 50×dNTP 2μl P2 primers 2μl R2 primers 2μl First round amplified production 2μl H₂O 70μl

The second wheel is carried out in step (5) as primer using P2, R2 to expand, amplification program is：95℃1min；95 DEG C of 15s, 68 DEG C 5min, 20 cycles；68℃5min；Amplified production electrophoresis, purified pool 400bp products, sets -20 DEG C and saves backup.

7. a kind of construction method of the yeast cDNA library of anti-Sai Nika paddy virus VHH antibody as described in claim 1, special Sign is：In the step (6), the structure and harvest in library：Competent yeast cells are prepared, every 600 μ L competent cells turn Change 20 μ l second wheel amplified productions and 6 μ l pGADT7-Rec carriers, the bacterium solution of conversion be spread evenly across on SD/-Leu tablets, 30 DEG C are inverted culture 3-5d until clone occurs；Tablet is placed in 4 DEG C of refrigerators again, places 3-4h, is contained to every piece of tablet addition The YPDA fluid nutrient mediums of 25% (v/v) glycerine, and sterile glass beads are added, horizontal direction is shaken repeatedly, collects bacterium solution later, Up to the yeast cDNA library of anti-Sai Nika paddy virus VHH antibody.

8. according to the yeast cDNA for the anti-Sai Nika paddy virus VHH antibody that any one of claim 1-7 the methods are built Library.

9. the yeast cDNA library of anti-Sai Nika paddy virus VHH antibody as claimed in claim 8, it is characterised in that：Described CDNA is distributed between 400-2000bp in library, and titre is 7.2 × 10⁶Cf μ/ml, recombination fraction 100%.

10. the yeast cDNA library of the anti-Sai Nika paddy virus VHH antibody as described in claim 1,2,3,4,5,6,7 or 9 exists Screen the purposes in anti-Sai Nika paddy virus VHH antibody.