CN103833851B - Be directed to single domain antibody and the application thereof of Apolipoprotein A1 - Google Patents

Abstract

The invention discloses a kind of VHH chain of single domain antibody of Apolipoprotein A1, comprise framework region FR and complementary determining region CDR, disclose aminoacid sequence and complementary determining region cdr amino acid sequence that framework region FR is selected from the FR of lower group, the invention also discloses two kinds of Apolipoprotein A1 single domain antibodies, also disclose two kinds of DNA moleculars, it is encoded the VHH chain of single domain antibody of Apolipoprotein A1 of the present invention or Apolipoprotein A1 single domain antibody of the present invention, also disclose a kind of host cell, it can the single domain antibody of expression of apolipoprotein A1, also disclose this Apolipoprotein A1 single domain antibody for detecting the purposes of Apolipoprotein A1.The single domain antibody gene order announced by the present invention and host cell, this single domain antibody can in intestinal bacteria high expression, be applied to the research and development of Apolipoprotein A1 detection reagent.

Description

Be directed to single domain antibody and the application thereof of Apolipoprotein A1

Technical field

The invention belongs to biomedicine or biological pharmacy technical field, relate to the single domain antibody being directed to Apolipoprotein A1.

Background technology

Lipophorin has important physiological function in lipoprotein metabolism.Lipophorin mainly divides A, B, C, D, E five classes, mainly in liver (part is at small intestine) synthesis, is the important component forming plasma lipoprotein.Apolipoprotein A1 level affects by factors such as sex, race, age, constitutional index, Ethanol intake amount, hormone, smokings.Result of study display Apolipoprotein A1 in conjunction with surrounding tissue free cholesterol, can promote the removing of cholesterol in arterial wall cell, accelerates the metabolism of liver inner cholesterol.So aPoA is identical with the clinical meaning of high-density lipoprotein (HDL), it and coronary heart disease, atherosclerosis are negative correlation, and namely it raises and is conducive to preventing coronary heart disease; And low hdl mass formed by blood stasis can appear in reduction, easily bring out arteriosclerosis and coronary heart disease.

For the mensuration of ApoA1, now extensively adopt Immunity transmission turbidity; This method is fast easy, be convenient to sample in enormous quantities and measure, and more this detection method realizes based on resisting of being obtained by Apolipoprotein A1 immune sheep.But this traditional Antibody stability is poor, sensitivity is low, production cost is high, and all factors all limit the detection for Apolipoprotein A1.Within 1993, Belgian scientist reports at Nature first: the antibody in camel blood, half is had not have light chain, and more allow people pleasantly surprised be, " heavy chain antibody " of these disappearance light chains can combine closely with the target such as antigen as normal antibody, to stick mutually unlike scFv in addition, be even gathered into block.This antibody only comprises a variable region of heavy chain and two conventional CH2 and CH3 districts, the more important thing is and to clone separately and the VHH district expressed has good structural stability and antigen-binding activity, molecular weight is 1/10 of common antibody, so VHH also claims Nanobody(single domain antibody); Meanwhile single domain antibody chemical property is also more flexible, good stability, solubility is high, expresses easily and utilizes microorganism to get final product a large amount of acquisitions, other molecules of easy coupling, therefore apply single domain antibody and have broad prospects for researching and developing Apolipoprotein A1 detection reagent.

Summary of the invention

Goal of the invention: technical problem to be solved by this invention is to provide the single domain antibody for Apolipoprotein A1, provides the application that the encoding sequence of this single domain antibody and this single domain antibody detect in preparation simultaneously.

Technical scheme: for achieving the above object, a first aspect of the present invention, provide a kind of single domain antibody of Apolipoprotein A1, comprise framework region FR and complementary determining region CDR, described framework region FR to be selected from the aminoacid sequence of the FR of lower group any one: the FR1 shown in SEQIDNO:1, FR2 shown in SEQIDNO:2, the FR3 shown in SEQIDNO:3, the FR4 shown in SEQIDNO:4; Or the FR1 shown in SEQIDNO:5, the FR2 shown in SEQIDNO:6, the FR3 shown in SEQIDNO:7, the FR4 shown in SEQIDNO:8;

Described complementary determining region CDR to be selected from the aminoacid sequence of the CDR of lower group any one:

CDR1 shown in SEQIDNO:9, the CDR2 shown in SEQIDNO:10, the CDR3 shown in SEQIDNO:11; Or the CDR1 shown in SEQIDNO:12, the CDR2 shown in SEQIDNO:13, the CDR3 shown in SEQIDNO:14;

Preferably, the VHH chain of the single domain antibody of described Apolipoprotein A1, it has the aminoacid sequence shown in SEQIDNO:15 and SEQIDNO:16.

Second aspect present invention, a kind of Apolipoprotein A1 single domain antibody, it is for the single domain antibody of Apolipoprotein A1 epi-position, comprises the VHH chain that two have aminoacid sequence shown in SEQIDNO:15 and SEQIDNO:16.

Third aspect present invention, provides a kind of DNA molecular, and its coding is selected from the protein of lower group: the VHH chain of the single domain antibody of Apolipoprotein A1 of the present invention, or Apolipoprotein A1 single domain antibody of the present invention.

Preferably, described DNA molecular, is characterized in that, it has the DNA sequence dna being selected from lower group:

SEQIDNO:17 and SEQIDNO:18

A fourth aspect of the present invention, provides a kind of expression vector, and it is containing the nucleotide sequence shown in SEQIDNO:17 and SEQIDNO:18.

A fifth aspect of the present invention, provides a kind of host cell, it is characterized in that, it contains described expression vector.

A sixth aspect of the present invention, provides Apolipoprotein A1 single domain antibody of the present invention for detecting the purposes of Apolipoprotein A1.

Beneficial effect: compared with prior art, advantage of the present invention is as follows: the Apolipoprotein A1 immunity Xinjiang dromedary that the present invention will extract in blood, this camel peripheral blood lymphocyte is utilized to establish the single domain antibody gene pool being directed to Apolipoprotein A1 subsequently, in test, Apolipoprotein A1 is coupled on enzyme plate, antigen in this format utilizes display technique of bacteriophage to screen the single domain antibody gene pool (camel heavy chain antibody phage display gene pool) of immunity, thus obtain for the specific single domain antibody gene of Apolipoprotein A1, this gene is gone in intestinal bacteria, thus establish can in the single domain antibody strain of E. coli.

Accompanying drawing explanation

Fig. 1 is the gene electrophorogram of single domain antibody; Wherein swimming lane 1 is DNA molecular standard, and swimming lane 2 is pcr amplification antibody heavy chain variable region fragments.

When Fig. 2 is the phage display library building single domain antibody, library storage capacity measures figure.

Fig. 3 is the bacterium colony PCR electrophorogram carried out for the specific single domain antibody library of constructed Apolipoprotein A1; Wherein swimming lane 1 is DNA molecular standard, and swimming lane 2-25 is picked clones random in constructed Apolipoprotein A1 single domain antibody library, and detected the insertion rate in library by bacterium colony PCR, calculation result shows library insertion rate to 100%.

Fig. 4 utilizes display technique of bacteriophage to sieve the sieve storehouse enrichment exploded view got in the process of Apolipoprotein A1.

Fig. 5 is the mode chart that enzyme-linked immunoassay method (ELISA) with phage screens the single positive colony of specificity; Wherein 1 is be coupled on enzyme plate by lipophorin, and 2 is single domain antibodies, and 3 is mouse-anti HA antibody, and 4 is the antibody of goat-anti-mouse alkaline phosphatase enzyme mark, and 5 is alkaline phosphatase nitrite ions.

Fig. 6 is the Apolipoprotein A1 single domain antibody of expressing, the electrophorogram of the SDS-PAGE after nickel post resin gel affinitive layer purification; Wherein swimming lane 1 is protein molecular standard, and swimming lane 2,3 is single domain antibodies that 250 mmole imidazole elution elute.

Fig. 7 is the mode chart that Apolipoprotein A1 single domain antibody detection specificity is analyzed.

Embodiment

The Apolipoprotein A1 immunity Xinjiang dromedary that first the present invention will extract in human blood, extracts this dromedary peripheral blood lymphocyte and constructs the special single domain heavy chain antibody library of Apolipoprotein A1 after 5 immunity.Apolipoprotein A1 is coupled on NUNC enzyme plate, the correct space structure of display protein matter, the epitope of Apolipoprotein A1 is come out, antigen in this format utilizes display technique of bacteriophage to screen the single domain antibody gene pool (camel heavy chain antibody phage display gene pool) of Apolipoprotein A1 immunity, and obtain can in the single domain antibody strain of E. coli.

Below in conjunction with specific embodiment, set forth the present invention further.

Embodiment 1: the structure being directed to the single domain antibody library of Apolipoprotein A1:

(1) by the Apolipoprotein A1 extracted in human blood (purchased from Hui Biao bio tech ltd, Nanjing), concentration for immunity Apolipoprotein A1 used is 500 μ g/mL, 0.5mg lipoprotein A1 mixes with freund's adjuvant equal-volume by each immunity, an immunity Xinjiang dromedary (Jurong Sheng Long livestock culturing factory), once in a week, immunity 5 times altogether, except first time uses freund's adjuvant (purchased from sigma) completely, residue all uses not formula incomplete adjuvant (purchased from sigma), the specific single domain antibody of immunologic process moderate stimulation B cell antigen expressed several times.After (2) 5 immunity terminate, extract camel peripheral blood lymphocyte 100mL and the RNA extracting total serum IgE and provide with reference to QIAGEN company extracts test kit.(3) according to Super-ScriptIIIFIRSTSTRANDSUPERMIX test kit specification sheets, the RNA reverse transcription of extraction is become cDNA.Variable region fragment with nested PCR amplification heavy chain antibody:

First round PCR:

Upstream primer GTCCTGGCTGCTCTTCTACAAGGC

Downstream GGTACGTGCTGTTGAACTGTTCC

Amplification heavy chain antibody guides the fragment between peptide and antibody CH2,54 DEG C of annealing, 25 circulations; The size that result shows this fragment as Fig. 1 is about 700bp, and namely DNA band from left to right respectively: first is the molecule Marker of DNA, and the second single domain antibody gene electrophoresis band is about 700bp.

Second takes turns PCR:

Template is made with first round PCR primer,

Upstream primer: GATGTGCAGCTGCAGGAGTCTGGRGGAGG

Downstream primer: GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT

Fragment (long segment and short-movie section) between amplification heavy chain antibody FR1 district and long and short hinge area, 60 DEG C of annealing, 17 circulations, reclaim object fragment, result such as Fig. 1 shows, the size of this fragment is about 500bp, and namely DNA band from left to right respectively: first is DNA molecular Marker, and the second single domain antibody gene electrophoresis band is about 500bp.(4) restrictive restriction endonuclease (purchased from NEB) PstI and NotI enzyme is used to cut 20 μ gpComb3 Vector for Phage Display (Biovector supply) and 10 μ gVHH and connect two fragments with T4DNA ligase enzyme (purchased from TaKaRa company).(5) connection product electricity is converted into electricity and turns Divine Land, competent cell TG1(Beijing red autumnal leaves Science and Technology Ltd.) in, build the single domain antibody phage display library of Apolipoprotein A1 and measure storage capacity, the size of storage capacity is 3.1 × 10 ⁸; Result such as Fig. 2 shows.Meanwhile, detecting primer by bacterium colony PCR uses second to take turns PCR primer, Tm55 DEG C.Fig. 3 shows bacterium colony PCR result.After library construction completes, for detecting the insertion rate in library, random selecting 24 clones are cooked bacterium colony PCR.Result display insertion rate reaches more than 90%.

Embodiment 2: the single domain antibody screening process for Apolipoprotein A1:

(1) be coated on NUNC enzyme plate by the Apolipoprotein A1 of the 100 μ g/mL be dissolved in PBS, 4 DEG C of placements are spent the night, and set up negative contrast simultaneously.Add 200 μ L1% milk in (2) second days two holes respectively, room temperature closes 2 hours.After (3) 2 hours, add 100 μ L phages (8 × 10 ¹¹tfu immunity camel single domain antibody phage display gene pool), at room temperature act on 1 hour.(4) 0.05% polysorbas20 is contained with in PBST(PBS) wash 5 times, to wash uncombined phage off.(5) dissociated down by the phage with Apolipoprotein A1 specific binding with triethylamine (100mM), and infect the e. coli tg1 being in logarithmic phase growth, produce also purified phage and be used for the screening of next round, identical screening process repeats 2 and takes turns.Result such as Fig. 4 shows: in the process of constantly screening, and positive clone will constantly by enrichment, thus reaches the object utilizing display technique of bacteriophage sieve to get Apolipoprotein A1 specific antibody in antibody library.

Embodiment 3: screen the single positive colony of specificity with the enzyme-linked immunoassay method (ELISA) of phage:

As shown in Figure 5, concrete detection is as follows for the principle modes figure of this experiment:

(1) contain the Tissue Culture Dish of phage after the screening of 3-4 wheel, select 96 single bacterium colonies and the TB substratum being inoculated in the penbritin containing 100 μ g/mL (containing 2.3g potassium primary phosphate in 1LTB substratum, 12.52g dipotassium hydrogen phosphate, 12g peptone, 24g yeast extract, 4mL glycerine) in, after growing to logarithmic phase, add the IPTG of final concentration 1mmol, 28 DEG C of overnight incubation.(2) utilize osmose process to obtain and slightly carry antibody, and antibody is transferred in antigen coated elisa plate, at room temperature place 1 hour.(3) wash away unconjugated antibody with PBST, add the anti-HA antibody of primary antibodie mouseanti-HAtagantibody(against murine, purchased from Beijing CoWin Bioscience Co., Ltd.), at room temperature place 1 hour.(4) wash away unconjugated antibody with PBST, add two anti-anti-mousealkalinephosphataseconjugate(goat-anti-mouse alkaline phosphatase enzyme mark antibody, purchased from the prompt Science and Technology Ltd. of Amy), at room temperature place 1 hour.(5) wash away unconjugated antibody with PBST, add alkaline phosphatase nitrite ion, on ELISA instrument, at 405nm wavelength, read absorption value.(6) when sample well OD value is greater than control wells OD value more than 3 times, positive colony hole is judged to.(7) bacterium in positive colony hole is turned shake containing 100 μ g/mL LB liquid in extract plasmid and to check order.

According to the gene order of sequence alignment program VectorNTI and each clone strain of IMGT analysis software, strain identical for CDR1, CDR2, CDR3 sequence is considered as same clone strain, and the different strain of its sequence is considered as different clone strain, finally has the antibody that 2 strains are different.The aminoacid sequence of the VHH chain of its antibody is respectively as shown in SEQIDNO:15, SEQIDNO:16.

Embodiment 4: single domain antibody is at Host Strains expression in escherichia coli, purifying:

(1) by sequencing analysis above obtain two kinds of single domain antibody subclones in the carrier PET32a of expressivity, and by recombinant plasmid transformed correct for order-checking qualification in expression type Host Strains DE3, it is coated on the plate of the LB solid medium containing 100 μ g/mL penbritins, and 37 DEG C are spent the night.(2) selecting single colony inoculation contains in the LB nutrient solution of penbritin at 15mL, 37 DEG C of shaking table overnight incubation.(3) inoculate the bacterial classification that spends the night of 1mL in 330mLLB substratum, 37 DEG C of shaking tables are cultivated, and when cultivation reaches 0.6-1 to OD value, add IPTG, 28 DEG C of shaking table overnight incubation.(4) second days, centrifugal receipts bacterium.(5) by bacterial cell disruption to obtain antibody crude extract.(6) through nickel post ion affinity chromatography antibody purification albumen, for obtaining highly purified antibody, adopt imidazole gradient elution method, lower concentration imidazole elution (50mmol) is for washing away assorted band, high density imidazole elution (250mmol, 500mmol) finally can prepare the albumen that purity reaches more than 90%.Band from left to right shown in Fig. 6 is respectively: first is the protein sample of standard protein molecule, the elution of the two or three 250mmol imidazoles; Result shows, and single domain antibody is after this purifying, and its purity can reach more than 95%.

Embodiment 5: Apolipoprotein A1 single domain antibody detection specificity is analyzed:

(1) by Apolipoprotein A1, prealbumin is coated on enzyme plate, does blank well contrast simultaneously, respectively wraps by two holes, lipophorin single domain antibody and control antibodies prealbumin single domain antibody are transferred to respectively in antigen coated elisa plate, at room temperature places 1 hour.(2) wash away unconjugated antibody with PBST, add the anti-HA antibody of primary antibodie mouseanti-HAtagantibody(against murine, purchased from Beijing CoWin Bioscience Co., Ltd.), at room temperature place 1 hour.(3) wash away unconjugated antibody with PBST, add two anti-anti-mousealkalinephosphataseconjugate(goat-anti-mouse alkaline phosphatase enzyme mark antibody, purchased from the prompt Science and Technology Ltd. of Amy), at room temperature place 1 hour.(4) wash away unconjugated antibody with PBST, add alkaline phosphatase nitrite ion, on ELISA instrument, at 405nm wavelength, read absorption value.Result display Apolipoprotein A1 single domain antibody can specific identification lipophorin.Mode chart is shown in Fig. 7, and result is as follows:

The above is only the preferred embodiment of the present invention; be noted that for those skilled in the art; under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

SEQUENCELISTING

<110> Southeast China University

The single domain antibody of a <120> Apolipoprotein A1, its encoding sequence and application

<130>

<160>18

<170>PatentInversion3.3

<210>1

<211>25

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<213> artificial sequence

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GlnValGlnLeuGlnGluSerGlyGlyGlySerValGlnAlaGlyGly

151015

SerLeuLysLeuSerCysAlaAlaSer

2025

<210>2

<211>17

<212>PRT

<213> artificial sequence

<400>2

MetGlyTrpPheArgGlnThrProGlyLysGluArgGluArgValAla

151015

Thr

<210>3

<211>38

<212>PRT

<213> artificial sequence

<400>3

IleTyrAlaAspSerValLysGlyArgPheThrIleSerLysAspAsn

151015

ValLysAsnThrLeuTyrLeuGlnMetAsnSerLeuLysProGluAsp

202530

ThrAlaMetTyrTyrCys

35

<210>4

<211>11

<212>PRT

<213> artificial sequence

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TrpGlyGlnGlyThrGlnValThrValSerSer

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<210>5

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<213> artificial sequence

<400>5

GlnValGlnLeuGlnGluSerGlyGlyGlySerValGlnAlaGlyGly

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SerLeuThrLeuSerCysAlaAlaSer

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<210>6

<211>17

<212>PRT

<213> artificial sequence

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MetGlyTrpPheArgGlnAlaProGlyLysGluArgGluArgValAla

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Ala

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TyrTyrAlaAspSerValLysGlyArgPheThrIleSerArgAspAsn

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GlyLysAsnThrLeuPheLeuGlnMetAsnSerLeuLysProGluAsp

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ThrAlaMetTyrTyrCys

35

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TrpGlyGlnGlyThrGlnValThrValSerSer

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<213> artificial sequence

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GlyTyrThrAspTyrThrTyrAsnThrSerCys

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<210>10

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<213> artificial sequence

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ValAsnLysValGlySerThr

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<213> artificial sequence

<400>11

AlaAlaArgGlySerTrpSerCysSerGlnPheTrpGlyAspPheGly

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Tyr

<210>12

<211>8

<212>PRT

<213> artificial sequence

<400>12

GlyTyrAlaAsnSerAsnThrCys

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<210>13

<211>8

<212>PRT

<213> artificial sequence

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IleSerGlyValGlyThrGlyThr

15

<210>14

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<213> artificial sequence

<400>14

AlaAlaAlaProGluGlyArgAlaTrpCysSerArgAspProSerGly

151015

TyrAsnTyr

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<212>PRT

<213> artificial sequence

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GlnValGlnLeuGlnGluSerGlyGlyGlySerValGlnAlaGlyGly

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SerLeuLysLeuSerCysAlaAlaSerGlyTyrThrAspTyrThrTyr

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AsnThrSerCysMetGlyTrpPheArgGlnThrProGlyLysGluArg

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GluArgValAlaThrValAsnLysValGlySerThrIleTyrAlaAsp

505560

SerValLysGlyArgPheThrIleSerLysAspAsnValLysAsnThr

65707580

LeuTyrLeuGlnMetAsnSerLeuLysProGluAspThrAlaMetTyr

859095

TyrCysAlaAlaArgGlySerTrpSerCysSerGlnPheTrpGlyAsp

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<212>PRT

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GlnValGlnLeuGlnGluSerGlyGlyGlySerValGlnAlaGlyGly

151015

SerLeuThrLeuSerCysAlaAlaSerGlyTyrAlaAsnSerAsnThr

202530

CysMetGlyTrpPheArgGlnAlaProGlyLysGluArgGluArgVal

354045

AlaAlaIleSerGlyValGlyThrGlyThrTyrTyrAlaAspSerVal

505560

LysGlyArgPheThrIleSerArgAspAsnGlyLysAsnThrLeuPhe

65707580

LeuGlnMetAsnSerLeuLysProGluAspThrAlaMetTyrTyrCys

859095

AlaAlaAlaProGluGlyArgAlaTrpCysSerArgAspProSerGly

100105110

TyrAsnTyrTrpGlyGlnGlyThrGlnValThrValSerSer

115120125

<210>17

<211>378

<212>DNA

<213> artificial sequence

<400>17

caggtgcagctgcaggagtctgggggaggctcggtgcaggctggagggtctctgaaactc60

tcctgtgcagcctctggatacaccgactacacctacaacaccagctgcatgggctggttc120

cgccagactccagggaaggagcgcgagagggttgcaactgttaataaggttggtagcaca180

atctacgcagactccgtgaagggccgattcaccatctccaaagacaacgtcaagaacact240

ctgtacctccaaatgaacagcctgaaacctgaggacactgccatgtactactgtgcggct300

cgtggtagttggtcctgttcccaattttggggcgactttggttactggggccagggaacc360

caggtcaccgtctcctca378

<210>18

<211>378

<212>DNA

<213> artificial sequence

<400>18

caggtgcagctgcaggagtctgggggaggctcggtgcaggctggagggtctctgacactc60

tcctgtgcagcctctggatacgccaatagtaatacctgtatgggctggttccgccaggct120

ccagggaaggagcgcgaacgggtcgctgctatttctggtgttggtactggcacatactat180

gccgactccgtgaagggccgattcaccatctcccgagacaacggcaagaacacactgttt240

ctacaaatgaacagcctgaaacctgaggacactgccatgtactactgtgcggcagcgcca300

gagggccgtgcgtggtgcagccgcgatccatcgggatataactattggggccaggggacc360

caggtcaccgtctcctca378

Claims (4)

1. an Apolipoprotein A1 single domain antibody, is characterized in that, its VHH chain-ordering is SEQIDNO:15.

2. a DNA molecular, is characterized in that, its coding is selected from the protein of lower group: according to claim 1 year fat

The VHH chain of the single domain antibody of albumin A 1.

3. DNA molecular according to claim 2, is characterized in that described DNA sequence dna is SEQIDNO:17.

4. Apolipoprotein A1 single domain antibody according to claim 1 is for the preparation of the application detected in Apolipoprotein A1 reagent.