CN109824776B - Single-domain heavy chain antibody Nb73 for vibrio fluvialis - Google Patents

Abstract

The invention provides a single-domain heavy chain antibody and polypeptide for vibrio fluvialis, which has the protein or polypeptide with the shown amino acid sequence and can be used in the fields of immunodetection, antigen enrichment and purification and the like. The amino acid sequence provided by the invention can be used as a precursor, and can be transformed by random or site-directed mutagenesis technology to obtain mutants with better properties (affinity, specificity, stability and the like) for developing proteins or polypeptides for further use in medicine, industry and agriculture.

Description

Single-domain heavy chain antibody Nb73 for vibrio fluvialis

Technical Field

The present invention relates to single domain heavy chain antibody technology, especially single domain heavy chain antibody or polypeptide for vibrio fluvialis, and gene engineering antibody technology.

Technical Field

Single domain antibodies refer to genetically engineered antibodies composed of the variable regions of common antibodies (VH or VL). A single domain heavy chain antibody (also called a nanobody, a VHH antibody) refers to a genetically engineered antibody consisting of only heavy chain antibody (heavy-chain antibody) Variable regions (Variable regions), wherein the heavy chain antibody (heavy-chain antibody) is an antibody that naturally lacks a light chain in animals such as camels and sharks. The single-domain heavy chain antibody has the characteristics of small molecular weight, good permeability and the like, and is widely applied to the fields of basic research, medical diagnosis and detection, antibody drug development and the like.

Vibrio Fluvialis (VF) is a common pathogenic bacterium in the ocean and is the pathogenic Vibrio next to Vibrio cholerae and Vibrio parahaemolyticus in the genus Vibrio. It is a gram-negative facultative anaerobic bacterium, can cause enteritis of human vibrio fluvialis, and has clinical symptoms very similar to cholera-like diarrhea. River vibrio was first isolated in 1975 from feces of a diarrhea patient in baclin, and was also isolated in the same year from feces of diarrhea patients in bangladesh and marine and aquatic products in the uk. Vibrio fluvialis can cause various diseases such as bacteremia, intestinal infection, acute diarrhea and the like for patients with immune function perfection or AIDS patients, and the diseases caused by pathogenic bacteria also include suppurative cholangitis and peritonitis. One study by cuba showed that vibrio fluvialis is one of the major pathogenic bacteria in different parenteral samples.

At present, monoclonal antibodies aiming at vibrio fluvialis are reported in public, but compared with single-domain heavy chain antibodies, the existing monoclonal antibodies aiming at vibrio fluvialis have the defects of relatively high production cost, complex preparation process and the like. The single-domain heavy chain antibody has the characteristics of small molecular weight, easy expression and the like, and shows wide application prospect.

Disclosure of Invention

The invention aims to provide a single-domain heavy chain antibody (comprising protein or polypeptide containing all or part of functional regions of the single-domain heavy chain antibody) for vibrio fluvialis and an amino acid sequence thereof, which can be used for preparing reagents and tools for detecting the vibrio fluvialis.

The invention provides a single-domain heavy chain antibody Nb73 for Vibrio fluvialis, which has the amino acid sequence shown in SEQ ID NO: 8.

The amino acid sequence is divided into IMGT numbering and domains by standardized amino acid numbering, as shown in FIG. 1.

The single-domain heavy-chain antibody provided by the invention respectively comprises four Framework Regions (FR) and three complementary-determining regions (CDR). SEQ ID NO: in 8, the framework regions (FR1-FR4) are respectively selected from 1-34, 43-59, 68-105, 116-126 amino acid sequences, and the complementarity determining regions (CDR1-CDR3) are respectively selected from 35-42, 60-67 and 106-115 amino acid sequences, wherein FR1 sequence is shown in SEQ ID NO:1, the FR2 sequence is shown as SEQ ID NO:2, the FR3 sequence is shown as SEQ ID NO:3, the FR4 sequence is shown as SEQ ID NO:4, the CDR1 sequence is shown in SEQ ID NO:5, the CDR2 sequence is shown in SEQ ID NO:6, the CDR3 sequence is shown in SEQ ID NO:7 (see sequence table or figure 1). The complementarity determining region is mainly responsible for antigen recognition, and the framework region is relatively stable in structure and mainly plays a role in supporting and maintaining the protein structure.

The invention also relates to a nucleotide for coding the single-domain heavy chain antibody Nb73 amino acid sequence aiming at Vibrio fluvialis, and the sequence is shown as SEQ ID NO: shown at 9.

The invention also provides a preparation method of the single-domain heavy chain antibody Nb73 for Vibrio fluvialis, which comprises the following steps:

(1) designing a specific primer, and amplifying from a cDNA library derived from alpaca by a PCR (polymerase chain reaction) technology;

(2) cloning the gene segment of the obtained PCR product to an expression vector pET25b-CTB, transforming the sufficient plasmid into a host cell, inducing and expressing a nano antibody Nb73 aiming at vibrio fluvialis, and purifying the nano antibody.

The invention also provides a nucleic acid molecule encoding the polypeptide of SEQ ID NO:8 or SEQ ID NO: 9, the specific sequence of the nucleic acid molecule can be obtained at any time by genetic code.

The invention also provides a nucleic acid molecule encoding the polypeptide of SEQ ID NO:8 or SEQ ID NO: 9, the specific sequence of the nucleic acid molecule can be obtained at any time by genetic code.

The nucleotide sequence or at least a part of the sequence provided by the invention can be expressed by a suitable expression system to obtain the corresponding protein or polypeptide. These expression systems include bacterial, yeast, filamentous fungi, animal cells, insect cells, plant cells, or cell-free expression systems.

The invention also provides a composition comprising the nucleic acid sequence and optionally a vector. The composition can be used in various applications such as diagnosis. Due to the degeneracy of the genetic code, the nucleic acid sequence can vary depending on the intended use.

The invention also provides a host cell comprising the protein or expression vector.

The invention also provides a method for enriching river vibrio, which is characterized by comprising the protein or the polypeptide. Based on the binding capacity of the protein or polypeptide and the vibrio fluvialis provided by the invention, the established enrichment method of the vibrio fluvialis is established, and the established enrichment method can be combined with immunofluorescence quantitative PCR (polymerase chain reaction) to be used for rapidly detecting the vibrio fluvialis.

The amino acid sequence provided by the invention can be used as a precursor, and can be transformed by random or site-directed mutagenesis technology to obtain mutants with better properties (water solubility, stability, affinity, specificity and the like) for developing further proteins or polypeptides for medicine, industry and agriculture.

Some of the terms described herein have the following meanings:

domain (b): the basic structural unit of the tertiary structure of a protein generally has a certain function.

IMGT number: a standardized numbering scheme for The amino acid sequence of antibodies in The IMGT database (The International ImmunoGeneTiCs Datbase). Specific numbering methods can be described in the literature (Ehrenman, F., Q. Kaas, et al (2010). "IMGT/3D structure-DB and IMGT/DomainGapAlign: a Database and a tool for immunoglobulin or antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic Acids Res38(Database subsystem) D301-307.Lefranc, M.P., C.Pommie, et al (2003), "GT unequique number for immunoglobulin and T cell receptor domains and Igsuperfamity V-domains" Dev 27 (1): 55-77).

The invention has the beneficial effects that:

the single-domain heavy chain antibody prepared by the invention has the excellent properties of small molecular weight, easy expression and the like, and meanwhile, compared with the preparation of the traditional monoclonal antibody, the single-domain heavy chain antibody has the advantages of relatively low production cost, relatively simple preparation process and wide application prospect.

Drawings

FIG. 1 amino acid numbering and Structure and schematic

FIG. 2 indirect ELISA for Nb73 binding activity and specificity

FIG. 3 is an expression and identification diagram of a Nb73 single-domain heavy chain antibody, lane M on the right is a protein molecular weight standard, lane 1 is a Rosetta-pET25b-CTB-Nb73 recombinant bacterium lysate induced without IPTG, lane 2 is a Rosetta-pET25b-CTB-Nb73 recombinant bacterium lysate induced by IPTG, lane 3 is a supernatant after ultrasonication, and lane 4 is precipitated after ultrasonication.

Detailed Description

The present invention is further illustrated below by the preparation, analysis and use of single domain heavy chain antibodies (polypeptides), and these specific examples should not be construed in any way as limiting the scope of the invention.

Application example 1

Panning and identification of anti-vibrio fluvialis single-domain heavy chain antibody

And panning the single-domain heavy-chain antibody aiming at the vibrio fluvialis from the camel-source natural antibody phage display library by adopting a solid-phase panning method. Extracting vibrio fluvialis lipopolysaccharide by hot phenol water extraction, and panning by taking the vibrio fluvialis lipopolysaccharide as a target to obtain a single-domain heavy chain antibody. The specific panning of single domain heavy chain antibodies comprises the steps of:

diluting the extracted vibrio fluvialis lipopolysaccharide to 100 mu g/mL by using sterile phosphate buffer solution (PBS, pH7.4), adding the diluted vibrio fluvialis lipopolysaccharide into a hole of an enzyme-labeled plate, coating the vibrio fluvialis overnight at 4 ℃, performing first round of panning and coating 3 holes, performing subsequent panning and coating 1 hole, sucking out the coating solution, and washing the plate for 3 times by using the PBS. In each round of panning, 1% gelatin was used as blocking agent, blocked at 37 ℃ for 2h, washed with PBS 3 times, and 100. mu.L phage antibody library (approximately 2X 10 contained)¹¹PFU), incubation for 1.5h at 37 ℃, first round of panning washed with PBS only, followed by 3 washes (1 round-by-round increase) with PBST (1% Tween-20) and 3 washes (2 round-by-round increase) with PBS for each subsequent round of panning. Bound phage were eluted with 100. mu.L glycine-HCl (Gly-HCl, pH2.2) eluent, the eluate was neutralized with 20. mu.L Tris-HCl (pH9.1), 10. mu.L was taken for titer determination, the remaining eluate was amplified, and the amplified product was used for the next round of panning.

After three rounds of panning, randomly selected monoclonals are rescued by using an auxiliary phage KM13 to respectively obtain phage particles displaying antibody variable regions, and specific single positive clones are screened by using a phage enzyme-linked immunosorbent assay (phase-ELISA). And when the OD value of the sample hole is more than 3 times larger than that of the control hole, judging the sample hole to be a positive cloning hole.

TABLE 1 Indirect phase-ELISA sample application Table

And (3) sending the ELISA positive clone to a sequencing company for sequence determination to obtain the DNA sequence of the insert of the positive clone of the anti-vibrio fluvialis single-domain heavy chain antibody phage, wherein the Nb73 nucleotide sequence is shown as SEQ ID NO: 9, and the Nb73 amino acid sequence is shown as SEQ ID NO:8, wherein the Nb73 framework regions (FR1-FR4) are respectively selected from 1-34, 43-59, 68-105 and 116-126 amino acid sequences, and the complementarity determining regions (CDR1-CDR3) are respectively selected from 35-42, 60-67 and 106-115 amino acid sequences, wherein the FR1 sequence is shown as SEQ ID NO:1, the FR2 sequence is shown as SEQ ID NO:2, the FR3 sequence is shown as SEQ ID NO:3, the FR4 sequence is shown as SEQ ID NO:4, the CDR1 sequence is shown in SEQ ID NO:5, the CDR2 sequence is shown in SEQ ID NO:6, the CDR3 sequence is shown in SEQ ID NO: shown at 7. See in particular the table below.

Application example 2

Expression of the anti-Vibrio fluvialis single-domain heavy-chain antibody in E.coli.

Obtaining of a DNA fragment encoding Vibrio fluvialis: designing specific primer, and amplifying the positive cloned nanometer antibody sequence. The gene fragment of the obtained PCR product is cloned to an expression vector pET25b-CTB, and the sufficient plasmid is transformed into a host cell, and after sequencing verification, the host cell is named as pET25-CTB-Nb 73.

The recombinant plasmid pET25-CTB-Nb73 is transformed into Escherichia coli Rosetta, and a single colony is picked for induction expression. Inoculating the single colony into a test tube containing 5mL of liquid LB culture medium, and carrying out shaking culture at 37 ℃ and 220rpm/min overnight; the cells were inoculated in 150mL of LB liquid medium at 1%, shake-cultured at 37 ℃ and 220rpm/min until the OD was about 0.5, and then IPTG was added to the cells at a final concentration of 0.1mM, and the cells were induced overnight at 20 ℃ and 180 rpm/min.

The bacterial liquid is centrifuged at 6000rpm/min for 5 minutes to obtain thalli, the thalli is washed for 3 times by using sterile PBS, 15mL of sterile PBS is used for carrying out heavy suspension on the thalli, the thalli is ultrasonically crushed on ice under 200W for 2s and 3s at intervals for 40 cycles, cell lysate is centrifuged at 4 ℃, the centrifugation condition is 8000rpm/min and the time is 10min, and supernatant is taken for SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis, and the result is shown in figure 3.

The expression quantity of the target protein (single-domain heavy chain antibody) can be further improved by optimizing induction expression conditions (such as host bacteria, expression vectors, induction time, induction temperature, IPTG concentration and the like), and a way is provided for preparing a large amount of single-domain heavy chain antibodies of the vibrio fluvialis.

Application example 3

Mutation and affinity improvement of anti-vibrio fluvialis single-domain heavy chain antibody

Designing a specific primer, using a recombinant plasmid pET25-CTB-Nb73 as a template, carrying out random mutation by using error-prone PCR (polymerase chain reaction), carrying out 4 groups in total, wherein the system of each group is different, the concentration of dNTP is adjusted, and each tube is a 100 mu L system, which is shown in Table 1. The 4 groups of error-prone PCR products were pooled and recovered with a DNA fragment recovery kit. Connecting part of recovered product to vector pHEN-1, electrically transforming Escherichia coli TG1 competent cells, coating 5 ampicillin-containing plates, adding 1mL 2 XYT culture medium to each plate, eluting bacteria with sterilized spoons, mixing bacterial suspensions of all plates, and mixing. Transferring 5mL of the bacterial suspension into a 50mL centrifuge tube, adjusting OD600 to 0.3 by using a culture medium containing antibiotics and glucose, and carrying out shake culture at 37 ℃ for 1 h; according to the formula of phase: cell 20: 1, adding helper phage, and carrying out shake culture at 37 ℃ for 2 h; centrifuging, removing supernatant, gently suspending cells by using 50mL of culture medium, and performing shaking culture at 30 ℃ for 8 h; centrifuging at 10000g for 20min, transferring the supernatant to a 10mL centrifuge tube, adding 1/5 volume of PEG/NaCl, mixing well, and precipitating at 4 ℃ overnight; centrifuging, discarding the supernatant, adding a PBS solution, and resuspending the phage to obtain an Nb73 random mutation phage library; the panning of the affinity-enhanced mutein was performed according to the protocol in application example 1, with lipopolysaccharide as the coating antigen, the OD of the ELISA reaction was higher than the phage-displayed Nb73, which was a positive clone, which was sent to sequencing companies for sequence determination to obtain the mutated DNA sequence, which was translated into a protein sequence for comparison with the Nb73 sequence.

TABLE 1 error-prone PCR reaction System

Sequence listing

<110> university of Nanchang

<120> Single Domain heavy chain antibody Nb73 against Vibrio fluvialis

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 34

<212> PRT

<213> alpaca (Lama pacos)

<400> 1

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

1 5 10 15

Gly Gly Ser Leu Val Gln Ala Gly Asp Ser Leu Arg Leu Ser Cys Ala

20 25 30

Ala Ser

<210> 2

<211> 17

<212> PRT

<213> alpaca (Lama pacos)

<400> 2

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

1 5 10 15

Ala

<210> 3

<211> 38

<212> PRT

<213> alpaca (Lama pacos)

<400> 3

Ala Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp

20 25 30

Thr Ala Asp Tyr Phe Cys

35

<210> 4

<211> 11

<212> PRT

<213> alpaca (Lama pacos)

<400> 4

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 5

<211> 8

<212> PRT

<213> alpaca (Lama pacos)

<400> 5

Gly Arg Thr Phe Ser Leu Tyr Ala

1 5

<210> 6

<211> 8

<212> PRT

<213> alpaca (Lama pacos)

<400> 6

Val Ser Arg Phe Gly Gly Arg Thr

1 5

<210> 7

<211> 10

<212> PRT

<213> alpaca (Lama pacos)

<400> 7

Ala Ser Thr His Thr Gly Ile Trp Lys Tyr

1 5 10

<210> 8

<211> 126

<212> PRT

<213> alpaca (Lama pacos)

<400> 8

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

1 5 10 15

Gly Gly Ser Leu Val Gln Ala Gly Asp Ser Leu Arg Leu Ser Cys Ala

20 25 30

Ala Ser Gly Arg Thr Phe Ser Leu Tyr Ala Met Gly Trp Phe Arg Gln

35 40 45

Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Val Ser Arg Phe Gly

50 55 60

Gly Arg Thr Ala Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser

65 70 75 80

Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys

85 90 95

Pro Glu Asp Thr Ala Asp Tyr Phe Cys Ala Ser Thr His Thr Gly Ile

100 105 110

Trp Lys Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 9

<211> 398

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ccatggccca tggcccagtt gcagctcgtg gagtcagggg gaggattggt gcaggctggg 60

gactctctga cactctcctg tgcagcctct ggacgcacct tcagcttgta tgccatgggc 120

tggtttcacc aggctccagg gaaggagcgc gagtttgtag cagctgttag caggtttggt 180

ggacgcacag cctatgcaga ctccgcgaag ggccgattca ccatctcccg agacaacgcc 240

aagaacatgg tgtatctgca aatgaacagc ctgaaacctg aggacacggc cgtttatttt 300

tgtgcatcaa cgcacaccgg gatatggaag tactggggcc acgggaccca ggtcaccgtc 360

tcctcagaac ccaagacacc aaaaccacaa gcggccgc 398

Claims (4)

1. A nano-antibody Nb73 for Vibrio fluvialis, wherein the nano-antibody Nb73 for Vibrio fluvialis comprises a framework region and a complementarity determining region, wherein the framework region comprises the following 4 amino acid sequences: FR1 shown in SEQ ID NO. 1, FR2 shown in SEQ ID NO. 2, FR3 shown in SEQ ID NO. 3 and FR4 shown in SEQ ID NO. 4, and the complementarity determining region comprises the following 3 amino acid sequences: CDR1 shown in SEQ ID NO. 5, CDR2 shown in SEQ ID NO. 6 and CDR3 shown in SEQ ID NO. 7.

2. The nano antibody Nb73 for the vibrio fluvialis is characterized in that the nano antibody Nb73 for the vibrio fluvialis has an amino acid sequence shown in SEQ ID NO. 8.

3. A nucleotide molecule encoding the amino acid sequence of claim 2.

4. The method for preparing the nanobody Nb73 for Vibrio fluvialis of any one of claims 1 to 2, comprising the steps of:

(1) designing a specific primer, and amplifying from a cDNA library derived from alpaca by a PCR (polymerase chain reaction) technology;

(2) cloning the gene segment of the obtained PCR product to an expression vector pET25b-CTB, transforming sufficient plasmids into host cells, and inducing and expressing a nano antibody Nb73 aiming at vibrio fluvialis.

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