CN105037546A - AFP nanometer antibody A65 based on AFP antigen - Google Patents

Abstract

The invention belongs to the technical field of biology, and relates to a camel source immunity nanometer antibody (single-domain heavy-chain antibody, VHH) specifically bound with AFP and application of the antibody. The amino acid sequence of the antibody is SEQ ID NO.:1. The invention further relates to nucleotides for encoding amino acids. The AFP nanometer antibody can be used as the antibody to be applied to immunological detection of AFP not for disease diagnosis and treatment. Compared with a Scfv antibody and a Fab antibody, the camel source immunity VHH has the higher specificity and affinity, more stable in structure, resistant to acid, base and high temperature, high in detection flexibility and the like, so that the immunity detection stability is greatly improved, and meanwhile the environment resistance is greatly improved.

Description

Based on the AFP nano antibody A65 of AFP antigen

Technical field

The invention belongs to biological technical field, be specifically related to specific binding alpha-fetoprotein (Alpha-FetalProtein, AFP) nano antibody (single domain heavy chain antibody, Variabledomainofheavychainofheavychainantibody, VHH) and application thereof.

Background technology

Alpha-fetoprotein is a kind of glycoprotein, produced by tire liver or adult human liver, be common in the blood of pregnant woman and baby, but content is little in health adult's blood, being mainly used in the early diagnosis of primary hepatocarcinoma, curative effect evaluation and prognostic evaluation at present, is the important serologic marker thing of primary hepatocarcinoma (PHC).Alpha-fetoprotein in kinds of tumors, especially has excessive performance in hepatocellular carcinoma (HCC) as a kind of mark.Along with the further further investigation to AFP gene structure and function, effect more and more important in its early diagnosis at HCC and biologic treatment.In other enterogastric tube tumours as rising in various degree also appears in carcinoma of the pancreas or the patient such as lung cancer and liver cirrhosis.When liver cell generation canceration, can produce this AFP, and its content in serum sharply can increase along with sb.'s illness took a turn for the worse, alpha-fetoprotein has just become a specific clinical index of diagnosing primary liver cancer.

The ordinary method of clinical detection patient AFP has radioimmunoassays (RIA), enzyme immunoassay (EIA) and colloidal gold method, reaction is fast, highly sensitive, accuracy is high because it has for current chemiluminescence immunoassay detection technique (CLIA), marker is stable, the feature such as "dead" or toxicity hazard and high specificity to human body, is generally acknowledge best micro quantitative determination detection method so far.In the middle of detect delay experiment, a large amount of antibody is needed to be applied to research that is novel, efficient detection method.Nano antibody has possessed special antigen binding capacity and high-affinity, shows peculiar property and huge applications potential in the field of study.And antibody and antigen standard are very expensive, and some toxin has danger.

Summary of the invention

The present invention utilizes display technique of bacteriophage, from the single domain heavy chain antibody storehouse of hunchbacked source immunity screening can with single domain heavy chain antibody (VHH) phage clone of target molecule (AFP antigen) specific binding, be applied to immune analysis detection field using VHH as AFP antibody.The method is screened the VHH obtained and is had and AFP immune response characteristic, and specificity is good, high-affinity, can be applicable to the immunology detection of AFP.The present invention is with AFP standard substance for target molecule, and by target molecule solid-phase coating on enzyme plate, affine elutriation is carried out in the single domain heavy chain antibody storehouse of dropping into the immunity of hunchbacked source, obtains a kind of anti-AFP nano antibody, has the aminoacid sequence shown in SEQIDNO.:1.The IMGT numbering of its aminoacid sequence and structural domain divide as shown in Figure 1.

AFP nano antibody (VHH) mentioned by the present invention comprises four framework regions (Frameworkregion, FR) and three complementary determining regions (Complementarity-determiningregion, CDR).Wherein, framework region (FR1-FR4) is selected from SEQIDNO.:2, SEQIDNO.:4, SEQIDNO.:6 and SEQIDNO.:8 respectively, and complementary determining region (CDR1-CDR3) is selected from SEQIDNO.:3, SEQIDNO.:5 and SEQIDNO.:7 respectively.Framework region structure is relatively conservative, mainly plays a part Protein requirement structure; Complementary determining region structure is relatively diversified, the identification of primary responsibility antibody.

The invention provides a kind of protein or polypeptide, comprise SEQIDNO.:2, one or two and above aminoacid sequence in SEQIDNO.:4, SEQIDNO.:6 and SEQIDNO.:8, and at least have 90% homology with the aminoacid sequence of one of them.

The invention provides a kind of protein or polypeptide, comprise SEQIDNO.:3, one or two and above aminoacid sequence in SEQIDNO.:5 and SEQIDNO.:7, and at least have 80% homology with the aminoacid sequence of one of them.

The invention still further relates to the Nucleotide of coding AFP antibody amino acids sequence, its sequence is SEQIDNO.:9.

The nano antibody combined based on AFP that the present invention mentions is prepared in a large number by Phage amplification or the recombinant expressed mode of genetically engineered.Phage amplification refers to phage displaying being had AFP nano antibody (VHH), the mode increased by biology, and amount reproduction produces the bacteriophage particles shown and have the nano antibody (VHH) of AFP.The recombinant expressed mode of genetically engineered refers to the gene of coding nano antibody, by being cloned into expression vector, carries out a large amount of preparations of AFP antibody with the form of protein expression.

The invention still further relates to the application of described AFP nano antibody in non-diseases diagnoses and treatment object immunology detection is analyzed.The type of immunology detection comprises the immune analysis type of detection based on Ag-Ab specific reaction such as MBP enzyme linked immuno-adsorbent assay, colloidal gold immunochromatographimethod, immunodotting hybridization.

AFP nano antibody of the present invention is when applying, and the displaying that can be obtained by Phage amplification is had the bacteriophage particles of AFP nano antibody to be directly used in analyzing and testing and AFP nano antibody is carried out immunology detection analysis with the form of albumen after prokaryotic organism or eukaryote are expressed.

The invention still further relates to the application of AFP nano antibody in non-diseases diagnoses and treatment object immunology detection is analyzed, be specially the application of AFP nano antibody in the reagent of preparation absorption AFP.Such as, AFP nano antibody of the present invention can be made immune affinity column, absorption captures AFP, enrichment, for further studying.

Aminoacid sequence of the present invention as precursor, can be transformed by random or site-directed mutagenesis technique, can obtain character (avidity, specificity and stability etc.) better mutant.

In the present invention some terms of describing there is following implication:

Homology: the similarity degree describing two or more aminoacid sequence, percent homology between first aminoacid sequence and second aminoacid sequence can be multiplied by [100%] to calculate divided by [in first aminoacid sequence amino acid sum] by [quantity of amino-acid residue identical with the amino-acid residue of corresponding position in the second aminoacid sequence in the first aminoacid sequence] again, and certain the amino acid whose disappearance wherein in the second aminoacid sequence, insertion, replacement or interpolation (compared with the first amino acid) are considered to there is difference.In addition, percent homology also can utilize the known Computing program (as: NCBIBlast) for sequence alignment to obtain.

Structural domain: the fundamental structural unit of tertiary protein structure, has certain function usually.

IMGT numbers: the one in IMGT database (TheInternationalImMunoGeneTicsDatabase) is standardized antibody amino acids sequence method for numbering serial.Concrete method for numbering serial can reference (Ehrenmann, F., Q.Kaas, etal. (2010). " IMGT/3Dstructure-DBandIMGT/DomainGapAlign:adatabaseandat oolforimmunoglobulinsorantibodies, Tcellreceptors, MHC, IgSFandMhcSF. " NucleicAcidsRes38 (Databaseissue): D301-307.Lefranc, M.P., C.Pommie, etal. (2003). " IMGTuniquenumberingforimmunoglobulinandTcellreceptorvari abledomainsandIgsuperfamilyV-likedomains. " DevCompImmunol27 (1): 55-77.) in description.

Codon (codon): also known as triplet code (tripletcode), refers to correspond to certain amino acid whose nucleotide triplet.In the process of translating, determine that this seed amino acid inserts the position of polypeptide chain in growth.

The invention has the beneficial effects as follows: nano antibody molecular structure is unique, and molecular weight is little, stable easily expression, and specificity is good, can replace traditional monoclonal antibody, avoids loaded down with trivial details preparation process, and cost-saving.AFP antibody prepared by the present invention can be widely used in AFP fundamental research, medical diagnosis and detection, antibody drug exploitation etc.Nano antibody method of the present invention has general applicability, can be used for screening and the preparation of other small-molecule substance analogue antigens, has very high using value.

Accompanying drawing explanation

Fig. 1 is amino acid number and the structural domain schematic diagram of AFP nano antibody (VHH).

Fig. 2 is AFP double crush syndrome typical curve and CAE, NSE, CA-125 cross reaction curve.The lowest detectable limit that phage clone detects AFP is respectively 0.39ng/mL, and linear relationship is respectively 0.9973, and linearity range is 1-200ng/mL.

Embodiment

The structure in immunity single domain heavy chain antibody storehouse, embodiment 1. hunchbacked source

1) immunity of alpaca:

Choose a healthy adult alpaca, take back intracutaneous, the mode of subcutaneous multi-point injection carries out immunity, initial immunity, not formula Freund's complete adjuvant, immunogenic dose 1mg; 28th day second immunisation, not formula Freund's incomplete adjuvant, immunogenic dose 0.5mg, exempts from, not formula Freund's incomplete adjuvant, immunogenic dose 0.25mg for the 49th day three; Within 70th day four, exempt from, not formula Freund's incomplete adjuvant, immunogenic dose 0.25mg.After third time booster immunization the 7th day, gather alpaca peripheral blood, for building phage display library.

2) the leukocytic separation in hunchbacked source: add lymphocyte separation medium in centrifuge tube, then add blood sample, the centrifugal 20min of 700g; In the centrifuge tube that the white corpuscle to that careful absorption middle layer suspends is new, add the PBS of 1/2 volume, the centrifugal 15min of 800g; Abandon supernatant, wash the white corpuscle on lower centrifugal tube wall with PBS, the centrifugal 10min of 850g; Abandon supernatant, the resuspended white corpuscle of 300 μ LPBS also counts; Add lysate (RNAiso) with volume ratio 1:10 to save backup.

3) Total RNAs extraction: the chloroform adding 1/5 volume in above-mentioned lysate, concuss 15s makes it fully emulsified, and room temperature leaves standstill 5min; 4 DEG C of centrifugal 15min of 12000g, get supernatant and are transferred in another fresh centrifuge tube; Add isopyknic Virahol, fully after mixing, room temperature leaves standstill 10min; 4 DEG C of centrifugal 10min of 12000g, abandon supernatant, slowly add 75% ethanol 1mL, careful washing centrifuge tube tube wall; 4 DEG C, after the centrifugal 5min of 12000g, discard ethanol; Drying at room temperature precipitation 5min, adds appropriate RNase-free water dissolution precipitation, in-80 DEG C of preservations after RNA precipitation is dissolved completely.

4) synthesis of cDNA: the primer of reverse transcription, dNTPMixture and template ribonucleic acid are mixed, 65 DEG C of 5min, rapid ice bath.Specifically be formulated as follows:

Then in above-mentioned Microtube, following inverse transcription reaction liquid is configured:

PCR instrument carries out reverse transcription reaction by following condition: 42 DEG C of 30min, 50 DEG C of 30min, 70 DEG C of 10min.PCR primer saves backup in-20 DEG C.

5) antibody variable gene amplification: cDNA reverse transcription obtained carries out PCR reaction as template.

First round PCR:

PCR reaction conditions: 94 DEG C of 4min; 98 DEG C of 10s, 55 DEG C of 15s, 72 DEG C of 45s, 30cycles; 72 DEG C of 7min.

Adopt test kit to reclaim pcr amplification product, suitably after dilution, take turns the template of PCR as second.

Second takes turns PCR:

PCR reaction conditions: 94 DEG C of 4min; 98 DEG C of 10s, 50 DEG C of 15s, 72 DEG C of 45s, 5cycles; 98 DEG C of 10s, 68 DEG C of 40s, 30cycles; 72 DEG C of 7min.

6) library construction:

The double digestion of A.VHH encoding gene and carrier

SfiI and NotI enzyme is adopted to carry out double digestion to VHH encoding gene and pHEN1 carrier respectively.

B. enzyme cuts the connection of after product

By carrier pHEN1 and VHH fragment mixing (mol ratio 1: 5), connect 12h in 16 DEG C.Successively add the 3MCH of 5 μ L (1/10 amount) ₃the cold dehydrated alcohol of COONa (pH5.2) and 125 μ L (2.5 times amount) ,-20 DEG C of centrifugal recovery precipitations of standing 1h, 12000rpm, by the cold ethanol purge precipitation of 70%, drying at room temperature, is dissolved in 10 μ L deionized waters.

C. electricity transforms

Get 5 μ L connection products to add in 80 μ L competent cell E.coliTG1, fully mix, place 1min on ice.Proceed to electroporated in the electric shock cup of 0.1cm (voltage is 1.8kV), add 900 μ LSOC substratum immediately in electric shock cup, 37 DEG C of 180rpm cultivate 1h.Bacterium liquid is coated SOB-AG flat board, is inverted overnight incubation for 37 DEG C.

7) rescue in library: to inoculate more than the cell of 10 times of storage capacities in 100mL2 × YT/amp/2% glucose, be cultured to OD600 and reach 0.5; Add helper phage (20:l infection multiplicity), 37 DEG C, after leaving standstill 15min, 220rpm cultivates 45min; 4 DEG C, the centrifugal 10min of 3000g; Abandon supernatant, add the resuspended precipitation of 2 × YT/amp/kan substratum that 100mL is fresh, 30 DEG C of overnight incubation; 4 DEG C of centrifugal 10min of 3000g, get supernatant; Add the PEG-NaCl solution of 1/5 volume, 4 DEG C of standing 2h; 4 DEG C of centrifugal 20min of 10000rpm, abandon supernatant, precipitation 1mLPBS is resuspended; Get 10 μ L and measure storage capacity, all the other add final concentration 50% glycerine ,-80 DEG C of preservations.

The affine elutriation of embodiment 2.AFP nano antibody and qualification thereof

1) the affine elutriation of AFP nano antibody: first round elutriation, dilutes anti-AFP antigen with PBS (pH7.4), and with final concentration 100 μ g/mL coated elisa plate, 4 DEG C are spent the night.After PBST (10mMPBS, 0.5%Tween-20 (v/v)) washing 3 times, close 1 hour for 3%BSA-PBS37 DEG C that adds 300 μ L.Abandon confining liquid after 1 hour, wash 5 times with PBST, every hole adds 100 immunity single domain heavy chain antibody storehouse, μ L camel source (titres about 1.0 × 10 ¹¹cfu), 1 hour is hatched for 37 DEG C.The unconjugated phage of sucking-off, washs 10 times with PBST, adds 0.2MGlycine-HCl (pH2.2) wash-out of 100 μ L, gentle shake 15min, uses 15 μ L1MHCl (pH9.1) neutralizations immediately.Get 10 μ L wash-out bacteriophages and measure titre, remaining E.coliTG1 bacterial strain growing to logarithmic phase for infecting 20mL increases.With the phage after the amplification of PEG/NaCl precipitation, and measure the titre of phage.

Same procedure carries out the elutriation of second, third and fourth round.The AFP antigen concentration of bag quilt is respectively 80 μ g/mL, 50 μ g/mL and 20 μ g/mL.

2) qualification of positive phage clones: random picking 75 clone the flat board measuring phage titre after third round and fourth round elutriation, carry out the amplification of phage, Immunofluorescent antibody detection method (IndirectEnzymeLinkedimmunoasorbentassay, I-ELISA) is adopted to carry out the qualification of positive phage clones.Concrete grammar is: first, and with PBS (pH7.4) by AFP antigen diluent to 2 μ g/mL, 37 DEG C of bags are by 2h.Within second day, with after PBST (10mMPBS, 0.05%Tween-20 (v/v)) washing 3 times, add the 3%BSA-OVA of 300 μ L, 37 DEG C of closed 1h; Abandon confining liquid, after PBST washs 4 times, add 100 μ L Phage amplification liquid (2.0 × 10 ¹¹cfu), using naive phage peptide storehouse as negative control, hatch 1 hour for 37 DEG C; Add the HRP/anti-M13100 μ L that 1:5000 doubly dilutes, hatch 1 hour for 37 DEG C; Add 100 μ LTMB substrate solutions, lucifuge colour developing 5min; Add 50 μ L stop buffer (2MH ₂sO ₄) termination reaction; The absorption value at 450nm place is measured by microplate reader (ThermoScientificMultiskanFC).Choose OD ₄₅₀the phage clone being greater than negative control 2 times is positive colony.

3) phage display VHH is for AFP specificity identification: CAE/CA-125/NSE cross reaction.

AAnti-AFP monoclonal antibody 2.0 μ L/mL100 μ L, 37 DEG C of bags are by 2h;

BPBS washes plate 3 times, and the skimmed milk of 300 μ L3% is closed, 37 DEG C of closed 1h;

C add respectively CAE, CA-125, NSE antigen 0,1,2,4,8,16,32,64,128,256ng/mL in every hole, hatch 30min for 37 DEG C;

D adds 2 × 10 ⁹cfu phage, hatches 0.5h for 37 DEG C;

EPBST washes plate 6 times, adds HRP/anti-M13 (working concentration 1/5000), 100 μ L/ holes, 37 DEG C of 0.5h;

F 0.05%PBST washes plate 6 times, adds nitrite ion 100 μ L/ hole, 37 DEG C of reaction 5min;

G adds the 2M sulfuric acid termination reaction in 50 μ L/ holes, measures the absorbancy that 450nm goes out;

The order-checking of embodiment 3.AFP nano antibody encoding gene and the determination of aminoacid sequence thereof

Positive phage clones after the qualification of double antibodies sandwich enzyme-linked immunoassay method is carried out DNA sequencing, can aminoacid sequence be obtained according to DNA sequencing result and password sublist.

A large amount of preparations of embodiment 4.AFP nano antibody phage

(1) be prepared in the mode of Phage amplification

The nano antibody phage of displaying being added to 20mL inoculation has in the culture of E.coliTG1,37 DEG C of 220rpm shaking culture 6h.And adding M13K07 phage, 37 DEG C of shaking tables infect 1h.Proceeded to by culture in another centrifuge tube, 4 DEG C of 3000rpm, centrifugal 10min, removes supernatant.By the resuspended precipitation of 1mL substratum, resuspended bacterium liquid is added in 20mL substratum, the ammonia benzyl of 1/1000 and kantlex, 30 DEG C of overnight incubation.Go that bacterium liquid 4 DEG C of 7500rpm are centrifugal to be proceeded to the top of supernatant 80% in a fresh centrifuge tube, add the PEG/NaCl of 1/6 volume, after 4 DEG C of standing 120min, 4 DEG C of centrifugal 10min of 10000rpm, abandon supernatant; Add a small amount of PBS again and clean phage.4 DEG C of centrifugal 10min of 10000rpm, abandon supernatant, add 1mLPBS and carry out resuspended, be Phage amplification liquid.

(2) be prepared with the form of protein expression

The external source code VHH gene of NcoI and NotI enzyme and expression vector (pET-25b) is adopted to carry out double digestion respectively, VHH encoding gene is cloned into expression vector pET-25b, after PCR and digestion verification, recombinant expression vector is proceeded to intestinal bacteria Rosetta (DE3).Choose a single colony inoculation in 5mLLB liquid nutrient medium from transformation plate, 37 DEG C, 220r/min shaking culture is spent the night, overnight culture is inoculated in the LB/Amp of 50mL by 1% inoculum size (v/v), in 2% dextrose culture-medium, 37 DEG C, 220r/min shaking culture; When culture cell concentration OD600 reaches 0.5, in culture, add the IPTG of 0.1mM, 30 DEG C, 220r/min shaking culture 8-12h; By culture in 4 DEG C, 8000rpm, centrifugal 20min collects bacterial sediment.Re-suspended cell is in the PBS solution of 5mL precooling, and after ultrasonication 10min, 8000rpm is centrifugal, and 20min gets supernatant, and supernatant is carried out affinitive layer purification, obtains AFP nano antibody.

Embodiment 5.AFP nano antibody heat-resistant experiment

In order to determine the thermostability of expressing protein nano antibody, expressing protein of the present invention is placed in 45 DEG C of environment, respectively 0,1,2,4,8,16,32h measures the immunocompetence of albumen.Protein immunization activity before 45 DEG C hatch is set to 100%,

1) wrap by Anti-AFPMcAb in enzyme plate, 2 μ g/mL, 100 μ L/ holes, 4 DEG C of bags are spent the night;

2) 0.05%PBST washes plate three times, skimmed milk 37 DEG C of closed 1h of 3%;

3) add the AFP antigen of 0.5 μ g/mL, 100 μ L/ holes, hatch 0.5h for 37 DEG C;

4) add the target protein solution of dilution 10,25,50,100,200,400,800,1600 times, hatch 0.5h for 37 DEG C;

5) the anti-His bis-of HRP enzyme mark adding 1:2000 working concentration resists, and hatches 0.5h for 37 DEG C;

6) TMB nitrite ion is added, lucifuge colour developing 5min;

7) add the H2SO4 termination reaction of 50 μ L/ hole 2M, measure 450nm place ultraviolet absorptivity.

Result shows after VHH of the present invention is placed at 45 DEG C of water-baths, and its immunodetection performance (OD value) has no difference, demonstrates good resistance to heat energy.

Embodiment 6.AFP nano antibody detects the application of serum in ELISA

In order to verify that phage clone success is for detecting AFP antigen in serum, the random serum extracting 10 parts of Healthy Peoples from local Disease Control and Prevention Center (Qingshanhu District Disease Control and Prevention Center), dilute 4 times, respectively to adding 1 in serum, 5,10,25,50,100ng/mLAFP antigen (AFP medical science detection threshold is 400ng/mL), remove three parts of serum at random, calculate the recovery of standard addition of AFP.

Claims (6)

1., based on the AFP nano antibody of AFP antigen, it is characterized in that the aminoacid sequence had shown in SEQIDNO.:1.

2. the Nucleotide of AFP nano antibody aminoacid sequence described in coding claim 1, its sequence is SEQIDNO.:9.

3. the preparation method of AFP nano antibody as claimed in claim 1, to it is characterized in that in the single domain heavy chain antibody storehouse of hunchbacked source immunity screening can with the single domain heavy chain antibody of target molecule specific binding, and to be prepared in a large number by Phage amplification or the recombinant expressed mode of genetically engineered; Described Phage amplification refers to will show the phage having AFP single domain heavy chain, the mode increased by biology, and amount reproduction produces the bacteriophage particles shown and have AFP nano antibody; The recombinant expressed mode of described genetically engineered refers to the gene of nano antibody, by being cloned into expression vector, carries out a large amount of preparations of nano antibody with the form of protein expression.

4. the application of AFP nano antibody described in claim 1 in non-diseases diagnoses and treatment object immunology detection is analyzed.

5. apply as claimed in claim 4, it is characterized in that AFP nano antibody substitutes the application of AFP monoclonal antibody in non-diseases diagnoses and treatment object immunology detection is analyzed.

6. AFP nano antibody described in claim 1 combines the application in absorption AFP reagent in preparation.