CN113461816A

CN113461816A - Nano antibody aiming at green fluorescent protein GFP and application thereof

Info

Publication number: CN113461816A
Application number: CN202110761519.1A
Authority: CN
Inventors: 董春明; 毛桂燕; 张瑞; 许丽君
Original assignee: Beijing Lablead Commerce And Trade Co ltd; Tianjin University of Science and Technology
Current assignee: Beijing Lablead Commerce And Trade Co ltd; Tianjin University of Science and Technology
Priority date: 2021-07-06
Filing date: 2021-07-06
Publication date: 2021-10-01

Abstract

The invention discloses a nano antibody aiming at green fluorescent protein GFP, the amino acid sequence of which is SEQ ID NO. 1. The invention discloses a nano antibody gene sequence and a host cell, wherein the nano antibody is a single-domain antibody heavy chain antibody (namely nano antibody, the same below) which can be specifically combined with GFP, can also be efficiently expressed in escherichia coli, and can be applied to the purification and detection of GFP and GFP fusion protein.

Description

Nano antibody aiming at green fluorescent protein GFP and application thereof

Technical Field

The invention belongs to the technical field of single-domain heavy chain antibodies and genetic engineering antibodies (also called nano antibody technology), and particularly relates to a nano antibody aiming at Green Fluorescent Protein (GFP) and application thereof.

Background

Green fluorescent protein was first discovered in the jellyfish in 1962 by Nomura et al, and the protein produced from the gene fluoresces green when excited by light in the blue wavelength range. GFP consists of 238 amino acid residues, has a size of about 27KDa, serves as a label, and is widely applied to the fields of immunological detection, cell imaging, affinity purification, protein engineering and the like.

At present, most of monoclonal or polyclonal antibodies aiming at GFP are used for detection in the market, the research and development and production processes of the monoclonal antibodies in the traditional sense are extremely complicated and complicated, the stability of the antibodies is poor, the production cost is high, and the sources of the polyclonal antibodies are limited. And the traditional antibody contains an Fc segment, and is easy to generate nonspecific binding or pollution. The anti-observation nano antibody only consists of one structural domain, has the characteristics of high affinity, strong stability, good histocompatibility, easy screening, easy preparation and the like due to small molecular weight, and does not have nonspecific combination or pollution of heavy chains and light chains of common antibodies. The purification medium prepared by using the nano antibody as the ligand has the advantages of low cost, reusability and the like, and is widely researched and developed in the aspects of therapeutic drug antibodies, clinical detection antibodies, scientific and technical application antibodies and the like in recent years.

Through searching, no patent publication related to the present patent application has been found.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a nano antibody aiming at Green Fluorescent Protein (GFP) and application thereof.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a nano antibody aiming at green fluorescent protein GFP has an amino acid sequence of SEQ ID No. 1.

Further, the amino acid sequence of the nanobody is divided into four framework regions and three complementarity determining regions.

A nucleic acid molecule encoding an amino acid sequence as described above.

Further, the sequence of the nucleic acid molecule is SEQ ID NO. 2.

A vector comprising a nucleic acid molecule as described above.

A host cell comprising a vector as described above.

The application of the nano-antibody in immunodetection and/or enrichment purification of green fluorescent protein GFP is disclosed.

The nanobody is an antibody which is obtained by modifying through random or site-directed mutagenesis technology and can be specifically combined with green fluorescent protein GFP.

An immunoaffinity adsorption material of Green Fluorescent Protein (GFP) can use a nano antibody aiming at the Green Fluorescent Protein (GFP) as a ligand, and the amino acid sequence of the nano antibody is SEQ ID NO. 1.

Further, the preparation method comprises the following specific steps:

the material carrier is adopted to couple the anti-GFP nano antibody, and the specific preparation method is as follows:

washing dry glue activated by CNBr with 0.1M HCl for 10 times, and balancing for 5min each time; washing with coupling buffer solution of 10mM, NA2HPO4 and pH7.2 for 10 times, adding anti-GFP label nano antibody with the addition of 2 mg/g material carrier, reacting at room temperature for 3.5h, and covalently coupling the anti-GFP label nano antibody with the CNBr activated material carrier; washing with 10mM coupling buffer solution NA2HPO4, pH7.2 for 3 times, adding blocking solution, and reacting at room temperature for 2h to block unreacted active group; alternately washing the mixture for 3 times by using 10mM phosphate buffer solution with pH7.2 and 0.1M acetic acid buffer solution with pH4.5 which are 6 times of the volume of the gel to obtain the immunoaffinity adsorption material which is covalently coupled with the anti-GFP label nano antibody; and (3) placing the immunoaffinity adsorption material in a chromatographic column, washing with 8-10 times of column volume of 10mM PBS (phosphate buffer solution) with pH of 7.2, adding 20% ethanol solution to obtain the immunoaffinity adsorption material, and storing at 4 ℃.

The invention has the advantages and positive effects that:

1. the invention discloses a nano antibody gene sequence and a host cell, wherein the nano antibody is a single-domain antibody heavy chain antibody (namely nano antibody, the same below) which can be specifically combined with GFP, can also be efficiently expressed in escherichia coli, and can be applied to the purification and detection of GFP and GFP fusion protein.

2. The GFP nano antibody disclosed by the invention can be specifically combined with GFP, and compared with the traditional monoclonal antibody, the nano antibody has higher affinity and binding specificity and better and wider application effect, and can be used for combination and detection of GFP and GFP fusion protein.

3. The amino acid sequence SEQ ID NO.1 provided by the invention can be used as a precursor, and is transformed by random or site-directed mutagenesis technology to obtain a mutant with better properties (water solubility, stability, affinity, specificity and the like), and the mutant can be specifically combined with GFP.

Drawings

FIG. 1 is a diagram showing colony PCR electrophoresis performed on a single domain antibody library specific to GFP constructed in the present invention; wherein, Lane 1 is the DNA molecule standard, Lanes 2-5 are randomly picked clones in the GFP nanobody library constructed, the insertion rate of the library is detected by colony PCR, and the calculation result shows that the insertion rate of the library is up to 100%;

FIG. 2 is a schematic diagram of screening specific single positive clones by enzyme-linked immunosorbent assay (ELISA) using phage in the present invention; wherein, 1 is to couple the apolipoprotein on the enzyme label plate, 2 is a nano antibody, 3 is a mouse anti-HA antibody, 4 is an antibody marked by goat anti-mouse alkaline phosphatase, and 5 is an alkaline phosphatase developing solution;

FIG. 3 is an electrophoresis chart of SDS-PAGE of GFP nanobodies expressed in the present invention after purification by nickel column resin gel affinity chromatography; wherein, lane 1 is the protein molecule standard, and

lanes

2 and 3 are the SDS-PAGE gel images after the purification of the target protein.

Detailed Description

The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.

The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.

Preferably, the amino acid sequence of the nanobody is divided into four Framework Regions (FRs) and three Complementary Determining Regions (CDRs).

A nucleic acid molecule encoding an amino acid sequence as described above.

Preferably, the sequence of the nucleic acid molecule is SEQ ID NO. 2. The specific sequence of the nucleic acid molecule can be obtained at any time by genetic code.

A vector comprising a nucleic acid molecule as described above. Due to the degeneracy of the genetic code, the nucleic acid sequence can vary from application to application.

A host cell comprising a vector as described above.

Preferably, the preparation method comprises the following steps:

Specifically, the preparation and detection are as follows:

the method comprises the steps of immunizing a Xinjiang bactrian camel with GFP, extracting bactrian camel peripheral blood lymphocytes after 4 times of immunization, and constructing a GFP specific nano antibody library. GFP is coupled on an enzyme label plate to display the correct spatial structure of protein, so that the epitope of GFP is exposed, and the antigen in the form is used for screening a GFP immune nano antibody gene library (camel heavy chain antibody phage display gene library) by utilizing a phage display technology, so that a nano antibody strain capable of being efficiently expressed in escherichia coli is obtained.

The invention will be further illustrated with reference to the following specific examples.

Example 1: construction of nanobody library against GFP:

(1) the GFP concentration is 500 micrograms per milliliter, 1 milligram of GFP and Freund's adjuvant are mixed in equal volume for each immunization, a Xinjiang bactrian camel is immunized once a week for 4 times, except for completely Freund's adjuvant used for the first time, Freund's adjuvant is used for the rest of times, and B cells are stimulated to express the antigen-specific nano antibody in the immunization process.

(2) After completion of 4 immunizations, camel peripheral blood lymphocytes (100 ml) were extracted and total RNA was extracted, referring to an RNA extraction kit supplied by QIAGEN.

(3) Extracted RNA was reverse transcribed into cDNA and VHH strands were amplified using nested PCR, FIRST round PCR:

an upstream primer: GTCCTGGCTGCTCTTCTACAAGGC

A downstream primer: GGTACGTGCTGTTGAACTGTTCC

Amplifying the fragment between the heavy chain antibody leader peptide and antibody CH2, annealing at 54 ℃ for 25 cycles;

second round PCR:

using the first round PCR product as template:

an upstream primer: GATGTGCAGCTGCAGGAGTCTGGRGGAGG

A downstream primer: GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT

The fragments between the FR1 region and the long and short hinge regions of the heavy chain antibody (long and short fragments) were amplified, annealed at 60 ℃ for 17 cycles, and the desired fragment was recovered, with the results shown in FIG. 1, and the DNA bands from left to right were: the first is a molecular Marker of 100bP, and the gene electrophoresis band of the second nano antibody is about 500 bP.

(4) Mu.g of pComb3 phage display vector (supplied by Biovector) and 10. mu.g of VHH were digested with restriction enzymes PstI and NotI (from NEB), and the two fragments were ligated with T4 DNA ligase (from TaKaRa).

(5) Electrotransformation of the ligation products into electrotransformation competent cells TG1(Beijing Shenzhou Red leaf technologies Co Ltd) In (1), a nano-antibody phage display library of GFP was constructed and the size of the library was determined to be 1.8 x 10⁸(ii) a Meanwhile, the insertion rate of the constructed library was measured by colony PCR, and the insertion rate was about 100%, and FIG. 2 shows the colony PCR results. After the library construction was completed, 24 clones were randomly selected for colony PCR to check the library insertion rate. The results show that: namely, the insertion rate of the invention reaches 100%.

Example 2: nano antibody screening process against GFP:

(1) the solution was dissolved in 100mM pH 8.2NaHCO₃200 micrograms of GFP in (1) was coupled to the microplate and left overnight at 4 ℃ with a negative control.

(2) On the next day, 100. mu.l of 0.1% casein was added to each well and blocked for 2 hours at room temperature.

(3) After 2 hours, 100. mu.l phage (8X 10) was added¹¹tfu immune camel nanobody phage display gene bank) at room temperature for 1 hour.

(4) Wash 5 times with PBST (0.05% Tween 20 in PBS) to wash away unbound phage.

(5) Phages specifically bound to GFP were dissociated with triethylamine (100mM) and infected with E.coli TG1, which was grown in log phase, and phages were generated and purified for the next round of screening, and the same screening procedure was repeated for 3-4 rounds. In the process of continuous screening, positive clones are continuously enriched, thereby achieving the purpose of screening GFP specific antibodies in an antibody library by using a phage display technology. The schematic diagram of this experiment is shown in fig. 3.

Example 3: screening of specific single positive clones by phage enzyme-linked immunosorbent assay (ELISA):

(1) from the phage-containing cell culture dishes after the 3-4 rounds of selection described above, 96 individual colonies were picked and inoculated into TB medium containing 100. mu.g per ml of ampicillin (2.3 g of potassium dihydrogen phosphate, 12.52 g of dipotassium hydrogen phosphate, 12 g of peptone, 24 g of yeast extract, 4 ml of glycerol in 1L of TB medium), grown to a logarithmic phase, followed by incubation at 28 ℃ overnight with 1 mM IPTG.

(2) Crude antibody was obtained by permeation and transferred to an antigen-coated ELISA plate and left for 1 hour at room temperature.

(3) Unbound antibody was washed away with PBST, and a mouse anti-HAtag antibody (anti-mouse anti-HA antibody available from Beijing kang, century Biotechnology Co., Ltd.) was added and left at room temperature for 1 hour.

(4) Unbound antibody was washed away with PBST, and anti-mouse alkali line phosphatase conjugate (goat anti-mouse alkaline phosphatase-labeled antibody, available from Eimei technologies, Ltd.) was added and allowed to stand at room temperature for 1 hour.

(5) Unbound antibody was washed away with PBST, and absorbance was read on an ELISA instrument at 405nm by adding an alkaline phosphatase developing solution.

(6) And when the OD value of the sample well is more than 3 times larger than that of the control well, judging the sample well to be a positive clone well.

(7) The bacteria of the positive cloning wells were shaken in LB liquid containing 100. mu.g per ml to extract the plasmid and sequenced.

The gene sequences of the respective clones were analyzed by the sequence alignment software VectorNTI, and the strains having the same CDR1, CDR2, and CDR3 sequences were regarded as the same clones, while the strains having different sequences were regarded as different clones, and finally 1 strain antibody. The amino acid sequences of the VHH chains of the antibodies are respectively shown in SEQ ID NO: 1, and the nucleic acid sequence is shown as SEQ ID NO: 2, respectively.

Example 4: the nano antibody is expressed and purified in host bacterium escherichia coli:

(1) subcloning two kinds of nano-antibodies obtained by the previous sequencing analysis into an expressive carrier PET32b, and transforming a recombinant plasmid with correct sequencing identification into an expressive host bacterium DE3, which is coated on a plate of LB solid medium containing 100 micrograms per milliliter of ampicillin overnight at 37 ℃, (2) selecting a single colony to be inoculated into 15 milliliters of LB culture solution containing ampicillin, shaking-culturing overnight at 37 ℃, (3) inoculating 1ml of overnight strain into 330ml of LB culture medium, shaking-culturing at 37 ℃, adding IPTG when the OD value reaches 0.6-1, shaking-culturing overnight at 28 ℃, (4) centrifuging for collecting bacteria for the next day, (5) crushing the bacteria to obtain an antibody crude extract, (6) purifying the antibody protein by nickel column ion affinity chromatography, adopting an imidazole gradient elution method to obtain a high-purity antibody, eluting with low-concentration imidazole (50 mmol, 100 mmol) is used for washing the impurity band, and the high-concentration imidazole eluent (250 mmol, 500 mmol) can finally prepare the protein with the purity of more than 90 percent.

The bands from left to right in fig. 3 are: the first is standard protein molecule, the second and the third are target protein purified samples.

Example 5 preparation of GFP immunoaffinity adsorbing Material

Agarose microspheres are used as a carrier (the carrier material is not limited to agarose gel, and can also be selected from silica spheres, nano magnetic beads and the like) to couple the anti-GFP nano antibody, and the specific preparation method is as follows:

the CNBr activated dry gel was washed 10 times with 0.1M HCl and equilibrated for 5min each time. The coupling buffer (10mM,

NA2HPO4, pH7.2), adding anti-GFP label nano antibody (2 mg/g agarose microsphere), reacting for 3.5h at room temperature, and covalently coupling the anti-GFP label nano antibody and the CNBr activated agarose gel microsphere. After washing 3 times with coupling buffer (10mM, NA2HPO4, pH7.2), the unreacted active group was blocked by adding blocking solution for 2h at room temperature. And (3) alternately washing the mixture by using phosphate buffer (10mM, pH7.2) and acetic acid buffer (0.1M, pH4.5) with 6 times of glue volume to obtain the immunoaffinity adsorbing material which is covalently coupled with the anti-GFP label nano antibody. 0.2ml of the immunoaffinity adsorption material is put into a chromatographic column with the capacity of 1ml, washed by PBS (10mM, pH7.2) with the volume of 8-10 times of the column volume, added with 20% ethanol solution and stored at 4 ℃.

The sequences used in the present invention may be as follows:

SEQ ID NO.1

QVQQHKSRGGLLEARGSLWHWCRGWGYWCSRCHMMWLRLARGNGPDSVWGPTTGRGPWYYDDPVEAPIHHLPRQRQEHGVSAGVQPGTWPPSHVLLCCPTRFPMGWAPPLAKILWLLGPGDPGHRLL

SEQ ID NO.2

CAGGTGCAGCAGCACAAGTCTCGCGGTGGGTTGCTGGAGGCTCGGGGGTCACTGTGGCACTGGTGTCGAGGCTGGGGATACTGGTGCAGTAGATGCCACATGATGTGGCTGCGGCTGGCTCGAGGGAACGGGCCCGACTCGGTCTGGGGCCCTACAACTGGTCGTGGGCCATGGTACTACGACGACCCCGTGGAGGCGCCGATTCACCATCTCCCGAGACAACGCCAAGAACATGGTGTATCTGCGGGTGTTCAGCCTGGGACCTGGCCACCCAGCCATGTACTACTGTGCTGCCCGACCAGGTTCCCTATGGGTTGGGCTCCCCCTCTCGCGAAGATTTTATGGTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTC

although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.

Sequence listing

<110> Tianjin science and technology university Beijing Lanbobedrid commercial and trade Co., Ltd

<120> nano antibody aiming at green fluorescent protein GFP and application thereof

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 127

<212> PRT

<213> Nanobody (Unknown)

<400> 1

Gln Val Gln Gln His Lys Ser Arg Gly Gly Leu Leu Glu Ala Arg Gly

1 5 10 15

Ser Leu Trp His Trp Cys Arg Gly Trp Gly Tyr Trp Cys Ser Arg Cys

20 25 30

His Met Met Trp Leu Arg Leu Ala Arg Gly Asn Gly Pro Asp Ser Val

35 40 45

Trp Gly Pro Thr Thr Gly Arg Gly Pro Trp Tyr Tyr Asp Asp Pro Val

50 55 60

Glu Ala Pro Ile His His Leu Pro Arg Gln Arg Gln Glu His Gly Val

65 70 75 80

Ser Ala Gly Val Gln Pro Gly Thr Trp Pro Pro Ser His Val Leu Leu

85 90 95

Cys Cys Pro Thr Arg Phe Pro Met Gly Trp Ala Pro Pro Leu Ala Lys

100 105 110

Ile Leu Trp Leu Leu Gly Pro Gly Asp Pro Gly His Arg Leu Leu

115 120 125

<210> 2

<211> 381

<212> DNA/RNA

<213> nucleic acid molecule (Unknown)

<400> 2

caggtgcagc agcacaagtc tcgcggtggg ttgctggagg ctcgggggtc actgtggcac 60

tggtgtcgag gctggggata ctggtgcagt agatgccaca tgatgtggct gcggctggct 120

cgagggaacg ggcccgactc ggtctggggc cctacaactg gtcgtgggcc atggtactac 180

gacgaccccg tggaggcgcc gattcaccat ctcccgagac aacgccaaga acatggtgta 240

tctgcgggtg ttcagcctgg gacctggcca cccagccatg tactactgtg ctgcccgacc 300

aggttcccta tgggttgggc tccccctctc gcgaagattt tatggttact ggggccaggg 360

gacccaggtc accgtctcct c 381

<210> 3

<211> 24

<212> DNA/RNA

<213> first round PCR upstream primer (Unknown)

<400> 3

gtcctggctg ctcttctaca aggc 24

<210> 4

<211> 23

<212> DNA/RNA

<213> first round PCR downstream primer (Unknown)

<400> 4

ggtacgtgct gttgaactgt tcc 23

<210> 5

<211> 29

<212> DNA/RNA

<213> second round PCR Forward primer (Unknown)

<400> 5

gatgtgcagc tgcaggagtc tggrggagg 29

<210> 6

<211> 35

<212> DNA/RNA

<213> second round PCR downstream primer (Unknown)

<400> 6

ggactagtgc ggccgctgga gacggtgacc tgggt 35

Claims

1. A nanobody against green fluorescent protein, characterized in that: the antibody has an amino acid sequence shown in SEQ ID NO. 1.

2. The nanobody against green fluorescent protein according to claim 1, characterized in that: the amino acid sequence of the antibody can be divided into four framework regions and three complementarity determining regions.

3. A nucleic acid molecule encoding the amino acid sequence of claim 1 or 2.

4. The nucleic acid molecule of claim 3, wherein: the sequence of the nucleic acid molecule is SEQ ID NO. 2.

5. A vector comprising the nucleic acid molecule of claim 3 or 4.

6. A host cell comprising the vector of claim 5.

7. The use of the nanobody against green fluorescent protein of claim 1 or 2 in immunodetection, or enrichment, detection and purification of green fluorescent protein and green fluorescent protein fusion protein.

8. The nanobody against green fluorescent protein of claim 1 or 2, which is an antibody capable of specifically binding to GFP, obtained by modification by random or site-directed mutagenesis techniques.

9. An immunoaffinity adsorption material of Green Fluorescent Protein (GFP), which is characterized in that: the adsorbing material can use a nano antibody aiming at green fluorescent protein GFP as a ligand, and the amino acid sequence of the nano antibody is SEQ ID NO. 1.

10. The immunoaffinity adsorbent material according to claim 9, wherein: the preparation method comprises the following specific steps: