CN104558168A - Bt toxalbumin targeted Cry1B nano antibody as well as coding sequence and application thereof - Google Patents

Abstract

The invention discloses a Bt toxalbumin targeted Cry1B nano antibody and further discloses a gene sequence for coding the nano antibody. The Cry1B nano antibody disclosed by the invention can be expressed efficiently in escherichia coli and can be used for researching and developing Cry1B nano antibody molecule detection reagents.

Description

A kind of for Bt toxalbumin Cry1B nano antibody and encoding sequence thereof and application

Technical field

The present invention relates to biomedicine or biological pharmacy technical field, particularly relate to a kind of nano antibody for Bt toxalbumin Cry1B and encoding sequence thereof and application.

Background technology

Bacillus thuringiensis (Bacillus thuringiensis, Bt) is gram-positive microorganism, and to be a class to strong, the lethal speed of insect virulence fast and to the avirulent entomopathogen of pest natural enemy.Thalline can form insecticidal crystal proteins matter in the stable growth phase, also known as insecticidal crystal protein matter (Insecticidal crystal protein, ICP).Insecticidal crystal protein confrontation target pest has stronger toxicity.According to Bt toxoprotein gene homology, the Bt toxalbumin of expression is divided into broad variety, names with Cry toxin.These toxin only have activity and should without any effect to Mammals and the mankind to specific insect originally, but there is the growth tool of report display trans Bt gene crops to the mycorrhizal fungi of rhizosphere soil to have a significant effect, the bone of Bt protein to young mouse of high dosage ossify and has certain retarding action, if long-term, high-dose uses, blood middle leukocytes sum and oxyphorase matter content significantly decline, and show that Bt protein has obvious immunosuppression toxicity.In recent years, along with transgenic Bt crops large-scale commercial, the food safety caused by it and environmental safety cause people to pay close attention to widely.Increasing national requirements carries out labeling system to transgenic product, carries out detection of GMOs how many containing, content and containing the transgenosis of what kind to determine whether to product.Therefore set up detection method fast and accurately and seem particularly important.

Summary of the invention

The object of the invention is to provide a kind of Cry1B nano antibody, provides the encoding sequence of this nano antibody and this nano antibody for detecting the purposes of Cry1B molecule simultaneously.

The present invention is by the following technical solutions:

First aspect present invention, provide a kind of VHH chain of Cry1B nano antibody, comprise framework region FR and complementary determining region CDR, described framework region FR comprises the aminoacid sequence of the FR1 ~ FR4 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 comprises the aminoacid sequence of the CDR1 ~ CDR3 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 described Cry1B nano antibody, its aminoacid sequence is as shown in SEQ ID NO:8.

Second aspect present invention, provides a kind of Cry1B nano antibody, and it is for the nano antibody of Bt toxalbumin Cry1B, comprises the VHH chain of an aminoacid sequence as shown in SEQ ID NO:8.

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 Cry1B nano antibody described in claim 1 or 2, or Cry1B nano antibody according to claim 3.

Preferably, described DNA molecular, its nucleotide sequence is as shown in SEQ ID NO:9.

Fourth aspect present invention, provides a kind of expression vector, and it is containing the nucleotide sequence shown in SEQ ID NO:9.

Fifth aspect present invention, provides a kind of host cell, and it can express Cry1B nano antibody of the present invention.

Sixth aspect present invention, provides Cry1B nano antibody of the present invention for detecting the purposes of Cry1B molecule.

Beneficial effect of the present invention:

The present invention utilizes Cry1B protein immunization Xinjiang two-humped camel, after 6 immunity, collects this immune camel peripheral blood lymphocyte, establishes the phage display nano antibody gene pool for Cry1B.Cry1B antigen is coupled on ELISA enzyme plate, display technique of bacteriophage is utilized to screen the nano antibody gene pool of immunity, thus obtain with the bacterial strain of specificity for the nano antibody gene of Cry1B, proceed in intestinal bacteria by this gene, establishing can in the nano antibody strain of E. coli.The Cry1B nano antibody molecular weight that the present invention obtains is little, be only 1/10 of common antibody, stable chemical nature is good, output is high, high specificity, to other Cry toxin no cross reactions, to Cry1B, there is good immunodetection effect, for the immunological detection method setting up fast, accurately detect Cry1B poisonous protein lays the foundation.

Accompanying drawing explanation

Fig. 1 is Cry1B nano antibody purifying figure, and left road is protein standard molecule, and right road is the Cry1B nano antibody after nickel post resin gel affinitive layer purification.

Embodiment

First the present invention adopts a Cry1B immunity Xinjiang two-humped camel, extracts this two-humped camel peripheral blood lymphocyte and construct the special single domain heavy chain antibody library of Cry1B after 6 immunity.Cry1B antigen is coupled on enzyme plate, the correct space structure of display protein matter, the epitope of Cry1B is come out, antigen in this format utilizes display technique of bacteriophage to screen the nano antibody gene pool (camel heavy chain antibody phage display gene pool) of Cry1B immunity, and obtain and in the nano antibody strain of E. coli, and high degree of specificity can be had to Cry1B.

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

Embodiment 1: the structure being directed to the nano antibody library of Cry1B:

(1) 1mg Cry1B antigen (purchased from You Long bio tech ltd, Shanghai) is mixed with freund's adjuvant equal-volume, an immunity Xinjiang two-humped camel, once in a week, immunity 6 times altogether, first time Freund's complete adjuvant, the rear full adjuvant that cannots be used up for five times, stimulates the specific nano antibody of B cell antigen expressed; After (2) 6 immunity terminate, extract 100mL camel peripheral blood lymphocyte and extract total serum IgE; (3) synthesize cDNA and utilize secondary PCR amplification VHH; (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 Cry1B nano antibody library and measure storage capacity, storage capacity size is 2.0 × 10 ⁹cFU/mL.(6) random picking 24 clone, by bacterium colony PCR detection build library insertion rate detected result insertion rate be 100%.

Utilize the actual conditions of secondary PCR amplification VHH as follows:

PCR system comprises template cDNA, upstream primer CALL001, downstream primer CALL002 for the first time;

Second time PCR system comprises template first time PCR rubber tapping and reclaims product, upstream primer BACK-1, downstream primer BACK-2.

First time PCR and second time PCR program are: denaturation 94 DEG C, 7min, sex change 94 DEG C, 1min, anneal 55 DEG C, 1min, extends 72 DEG C, 1min.

Primer sequence is as follows:

CALL001 is as shown in SEQ ID NO:10: 5 '-GTCCTGGCTGCTCTTCTACAAGG-3 '

CALL002 is as shown in SEQ ID NO:11: 5 '-GGTACGTGCTGTTGAACTGTTCC-3 '

BACK-1 is as shown in SEQ ID NO:12: 5 '-GATGTGCAGCTGCAGGAGTCTGGAGGAGG-3 '

BACK-2 is as shown in SEQ ID NO:13: 5 '-GATGTGCAGCTGCAGGAGTCTGGGGGAGG-3 '

Embodiment 2: the nano antibody screening process for Cry1B:

(1) get in 200 μ L restructuring TG1 cell to 2 × TY substratum and cultivate, period adds 40 μ L helper phage VCSM13 and infects TG1 cell, and overnight incubation is with phage of increasing, and utilizes PEG/NaCl precipitating phage next day, collected by centrifugation amplification phage; (2) 100mM pH8.2NaHCO will be dissolved in ₃in 10 μ g Cry1B and His-tag be coupled in NUNC enzyme plate hole respectively, 4 DEG C of placements are spent the night; Within (3) second days, add 100 μ L 0.1% caseins, room temperature closes 2h; (4), after 2h, 100 μ L amplification phages (2 × 10 are added ¹¹tfu immunity camel nano antibody phage display gene pool), room temperature effect 1h; (5) 5 times are washed with PBS+0.05%Tween-20, to wash uncombined phage off; (6) with 100mM trolamine TEA (triethylamine), the phage with Cry1B specific binding is dissociated down, and infect the e. coli tg1 cell being in logarithmic phase growth, cultivate 1h for 37 DEG C, produce and collect the screening of phage for next round, identical screening process repeats 2 and takes turns, and obtains enrichment.

Embodiment 3: screen specific positive clone with enzyme-linked immunoassay method (ELISA):

(1) take turns the rear Tissue Culture Plate of screening from above-mentioned 2, select 95 single bacterium colonies respectively and be inoculated in TB substratum containing 100 μ g/mL penbritins (containing 2.3g potassium primary phosphate in 1L TB substratum, 12.52g dipotassium hydrogen phosphate, 12g peptone, 24g yeast extract, 4mL glycerine) 96 orifice plates in, and a blank is set, 37 DEG C be cultured to logarithmic phase after, add the IPTG that final concentration is 1mM, 28 DEG C of overnight induction.(2) utilize osmose process to obtain and slightly carry antibody, and antibody is transferred to Cry1B antigen and His-tag and bears contrast and spend the night in the elisa plate of coupling, incubated at room temperature 1h.(3) wash away unconjugated antibody with PBST, add the Mouseanti-HA tag antibody (against murine anti-HA antibody, purchased from Beijing CoWin Bioscience Co., Ltd.) of 100 μ L after 1:2000 dilution, at room temperature place 1h.(4) unconjugated antibody is washed away with PBST, add Anti-mousealkaline phosphatase conjugate (the goat-anti-mouse alkaline phosphatase enzyme mark antibody of 100 μ L after 1:2000 dilution, purchased from the prompt Science and Technology Ltd. of Amy), at room temperature place 1h.(5) wash away unconjugated antibody with PBST, add alkaline phosphatase nitrite ion, reaction 5 ~ 10min after in microplate reader 405nm wavelength place, 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 in LA substratum (LB+100 μ g/mL penbritin) to extract plasmid and to check order.

Analyze the gene order of each clone strain 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, finally obtain 1 strain Cry1B specific nano antibody.The aminoacid sequence of its antibody is the FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 district shown in SEQ ID NO:8, forms whole VHH.

Embodiment 4:Cry1B nano antibody is at Host Strains expression in escherichia coli and purifying:

(1) by above-mentioned sequencing analysis 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 5mL, and 37 DEG C of shaking tables are cultured to OD _600nm=0.6 ~ 0.9; (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 ~ 0.9 to OD value, add 1M IPTG, 28 DEG C of shaking table overnight incubation; (4) centrifugal, receive bacterium; (5) utilize osmose process, obtain antibody crude extract; (6) be purified into antibody by affinity chromatography method, its output be about 21.6mg/L and purity reach more than 90% Cry1B nano antibody, antibody size is 15.7kD, and iso-electric point is 6.04, as shown in Figure 1.

Embodiment 5:Cry1B nano antibody detection specificity is analyzed

Cry1B and other Cry toxin Cs ry1Aa, Cry1Ab, Cry1C, Cry1F are dissolved in 100mM NaHCO respectively ₃in, equivalent (2 μ g/mL) is coated in same 96 hole elisa plates, and arranges blank, and 4 DEG C are spent the night.After within second day, closing 1h with 1% milk, add 100 μ L 2 μ g/mL Cry1B antibody incubation 1h.Unconjugated antibody is washed away with PBST, add the Mouse anti-HA tag antibody 1h of 100 μ L after 1:2000 dilution, after PBST is washed, then add the Anti-mouse alkaline phosphatase conjugate of 100 μ L after 1:2000 dilution, at room temperature place 1h.(5) wash away unconjugated antibody with PBST, add alkaline phosphatase nitrite ion, 405nm wavelength place in microplate reader, read absorption value, as shown in the table:

	Cry1Aa	Cry1Ab	Cry1B	Cry1C	Cry1F	Blank
							Mean value	0.2915	0.359	1.7095	0.3855	0.254	0.315
Standard error	0.02192	0.012728	0.086974	0.003536	0.004243	0.001414

To Cry1B toxin, there is high degree of specificity by this nano antibody of ELISA experimental verification, to other Cry toxin no cross reactions.

More than show and describe ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.

Claims (8)

1. the VHH chain of a Cry1B nano antibody, comprise framework region FR and complementary determining region CDR, it is characterized in that, described framework region FR comprises the aminoacid sequence of the FR1 ~ FR4 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 comprises the aminoacid sequence of the CDR1 ~ CDR3 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 Cry1B nano antibody according to claim 1, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO:8.

3. a Cry1B nano antibody, is characterized in that, it is for the nano antibody of Bt toxalbumin Cry1B, comprises the VHH chain of an aminoacid sequence as shown in SEQ ID NO:8.

4. a DNA molecular, is characterized in that, its coding is selected from the protein of lower group: the VHH chain of the Cry1B nano antibody described in claim 1 or 2, or Cry1B nano antibody according to claim 3.

5. DNA molecular according to claim 4, is characterized in that, its nucleotide sequence is as shown in SEQ ID NO:9.

6. an expression vector, is characterized in that, it is containing the nucleotide sequence shown in SEQ ID NO:9.

7. a host cell, is characterized in that, it can express Cry1B nano antibody according to claim 3.

8. Cry1B nano antibody according to claim 3 is for detecting the purposes of Cry1B molecule.