CN111574629A

CN111574629A - CD 20-resistant nano antibody, encoding gene, recombinant nano antibody, recombinant vector, recombinant strain and application

Info

Publication number: CN111574629A
Application number: CN202010486402.2A
Authority: CN
Inventors: 张爱君; 徐广贤; 李艳宁; 楚元奎
Original assignee: Ningxia Medical University
Current assignee: Ningxia Medical University
Priority date: 2020-06-01
Filing date: 2020-06-01
Publication date: 2020-08-25
Anticipated expiration: 2040-06-01
Also published as: CN111574629B

Abstract

The invention provides a CD 20-resistant nano antibody, an encoding gene, a recombinant nano antibody, a recombinant vector, a recombinant strain and application, and belongs to the technical field of immunology, wherein the amino acid sequence of the nano antibody is shown as SEQ ID No.1, and the nucleotide sequence of the gene is shown as SEQ ID No. 2; the amino acid sequence of the recombinant nano antibody is shown in SEQ ID No. 3. The recombinant nano antibody for resisting CD20 provided by the invention is expressed by fusion of a camel-derived nano antibody gene and a human IgG Fc segment gene, can be stably expressed by using the recombinant strain, and can specifically recognize a CD20 antigen.

Description

CD 20-resistant nano antibody, encoding gene, recombinant nano antibody, recombinant vector, recombinant strain and application

Technical Field

The invention belongs to the technical field of immunology, and particularly relates to a CD 20-resistant nano antibody, a coding gene, a recombinant vector, a recombinant strain and application.

Background

Human leukocyte differentiation antigen 20(CD20 antigen) is a B lymphocyte surface differentiation antigen, is expressed in 95% of B cell tumors, is not expressed in normal tissues, and has no obvious internalization and shedding after being combined with an antibody thereof, so the human leukocyte differentiation antigen becomes an ideal target point for treating B cell lymphoma.

The nanobody is the first report in Nature by belgium scientist in 1993 that an antibody (VHH) naturally lacking a light chain exists in the peripheral blood of alpaca, and is found in 1995 in cartilage fish such as nurse shark, zebra shark and mackerel, and is the smallest unit which is known to bind to a target antigen. VHH has a molecular weight of only 15KD and is therefore also called Nanobody (Nb). The nano antibody has unique advantages of small molecular weight, good solubility, strong stability, high affinity, low immunogenicity, good in vivo tissue permeability, easy to pass through blood vessels or tissues to reach target sites and the like, has a large development space, is widely applied, and can be clinically used for tumor treatment and also used as a diagnostic tool.

There are over ten antibody drugs targeting CD20 that have been successfully developed at present, Rituximab is the first global CD 20-targeting monoclonal antibody approved by FDA in 1997 for the treatment of B-cell non-hodgkin lymphoma (B-NHL), belongs to a human-mouse chimeric monoclonal antibody, possesses a variable region of murine origin and a constant region of human origin, is the CD20 monoclonal antibody drug that has been most successful in commercialization so far, and was developed by Rituximab. Although it has shown better efficacy in clinical treatment, part of patients have ineffective treatment, drug resistance or adverse reaction due to low humanization degree. Moreover, since the antibody is of murine origin, it causes a human anti-mouse antibody reaction after long-term use and cannot be used continuously.

Disclosure of Invention

In view of the above, the present invention aims to provide a CD 20-resistant nanobody, a coding gene, a recombinant vector, a recombinant strain and applications thereof; the recombinant nano antibody for resisting CD20 is expressed by fusion of a camel-derived nano antibody gene and a human IgG Fc segment gene, and can specifically recognize a CD20 antigen.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a nano antibody for resisting CD20, wherein the amino acid sequence of the nano antibody is shown in SEQ ID No. 1.

The invention provides a gene for coding the nano antibody, and the nucleotide sequence of the gene is shown as SEQ ID No. 2.

The invention provides a recombinant nano antibody for resisting CD20, which comprises the nano antibody for resisting CD20 and a human IgG Fc segment.

Preferably, the amino acid sequence of the recombinant nanobody is shown in SEQ ID No. 3.

The invention provides a gene for coding the recombinant nano antibody, which comprises the gene of the nano antibody and a human IgG Fc segment gene; the nanometer antibody gene and the human IgG Fc segment gene are connected through a connecting segment, and the nucleotide sequence of the connecting segment is shown as SEQ ID No. 4.

Preferably, the nucleotide sequence of the gene of the recombinant nano antibody is shown in SEQ ID No. 5.

The invention provides a recombinant vector for expressing a nano antibody of anti-CD 20, which comprises a gene and an initial vector of the recombinant nano antibody.

Preferably, the initial vector is a pCZN1 plasmid vector.

The invention provides a recombinant strain for expressing a CD 20-resistant nano antibody, which comprises a recombinant vector and escherichia coli Arctic Express.

The invention provides the CD 20-resistant nano antibody, the gene for encoding the CD 20-resistant nano antibody, the recombinant nano antibody, the gene for encoding the recombinant nano antibody, the recombinant vector and the application of the recombinant strain in preparing an anti-tumor medicament or a tumor diagnosis reagent.

The invention has the beneficial effects that: the invention provides an anti-CD 20 nanobody, which is directed to the heavy chain of CD20 and comprises a framework region FR and an antigenic determinant complementary CDR; the recombinant nano antibody expressed by fusing the gene of the anti-CD 20 nano antibody and the human IgG Fc segment gene can specifically identify the CD20 antigen, and can be applied to molecular diagnosis of tumors and preparation of antitumor drugs.

Drawings

FIG. 1 is a first round DNA electrophoresis of the nanobody, the DNA bands from left to right gel pores are: the sixth lane is a Marker of 1000bp (the sizes of the bands are 1000, 700, 500, 400, 300, 200 and 100bp in sequence), the first, second, third and fifth lanes are PCR products, the bands are about 700bp, and the fourth lane is empty;

FIG. 2 is a second round DNA electrophoresis of the nanobody, the DNA bands from left to right gel wells are: the first lane is a Marker of 1000bp (the size of the band is the same as above), the second, fourth and sixth lanes are PCR products, the band is about 400bp, and the third and fifth lanes are empty;

FIG. 3 is an anti-CD 20 nanobody screening enrichment sequence;

FIG. 4 is a schematic diagram of screening of specific single positive clones by phage enzyme-linked immunosorbent assay (phase-ELISA): wherein 1 is to coat CD20 antigen on an enzyme label plate, 2 is phage supernatant, 3 is a mouse anti-km 13107 antibody, 4 is a goat anti-mouse IgG (AP) antibody, and 5 is TMB color development liquid;

FIG. 5 is a SDS-PAGE electrophoretogram of purified anti-CD 20 nanobody, wherein the left image is purified anti-CD 20 nanobody; the right picture is a western blot of the purified anti-CD 20 nanobody;

FIG. 6 is a SDS-PAGE electrophoresis of the CD20 antigen and the total protein of CD20 positive Daudi cells;

FIG. 7 is a whole protein western blot of CD20 antigen and CD20 positive Daudi cells.

Detailed Description

The invention provides a nano antibody for resisting CD20, wherein the amino acid sequence of the nano antibody is shown as SEQ ID No. 1; the method comprises the following specific steps:

QVQLQESGGGSVQAGGSLRLRCIISRYGVTLPYMAWFRQGPGEERE GVAATTLRGSTLYADNVKGRFTLSQDPPKRALFLQMNNLQPEDSGMYYC AAGTSARSLSPSDYGYRGRGTQVTVSS。

in the present invention, the nanobody against CD20 comprises a framework region FR and an epitope-complementary region CDR; in the present invention, the framework regions FR include FR1, FR2, FR3 and FR 4; the specific amino acid sequence is as follows:

FR1:QVQLQESGGGSVQAGGSLRLRCIIS(SEQ ID No.6)

FR2:WFRQGPGEEREGVAA(SEQ ID No.7)

FR3:LYADNVKGRFTLSQDPPKRALFLQMNNLQPEDSGMYYC(SEQ ID No.8)

FR4:RGRGTQVTVSS(SEQ ID No.9)

the corresponding nucleotide sequences are as follows:

FR1:CAGGTCCAACTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGC TGGAGGGTCTCTGAGGCTCCGCTGTATAATCTCG(SEQ ID No.10)

FR2:TGGTTTCGCCAGGGCCCAGGCGAGGAGCGCGAGGGGGTCGC GGCC(SEQ ID No.11)

FR3:CTCTACGCTGACAACGTGAAGGGCCGATTCACCCTCTCCCAA GACCCGCCCAAGCGCGCTCTATTTCTCCAGATGAACAACCTTCAACCT GAAGATTCTGGCATGTACTACTGT(SEQ ID No.12)

FR4:CGGGGCCGGGGGACCCAGGTCACCGTCTCCTCA(SEQ ID No.13)。

in the present invention, the epitope-complementing region CDRs include CDR1, CDR2 and CDR 3; the specific amino acid sequence is as follows:

CDR1:RYGVTLPYMA(SEQ ID No.14)

CDR2:TTLRGST(SEQ ID No.15)

CDR3:AAGTSARSLSPSDYGY(SEQ ID No.16)。

in the present invention, the nucleotide sequences of the CDR1, CDR2 and CDR3 are specifically as follows:

CDR1:CGATACGGCGTAACTCTGCCCTACATGGCC(SEQ ID No.17)

CDR2:ACCACCCTGCGTGGAAGCACT(SEQ ID No.18)

CDR3:GCGGCAGGCACTAGCGCCCGTTCACTGAGCCCCAGCGACTA TGGCTAC(SEQ ID No.19)。

in the invention, the anti-CD 20 nanobody is obtained by screening according to the following method: a natural camel source nano antibody phage display library is constructed by using a phage surface display technology, and then screening is carried out on the basis of biotinylated CD20 antigen to obtain a CD20 specific nano antibody gene sequence.

In the present invention, the method for constructing the phage display gene library of the natural camel heavy chain antibody preferably comprises the following steps: 1) extracting total RNA of camel spleen tissues and carrying out reverse transcription on the total RNA to obtain cDNA; 2) performing nested PCR amplification by using the cDNA as a template to obtain a variable region fragment of the heavy chain antibody; 3) respectively carrying out enzyme digestion on the variable region fragment of the heavy chain antibody and a pcantab5e phage vector, and then connecting to obtain a connection product; 4) and transferring the ligation product into a competent cell to obtain a natural camel source nano antibody phage display library.

In the invention, total RNA of camel spleen tissues is extracted and is subjected to reverse transcription to obtain cDNA. The extraction method of the camel spleen tissue total RNA is not particularly limited, and the conventional extraction method of animal tissue total RNA in the field can be adopted. In the present invention, the reverse transcription is preferably carried out by a Thermo Scientific reverse aid first Strand cDNA Synthesis Kits.

After the cDNA is obtained, the variable region fragment of the heavy chain antibody is obtained by nested PCR amplification by taking the cDNA as a template. In the present invention, the nested PCR preferably comprises two rounds of PCR; the first round of PCR is used for amplifying a fragment between the heavy chain antibody leader peptide and the antibody CH2, and the primer sequences of the first round of PCR are preferably shown as SEQ ID No.20 and SEQ ID No. 21; a second round of PCR, the primer sequences of which are preferably shown in SEQ ID No.22 and SEQ ID No.23, was used to amplify the fragment between the FR1 region and the long and short hinge regions of the heavy chain antibody.

After the variable region fragment of the heavy chain antibody is obtained, the variable region fragment of the heavy chain antibody and a pcantab5e phage vector are respectively enzyme-cut and then are connected to obtain a connection product. In the present invention, the enzyme is preferably a double enzyme, and the enzyme is preferably performed by using restriction enzymes SifI and Not I. In the present invention, the procedure of the enzyme cleavage is preferably as follows: enzyme digestion is carried out for 1h at 37 ℃; the enzyme was cleaved at 50 ℃ for 1 h. In the present invention, the temperature of the joining is preferably 16 ℃ and the time of the joining is preferably 4 hours.

After the connecting product is obtained, the connecting product is transferred into competent cells to obtain a natural camel source nano antibody phage display library. In the invention, the competent cell is preferably escherichia coli competent cell TG1, and the escherichia coli competent cell TG1 is preferably self-made by a glycerol resuspension method; the method of transformation is preferably electrotransformation. In the present invention, the transformation also includes a helper phage rescue process, and the process of the electric transformation and helper phage rescue is not particularly limited, and the detailed procedures are described in the examples.

The specific steps of screening the anti-CD 20 nano antibody by the biotinylated CD20 antigen are not particularly limited, and the steps are described in the examples by adopting a conventional nano antibody screening method in the field.

The invention also provides a gene for coding the nano antibody, wherein the nucleotide sequence of the gene is shown as SEQ ID No.2, and the gene specifically comprises the following components:

CAGGTCCAACTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGG AGGGTCTCTGAGGCTCCGCTGTATAATCTCGCGATACGGCGTAACTCTG CCCTACATGGCCTGGTTTCGCCAGGGCCCAGGCGAGGAGCGCGAGGG GGTCGCGGCCACCACCCTGCGTGGAAGCACTCTCTACGCTGACAACGT GAAGGGCCGATTCACCCTCTCCCAAGACCCGCCCAAGCGCGCTCTATT TCTCCAGATGAACAACCTTCAACCTGAAGATTCTGGCATGTACTACTGT GCGGCAGGCACTAGCGCCCGTTCACTGAGCCCCAGCGACTATGGCTAC CGGGGCCGGGGGACCCAGGTCACCGTCTCCTCA。

the invention provides a recombinant nano antibody for resisting CD20, which comprises the nano antibody for resisting CD20 and a human IgGFc segment. In the invention, the amino acid sequence of the recombinant nano antibody is shown as SEQ ID No. 3.

The invention also provides a gene for coding the recombinant nano antibody, which comprises the gene of the nano antibody and a human IgG Fc segment gene. In the invention, the nano antibody gene and the human IgG Fc segment gene are connected through a connecting fragment, and the nucleotide sequence of the connecting fragment is shown as SEQ ID No.4, and specifically comprises the following steps: GGTGGTGGTGGTAGT are provided.

In the invention, the nucleotide sequence of the gene of the recombinant nano antibody is shown as SEQ ID No. 5; wherein, the nucleotide sequence of the human IgG Fc segment gene is shown as SEQ ID No.24, and specifically comprises the following steps:

GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCA GCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAA CCCAAGGACACCCTCATGATCTCCCGGACCCCTGGGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTA CGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGG AGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGC ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGG GCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGA GCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGA ACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCT TCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA。

the invention also provides a recombinant vector for expressing the anti-CD 20 nano antibody, which comprises the gene and the initial vector of the recombinant nano antibody.

In the present invention, the initial vector is preferably a pCZN1 plasmid vector; the insertion site of the gene of the recombinant nanobody is preferably located at NdeI and XbaI sites of the pCZN1 plasmid vector. The method for preparing the recombinant vector is not particularly limited in the present invention, and a conventional method for preparing a recombinant vector in the art may be used.

The invention also provides a recombinant strain for expressing the anti-CD 20 nano antibody, which comprises the recombinant vector and Escherichia coli Arctic Express. In the present invention, it is preferable that the recombinant strain is obtained by transferring the recombinant vector expressing the nanobody against CD20 into the escherichia coli Arctic Express. The specific preparation method of the recombinant strain is not particularly limited, and a conventional preparation method of the recombinant strain in the field can be adopted.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Aiming at the construction of a natural camel source nano antibody gene bank:

extracting total RNA of camel spleen tissues, which comprises the following steps:

taking out camel spleen tissue from-80 ℃, adding liquid nitrogen to crush a sample specimen, adding 1ml of Trizol to every 100mg of crushed tissue, and homogenizing by a glass homogenizer until no tissue sample particles can be seen by naked eyes. Then, the mixture was transferred into a 1.5ml centrifuge tube and left at room temperature for 15 min.

② adding 0.2ml chloroform into 1ml Trizol, shaking violently for 15s, and standing for 3min at room temperature.

③ the sample was centrifuged at 12000rpm at 4 ℃ for 15min and the upper layer was transferred to a new Ep tube.

And fourthly, adding isopropanol with the same volume, mixing and placing for 20min on ice.

Fifthly, centrifuging the solution at 12000rpm and 4 ℃ for 10min, and discarding the supernatant.

Sixthly, adding 75% ethanol prepared by 1ml of DEPC water to wash the precipitate (at least 1ml of ethanol is added to each 1ml of Trizol).

Seventhly, centrifuging the solution in the previous step at 10000rpm for 10min at 4 ℃, removing the supernatant, and repeating the previous step for repeated washing once.

Eighty percent (10000 rpm), centrifuging at 4 ℃ for 10min, discarding the supernatant, and drying for 10-15 min.

⑨ adding appropriate amount of ddH₂The RNA pellet was dissolved by O, the RNA concentration was determined, and the pellet was stored at-80 ℃.

The cDNA was obtained by reverse transcription using RNA purification kit provided by TIANGEN, according to Thermo Scientific reverse Aidfirst Strand cDNA Synthesis kit.

(2) Using cDNA as template, using nested PCR amplification to obtain variable region fragment of heavy chain antibody;

first round PCR:

an upstream primer: 5'-GTCCTGGCTGCTCTTCTACAAAG-3' (SEQ ID No.20)

A downstream primer: 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID No.21)

First round PCR reaction system

The conditions of the first round of PCR amplification reaction are as follows: 5min at 95 ℃; 30s at 95 ℃, 30s at 55 ℃, 45s at 72 ℃ and 32 cycles; 10min at 72 ℃.

The fragment between the heavy chain antibody leader peptide and the antibody CH2 was amplified, and the result showed that the size of the fragment was about 700bp, i.e., the gene electrophoresis band of the nanobody was about 700 bp.

Second round PCR:

taking the first round PCR product as a template,

an upstream primer:

5'-TCGCGGCCCAGCCGGCCCAGGTCCAACTGCAGGAGTCTGGGG-3 '(SEQ ID No.22)

a downstream primer:

5'-ATAAGAATGCGGCCGCTGAGGAGACGGTGACCTGGGTCCCC-3' (SEQ ID No.23)

second round PCR reaction system

The conditions of the second round of PCR amplification reaction are as follows: 5min at 94 ℃; 94 ℃ for 40s, 55 ℃ for 40s, 72 ℃ for 40s, 25 cycles; 10min at 72 ℃. The amplification of the fragment between the FR1 region and the long and short hinge regions of the heavy chain antibody (long fragment and short fragment) revealed that the size of the fragment was about 400bp, i.e., the gene electrophoresis band of the nanobody was about 400 bp.

The pcantab5e phage vector and the VHH fragment were digested with restriction enzymes (purchased from Takara) SifI and Not I, and the two fragments were ligated using T4 DNA ligase (purchased from Takara). The specific enzyme digestion system and the connection system are as follows:

enzyme digestion system

Enzyme cutting conditions are as follows: after digestion at 37 ℃ for 1h, digestion was carried out at 50 ℃ for 1 h.

Connection system

Connection conditions are as follows: after ligation at 16 ℃ for 4h, ligation was performed overnight at 4 ℃.

The ligation product is electrically transformed into an electrotransformation competent cell TG1 to construct a natural camel source nano antibody phage display library, and the library capacity reaches 9.0 × 10 after the phage display library is rescued by auxiliary phage¹³。

The preparation method of the competent cell TG1 comprises the following steps:

escherichia coli TG1 glycerol was taken out from a-80 ℃ freezer, streaked on a 2 XYT solid plate and cultured at 37 ℃ for 10 hours, and a single colony was picked and inoculated in 3ml of 2 XYT liquid medium and cultured overnight at 37 ℃ with shaking at 200 rpm. The next day, the bacterial solution was cultured in 200ml of 2 XYT medium in an enlarged scale at a ratio of 1:100 in a conical flask, and when the culture was continued at 37 ℃ until the OD600 was about 0.4, the bacterial solution was collected in a 50ml centrifuge tube, allowed to stand on ice for 1 hour, centrifuged at 9000rpm at 4 ℃ for 10min, and the supernatant was discarded, and the pellet was resuspended in the same volume of cold pure water, centrifuged, and repeated once. The cells were then resuspended in 10% glycerol pre-chilled and centrifuged. Suspending the thallus precipitate with 1ml 10% glycerol (prepared by pre-cooled pure water), packaging in pre-cooled 1ml Ep tubes (100 μ l each), and immediately transferring to-80 deg.C refrigerator for storage to obtain competent cell TG 1.

The helper phage rescue steps are as follows:

firstly, 100 mu L of the library is inoculated into 50ml of 2 XYT/Amp/Glu culture medium, cultured at 37 ℃ and 200rpm with shaking until the logarithmic phase OD600 is about 0.4-0.5.

② adding the helper phage M13KO7 with the multiplicity of infection of 20:1 into the culture solution, mixing uniformly and standing for 30min at 37 ℃.

③ the culture solution is centrifuged at 9000rpm for 10min at room temperature, the supernatant is discarded to precipitate the thalli, 200mL of 2 XYT/Amp/Kana culture solution is used for resuspension, and the culture is carried out at 200rpm at 37 ℃ overnight.

Fourthly, the culture solution is centrifuged for 10min at 9000rpm at 4 ℃, the supernatant is taken, and 1/5 volume of PEG/NaCl is added, and the mixture is kept stand for 6h at 4 ℃.

And fifthly, centrifuging at 9000rpm for 20min, discarding the supernatant, resuspending the precipitate with PBS (1mL) to obtain a recombinant phage antibody library, subpackaging the recombinant phage antibody library in 1.5mL Ep tubes, and storing at 4 ℃.

At the same time, the library was tested for insertion rate by colony PCR using the second PCR primer at 55 ℃ annealing temperature, showing that the insertion rate was 95% or more (the desired fragment insertion rate is the number of colonies containing the desired fragment/the number of all colonies).

Screening process for anti-CD 20 nanobody:

phage library (1 × 10)¹²Phage) and 50 mul streptavidin magnetic beads are incubated for 1h at room temperature on a rotating platform, and then phage antibodies are collected; mu.l of the pre-reduced phage antibody was added to 21 ml centrifuge tubes blocked with 2% PBSM, 500. mu.l of 5. mu.g biotinylated CD20 antigen diluted with PBS was added to one of the tubes, 500. mu.l of PBS buffer was added to the other tube as a negative control, the tubes were incubated on a rotating platform at room temperature for 1h, 50. mu.l of pre-blocked streptavidin magnetic beads were added, and the tubes were incubated on the rotating platform at room temperature for 30min and the magnetic beads were collected. The beads were washed 7 times with PBST, 2 times with PBSM and 1 time with PBS. Glycine at pH2.7 was added for elution and 1mol/L Tris-HCl at pH9.1 was used for neutralization. The above neutralization solution was added to 5ml of TG1(OD 0.5) in the logarithmic growth phase, phages were generated and purified for the next round of selection, and 4 rounds of selection were performedAnd positive clones are continuously enriched, so that the purpose of screening CD20 specific antibodies in an antibody library by using a phage display technology is achieved.

Phage enzyme-linked immunosorbent assay (phase-ELISA) screening of specific single positive clones:

the screening principle mode diagram is shown in fig. 4, and the specific method is as follows:

a VHH phage monoclonal supernatant was first prepared: selecting 90 single colonies from solid plates after 3-4 rounds of screening, inoculating the colonies in a 2 XYT culture medium containing 100 mu g/ml of ampicillin and 2% of glucose, culturing at 220rpm37 ℃ overnight, taking 50 mu l of bacterial liquid the next day to a new 96-deep-well plate, adding 800 mu l of a 2 XYT culture medium containing 100 mu g/ml of ampicillin and 2% of glucose into each well, after the colonies grow to logarithmic phase, adding an auxiliary phage M13K07 with the infection number of 20:1, infecting at 37 ℃ for 30min, centrifuging at 10000rpm for 5min, discarding the supernatant, resuspending the bacteria in 800ul of a fresh 2 XYT culture medium containing 100ug/ml of ampicillin and 50ug/ml of kanamycin, culturing at 37 ℃, culturing at 220rpm for 12h, centrifuging at 10000rpm for 5min, and obtaining the bacterial liquid of which is VHH monoclonal supernatant.

The CD20 antigen was diluted to 10. mu.g/ml with coating solution, 100. mu.l was added per well, coated overnight at 4 ℃ and negative and positive controls were set up. The next day, three washes with PBST, blocking with 2% PBSM at 37 ℃ for 2h, three washes with PBST, addition of 200. mu.l of pretreated VHH phage monoclonal supernatant, and incubation at 37 ℃ for 1 h. Add 1:5000 secondary antibody of murine anti-M13 KO7/HRP diluted with 0.1% PBST, incubate 1h at 37 deg.C, wash away unbound antibody, add TMB developing solution, read absorbance values at 450nm wavelength on a microplate reader. And when the OD value of the sample hole is more than twice of the OD value of the control hole, determining the sample hole as a positive control hole, and taking the positive bacterial liquid to perform gene sequencing.

The DNAMAN software was used for sequence analysis and blast alignment, and strains with the same CDR1, CDR2, and CDR3 sequences were regarded as the same clones. FIG. 3 is the enrichment sequence of anti-CD 20 nano antibody screening. Finally, the nano antibody sequence shown by the amino acid sequence SEQ IDN0.1 is adopted for subsequent experiments.

Example 2

And (3) expression and purification of the nano antibody in host bacterium escherichia coli:

(1) connecting a nano antibody sequence obtained by sequencing analysis with a human IgG Fc segment and subcloning a pCZN1 plasmid vector, converting the sequence into escherichia coli Arctic Express, selecting a monoclonal on a conversion plate, inoculating the monoclonal on the conversion plate into a test tube containing 3mL of LB culture solution with the concentration of 50 mu g/mLAmp, and shaking at 37 ℃ and 220rpm overnight; (2) inoculating the cells into 30mL LB culture solution of 50 μ g/mL Amp according to the ratio of 1:100 the next day, shaking at 37 ℃ and 220rpm until the OD600 of the cells is 0.6-0.8, adding IPTG until the final concentration is 0.5mM, shaking at 20 ℃ and 220rpm overnight, and inducing the expression of the fusion protein; (3) collecting thalli, carrying out ultrasonic crushing to obtain an inclusion body protein crude body fluid, and then carrying out Ni column affinity purification to obtain the fusion protein. FIG. 5 is a SDS-PAGE electrophoretogram of purified anti-CD 20 nanobody, wherein the left panel is purified anti-CD 20 nanobody: wherein lane 1 is a protein molecular standard and lane 2 is a purified anti-CD 20 nanobody; the right panel is a western blot of purified anti-CD 20 nanobody: wherein lane 1 is a protein molecular standard and lane 2 is a purified anti-CD 20 nanobody (wherein the primary antibody is a murine his-tag-labeled antibody and the secondary antibody is goat anti-mouse IgG/HRP).

Example 3

Specificity verification of anti-CD 20 nanobody:

the CD20 antigen and the whole protein of the human leukemia cell Daudi cell are subjected to SDS-PAGE electrophoresis, wherein one gel is stained, the other gel is subjected to membrane transfer, and a western blot is prepared (wherein one antibody is a purified anti-CD 20 nano antibody, and the second antibody is anti-human IgG Fc/HRP). FIG. 6 is a SDS-PAGE staining pattern of the CD20 antigen and the total protein of the CD20 positive Daudi cells, wherein lane 1 is the protein molecular standard, lane 2 is the CD20 antigen, and lane 3 is the total protein of the Daudi cells; FIG. 7 is a western blot of whole protein and CD20 antigen of CD20 positive Daudi cells: wherein lane 1 is a protein molecular standard, lane 2 is the whole protein of Daudi cells, and lane 3 is the CD20 antigen. Therefore, the anti-CD 20 nano antibody provided by the invention can specifically recognize the CD20 antigen.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Ningxia medical university

<120> CD 20-resistant nano antibody, encoding gene, recombinant nano antibody, recombinant vector, recombinant strain and application

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<210>1

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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

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Ser Leu Arg Leu Arg Cys Ile Ile Ser Arg Tyr Gly Val Thr Leu Pro

20 25 30

Tyr Met Ala Trp Phe Arg Gln Gly Pro Gly Glu Glu Arg Glu Gly Val

35 40 45

Ala Ala Thr Thr Leu Arg Gly Ser Thr Leu Tyr Ala Asp Asn Val Lys

50 55 60

Gly Arg Phe Thr Leu Ser Gln Asp Pro Pro Lys Arg Ala Leu Phe Leu

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Gln Met Asn Asn Leu Gln Pro Glu Asp Ser Gly Met Tyr Tyr Cys Ala

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Ala Gly Thr Ser Ala Arg Ser Leu Ser Pro Ser Asp Tyr Gly Tyr Arg

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Gly Arg Gly Thr Gln Val Thr Val Ser Ser

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caggtccaac tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgaggctc 60

cgctgtataa tctcgcgata cggcgtaact ctgccctaca tggcctggtt tcgccagggc 120

ccaggcgagg agcgcgaggg ggtcgcggcc accaccctgc gtggaagcac tctctacgct 180

gacaacgtga agggccgatt caccctctcc caagacccgc ccaagcgcgc tctatttctc 240

cagatgaaca accttcaacc tgaagattct ggcatgtact actgtgcggc aggcactagc 300

gcccgttcac tgagccccag cgactatggc taccggggcc gggggaccca ggtcaccgtc 360

tcctca 366

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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Arg Cys Ile Ile Ser Arg Tyr Gly Val Thr Leu Pro

20 25 30

Tyr Met Ala Trp Phe Arg Gln Gly Pro Gly Glu Glu Arg Glu Gly Val

35 40 45

Ala Ala Thr Thr Leu Arg Gly Ser Thr Leu Tyr Ala Asp Asn Val Lys

50 55 60

Gly Arg Phe Thr Leu Ser Gln Asp Pro Pro Lys Arg Ala Leu Phe Leu

65 70 75 80

Gln Met Asn Asn Leu Gln Pro Glu Asp Ser Gly Met Tyr Tyr Cys Ala

85 90 95

Ala Gly Thr Ser Ala Arg Ser Leu Ser Pro Ser Asp Tyr Gly Tyr Arg

100 105 110

Gly Arg Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

130 135 140

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

145 150 155 160

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Gly Val

165 170 175

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

180 185 190

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

195 200 205

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

210 215 220

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

225 230 235 240

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

245 250 255

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

260 265 270

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

275 280 285

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

290 295 300

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

305 310 315 320

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

325 330 335

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

340 345 350

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

355 360

<210>4

<211>15

<212>PRT

<213>Artificial Sequence

<400>4

Gly Gly Thr Gly Gly Thr Gly Gly Thr Gly Gly Thr Ala Gly Thr

1 5 10 15

<210>5

<211>1077

<212>DNA

<213>Artificial Sequence

<400>5

caggtccaac tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgaggctc 60

cgctgtataa tctcgcgata cggcgtaact ctgccctaca tggcctggtt tcgccagggc 120

ccaggcgagg agcgcgaggg ggtcgcggcc accaccctgc gtggaagcac tctctacgct 180

gacaacgtga agggccgatt caccctctcc caagacccgc ccaagcgcgc tctatttctc 240

cagatgaaca accttcaacc tgaagattct ggcatgtact actgtgcggc aggcactagc 300

gcccgttcac tgagccccag cgactatggc taccggggcc gggggaccca ggtcaccgtc 360

tcctcaggtg gtggtggtag tgagcccaaa tcttgtgaca aaactcacac atgcccaccg 420

tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 480

gacaccctca tgatctcccg gacccctggg gtcacatgcg tggtggtgga cgtgagccac 540

gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 600

acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 660

ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 720

ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 780

tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 840

gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 900

aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 960

aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1020

catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaa 1077

<210>6

<211>25

<212>PRT

<213>Artificial Sequence

<400>6

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Arg Cys Ile Ile Ser

20 25

<210>7

<211>15

<212>PRT

<213>Artificial Sequence

<400>7

Trp Phe Arg Gln Gly Pro Gly Glu Glu Arg Glu Gly Val Ala Ala

1 5 10 15

<210>8

<211>38

<212>PRT

<213>Artificial Sequence

<400>8

Leu Tyr Ala Asp Asn Val Lys Gly Arg Phe Thr Leu Ser Gln Asp Pro

1 5 10 15

Pro Lys Arg Ala Leu Phe Leu Gln Met Asn Asn Leu Gln Pro Glu Asp

20 25 30

Ser Gly Met Tyr Tyr Cys

35

<210>9

<211>11

<212>PRT

<213>Artificial Sequence

<400>9

Arg Gly Arg Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210>10

<211>75

<212>DNA

<213>Artificial Sequence

<400>10

caggtccaac tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgaggctc 60

cgctgtataa tctcg 75

<210>11

<211>45

<212>DNA

<213>Artificial Sequence

<400>11

tggtttcgcc agggcccagg cgaggagcgc gagggggtcg cggcc 45

<210>12

<211>114

<212>DNA

<213>Artificial Sequence

<400>12

ctctacgctg acaacgtgaa gggccgattc accctctccc aagacccgcc caagcgcgct 60

ctatttctcc agatgaacaa ccttcaacct gaagattctg gcatgtacta ctgt 114

<210>13

<211>33

<212>DNA

<213>Artificial Sequence

<400>13

cggggccggg ggacccaggt caccgtctcc tca 33

<210>14

<211>10

<212>PRT

<213>Artificial Sequence

<400>14

Arg Tyr Gly Val Thr Leu Pro Tyr Met Ala

1 5 10

<210>15

<211>7

<212>PRT

<213>Artificial Sequence

<400>15

Thr Thr Leu Arg Gly Ser Thr

1 5

<210>16

<211>16

<212>PRT

<213>Artificial Sequence

<400>16

Ala Ala Gly Thr Ser Ala Arg Ser Leu Ser Pro Ser Asp Tyr Gly Tyr

1 5 10 15

<210>17

<211>30

<212>DNA

<213>Artificial Sequence

<400>17

cgatacggcg taactctgcc ctacatggcc 30

<210>18

<211>21

<212>DNA

<213>Artificial Sequence

<400>18

accaccctgc gtggaagcac t 21

<210>19

<211>48

<212>DNA

<213>Artificial Sequence

<400>19

gcggcaggca ctagcgcccg ttcactgagc cccagcgact atggctac 48

<210>20

<211>23

<212>DNA

<213>Artificial Sequence

<400>20

gtcctggctg ctcttctaca aag 23

<210>21

<211>23

<212>DNA

<213>Artificial Sequence

<400>21

ggtacgtgct gttgaactgt tcc 23

<210>22

<211>42

<212>DNA

<213>Artificial Sequence

<400>22

tcgcggccca gccggcccag gtccaactgc aggagtctgg gg 42

<210>23

<211>41

<212>DNA

<213>Artificial Sequence

<400>23

ataagaatgc ggccgctgag gagacggtga cctgggtccc c 41

<210>24

<211>696

<212>DNA

<213>Artificial Sequence

<400>24

gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 60

gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120

acccctgggg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420

gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 480

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540

cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660

tacacgcaga agagcctctc cctgtctccg ggtaaa 696

Claims

1. The nanometer antibody for resisting CD20 is characterized in that the amino acid sequence of the nanometer antibody is shown as SEQ ID No. 1.

2. A gene for coding the nanobody of claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID No. 2.

3. A recombinant nanobody against CD20, comprising the nanobody against CD20 of claim 1 and a human IgG Fc fragment.

4. The recombinant nanobody according to claim 3, wherein the amino acid sequence of the recombinant nanobody is shown in SEQ ID No. 3.

5. A gene encoding the recombinant nanobody of claim 4, comprising the gene of the nanobody of claim 2 and a human IgG Fc fragment gene; the nanometer antibody gene and the human IgG Fc segment gene are connected through a connecting segment, and the nucleotide sequence of the connecting segment is shown as SEQ ID No. 4.

6. The gene of the recombinant nanobody of claim 5, wherein the nucleotide sequence of the gene of the recombinant nanobody is shown in SEQ ID No. 5.

7. A recombinant vector expressing nanobody against CD20, comprising the gene of the recombinant nanobody of claim 5 or 6 and an initial vector.

8. The recombinant vector according to claim 7, wherein the initial vector is a pCZN1 plasmid vector.

9. A recombinant strain expressing a nanobody against CD20, comprising the recombinant vector of claim 7 or 8 and Escherichia coli Arctic Express.

10. The nanobody against CD20 of claim 1, the gene encoding nanobody against CD20 of claim 2, the recombinant nanobody of claim 3 or 4, the gene encoding nanobody of claim 5 or 6, the recombinant vector of claim 7 or 8, or the recombinant strain of claim 9 for preparing antitumor drugs or tumor diagnostic reagents.