CN104628860A - CP4-EPSPS specific nano-antibody and application thereof - Google Patents
CP4-EPSPS specific nano-antibody and application thereof Download PDFInfo
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- CN104628860A CN104628860A CN201510048148.7A CN201510048148A CN104628860A CN 104628860 A CN104628860 A CN 104628860A CN 201510048148 A CN201510048148 A CN 201510048148A CN 104628860 A CN104628860 A CN 104628860A
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Abstract
The invention discloses a CP4-EPSPS specific nano-antibody and application thereof and also discloses amino acid sequences of the nano-antibody and a gene sequence encoding the nano-antibody. By adopting the CP4-EPSPS specific nano-antibody disclosed by the invention, a detection reagent for a CP4-EPSPS specific protein is researched and developed and can be used for detecting genetically modified foods and solving the problems of high cost, complex operation and poor specificity of the existing method for detecting the genetically modified foods.
Description
Technical field
The invention belongs to biomedicine or biological pharmacy technical field, relate to a kind of be directed to CP4-EPSPS albumen nano antibody and encoding sequence and its application in this field.
Background technology
5-enolpyruvyl shikimic acid-3-phosphate synthase (5-enolpyruvyl-shikimate-3-phosphate synthase, EPSPS:EC 2.5.1.19) be the target enzymes of herbicide glyphosate, and show high resistance from the glyphosate of EPSPS to high density of agrobacterium tumefaciens (Agrobacterium sp.) CP4 bacterial strain, therefore cp4-epsps gene is widely used in the transgenic research of various crop, the CP4-EPSPS of its coding is the key enzyme in shikimic acid pathway, can catalytic phosphatase enol pyruvic acid (phosphoenolpyruvate, PEP) with shikimic acid-3-phosphoric acid (shikimate-3-phosphate, SP3) 5-enolpyruvyl shikimic acid-3-phosphoric acid (EPSP) is generated.In farming object, proceed to this gene can make it obtain the resistance of herbicide glyphosate, but the safety problem of genetically modified food more and more receives the concern of human consumer, therefore, sets up a set of quick, special detection GMOs system and seems particularly important.1993, Belgium scientist C.Hamers-Casterman etc. are first at " Nature " upper report: the antibody in camel blood, half is had not have light chain, and, " heavy chain antibody " (heavy-chain antibodies of these disappearance light chains, HCAbs) can combine closely with targets such as antigens as normal antibody, and to stick mutually unlike single-chain antibody scFv, even be gathered into block.This antibody only comprises a variable region of heavy chain (variable domain of heavy chain of HCAb, VHH) 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.VHH be known at present can the least unit of combining target antigen, so VHH also claims nano antibody (Nanobody, Nb).Nano antibody has that unique character is as little in relative molecular mass, good water solubility, stability are strong, antigen binding ability is strong, be easy to production etc.Therefore, in biotechnology and medical use, than other antibody, there is superiority.So applying nano antibody technique research and development CP4-EPSPS detection reagent has broad prospects.
Summary of the invention
Goal of the invention: in order to overcome the deficiencies in the prior art, the invention provides a kind of nano antibody for CP4-EPSPS albumen, provides encoding sequence and the application of this nano antibody in preparation detection reagent of this nano antibody simultaneously.
Technical scheme: for achieving the above object, the technical solution used in the present invention is:
A kind of CP4-EPSPS specific nano antibody, its VHH chain comprises framework region and complementary determining region, described framework region FR comprises following aminoacid sequence: 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 comprises following aminoacid sequence: the CDR2 shown in the CDR1 shown in SEQ ID NO:5, SEQ ID NO:6, the CDR3 shown in SEQ ID NO:7.
Further, in the present invention, the VHH chain with aminoacid sequence shown in SEQ ID NO:8 is comprised.
A kind of DNA molecular, the VHH chain of its coding CP4-EPSPS specific nano antibody, or CP4-EPSPS specific nano antibody.
Further, in the present invention, described DNA molecular comprises nucleotide sequence as shown in SEQ ID NO:9.
Further, in the present invention, the described application of CP4-EPSPS specific nano antibody in CP4-EPSPS Protein Detection.
Beneficial effect: the nano antibody for CP4-EPSPS albumen provided by the invention, first by CP4-EPSPS albumen coupling on enzyme plate, 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, thus obtain the specific nano antibody gene of CP4-EPSPS, this gene is gone in intestinal bacteria, thus foundation can in the nano antibody strain of E. coli.The CP4-EPSPS specific nano antibody announced by the present invention, research and develop the detection reagent for CP4-EPSPS protein-specific, can be used for detecting genetically modified food, to solve, existing genetically modified food detection method cost is high, the problem of complicated operation, poor specificity.
Accompanying drawing explanation
Fig. 1 is the electrophorogram of the DNA of CP4-EPSPS specific nano antibody of the present invention; Wherein swimming lane M represents Protein Marker, and swimming lane 1 is the product of polymerase chain reaction PCR;
Fig. 2 is the electrophorogram of CP4-EPSPS specific nano antibody of the present invention; Wherein swimming lane M represents Protein Marker, and swimming lane 1 is CP4-EPSPS specific nano antibody.
Embodiment
The present invention by CP4-EPSPS albumen coupling on enzyme plate, the correct space structure of display protein matter, antigen selection immune nano Antibody geometric mean titer in this format, preferred camel heavy chain antibody phage display gene pool, and obtain can in the nano antibody strain of E. coli.
Below in conjunction with drawings and Examples, the present invention is further described.
Embodiment 1: the structure being directed to CP4-EPSPS specific nano antibody library:
(1-1) first mixed with freund's adjuvant equal-volume by 1mg CP4-EPSPS, an immunity Xinjiang dromedary, once in a week, immunity 7 times, stimulates the specific nano antibody of B cell antigen expressed altogether;
(1-2), after 7 immunity terminate, extract 100mL camel peripheral blood lymphocyte and extract total serum IgE;
(1-3) synthesize complementary DNA (cDNA) cDNA and utilize nested-polymerase chain reaction pcr amplification VHH chain;
(1-4) utilize restriction enzyme Pst I and Not I enzyme to cut 20ug pComb3 Vector for Phage Display and 10 μ gVHH chains, and connect two fragments;
(1-5) will connect product conversion in competent cell TG1, build CP4-EPSPS nano antibody library and measure storage capacity, storage capacity size is 3.6 × 10
9.
As shown in Figure 1, PCR primer electrophoretic band size is about 500bp.
Embodiment 2: the nano antibody screening process for CP4-EPSPS:
(2-1) the 100mM NaHCO of pH 8.2 will be dissolved in
320 μ g CP4-EPSPS albumen couplings in solution are on NUNC enzyme plate, and 4 DEG C of placements are spent the night;
(2-2) within second day, add 100 μ L 0.1% caseins, room temperature closes 2h;
(2-3) after 2h, 100 μ L phages are added, namely 5 × 10
11tfu immunity camel nano antibody phage display gene pool, room temperature effect 1h;
(2-4) 5 times are washed with 0.05%PBS+Tween-20 (PBST), to wash uncombined phage off;
(2-5) with 100mM trolamine (triethylamine, TEA) phage with CP4-EPSPS specific binding is dissociated down, and infect the e. coli tg1 being in logarithmic phase growth, cultivate 1h for 37 DEG C, produce also purified phage and be used for the screening of next round, identical screening process repeats 3 and takes turns, and progressively obtains enrichment.
Embodiment 3: screen the single positive colony of specificity with the enzyme-linked immunoassay method (ELISA) of phage:
(3-1) contain the Tissue Culture Dish of phage after taking turns screening from above-mentioned 3, select 96 single bacterium colonies and be inoculated in the inducible protein liquid nutrient medium of the penbritin containing 100 μ g/mL and TB substratum (containing 2.3g potassium primary phosphate in the TB substratum of 1L, 12.52g dipotassium hydrogen phosphate, 12g peptone, 24g yeast extract, 4mL glycerine) in, after growing to logarithmic phase, add isopropylthiogalactoside (IPTG) that final concentration is 1mM as inductor, 28 DEG C of overnight incubation;
(3-2) utilize osmose process to obtain and slightly carry antibody, and antibody is transferred in antigen coated enzyme-linked immunosorbent assay (ELISA) plate, at room temperature place 1h;
(3-3) wash away unconjugated antibody with PBST, add the anti-HA antibody of against murine (mouse anti-HA tag antibody, Beijing CoWin Bioscience Co., Ltd.), at room temperature act on 1h;
(3-4) wash away unconjugated antibody with PBST, add goat-anti-mouse alkaline phosphatase enzyme mark antibody (anti-mouse alkaline phosphatase conjugate, the prompt Science and Technology Ltd. of Amy), at room temperature act on 1h;
(3-5) wash away unconjugated antibody with PBST, add alkaline phosphatase nitrite ion, on ELISA instrument, at 405nm wavelength, read absorption value;
(3-6) when sample well OD value is greater than control wells OD value more than 2 times, positive colony hole is judged to;
(3-7) bacterium in positive colony hole is turned shake containing 100 μ g/mL penbritin LB substratum 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:8.
Embodiment 4:CP4-EPSPS specific nano antibody is at Host Strains expression in escherichia coli, purifying:
(3-1) by above-mentioned sequencing analysis obtain different clone strain plasmid electricity be transformed in intestinal bacteria WK6, and be coated on the culture plate of LA+glucose (namely containing penbritin and glucose), 37 DEG C of overnight incubation;
(3-2) selecting single colony inoculation contains in the LB substratum of penbritin at 5mL, 37 DEG C of shaking table overnight incubation;
(3-3) inoculate the bacterial classification that spends the night of 1mL in 330mL TB nutrient solution, 37 DEG C of shaking tables are cultivated, and when cultivation reaches 0.6 ~ 1.0 to OD value, add IPTG, 28 DEG C of shaking table overnight incubation;
(3-4) centrifugal, receive bacterium;
(3-5) utilize osmose process, obtain antibody crude extract;
(3-6) the higher CP4-EPSPS specific nano antibody of purity can be obtained through nickel post ion affinity chromatography.As shown in Figure 2, the molecular weight of CP4-EPSPS Nb is 14.7kDa, iso-electric point pI is 8.40, and the output expressed in intestinal bacteria WK6 is about 7.8mg/L.
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.
Claims (5)
1. a CP4-EPSPS specific nano antibody, it is characterized in that, its VHH chain comprises framework region and complementary determining region, described framework region FR comprises following aminoacid sequence: 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 comprises following aminoacid sequence: 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. CP4-EPSPS specific nano antibody according to claim 1, is characterized in that, comprises the VHH chain with aminoacid sequence shown in SEQ ID NO:8.
3. a DNA molecular, is characterized in that, the VHH chain of the CP4-EPSPS specific nano antibody of its coding described in claim 1 or 2, or CP4-EPSPS specific nano antibody according to claim 2.
4. DNA molecular according to claim 3, is characterized in that, comprises nucleotide sequence as shown in SEQ ID NO:9.
5. the application of CP4-EPSPS specific nano antibody according to claim 1 and 2 in CP4-EPSPS Protein Detection.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105717295A (en) * | 2016-01-15 | 2016-06-29 | 北京市农林科学院 | Test strip for rapidly detecting CP4-EPSPS transgenic plant and derivative thereof |
| WO2017128301A1 (en) * | 2016-01-29 | 2017-08-03 | 四川天豫兴禾生物科技有限公司 | Glyphosate-tolerant epsps gene screening method and use thereof |
| US11060126B2 (en) | 2016-02-19 | 2021-07-13 | Nutrasource Pharmaceutical And Nutraceutical Services Inc. | Methods for detecting genetically modified organisms (GMO) |
| CN117147830A (en) * | 2023-10-26 | 2023-12-01 | 德州国科医疗科技有限公司 | Fluorescent staining solution for detecting specific fungus D-glucan |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103342750A (en) * | 2013-06-21 | 2013-10-09 | 东南大学 | Apolipoprotein B nano antibody and coding sequence and application thereof |
| CN103497253A (en) * | 2013-09-26 | 2014-01-08 | 东南大学 | Nanometer antibody, encoding sequence and application of H2A.Z variant |
-
2015
- 2015-01-29 CN CN201510048148.7A patent/CN104628860A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103342750A (en) * | 2013-06-21 | 2013-10-09 | 东南大学 | Apolipoprotein B nano antibody and coding sequence and application thereof |
| CN103497253A (en) * | 2013-09-26 | 2014-01-08 | 东南大学 | Nanometer antibody, encoding sequence and application of H2A.Z variant |
Non-Patent Citations (1)
| Title |
|---|
| 李忠鹏: "转基因靶分子CP4-EPSPS单克隆抗体的制备及其ELISA检测方法研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105717295A (en) * | 2016-01-15 | 2016-06-29 | 北京市农林科学院 | Test strip for rapidly detecting CP4-EPSPS transgenic plant and derivative thereof |
| WO2017128301A1 (en) * | 2016-01-29 | 2017-08-03 | 四川天豫兴禾生物科技有限公司 | Glyphosate-tolerant epsps gene screening method and use thereof |
| US11060126B2 (en) | 2016-02-19 | 2021-07-13 | Nutrasource Pharmaceutical And Nutraceutical Services Inc. | Methods for detecting genetically modified organisms (GMO) |
| CN117147830A (en) * | 2023-10-26 | 2023-12-01 | 德州国科医疗科技有限公司 | Fluorescent staining solution for detecting specific fungus D-glucan |
| CN117147830B (en) * | 2023-10-26 | 2024-01-12 | 德州国科医疗科技有限公司 | Fluorescent staining solution for detecting specific fungus D-glucan |
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Application publication date: 20150520 |