CN107840884B

CN107840884B - Nano antibody for resisting avian infectious bronchitis virus and preparation method thereof

Info

Publication number: CN107840884B
Application number: CN201711131496.6A
Authority: CN
Inventors: 周景明; 王爱萍; 张改平; 耿玥; 祁艳华; 刘红亮; 胡小宏; 马文利; 刘燕凯
Original assignee: Henan Zhongze Biological Engineering Co ltd; Zhengzhou University
Current assignee: Henan Zhongze Biological Engineering Co ltd; Zhengzhou University
Priority date: 2017-11-15
Filing date: 2017-11-15
Publication date: 2020-12-08
Anticipated expiration: 2037-11-15
Also published as: CN107840884A

Abstract

The invention discloses a nano antibody for resisting avian infectious bronchitis virus, which is prepared by subculturing IBV virus on chick embryos, collecting allantoic fluid of the chick embryos containing the virus and purifying the allantoic fluid; extracting RNA from peripheral blood lymphocytes of healthy camels without immunization, specifically amplifying camel heavy chain antibody variable region genes, connecting the genes subjected to enzyme digestion with a phagemid vector pCANTAB5E, and then electrically transforming the genes into escherichia coli TG1 to obtain a VHH antibody gene library; after library capacity and diversity of the antibody are analyzed, a nano antibody VHH-A2 fragment capable of being specifically combined with IBV is screened; and (3) constructing a pet28a-A2 expression vector, and carrying out prokaryotic expression, purification and identification on the expression vector to obtain the required nano antibody. The nano antibody prepared by the invention is used as a novel genetic engineering antibody, has strong antigen recognition capability due to the unique structural characteristics, can be used for quickly and accurately detecting the infectious bronchitis of poultry (such as chicken), and has short preparation period and simple method.

Description

Nano antibody for resisting avian infectious bronchitis virus and preparation method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a nano antibody for resisting avian infectious bronchitis viruses and a preparation method of the nano antibody.

Background

Infectious bronchitis of chicken is an acute and highly contagious disease of chicken caused by Infectious Bronchitis Virus (IBV), and is one of the major infectious diseases seriously harming the poultry industry. IBV is a very variable coronavirus of birds, with a membrane, and is a single negative strand RNA virus.

Most of the traditional methods for culturing IBV are to inoculate and culture the allantoic cavity of SPF chick embryos of 9-11 days old, and inoculate PBS as a control group under the same conditions. Capto^TMCore 700 is designed for purification of viruses and other macromolecules and binds proteins with molecular weights up to 660000, while proteins with larger molecular weights are excluded from the microspheres. Mixing allantoic fluid containing virus with Capto^TMCore 700 is subjected to virus purification to obtain IBV antigen.

Within the camelid immune system there is an antibody consisting of only a single folded unit of the heavy chain, which is called a single domain of heavy-chain antibodies (VHH) or nanobodies (Nbs). The nano antibody only consists of a variable region of a heavy chain antibody, has certain unique properties compared with a common antibody, such as small molecular weight, high stability, high affinity, weak immunogenicity, good and wide antigen binding capacity, good water solubility and the like, and is widely applied to the fields of pathogen detection, food safety analysis and the like.

The immunological detection method has many advantages, mainly including simple operation, fast detection speed, etc., among which there are ELISA method, fluorescence immunoassay method, etc. In the immunological detection method, the detection result is mainly influenced by the binding effect of the antibody and the antigen. The traditional antibody preparation needs animal immunization, which causes complex preparation process and higher experimental technical requirements on operators, thereby causing higher production cost and longer research and development period.

Disclosure of Invention

The invention aims to provide a nano antibody for resisting avian Infectious Bronchitis Virus (IBV) and a preparation method thereof.

In order to achieve the purpose, the invention can adopt the following technical scheme:

the amino acid coding sequence of the nano antibody for resisting the avian infectious bronchitis virus is as follows: SEQ ID NO: 2, the DNA sequence encoding the amino acid sequence is: SEQ ID NO: 1.

the nano antibody only consists of a heavy chain, naturally lacks a light chain, and has the molecular weight of about 15 kD.

The preparation method of the nano antibody for resisting avian infectious bronchitis viruses comprises the following steps:

first, IBV virus is inoculated to 9-11 day-old SPF chick embryo for subculture according to conventional method, allantoic fluid of chick embryo containing virus is collected and is applied to Capto^TMPurifying Core 700, and detecting a virus purification result to be good for later use;

secondly, extracting RNA from peripheral blood lymphocytes of the healthy camel which is not immunized, and identifying the good integrity of the RNA; taking RNA as a template, carrying out reverse transcription to obtain cDNA, specifically amplifying a camel heavy chain antibody variable region gene, connecting the enzyme-digested gene with a phagemid vector pCANTAB5E, and then electrically transforming the phagemid vector into escherichia coli TG1 to obtain a VHH antibody gene library; after library capacity and diversity of the antibody are analyzed, a nano antibody VHH-A2 fragment capable of being specifically combined with IBV is screened; constructing a pet28a-A2 expression vector, and carrying out prokaryotic expression, purification and identification on the expression vector to obtain an amino acid coding sequence as follows: SEQ ID NO: 2, the DNA sequence encoding the amino acid sequence is: SEQ ID NO: 1.

The invention has the advantages that the lymphocytes of the camel without immunization are used as initial experimental materials, a whole set of single domain antibody genes are amplified, a camel non-immune phage antibody library is constructed, and the library capacity and diversity of the antibody library are identified. The IBV antigen is utilized to screen a camel natural single domain antibody phage display library, and the anti-IBV nano antibody is screened from an antibody library, thereby providing a new idea for IB detection.

The nano antibody prepared by the invention is used as a novel genetic engineering antibody, has strong antigen recognition capability due to the unique structural characteristics, can be used for quickly and accurately detecting the infectious bronchitis of poultry (such as chicken), and has short preparation period and simple preparation method.

Drawings

FIG. 1 shows the results of RNA identification in examples of the present invention.

FIG. 2 shows the results of VHH identification in the examples of the present invention.

FIG. 3 identification results of library PCR in the examples of the present invention.

FIG. 4 sequencing results of random clones in VHH antibody libraries according to the present example.

FIG. 5 purification results of IBV in the examples of the invention.

FIG. 6 shows the result of the purification and identification of IBV in the examples of the present invention.

FIG. 7 shows the result of the enzyme-linked immunosorbent assay for the IBV nanobody in the example of the present invention.

Detailed Description

The technical solution of the present invention is further described with reference to the following specific examples, but the scope of the present invention is not limited thereto. The raw materials and reagents used in the following examples are all commercially available unless otherwise specified; the detection method or the test method is a conventional method unless otherwise specified.

Example 1 construction of a Camel Natural Single Domain heavy chain phage display antibody library

BALB/c mice used in the experiments were purchased from the animal experiments center of medical college of Zhengzhou university. 9-11 day old chick embryos were incubated with hatching eggs purchased from Beijing Meiliya Viton laboratory animal technology, Inc. The main reagents used in the experiments are shown in table 1.

TABLE 1

。

1. Isolation of peripheral blood lymphocytes and extraction of Total RNA

Taking camel peripheral blood, and mixing with 10⁹And (5) anticoagulating mmol/L sodium citrate. By means of density laddersSeparating peripheral blood mononuclear cells by centrifugation, and counting the cells at a rate of 10⁷Cells were dissolved in 1mL of TRIzol reagent and total RNA was extracted using the instructions for TRIzol reagent. The extracted total RNA is dissolved by deionized water, the concentration is measured, and the total RNA is stored at the temperature of minus 80 ℃ for standby. The extracted RNA was identified as shown in FIG. 1, which demonstrates good integrity.

2. Amplification of camelid heavy chain antibody variable region genes

First strand cDNA was synthesized using total RNA as template and oligo (dT)18 as primer according to the reverse transcription kit instructions. The variable region gene VHH of the heavy chain antibody was PCR-amplified using cDNA as a template with reference to the PCR primers synthesized in Table 1. The reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; 35s at 95 ℃; 35s at 63 ℃; 35s at 72 ℃; a total of 32 cycles; extension at 72 ℃ for 7 min. The reaction product is verified by 1% agarose gel electrophoresis, and then is recovered and quantified by a DNA fragment recovery kit, and is stored at-20 ℃ for later use. The results of VHH identification are shown in FIG. 2, and a camel heavy chain variable region gene VHH with a fragment size of about 500bp was obtained.

3. Construction of phage Single Domain antibody libraries

Carrying out enzyme digestion reaction on the phagemid vector pCANTAB5E and the PCR amplification product VHH by using Not I and Sfi I respectively; recovering the enzyme digestion fragment by using a DNA recovery kit, and connecting the enzyme digestion fragment with the pCANTAB5E vector under the action of T4DNA ligase; the ligation product was electrically transformed into competent Escherichia coli TG1, and the transformed broth was cultured in LB liquid medium at 37 ℃ and 220rpm for 1 hour. The transformed bacterial liquid was concentrated, applied to several plates (2 XYT plates containing 2% glucose and Amp antibiotics), diluted 10. mu.L at double ratio to calculate the storage capacity, and cultured overnight at 37 ℃. The next day, Colony Forming Units (CFU) were calculated as the titer of the phage antibody library, and 20 single colonies were randomly selected from the plate and subjected to PCR assay to determine the recombination rate of the antibody library, as shown in fig. 3, with 18 positive clones, demonstrating that the recombination rate of the natural antibody library was about 90%. 8 single clones were picked and sent to biotechnology service companies for sequencing, the sequencing results are shown in FIG. 4, and the results show that the exogenous fragments of the 8 clones are all genes encoding the variable region of the heavy chain antibody. The remaining bacterial liquid was cultured overnight at 37 ℃ in an appropriate amount of 2 XYT/Amp liquid medium and M13K 07. The following day, phages were isolated from the supernatant of the inoculum with 20% PEG8000/NaCl and suspended in PBS.

Example 2 propagation and purification of IBV

1. IBV subculture on chick embryo

And (3) disinfecting the hatching eggs and the incubator by using a potassium permanganate-formalin fumigation method, and placing the hatching eggs with the big ends upwards in the incubator for incubation. The standard strain freeze-dried powder is diluted by PBS according to the ratio of 1:1000, 200ul of SPF chick embryos of 9-11 days old are inoculated, and the same amount of PBS is injected into the control group of the chick embryos. Abnormal dead chick embryos within 24 hours are discarded, and chick embryo allantoic fluid is harvested after 48 hours. Centrifuging at 6000rpm/min at 4 deg.C for 15min, and filtering at 0.22 μm. Continuously uploading for 3 generations on chick embryo, storing allantoic fluid containing virus at-80 deg.C.

2. Purification of IBV

Capto was equilibrated with 10 column volumes of ddw followed by PBS^TMCore 700 column, flow rate 1 ml/min. And (4) loading, starting sample inoculation when a peak is observed, passing through PBS, and stopping sample inoculation when the OD value is reduced to 0. The impure proteins were eluted with an eluent (1M NaOH containing 30% isopropanol) and the column was kept in 20% ethanol. FIG. 5 shows the purification of IBV, where IBV is excluded from the microspheres and eluted first, and the hetero-protein is bound to the column.

3. Purification result identification of IBV

As shown in the left panel of FIG. 6, the results of purification were examined by SDS-PAGE, and the control was allantoic fluid containing no virus. The results show that the virus integrity before and after purification is better.

As shown in the right panel of FIG. 6, the results of the purification were examined by Western-blot analysis, and the allantoic fluid was used as a control without virus. Cutting off the gel to be used for Western, soaking in membrane conversion buffer solution for 20min, soaking filter paper in advance for 30min, taking PVDF membrane, soaking in methanol for 30s, and soaking in membrane conversion buffer solution for 3-5 min. And sequentially placing filter paper, a PVDF film, glue and filter paper on the electrotransfer to expel air bubbles. And (5) stopping film rotation after 15V 1h 20 min. Sealing PVDF membrane with 2.5% skimmed milk for 2h, wherein the primary antibody is specific serum of infectious bronchitis, the secondary antibody is goat anti-chicken IgG-HRP, and developing with ECL method. The results showed that the major structural proteins were not lost and were able to bind to IBV positive sera.

Example 3 panning of IBV-specific Nanobodies

1. Enrichment screening recombinant phage

With NaHCO₃IBV was diluted with buffer to a final concentration of 100. mu.g/mL, coated in 100. mu.L/well on an ELISA plate, and incubated overnight at 4 ℃. The next day, the plate coating was discarded, washed 4 times with TBST, blocking buffer was added, 200. mu.L/well, and blocked at 37 ℃ for 2 h. The blocking solution was decanted and washed with TBST (TBS + 0.1%)<v/v>Tween-20), quick washing 10 times. Then, throwing and beating on a clean paper towel to remove the washing liquid; phage was added, 100. mu.L/well and incubated for 1h at 37 ℃. Unbound phage were discarded and washed 10 times with TBST and 5 times with TBS. Add buffer eluent, 100. mu.L/well, elute bound phage into the eluent with gentle shaking for 8 min. The eluate was transferred to a microcentrifuge tube and 100. mu.L of a neutralization buffer was added to make the eluate neutral. A small amount of neutralized eluate was collected for titer determination, and the remainder was used for amplification. 400 μ L of the eluate was infected with 4mL of log-phase TG1 cells, shaken well and allowed to stand at 37 ℃ for 30min, 16mL of 2 XYT/AMP-GLU was added and cultured at 37 ℃ and 220rpm until log-phase. 20 μ L of M13KO7 helper phage was added to the culture medium, shaken well and allowed to stand at room temperature for 30min, 2800g was centrifuged at room temperature for 10min, the supernatant was discarded, and the cells were resuspended in 100 μ L of 2 XYT/AMP-KANA medium at 37 ℃ and 220rpm for 14 h. The phage pellet was resuspended by concentrating and purifying with PEG8000/NaCl solution and adding 0.5mLPBS per tube.

And repeating the steps to finish the second round and the third round of panning. The IBV coating antigen concentration is reduced in turn and is respectively 100 mug/mL, 50 mug/mL and 25 mug/mL. The content of Tween20 in the washing solution was increased in the order of 0.1%, 0.3%, and 0.5%.

2. Positive clone's phase-ELISA identification

And selecting single colonies from a large plate in the third round of screening, putting the single colonies into a 96-well plate, respectively amplifying and purifying, taking a phage library amplified after each round of screening, diluting IBV to 10 mu g/mL by using PBS buffer solution to serve as a coating antigen, and detecting the specific recombinant phage by using phage-ELISA.

3. The ELISA positive clone was sent to biotechnology service company for sequencing to obtain the DNA sequence of the insert, which encodes a single domain heavy chain antibody against IBV as follows (SEQ ID NO: 1):

CAGGTCCAACTGCAGGAGTCTGGGGGAGGCTCGGTGCATCTGGAGGGTCTCTAAGACTCTCCTGTGTAGCCTCTGGATACCGCCTCAATTTAAACTGCATGGGCTGGTTCCGCCAGGCTCCTGGAAAGGAGCGCGAGGGGGTCGCAACTATTGCAAGTGATTCGTATGGGAGGTACGCAGACTCCGTGAGGGGCCGATTCGCCATCTCCCAAGACAACGCCAAGAACACGCTGTACCTGCAAATGAACACCCTGAACCCTGAGGACACGGCCACGTATTATTGTGCGGCCTGGCGGGGAGATTGTGGCATACCGGATATGTTAAGAACCGACAACTACCGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA。

the amino acid sequence of the single domain heavy chain antibody against IBV was obtained from the sequencing results and codon table (SEQ ID NO: 2)

QVQLQESGGGSVHLEGLDSPVPLDTASITAWAGSARLLERSARGSQLLQVIRMGGTQTPGADSPSPKTTPRTRCTCKTPTLRTRPRIIVRPGGEIVAYRICEPTTTGARGPRSPSPH。

Example 4 expression and purification of Nanobodies in E.coli

1. Construction of expression vectors

According to the gene sequence of the antibody, a pair of specific primers is designed, and the VHH-A8 gene is amplified by adopting a PCR technology. The DNA fragment of the nano antibody and an expression vector pET28a are subjected to double enzyme digestion, purified and recovered, connected by T4 ligase and transformed into an expression host bacterium. The expression vector was digested under conditions of 1ug (8. mu.L) of the vector, 5. mu.L of 10 Xfast-cutting enzyme buffer, 1. mu.L of each of the two enzymes NdeI and NotI, 35. mu.L of water, incubated at 37 ℃ for 60min, and the plasmid was purified and recovered from the reaction mixture. The digestion conditions of the DNA fragment were 1ug (10. mu.L) of the DNA fragment, 5. mu.L of 10 Xfast-cutting enzyme buffer, 1. mu.L of each of the two enzymes NdeI and NotI, 33. mu.L of water, and 60min in a 37 ℃ water bath, and the purified DNA was recovered from the reaction mixture. Connecting conditions of the expression vector and the DNA fragment: 3 uL of expression vector (100ng), 5 uL of DNA fragment (120ng), 1 uL of 10 XT 4 buffer, 1 uL of T4 ligase, 16 ℃ overnight ligation, 65 ℃ incubation for 10min to inactivate ligase, transferring intoEcoliBL21 is competent.

2. Inducible expression

Selecting positive clones, and performing induced expression on a sample with correct sequencing, wherein the induced expression steps are as follows: inoculating the sample bacterial liquid with correct sequencing into a 2YT culture medium according to the ratio of 1:100, activating at 37 ℃ and 220rpm until the sample bacterial liquid is activatedBacterial liquid OD₆₀₀When the value is 0.6-0.8, adding IPTG (isopropyl-beta-D-thiogalactoside) with the final concentration of 0.8mmol/L, inducing at 20 ℃ and 220rpm for 5-6 h, collecting bacterial liquid, and operating the empty carrier pet28a bacterial liquid according to the induced expression step; SDS-PAGE gel electrophoresis is carried out on the correct sample bacterial liquid before and after induction and the empty carrier pet28a bacterial liquid before and after induction, and the result shows that IPTG can induce the expression of the anti-IBV nano antibody in pet28 a.

3. Purification of proteins

Centrifuging the bacteria liquid after induction expression at 5000rpm for 15min, and discarding the supernatant; the suspension was resuspended in ice 1 XPBS (pH7.4) buffer, 3500g, centrifuged for 10min and the supernatant discarded. And (3) carrying out vortex shaking and resuspension on 40mL of lysate, placing the lysate on ice, placing the lysate in an ultrasonic cell crusher, carrying out ultrasonic crushing for 20min according to the program of ultrasonic vibration time 3s and gap time 3s, centrifuging the crushed bacterium liquid at 12000rpm for 20min, carrying out affinity chromatography purification on the supernatant, and storing the supernatant for later use.

Example 5 detection of enzyme-linked immunosorbent assay for IBV Nanobody

The purified IBV antigen was diluted to 2 ng/. mu.L with CBS buffer pH (9.6), added to the microplate at 100. mu.L per well, overnight at 4 ℃ and subjected to ELISA next day with the following steps:

1) adding 200 mu L PBST per hole into the coated ELISA plate for cleaning, washing for 5 times, 1min each time, throwing off the washing liquid, and drying on a paper towel;

2) adding 1% BSA blocking solution into an enzyme label plate according to 200 mu L per hole, and blocking for 2h at 37 ℃;

3) washing the plate according to the step 1, sequentially adding 100 mu L of blank control, negative control and purified anti-IBV nano antibody into the ELISA plate, and incubating for 1h at 37 ℃;

4) washing the plate according to the step 1, then adding HRP-labeled His tag antibody into the ELISA plate according to 100 mu L of each hole, and incubating for 1h at room temperature;

5) washing according to the step 1, then adding 100 mu L of TMB into each hole, and standing for 5min in a dark place;

6) add 100. mu.L of 2mol/L sulfuric acid solution to each well, stop the reaction, place the microplate in a microplate reader, OD_450nmAnd (6) reading.

Wherein, the negative control is the empty carrier culture solution which is not connected with the antibody gene, and the control of the pore plate is PBS washing liquid. The identification result is shown in fig. 7, and the nanobody has the capability of binding to IBV.

SEQUENCE LISTING

<110> Zhengzhou university

<120> Nanobody against IBV

<141> 2017-11-15

<170> PatentIn version 3.3

<210> 1

<211> 372

<212> DNA

<213> Artificial sequence

<400> 1

caggtccaac tgcaggagtc tgggggaggc tcggtgcagt ctggagggtc tctaagactc 60

tcctgtgtag cctctggata ccgcctcaat ttaaactgca tgggctggtt ccgccaggct 120

cctggaaagg agcgcgaggg ggtcgcaact attgcaagtg attcgtatgg gaggtacgca 180

gactccgtga ggggccgatt cgccatctcc caagacaacg ccaagaacac gctgtacctg 240

caaatgaaca ccctgaaccc tgaggacacg gccacgtatt attgtgcggc ctggcgggga 300

gattgtggca taccggatat gttaagaacc gacaactacc ggggccaggg gacccaggtc 360

accgtctcct ca 372

<210> 2

<211> 110

<212> PRT

<213> Artificial sequence

<400> 2

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val His Leu Glu Gly Leu Asp Ser Pro

1 5 10 15 20

Val Pro Leu Asp Thr Ala Ser IIe Thr Ala Trp Ala Gly Ser Ala Arg Leu Leu Glu Arg

25 30 35 40

Gly Ser Gln Leu Leu Gln Val IIe Arg Met Gly Gly Thr Gln Thr Pro Gly Ala Asp Ser

45 50 55 60

Pro Ser Pro Lys Thr Thr Pro Arg Thr Arg Cys Thr Cys Lys Thr Pro Thr Leu Arg Thr

65 70 75 80

Arg Pro Arg IIe IIe Val Arg Pro Gly Gly Glu IIe Val Ala Tyr Arg IIE Cys Glu Pro Thr

85 90 95 100

Thr Thr Gly Ala Arg Gly Pro Arg Ser Pro Ser Pro His

105 110

Claims

1. A nanometer antibody for resisting avian infectious bronchitis virus is characterized in that: the amino acid coding sequence of the nano antibody is as follows: SEQ ID NO: 2, the DNA sequence encoding the amino acid sequence is: SEQ ID NO: 1.

2. the nanobody against avian infectious bronchitis virus of claim 1, which is characterized by: the nanobody consists of only heavy chains and has a molecular weight of 15 kD.