CN104628852A - Cry1Ab toxin resisting nanometer antibody, coding sequence and application thereof - Google Patents

Abstract

The invention discloses a Cry1Ab toxin resisting nanometer antibody, a coding sequence thereof, a host cell capable of expressing the nanometer antibody and application of the nanometer antibody. The nanometer antibody is obtained by panning in a phage antibody library, the antibody is characterized in that the specificity of the nanometer antibody is decided by a CDR region sequence in a heavy chain variable region sequence. The nanometer antibody can be efficiently expressing in the escherichia coli and has large utilization value.

Description

Anti-Cry1Ab toxin nano antibody, its encoding sequence and application

Technical field

The invention belongs to Food science or technical field of food biotechnology, relate to a kind of be directed to Cry1Ab toxin nano antibody, its encoding sequence and application.

Background technology

The insecticidal crystal protein that Bt toxin is produced by bacillus thuringiensis (Bacillus thuringiensi is called for short Bt) as a class, is widely known by the people in recent years.Because it has efficient, special toxic action to some insects of lepidopteran and Coleoptera, current Bt toxin has been widely used in the control of insect as microbial pesticide or has been expressed in transgenic plant.Wherein Cry toxin is most widely used Bt albumen, such as Cry1Ab and Cry1Ac.But since first commercialization Bt product comes out decades ago, people further pay close attention to the security of transgenic plant and limitation and worry.Have report to point out, some nutritive property of transgenic plant is existing to be changed; Other studies proof, and Cry toxin can cause hematotoxicity in Mice Body.The application prospect that Cry toxin is wide and the potential risk to human health thereof and the long-term effect to ecotope will cause the public give more sustained attention.

At present, the detection major part of transgenic plant and products thereof is based on nucleic acid and protein level.Whether nucleic acid level detects containing the exotic toxin gene inserted in genetic material, as PCR, quantitative PCR etc.; Protein level utilizes the protein of insertion exotic toxin genetic expression or its function to detect, as Enzyme-Linked Immunospot, Western blot method, test strip method etc.In transgenic plant detection, adopting enzyme linked immunosorbent detection technology (ELISA) to detect Cry toxin is a kind of technology with clear superiority of generally acknowledging at present, it have quick, accurate, highly sensitive, the advantages such as sample can be detected in a large number.But, obtain there is strong avidity, the Cry toxin antibody (mono-clonal, polyclone, single-chain antibody) of high specific is but a difficult job.

Single domain antibody, also nano antibody (VHH) is, the peptide section be made up of about 120 amino acid, it is minimum natural antibody fragment known at present, some site that on energy identifying purpose antigen, conventional antibody can not identify, and there is the features such as thermostability is high, expression amount is high, avidity is strong, specificity is good, with short production cycle.Its molecular structure only containing a variable region of heavy chain (VHH) and two conventional CH2 and CH3 districts, but compared to engineered single chain antibody fragments (scFv), which overcomes inter-adhesive, to be even gathered into block shortcoming.The more important thing is, to clone separately and the VHH structure expressed possesses the structural stability suitable with original weight chain antibody and antigen-binding activity, be known at present can the least unit of combining target antigen.Along with the development of biotechnology, nano antibody has started to be widely used in the fields such as detection, analysis, treatment, compensate for the weak point of other antibody species.Such as based on the research etc. of nano antibody technology for detection human body prealbumin, apolipoprotein B.Therefore applying nano antibody technique research and development Cry1Ab detection reagent has broad prospects.

Summary of the invention

Goal of the invention: technical problem to be solved by this invention is to provide a kind of nano antibody for Cry1Ab epi-position, provides the encoding sequence of this nano antibody simultaneously.

Technical scheme: for achieving the above object, a first aspect of the present invention, provide the VHH chain of the nano antibody of a kind of Cry1Ab, comprise framework region FR and complementary determining region CDR, described framework region FR is selected from the aminoacid sequence of the FR of lower group: the FR1 shown in SEQ ID NO:1, FR3 shown in FR2 shown in SEQ ID NO:2, SEQ ID NO:3, the FR4 shown in SEQ ID NO:4; Described complementary determining region CDR is selected from the aminoacid sequence of the CDR of lower group: the CDR2 shown in the CDR1 shown in SEQ ID NO:5, SEQ ID NO:6, the CDR3 shown in SEQ ID NO:7.

Preferably, the VHH chain of the nano antibody of described Cry1Ab, it has the nucleotide sequence shown in SEQ ID NO:8.

Second aspect present invention, a kind of Cry1Ab nano antibody, it is for the nano antibody of Cry1Ab epi-position, comprises the VHH chain that two have aminoacid sequence shown in SEQ ID NO:9.

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 nano antibody of Cry1Ab of the present invention, or Cry1Ab nano antibody of the present invention.

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

SEQ ID NO：8。

A fourth aspect of the present invention, provides a kind of expression vector, and it is containing the nucleotide sequence shown in SEQ ID NO:8.

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 the purposes that Cry1Ab nano antibody of the present invention detects Cry1Ab molecule.

Beneficial effect: compared with prior art, advantage of the present invention is as follows: first Cry1Ab lps molecule is coupled on enzyme plate by the present invention, show the correct space structure of this albumen, antigen in this format utilizes display technique of bacteriophage to screen immune nano Antibody geometric mean titer (camel heavy chain antibody phage display gene pool), thus obtain the specific nano antibody gene of Cry1Ab, this gene is gone in intestinal bacteria, thus establish can in the nano antibody strain of E. coli.

Accompanying drawing explanation

Fig. 1 is the DNA electrophorogram of nano antibody, and from left to right the DNA band of gel pore respectively: first is the molecule marker of 1000bp, and all the other ducts are PCR primer, and PCR primer band is about 500bp;

Fig. 2 is Cry1Ab nano antibody, the electrophorogram of the SDS-PAGE after nickel post resin gel affinitive layer purification, and result shows, and Cry1Ab nano antibody is through this purge process, and its purity can reach more than 95%.

Embodiment

The present invention's application is coupled on enzyme plate for Cry1Ab, the correct space structure of display protein matter, antigen selection immune nano Antibody geometric mean titer (camel heavy chain antibody phage display gene pool) in this format, and obtain can in the nano 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 nano antibody library of Cry1Ab:

(1) first obtain Cry1Ab albumen (purchased from You Long bio tech ltd, Shanghai), mixed by 1 mg Cry1Ab with freund's adjuvant equal-volume, an immunity dromedary, once in a week, immunity 6 times, stimulates the specific nano antibody of B cell antigen expressed altogether; After (2) 6 immunity terminate, extract 100 mL camel peripheral blood lymphocytes and extract total serum IgE; (3) synthesizing cDNA(utilizes Superscript reverse transcriptase CAT:18064-014 to synthesize) and utilize nested PCR amplification VHH, the first step PCR concrete steps are: 94 ° of C 7 min, 1 circulation, 94 ° of C 1 min, 30 circulations, 55 ° of C 1 min, 72 ° of C 1 min, 72 ° of C 10 min, second step PCR concrete steps are with the first step PCR, and just cycle index is down to 17 times by 30 times; (4) restriction enzyme PstI and NotI enzyme is utilized to cut 20 μ g pComb3 Vector for Phage Display (Biovector supply) and 10 μ g VHH and connect two fragments; (5) turn in competent cell TG1 by connection product conversion to electricity, build Cry1Ab nano antibody library and measure storage capacity, storage capacity size is 2 × 10 ⁹.

Embodiment 2: the nano antibody screening process for Cry1Ab:

(1) 100 mM NaHCO will be dissolved in ₃, 20 μ g Cry1Ab in pH 8.2 are coupled on NUNC enzyme plate,

4 DEG C of placements are spent the night; Within (2) second days, add 100 μ L 0.1% caseins, room temperature closes 2 h; After (3) 2 h, add 100 μ L phages (5 × 10 ¹¹tfu immunity camel nano antibody phage display gene pool, concrete procurement process is shown in embodiment 1), room temperature effect 1 h; (4) 5 times are washed with 0.05% PBS+Tween-20, to wash uncombined phage off; (5) with 100 mM TEA(triethylamine) phage with Cry1Ab specific binding is dissociated down, and infect the e. coli tg1 being in logarithmic phase growth, cultivate 1 h for 37 DEG C, produce also purified phage and be used for the screening of next round, identical screening process repeats 3-4 wheel, progressively obtains enrichment.

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

(1) contain in the Tissue Culture Dish of phage after the screening of 3-4 wheel from above-described embodiment 2, select 96 single bacterium colonies and the TB substratum being inoculated in the penbritin containing 100 μ g/mL (containing 2.3 grams of potassium primary phosphates in 1LTB substratum, 12.52 gram dipotassium hydrogen phosphate, 12 grams of peptones, 24 grams of yeast extracts, 4mL glycerine) in, after growing to logarithmic phase, add the IPTG of final concentration 1 mmole, 28 DEG C of overnight incubation.(2) osmose process (Zhu is utilized, M., Hu, Y., Li, G., Ou, W., Mao, P., Xin, S., Wan, Y., 2014. Combining magnetic nanoparticle with biotinylated nanobodies for rapid and sensitive detection of influenza H3N2. Nanoscale research letters 9 (1), 528. P3-4) 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 a mouse anti-HA tag antibody(against murine, purchased from Beijing CoWin Bioscience Co., Ltd.), at room temperature place 1 hour.(4) wash away unconjugated antibody with PBST, add anti-mouse alkaline phosphatase conjugate(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 micrograms per millilitre LB liquid in extract plasmid and to check order.

The gene order of each clone strain is analyzed according to sequence alignment program Vector NTI, 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, the aminoacid sequence of the VHH chain of its antibody is as shown in SEQ ID NO:9.

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

1. by sequencing analysis above obtain different clone strain plasmid electricity be transformed in intestinal bacteria WK6, and be coated on LA+glucose namely containing on the culture plate of penbritin and glucose, 37 DEG C of overnight incubation; (2) selecting single colony inoculation contains in the LB nutrient solution of penbritin at 5 mL, 37 DEG C of shaking table overnight incubation; (3) that inoculates 1 mL spends the night in bacterial classification to 330 mL TB nutrient solution, and 37 DEG C of shaking tables are cultivated, and cultivates when reaching 0.6 ~ 1 to OD value, adds IPTG, 28 DEG C of shaking table overnight incubation; (4) centrifugal, receive bacterium; (5) utilize osmose process, obtain antibody crude extract; (6) can prepare through nickel post ion affinity chromatography the albumen that purity reaches more than 90%.

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.

SEQUENCE LISTING

 

<110> Southeast China University

 

<120> anti-Cry1Ab toxin nano antibody, its encoding sequence and application

 

<130>

 

<160> 9

 

<170> PatentIn version 3.3

 

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<212> PRT

<213> artificial sequence

 

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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

 

 

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

 

 

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<213> artificial sequence

 

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Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly

1 5 10 15

 

 

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<211> 38

<212> PRT

<213> artificial sequence

 

<400> 3

 

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

1 5 10 15

 

 

Lys Asn Thr Leu Tyr Leu Gln Leu Asn Ser Leu Ser Thr Glu Asp Thr

20 25 30

 

 

Ala Met Tyr Tyr Cys Val

35

 

 

<210> 4

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<213> artificial sequence

 

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Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

 

 

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<212> PRT

<213> artificial sequence

 

<400> 5

 

Gly Phe Thr Phe Ser Glu Tyr Gly Met Thr

1 5 10

 

 

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Ile Ser Ser Gly Ala Asp Ile Thr Ser

1 5

 

 

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Lys Pro Glu Gln Ser His Cys Thr Ala Tyr Cys Arg Val Asp Ser

1 5 10 15

 

 

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caggtgcagc aggtgcagct gcaggagtct gggggaggct tggtgcagcc tggggggctc 60

 

tcctgtgcag cctctggatt caccttcagt gaatatggca tgacctgggt ccgccaggct 120

 

ccaggcaagg gactcgaatg ggtctcagga attagtagtg gtgcggatat cacatcctat 180

 

gcagagtccg tgaagggccg attcaccatc tccagagaca acggcaagaa tacgctgtat 240

 

ctgcaattga acagcctgag tactgaggac acggccatgt attactgtgt aaagccagag 300

 

cagagccact gtactgctta ctgccgcgtt gactcgagag gccagggaac ccaggtcacc 360

 

gtctcctca 369

 

 

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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

 

 

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Glu Tyr

20 25 30

 

 

Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

 

 

Ser Gly Ile Ser Ser Gly Ala Asp Ile Thr Ser Tyr Ala Glu Ser Val

50 55 60

 

 

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Asn Thr Leu Tyr

65 70 75 80

 

 

Leu Gln Leu Asn Ser Leu Ser Thr Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

 

 

Val Lys Pro Glu Gln Ser His Cys Thr Ala Tyr Cys Arg Val Asp Ser

100 105 110

 

 

Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

 

Claims (9)

1. the VHH chain for Cry1Ab nano antibody, comprise framework region FR and complementary determining region CDR, it is characterized in that, described framework region FR is selected from the aminoacid sequence of the FR of lower group: the FR1 shown in SEQ ID NO:1, FR2 shown in SEQ ID NO:2, FR4 shown in FR3 shown in SEQ ID NO:3, SEQ ID NO:4; Described complementary determining region CDR is selected from the aminoacid sequence of the CDR of lower group: the CDR2 shown in the CDR1 shown in SEQ ID NO:5, SEQ ID NO:6, the CDR3 shown in SEQ ID NO:7.

2. the VHH chain of Cry1Ab nano antibody according to claim 1, is characterized in that, it has the nucleotide sequence shown in SEQ ID NO:8.

3. a Cry1Ab nano antibody, is characterized in that, it is for the nano antibody of Cry1Ab epi-position, comprises the VHH chain that two have aminoacid sequence shown in SEQ ID NO:9.

4. a DNA molecular, is characterized in that, its coding is selected from the protein of lower group: the VHH chain of the Cry1Ab nano antibody described in claim 1 or 2, or Cry1Ab nano antibody according to claim 3.

5. DNA molecular according to claim 4, is characterized in that, it have be selected from DNA sequence dna as

Shown in SEQ ID NO:8.

6. an expression vector, is characterized in that, it is containing the nucleotide sequence shown in SEQ ID NO:8.

7. a host cell, is characterized in that, it can express the nano antibody of Cry1Ab.

8. Cry1Ab nano antibody according to claim 3 is for detecting the purposes of Cry1Ab molecule.

9., for detecting a test kit for Cry1Ab molecule, it comprises Cry1Ab nano antibody according to claim 3.