CN106188283B - Nano antibody of avian influenza A H7N2 and application thereof - Google Patents

Abstract

The invention discloses a nano antibody aiming at an influenza A virus H7N2 epitope and a gene sequence for encoding the nano antibody, and also discloses a host cell capable of expressing the influenza A virus H7N2 nano antibody. The invention obtains the nano antibody of the influenza A virus H7N2 and the coding sequence thereof for the first time, the nano antibody can be efficiently expressed in escherichia coli, specifically identifies the influenza A virus H7N2, has high detection sensitivity, shows good linear relation when being used for detecting the influenza A virus H7N2, and has application value for diagnosing the influenza A virus H7N 2.

Description

Nano antibody of avian influenza A H7N2 and application thereof

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to a nano antibody aiming at avian influenza A H7N2, a coding sequence thereof and application thereof.

Background

The genome of the Influenza a Virus (AIV) is eight segmented negative strand RNA, belongs to the orthomyxoviridae, encodes 12-13 proteins in total, can be divided into different serotypes according to two surface proteins, namely HA (hemagglutinin) and NA (neuraminidase), and is separated from poultry into 81 Avian Influenza viruses with different serum subtypes, and new HA and NA are continuously discovered, so that the polymorphism of the Avian Influenza viruses brings great difficulty to prevention and treatment of Avian Influenza. According to the pathogenicity, the avian influenza viruses can be divided into three types, namely High Pathogenicity (HPAIV), Low Pathogenicity (LPAIV) and non-pathogenicity, and the non-pathogenicity avian influenza can not cause obvious symptoms; the low pathogenicity avian influenza can cause mild respiratory symptoms of poultry, which leads to the reduction of food intake, the reduction of egg production and even sporadic death; the highly pathogenic avian influenza has the most serious harm, is fast to spread, has high morbidity and mortality, is classified as a type A animal epidemic disease by the world animal health organization, and is also classified as a type A animal epidemic disease in China. As with all subtypes of AIVs, subtype H7 AIVs have also been directly transmitted by birds to humans, but have relatively low interest in humans infected with the H7 subtype of avian influenza virus. H7N2 was prevalent in the live bird market in the northeast U.S. for decades, with flu-like symptoms occurring in 80 professional populations in turkeys in large scale in 2002, and with a reoccurrence of avian influenza virus H7N2 in 2003. 3 reports that people infected with the novel reassortant H7N9 virus are found in Shanghai and Anhui of China at 31.3.3.2013, wherein 2 people die and 1 person seriously reminds people of the threat of the H7 subtype avian influenza virus to public health to be not ignored.

Effective vaccines provide the best protection against avian influenza, but current vaccine technology limits production to meet the demand for large amounts of effective vaccines in a short period of time. For the HPAIV H7N2 virus, since different strains of H7N2 virus prevail in the european asia, the selection of a population-specific vaccine strain is a great challenge for the design and selection of H7N2 virus vaccines. While the different antigenic H7 lineages are pathogenic to humans, human isolates of north american lineages H7N2, H7N3 and european lineages H7N2, H7N3 isolated from the population in recent years have been selected for suitable vaccine strains, but human H7 vaccines against both lineages are still under development.

An antiviral drug is one of the very effective methods for treating influenza virus infection, but H7 has a drug-resistant strain, in vitro experiments show that H7 which is outbreak in the Netherlands in 2003 is resistant to amantadine which is an M2 ion channel inhibitor, and H7 of the same North America lineage has also been reported to be resistant to amantadine, so that the monitoring of the drug-resistant strain is also important as the development of a novel drug.

The nano antibody has wide application prospect in the fields of basic research, new drug development, disease diagnosis and treatment and food science, and becomes an important research hotspot. The nano antibody aiming at the influenza A H7N2 virus epitope is obtained by using an antibody technology, so that the nano antibody has wide prospects in clinical diagnosis and targeted therapy. At present, no nano antibody aiming at the influenza A H7N2 virus epitope is reported.

Disclosure of Invention

The invention aims to provide a nano antibody aiming at influenza A virus H7N 2. The invention provides a nanobody (also called a heavy chain antibody, VHH) aiming at influenza A virus H7N2, which comprises a framework region FR and a complementarity determining region CDR, wherein the framework region FR is selected from the amino acid sequences of the FRs in the following groups: SEQ ID NO: FR1 as shown in 1, SEQ ID NO: FR2 as shown in 2, SEQ ID NO: FR3 as shown in SEQ ID NO: FR4 shown in fig. 4.

The CDR is selected from the amino acid sequence of CDR of the following group: SEQ ID NO: 5, CDR1 shown in seq id NO: 6, CDR2 shown in SEQ ID NO: CDR3 shown in FIG. 7.

Preferably, the VHH chain of the H7N2 nanobody has the amino acid sequence of SEQ ID NO: 8.

The nano antibody (Nb) is a novel antibody discovered in recent years, the antigen binding site of which is composed of only the variable domain of the heavy chain of the HCAbs (VHHs), also called VHH antibody, is the smallest antibody molecule fragment with complete function that can be obtained at present, and has the characteristics of small relative molecular mass, strong stability, good solubility, good antigen binding performance, high expression level, low immunogenicity and the like, and is wider in application compared with the conventional antibody.

Unlike conventional antibody molecules consisting of 4 polypeptide chains of heavy and light chains, a heavy chain antibody consists of two homologous heavy chain peptide segments, the heavy chain molecule comprises only the variable region, the CH2 region and the CH3 region, has a relative molecular mass of 90kDa, and is much smaller than the 150kDa of conventional IgG antibody molecules.

In a second aspect of the invention, there is provided a heavy chain antibody VHH directed against the surface antigen of influenza a virus H7N2, comprising a heavy chain having the sequence of SEQ ID NO: 8, or a VHH chain of the amino acid sequence set forth in 8.

In a third aspect of the invention, there is provided a DNA molecule encoding a protein selected from the group consisting of: the heavy chain antibody VHH of the influenza A virus H7N2 is disclosed.

Preferably, said DNA molecule is characterized in that it has a DNA sequence selected from the group consisting of: SEQ ID NO: 9.

in a fourth aspect of the invention, there is provided an expression vector comprising SEQ ID NO: 9, or a nucleotide sequence shown in the specification. Preferably, it is pMECS, pET32a or pET28 a.

In the fifth aspect of the invention, a host cell is provided, which contains the expression vector, the nano-antibody of the influenza A virus H7N 2. Preferably, it is WK6, TG1 or BL 21.

In a sixth aspect of the invention, a kit for detecting influenza a virus H7N2 is provided, which comprises the nanobody modified or unmodified, preferably, the modification is HRP, FITC or biotin coupling.

In a seventh aspect of the invention, the invention provides the use of the heavy chain antibody VHH of influenza a virus H7N2 in the preparation of a formulation for diagnosing influenza a H7N 2. The early diagnosis of the avian influenza has great significance for controlling epidemic situation, and the nano antibody of the influenza A virus H7N2 plays a role in the diagnosis of the avian influenza, thereby having positive influence on the control of the epidemic situation.

Compared with the prior art, the invention has the following advantages: the invention uses inactivated influenza A H7N2 virus to immunize bactrian camels, and then uses the camel peripheral blood lymphocytes to establish a heavy chain antibody VHH phage library aiming at the influenza A H7N 2. In the subsequent test, the inactivated influenza A H7N2 virus is coupled on an enzyme label plate, and a phage display technology is utilized to screen an immune nano antibody phage library, so that a specific nano antibody gene aiming at the influenza A H7N2 virus is obtained, and the gene is transferred into escherichia coli, thereby establishing a nano antibody strain capable of being efficiently expressed in the escherichia coli. After obtaining the nano antibody, biotinylating the nano antibody, coupling the biotinylated nano antibody with a streptavidin microporous plate, capturing an antigen, developing the other nano antibody which recognizes the other epitope of the antigen and is coupled with HRP to obtain the influenza A H7N2 with the detection sensitivity of 2.97 ng/ml, the detection linear range of 0-100 ng/ml, the linear equation of y = 0.0105x +0.3229 and the correlation coefficient of 0.9952, and the method is used for detecting the influenza A H7N2 to show good linear relation, thereby laying the foundation for applying the nano antibody of the avian influenza H7N2 to clinical diagnosis and developing a diagnostic kit.

Drawings

FIG. 1 is a gene electrophoresis diagram of a nano-antibody, 1, DNA molecular standard (bp); 2. the size of the nano antibody DNA fragment is about 500 bp;

FIG. 2 shows the results of detecting the insertion rate of the constructed single-domain antibody library, and the DNA bands from left to right gel wells are: the first path is DNA molecular standard, the rest pore paths are PCR products of monoclonal detection insert randomly picked from the established single domain antibody library, and the insertion rate of the library is 100% through detection;

FIG. 3 is an electrophoresis diagram of SDS-PAGE of H7N2 nano antibody obtained after nickel column purification, wherein the expressed protein corresponds to the protein shown in SEQ ID NO: 9, or a nucleotide sequence shown in the specification.

FIG. 4 is a schematic diagram of the detection of influenza A H7N2 using a double antibody sandwich method.

FIG. 5 is the sensitivity and detection linearity for influenza A H7N2 using the double antibody sandwich method.

Detailed Description

The invention discloses a nano antibody of avian influenza A H7N2 and application thereof, and a person skilled in the art can realize the nano antibody by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the techniques of the invention can be implemented and practiced with modification, or with appropriate modification, and combinations of the methods and applications described herein without departing from the spirit, scope, and spirit of the invention.

The invention firstly immunizes an inactivated influenza A H7N2 virus with bactrian camel, extracts the bactrian camel peripheral blood lymphocytes after 7 times of immunization and constructs a specific single-domain heavy chain antibody library of the influenza A H7N2 virus. The inactivated influenza A H7N2 virus is coupled on an enzyme label plate, the epitope on the surface of the inactivated influenza A H7N2 virus is exposed, and a nano antibody gene of the influenza A H7N2 virus immunity is screened from a 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 a heavy chain antibody VHH library directed against influenza A Virus H7N2

(1) Immunizing a bactrian camel with inactivated influenza A H7N2 virus, and mixing the inactivated influenza A H7N2 virus with Freund's adjuvant according to the ratio of 1: 1 volume mix, weekly, 7 total immunizations, first with complete Freund's adjuvant, and the remaining sixFreund's incomplete adjuvant was used next time to stimulate B cells to express the heavy chain antibody VHH specific to influenza A virus H7N2 during immunization. (2) After 7 times of immunization, 100 ml of camel peripheral blood lymphocytes are extracted and total RNA is extracted. (3) The extracted RNA was reverse transcribed into cDNA and the VHH strands were amplified using nested PCR, the size of the fragment being approximately 500bp as shown in FIG. 1. (4) Using restriction endonucleasesPstI andNoti (from NEB) 20. mu.g of pComb3 phage display vector (from Biovector) and 10. mu.g of VHH were digested and the two fragments were ligated. (5) Electrotransformation of the ligation products into electrotransformation competent cells TG1 (fromBeijing Shenzhou Red leaf technologies Co Ltd) In (1), a heavy chain antibody VHH phage display library of influenza A virus H7N2 was constructed and the size of the library was determined to be 3X 10⁸(ii) a Meanwhile, the insertion rate of the constructed library was measured by colony PCR, and the insertion rate was about 95%, and FIG. 2 shows the colony PCR results. After the library was constructed, we randomly selected 24 clones for colony PCR to check the library insertion rate. The results show that: the library insertion rate has reached 100%.

Example 2: heavy chain antibody VHH screening process against influenza A virus H7N2

(1) The solution was dissolved in 100 mM NaHCO, pH 8.2₃200 mu g of inactivated influenza A H7N2 virus is coupled on NUNC enzyme label plate, is placed at 4 ℃ overnight, and is simultaneously provided with negative control coated NaHCO₃. (2) On the next day, 100. mu.g of 1% skim milk was added to each well and sealed for 2 hours at room temperature. (3) After 2 hours, 100. mu.l of phage was added and allowed to act at room temperature for 1 hour. (4) The phage that failed to bind antigen were washed away with PBST (0.05% Tween 20 in PBS) 5 times. (5) Phages specifically binding to influenza a virus H1N1 were eluted with 100 mM triethanolamine and infected with e.coli TG1 in log phase growth, 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, so that the aim of screening the specific antibody of the influenza A virus H7N2 in the antibody library by using a phage display technology is fulfilled.

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 (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) containing 100. mu.g/mL ampicillin, grown to a logarithmic phase, followed by addition of IPTG at a final concentration of 1 mM and incubation at 28 ℃ overnight. (2) Crude antibodies were obtained by osmotic pressure and transferred to antigen-coated ELISA plates and left for 1 hour at room temperature. (3) Unbound antibody was washed away with PBST, and anti-mouse anti-HA 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 off with PBST, alkaline phosphatase developing solution was added, and absorbance was read at 405nm using a microplate reader. (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 medium containing 100. mu.g/mL ampicillin to extract plasmids and sequenced.

Analyzing the gene sequence of each clone strain according to the Vector NTI of the sequence alignment software, regarding the strains with the same CDR1, CDR2 and CDR3 sequences as the same clone strain, and regarding the strains with different sequences as different clone strains, and finally obtaining the amino acid sequence SEQ ID NO: 8, the amino acid sequences of the framework region and the complementarity determining region of the nano antibody are as follows:

FR1：QVQLQESGGGSVQAGGSLRLSCAES

FR2：WFRQAPGKEREGVA

FR3：RFTISRDNAKNTVYLQMSSLQPEDTAMYYCAA

FR4：WGQGTQVTVSS

CDR1：GYIGSNRPM

CDR2：GISTIGGYTYYADSVKG

CDR3：CYGLRADMAGDYSSLCTY。

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

(1) plasmids of different clones obtained by the previous sequencing analysis were electrically transformed into E.coli WK6, spread on LB plates containing ampicillin and glucose, and cultured overnight at 37 ℃; (2) selecting a single colony, inoculating the single colony in 5mL LB culture solution containing ampicillin, and carrying out shake culture at 37 ℃ overnight; (3) inoculating 1 mL of overnight strain into 330 mL of LTB culture solution, carrying out shake culture at 37 ℃, adding 1 mmol of IPTG when OD value reaches 0.6-1.0, and carrying out shake culture at 28 ℃ overnight; (4) centrifuging and collecting bacteria; (5) obtaining antibody crude extract by using an osmosis method; (6) the protein with purity of more than 90% can be prepared by nickel column ion affinity chromatography, and the result is shown in figure 3.

Example 5: the method for coupling the nano antibody with the HRP comprises the following steps:

(1) mixing 200 μ L5 mg/ml HPR with 100 μ L0.1 mol/L NaIO4, standing at 4 deg.C for 15-30 min, adding 200 μ L2.5% ethylene glycol, and standing at room temperature for 30-60 min; (2) 1 mL of 1mg/mL antibody to be labeled was added, and 1 mol/L of a solution of pH 9.5 CB (Na)₂CO₃、NaHCO₃Solution) to a pH of 9.0, mixing, and standing overnight at 4 deg.C; (3) add 20. mu.l of 5mg/mL NaHB to the overnight antibody and leave it at 4 ℃ for 3 hours; (4) and (4) replacing the nano antibody coupled with the HRP into the PBS solution.

Example 6: method for biotinylation of nano antibody

(1) Subcloning a nano antibody VHH fragment onto a pBAD vector, then co-transferring the constructed plasmid pBAD and the plasmid BirA into escherichia coli WK6, coating the escherichia coli WK6 on an LB culture plate containing ampicillin, chloramphenicol and glucose, and culturing at 37 ℃ overnight; (2) selecting a single colony to be inoculated in 5mL LB culture solution containing ampicillin and chloramphenicol, and shaking-culturing at 37 ℃ overnight; (3) inoculating 1 mL of overnight strain into 330 mL of TB culture solution containing ampicillin and chloramphenicol, shake culturing at 37 deg.C until OD reaches 0.4-0.5, adding 330 μ l of 50 mM D-biotion solution, and slowly shaking at 37 deg.C for 30 min; (4) adding 1 mM IPTG into the mixture, and performing shake culture at 28 ℃ overnight; (4) centrifuging and collecting bacteria; (5) obtaining antibody crude extract by using an osmosis method; (6) the biotin-coupled nanobody was purified using streptavidin magnetic beads.

Example 7: influenza A H7N2 (see figure 4 for schematic) was detected by double antibody sandwich method

(1) Coating 100 mu l of 1 mu g/mL biotinylated H7N2 nano antibody on a microplate coupled with streptavidin, and performing shake incubation for 1-2 hours at room temperature by a horizontal shaking table; (2) PBST washing 5 times, each hole is added with 100 u l 5% BSA for blocking, room temperature shake incubation for 1 hours; (3) PBST was washed 5 times, 100. mu.l of influenza virus H7N2 ( antigen concentration 0, 1 ng/mL, 5 ng/mL, 10 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500ng/mL, 1000 ng/mL, 2000 ng/mL, 5000 ng/mL) was added to each well in a gradient, and incubated at room temperature for 1-3 hours with shaking (or overnight incubation at 4 ℃ with standing); (4) PBST is washed for 5 times, 100 mul of nano antibody (1 mug/mL) which can be combined with another epitope of influenza virus H7N2 and is coupled with HRP is added into each hole, and the mixture is incubated for 1 hour at room temperature with shaking; (5) PBST was washed 5 times and TMD developed, and the results are shown in FIG. 5, showing a good linearity.

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> Beijing Kogyo clinical diagnostic reagent, Inc.;

<120> nano antibody of avian influenza A H7N2 and application thereof

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gactccgtga agggccgatt caccatctcc cgagacaacg ccaagaacac ggtgtatctg 240

caaatgagca gcctgcaacc cgaggacact gccatgtact actgtgcagc atgctacggg 300

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Claims (9)

1. A nanobody of influenza A virus H7N2, comprising a framework region FR and a complementarity determining region CDR, the framework region FR being an amino acid sequence of FRs of the group consisting of:

SEQ ID NO: FR1 as shown in 1, SEQ ID NO: FR2 as shown in 2, SEQ ID NO: FR3 as shown in SEQ ID NO: FR4 shown in FIG. 4;

the CDR is the amino acid sequence of the CDR of the following group:

SEQ ID NO: 5, CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: CDR3 shown in FIG. 7.

2. A DNA molecule encoding the nanobody of influenza a virus H7N2 of claim 1.

3. The DNA molecule of claim 2, characterized in that it has the DNA sequence of SEQ ID NO: shown at 9.

4. Comprises the amino acid sequence of SEQ ID NO: 9.

5. The expression vector according to claim 4, characterized in that it is pMECS, pET32a or pET28 a.

6. A host cell expressing the nanobody of influenza A virus H7N2 of claim 1.

7. The host cell of claim 6, which is WK6, TG1 or BL 21.

8. A kit for detecting influenza a virus H7N2, comprising the nanobody of claim 1 modified or unmodified, wherein the modification is HRP, FITC or biotin coupling.

9. Use of nanobody according to claim 1 for the preparation of a formulation for the diagnosis of influenza a H7N 2.

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