Phage display library panning method based on elastic-like tag recombinant protein

Abstract

The invention discloses a phage display library panning method based on elastic tag-like recombinant protein, and belongs to the technical field of biology. According to the method, the reversible phase change characteristic of elastin-like proteins (ELPs) is utilized, a target antigen sequence used for elutriation is fused with the ELPs, an antibody of an anti-specific antigen sequence displayed by phage is elutriated from a solution through multiple reversible phase change cycles, that is, the antibody displayed by the phage and combined with the specific antigen can be obtained through enrichment through multiple rounds of elutriation and amplification, and the specific antigen sequence of the ELPs is detected. The process only needs a conventional centrifugal machine and a constant-temperature water bath kettle, has the advantages of low cost, simplicity and convenience in operation and the like, and provides a new idea for elutriation of the phage display library.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a phage display library screening method based on elastic tag-like recombinant protein. Background Art

[0002] Elastin-Like Polypeptides (ELPs) are a type of polypeptide produced by genetic engineering technology based on the amino acid sequence composition of elastin. They are composed of pentapeptide repeating units Val-Pro-Gly-Xaa-Gly (VPGXG)n repeated in series, where Xaa is called the guest residue and can be any amino acid except Pro. Different Xaa and the number of repetitions n can constitute different ELPs. The temperature-sensitive reversible phase transition characteristics of ELPs are reflected in the fact that they are soluble below the phase transition temperature and condensed above the phase transition temperature, and this process is reversible. The reversible phase transition cycle (ITC) based on the ELPs tag is a new non-chromatographic technology for separating and purifying recombinant proteins. It utilizes the reversible phase transition characteristics of ELPs and specifically separates the fusion protein containing the ELPs tag from the soluble protein supernatant solution through multiple ITC cycles. This technology only requires a conventional centrifuge to obtain the target protein with a purity equivalent to that of affinity chromatography by chromatography. It is low-cost, easy to operate, and suitable for large-scale production needs.

[0003] At present, according to the different media, phage display library panning technology is mainly divided into solid phase panning and liquid phase panning. Solid phase panning is to fix the antigen on a solid carrier such as an ELISA plate or an affinity chromatography column, and after a series of "incubation-washing-elution-amplification" steps, the resulting antibodies often have higher affinity and stronger stability; liquid phase panning is to collect magnetic beads by magnetically collecting magnetic beads by labeling biotin or coupling avidin, thereby enriching phage display antibodies that specifically bind to the target antigen, and through repeated washing, elution and recovery steps, high-affinity antibodies can be effectively screened. Compared with the most classic solid phase screening, the advantage of liquid phase panning is that it does not need to fix the antigen on the surface medium, which avoids the loss of the antigen to a certain extent. At the same time, the liquid environment increases the probability of antigen-antibody binding, greatly improving the efficiency of panning.

[0004] However, so far, there have been no reports on the use of elastic-tagged recombinant proteins for phage display library panning. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a phage display library panning method based on an elastic-like tag recombinant protein. In order to avoid the disadvantage that the antigen binding site in solid phase panning cannot be fully exposed, resulting in low panning efficiency, the present invention is implemented through liquid phase. However, the conventional liquid phase magnetic bead method often requires the antigen to be coupled to the magnetic beads, and the method is cumbersome. Therefore, the present invention designs an elastic-like tag recombinant protein to display antigen, and relies on the reversible phase change property of elastic-like protein with temperature change to complete the panning of phage display library. The operation is simple and cost-saving, which provides a new idea and method for antibody discovery and screening.

[0006] To achieve the above object, the present invention is implemented through the following technical solutions:

[0007] In a first aspect, the present invention provides a recombinant negative sieve protein with an elastic tag, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] In a second aspect, the present invention provides a recombinant panning protein with an elastic tag, wherein the elastic tag in the recombinant panning protein with an elastic tag is connected to an antigen via a flexible chain, and the antigen includes a heart failure biomarker NT-proBNP.

[0009] Furthermore, the amino acid sequence of the elastic tag-like recombinant panning protein is shown in SEQ ID NO.3.

[0010] In a third aspect, the present invention provides a recombinant expression plasmid into which is inserted a gene encoding the above-mentioned elastic-tag-like recombinant negative screening protein or elastic-tag-like recombinant panning protein.

[0011] Furthermore, the vector is pET28a(+).

[0012] In a fourth aspect, the present invention provides an engineered bacterium carrying the above-mentioned recombinant expression plasmid.

[0013] Furthermore, the engineered bacteria is Escherichia coli BLR (DE3).

[0014] In the fifth aspect, the present invention provides a method for preparing the above-mentioned elastic-tag-like recombinant negative screening protein or elastic-tag-like recombinant panning protein, the above-mentioned recombinant expression plasmid is transformed into Escherichia coli BLR (DE3) competent cells by 42°C heat shock method, positive single clones are screened out by kanamycin, single clones are picked for expansion culture and induced to express recombinant protein, and after the Escherichia coli BLR (DE3) is broken, the elastic-tag-like recombinant negative screening protein or elastic-tag-like recombinant panning protein is obtained through reversible phase change cycle purification.

[0015] Furthermore, the induced expression conditions are automatic induction culture based on induced expression at 25°C for 22 to 24 hours; the reversible phase change cycle purification process is: 0.5M NaCl, 30 to 32°C water bath for 15 to 25 minutes to denature the protein, 30 to 32°C, 10,000 to 12,000 rpm, centrifugation for 5 to 15 minutes to obtain a protein precipitate, and then using 0.2 mol / L phosphate buffer to resuspend the precipitate, ice bath for 15 to 25 minutes to renature the protein, 4°C, 10,000 to 12,000 rpm centrifugation for 5 to 15 minutes to obtain a protein solution, and a total of 3 rounds of reversible phase change cycle purification are performed.

[0016] In a sixth aspect, the present invention provides a phage display library panning method based on a recombinant protein of a quasi-elastic tag, wherein the background of the phage display nanoantibody library constructed by immunization of alpaca is firstly subtracted by using the recombinant negative screening protein of the quasi-elastic tag, and then the phage display nanoantibody library after negative screening is mixed with the solution of the recombinant panning protein of the quasi-elastic tag to allow the antigen and antibody to bind, and the unbound phage display nanoantibody and weakly bound or non-specifically bound products are removed by repeated centrifugation and washing during the reversible phase change cycle of the recombinant panning protein of the quasi-elastic tag, and then the elution is performed. The specifically bound phage-displayed nanoantibody is eluted from the target antigen with a liquid, the pH value is adjusted to neutral, and then the elastic-tag-like recombinant panning protein is denatured and precipitated, and a phage-displayed nanoantibody solution is obtained after separation. The obtained phage-displayed nanoantibody solution is mixed with the solution of the elastic-tag-like recombinant panning protein again and the above panning process is repeated. Through multiple rounds of panning, a phage-displayed nanoantibody library that can specifically bind to the target antigen is obtained. After polyclonal and monoclonal PhageELISA detection, a high-affinity phage-displayed nanoantibody that can specifically bind to the target antigen is obtained.

[0017] Furthermore, the eluent is a 100 mM Gly-HCl solution with a pH of 2.2.

[0018] Furthermore, the number of rounds of panning is 3;

[0019] The parameters for the first round of selection were: antigen input 5-15 μg, total phage library input 2×10 11 ~9×10 11 , antigen-antibody binding time 40-60min, 0.5M NaCl, 30-32℃ water bath 5-15min denaturation, 30-32℃, 10000-12000rpm, centrifugation 5-15min, 0.5M NaCl / 0.1% PBST washing 9-11 times, 0.1% PBST resuspending precipitate, ice bath 20-40min renaturation;

[0020] The parameters for the second round of selection were: antigen input 5-15 μg, total phage library input 2×1011 ~9×10 11 , antigen-antibody binding time 20-40min, 0.5M NaCl, 30-32℃ water bath 5-15min denaturation, 30-32℃, 10000-12000rpm, centrifugation 5-15min, 0.5M NaCl / 0.5% PBST washing precipitate 14-16 times, 0.5% PBST resuspending precipitate, ice bath 20-40min renaturation;

[0021] The parameters for the third round of selection were: antigen input 3-7 μg, total phage library input 2×10 11 ~9×10 11 , antigen-antibody binding time 20-40min, denaturation in 0.5M NaCl, 30-32℃ water bath for 5-15min, centrifugation at 30-32℃, 10000-12000rpm for 5-15min, wash the precipitate with 0.5M NaCl / 1% PBST 14-16 times, resuspend the precipitate with 1% PBST, and renature in ice bath for 20-40min.

[0022] Furthermore, polyclonal Phage ELISA was performed on the phage-displayed nanoantibody library obtained from each round of panning amplification to evaluate the enrichment effect in order to reflect the differences in the efficiency of each round of panning.

[0023] Furthermore, in the phage display library panning method, several monoclonal colonies are randomly selected from the titer detection culture dish of the phage obtained in the third round of panning for monoclonal Phage ELISA, and their positive rate is calculated to evaluate the panning effect. At the same time, the positive clones are sequenced to obtain the target nanoantibody sequence.

[0024] In a sixth aspect, the present invention provides the use of the above-mentioned elastic tag-like recombinant negative screening protein, elastic tag-like recombinant panning protein, and phage display library panning method in the panning of phage display nanoantibody library.

[0025] The present invention takes the panning of NT-proBNP nanoantibody, an important biomarker of heart failure, as an example, and mainly comprises designing and synthesizing pET28a(+)-4WELP and pET28a(+)-4WELP-NT-proBNP recombinant plasmids, then expressing and purifying these two types of elastic tag recombinant proteins, and using them for panning of a phage-displayed nanoantibody library constructed by immunization of alpaca, using reverse screening for background subtraction, and enriching phage-displayed nanoantibodies specifically binding to NT-proBNP through three rounds of panning. The phage recovery rate of the third round of panning is about 30 times higher than that of the first round of panning, and the technical means for panning the phage-displayed library is successfully optimized; polyclonal Phage ELISA verifies the enrichment results of the three rounds of panning, and 40 monoclonal Phage ELISA tests obtain 14 phage-displayed nanoantibodies with good affinity, and the gene sequences thereof obtained by sequencing can be used for the subsequent expression and evaluation of the NT-proBNP nanoantibody.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The method of the present invention does not require the fixation of antigens, and the antigens can be completely exposed to the binding system, which avoids the loss of antigens to a certain extent, increases the probability of antigen-antibody binding, and significantly improves the panning efficiency.

[0028] 2. The method of the present invention is easy to operate and saves costs. It cleverly utilizes the reversible phase change characteristics of elastin-like proteins regulated by temperature. The purification of the selected protein and the selection of the library can be completed through simple operations and instruments such as warm water bath, ice bath and temperature-controlled centrifuge. The antigen does not need to be processed by biotinylation labeling and coupling magnetic beads, which reduces costs and saves time.

[0029] 3. The method of the present invention has high panning efficiency. By changing the parameters of each round of panning, from loose to strict, the panning pressure is gradually increased, so that phage-displayed nanoantibody molecules with strong specificity and high affinity are strictly selected, achieving a good enrichment effect.

[0030] 4. The framework design of the elastin-like tag recombinant protein is universal, and its antigen part is replaceable. By connecting the elastin-like tag with other different antigens or epitopes, a new selection protein can be recombined to be used for selection of different phage display libraries.

[0031] 5. A new liquid phase panning method for phage display libraries based on elastic-like tag recombinant proteins has been implemented and has significant advantages, which provides a new, effective and beneficial strategy for optimizing the panning system of phage display libraries. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are some embodiments of the present invention.

[0033] Figure 1 Schematic diagram of the gene structure design of the expression vector of the elastin-tagged recombinant protein 4WELP (A) and 4WELP-NT-proBNP (B).

[0034] Figure 2 This is the plasmid gene map of the elastic tag-like recombinant protein pET28a(+)-4WELP(A) and pET28a(+)-4WELP-NT-proBNP(B).

[0035] Figure 3 These are the expression and purification results of the elastic tag-like recombinant proteins 4WELP and 4WELP-NT-proBNP. A is the expression and purification of 4WELP protein, M: pre-stained protein Marker, 1: total protein after induction, 2: soluble protein after induction, 3-6: purified protein; B is the purification of 4WELP-NT-proBNP protein, M: pre-stained protein Marker, 1: purified protein.

[0036] Figure 4 The results are for monoclonal Phage ELISA. DETAILED DESCRIPTION

[0037] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solution of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Unless otherwise specified, the experimental equipment, materials, reagents, etc. involved in the present invention can be obtained from commercial channels.

[0038] Example 1 Design of recombinant protein expression vector with elastic tag

[0039] The gene encoding 4WELP protein (the gene sequence is shown in SEQ ID NO.2) and the gene sequence carrying the NT-proBNP (N-terminal pro-brain natriuretic peptide) antigen epitope (the gene sequence is shown in SEQ ID NO.4) were cloned into the pET28a(+) vector. In order to reduce the steric hindrance effect and fully expose the antigen epitope, the 4WELP and NT-proBNP antigens were connected through the amino acid residue sequence encoding the soft chain poly N, and the gene expression frame of the six histidine tags was removed. The gene structure of the recombinant expression vector is as follows: Figure 1 As shown, according to the design, the panning protein recombinant plasmids pET28a(+)-4WELP and pET28a(+)-4WELP-NT-proBNP were synthesized, and the plasmid gene map is shown in Figure 2shown.

[0040] The amino acid sequence of 4WELP is shown in SEQ ID NO.1.

[0041] The amino acid sequence of 4WELP-NT-proBNP is shown in SEQ ID NO.3.

[0042] Example 2 Expression and purification of elastic tag-like recombinant protein

[0043] (1) Preparation of Escherichia coli BLR (DE3) chemically competent cells

[0044] I. Streak out a single clone of BLR (DE3), activate it in 5 mL of LB medium, and culture it overnight at 37°C and 220 rpm;

[0045] II. On the second day, inoculate the bacterial solution at a ratio of 1:100, culture at 37°C and 220 rpm until the logarithmic phase, and place on ice for 30 minutes;

[0046] III. Pour the liquid in the shake flask into the bacteria collection bottle in the clean bench, centrifuge at 4°C and 4000 rpm for 8 minutes, discard the supernatant, and add 50 mL of 0.1 M CaCl2 liquid to resuspend the bacteria;

[0047] IV. After 10 minutes of ice storage, centrifuge at 4°C and 5000 rpm for 8 minutes, discard the supernatant, and add 50 mL of 0.1 M CaCl2 liquid to resuspend the cells;

[0048] V. After placing on ice for 10 min, centrifuge at 4°C and 5000 rpm for 8 min, discard the supernatant, add 600 μL of 0.1 M CaCl2 to resuspend the bacteria, add an equal volume of 20% glycerol, divide into 1.5 mL centrifuge tubes, and store at -80°C.

[0049] (2) Chemical transformation of recombinant plasmid

[0050] The recombinant plasmids pET28a(+)-4WELP and pET28a(+)-4WELP-NT-proBNP were transformed into E. coli BLR (DE3) respectively: 1 μL of plasmid with a concentration of 100 ng / μL was taken, the pipette tip was inserted into the bottom and 50 μL of E. coli BLR (DE3) competent cells prepared in the previous step were added, and the tube was gently mixed and placed on ice for 20 min. The centrifuge tube was placed in a 42°C water bath for heat shock for 80 s, and then placed on ice for 5 min. 200 μL of warm SOC medium was added, and the plate was incubated at 37°C for 30 min. The plate was transferred to a shaker and incubated at 37°C and 220 rpm for 1 h. 100 μL of the cultured bacterial solution was spread on an LB-Kan plate and incubated inverted in a 37°C incubator overnight.

[0051] (3) ZY (auto) induced expression

[0052] The positive monoclonal colonies on the above culture plates were picked and inoculated into 5 mL LB medium containing a final concentration of 50 μg / mL Kan for overnight shaking culture. The next day, they were inoculated into 500 mL automatic induction medium (containing 0.5 mL 1 M MgSO4, 10 mL 50×5052, 25 mL 20×NPS, 464.5 mL ZY culture medium) containing a final concentration of 50 μg / mL Kan at a ratio of 1:100. The expression was induced at 25°C for 22 to 24 h, and the colonies were centrifuged at 8000 rpm for 10 min at 4°C. The supernatant was discarded, and each 1 g of bacteria was resuspended and dissolved in 10 ml 0.2 mol / L phosphate buffer and stored at -80°C.

[0053] (4) ITC purification of recombinant protein

[0054] I. Freeze and thaw the bacterial solution stored at -80℃ for two times at 37℃ and -80℃ to break most of the bacteria;

[0055] II. Add lysozyme at a final concentration of 50 μg / mL according to the volume of bacterial solution and bathe in 37°C water for 30 min to allow lysozyme to work;

[0056] III. Place on ice for 15 minutes, and ultrasonically break the sample in an ice bath for 10 minutes (conditions: ultrasonic for 2 seconds, pause for 3 seconds, power 15%). After completing one ultrasonication, turn the sample upside down to mix, and ultrasonicate it again for 10 minutes to ensure sufficient breakage.

[0057] IV. Place on ice for 15 min, centrifuge at 12000 rpm for 10 min at 4°C, transfer the supernatant to a new 50 mL centrifuge tube, add DNase at a final concentration of 50 μg / mL according to the volume of the supernatant, mix by inversion, and place on ice for 10 min;

[0058] V. Centrifuge at 4°C, 12,000 rpm for 10 min, discard the precipitate, transfer the supernatant to a new tube, and centrifuge several times until there is no obvious precipitate at the bottom of the tube;

[0059] VI. Add 5M NaCl solution to the supernatant to a final concentration of 0.5 mol / L, incubate in a 30°C water bath for 20 min, and centrifuge at 30°C, 10,000 rpm for 10 min;

[0060] VII. A small amount of white precipitate can be seen at the bottom of the tube. Discard the supernatant and add 20 mL of 0.2 mol / L phosphate buffer to the tube to resuspend the precipitate. Place on ice for 20 min, centrifuge at 10,000 rpm for 10 min at 4°C, discard the precipitate and transfer the supernatant to a new tube.

[0061] Repeat step VI, discard the supernatant, add 10 mL of 0.2 mol / L phosphate buffer to the tube to resuspend the precipitate, place on ice for 20 min, centrifuge at 10000 rpm for 10 min at 4°C, and transfer the supernatant to a new tube;

[0062] IX. Repeat step VI again, discard the supernatant, add 5 mL of 0.2 mol / L phosphate buffer to the tube to resuspend the precipitate, place on ice for 20 min, centrifuge at 4°C, 10,000 rpm for 10 min, transfer the supernatant into new tubes, and store at 4°C.

[0063] (5) Determination of protein purity and concentration

[0064] Take 80 μL of purified protein sample and mix with 20 μL 5× Loading Buffer, cook the sample at 99℃ for 10 min, and then perform SDS-PAGE. The molecular weight and purity of the purified protein are determined by electrophoresis. Figure 3 As shown, the protein concentration was measured using a BCA quantitative detection kit for panning.

[0065] Table 1 Composition of 500 mL auto-induction medium

[0066]

[0067] *1.50×5052: ① 2.5g glucose, ② 10g α-lactose, ③ 73ml RO water, ④ 25g glycerol. When preparing, add ①, ②, ③ in sequence and then place in a 40℃ water bath. Stir intermittently with a small glass rod until the solution becomes clear and transparent, then add 25g glycerol and mix well. Filter and set aside.

[0068] *2.20×NPS: ①Prepare 160ml 1M Na2HPO4 solution; ②Prepare 50ml 1M KH2PO4 solution; ③Measure the pH of the above Na2HPO4 solution, and then use KH2PO4 solution to adjust the pH value of the Na2HPO4 solution to 7.2-7.5. If the pH is <7.2, add Na2HPO4; if the pH is >7.2, add KH2PO4; ④Calculate the amount of ammonium sulfate added by V*0.5mmol / L*132.14g / ml (V is the total volume of the solution after step ③); ⑤Measure the pH of the solution again and keep it in high pressure for later use.

[0069] *3.ZY culture medium: 4g tryptone, 2g yeast extract, dilute to 400ml with RO water, and place under high pressure for standby use.

[0070] Example 3 Phage display library panning

[0071] (1) Background subtraction

[0072] I. Closed phage display library: total input is 2×10 11 The phage-displayed nanoantibody library constructed by immunization of alpaca was mixed with 3% BSA blocking solution and placed at 37°C for 1 hour.

[0073] II. Block the centrifuge tube: Fill a 1.5 mL centrifuge tube with 3% BSA blocking solution and shake at 37°C, 120 rpm for 1 hour;

[0074] III. The phage display library solution (the closed phage display library in step I) was mixed with the 4WELP protein solution and added to a closed centrifuge tube, and the mixture was mixed by pipetting. The mixture was incubated at 37°C, 220 rpm for 1 h with a total volume of about 1 mL, so that the phage display library and the protein were fully combined. A small amount of 5 M NaCl solution was added to make the final concentration 0.5 M. After incubating at 30°C in a water bath for 10 min, the mixture was centrifuged at 30°C, 12000 rpm for 20 min. The supernatant was aspirated into a new closed 1.5 mL centrifuge tube.

[0075] IV. Add 4WELP protein solution to the supernatant again and repeat step III twice;

[0076] (2) 4WELP-NT-proBNP recombinant panning protein was subjected to three rounds of panning

[0077] First round of selection

[0078] I. After blocking the background-deducted library supernatant with 3% BSA blocking solution, add a total of 10 μg of 4WELP-NT-proBNP protein solution, mix by inversion, and incubate at 37°C on a shaker at 220 rpm for 1 hour to fully react;

[0079] II. Add 5M NaCl solution to the tube to make the final concentration 0.5M, incubate in a 30℃ water bath for 10min, centrifuge at 30℃, 12000rpm for 20min, remove the supernatant with a pipette, and do not poke the precipitate at the bottom of the tube. Wash the precipitate 8 times with 0.5M NaCl / 0.1% PBST. After each wash, centrifuge at 30℃, 12000rpm for 2min to remove the supernatant, add 0.1% PBST solution to resuspend the precipitate, and bathe on ice for 30min to allow the protein to renature and become soluble.

[0080] III. Add 5M NaCl solution to the tube to make the final concentration 0.5M, incubate in 30℃ water bath for 10min, centrifuge at 12000rpm for 10min, discard the supernatant, wash twice with 0.5M NaCl / 0.1% PBST, resuspend the precipitate with 0.1% PBST solution, and incubate on ice for 30min to renature the protein and make it soluble;

[0081] IV. Add 5M NaCl solution again to a final concentration of 0.5M, incubate in a 30°C water bath for 10min, centrifuge at 12000rpm for 10min, discard the supernatant, wash the precipitate at the bottom of the tube once with 0.5M NaCl / 1×PBS, add 400μL of eluent (100mM Gly-HCl) at pH=2.2, and place at room temperature for 10min on a rotating mixer at 20rpm to fully elute the phage-displayed nanoantibodies bound to the target antigen;

[0082] V. Add 200 μL of neutralizing solution (1M Tris-HCl, pH=8.0) to adjust the pH, then add 5M NaCl solution to a final concentration of 0.5M, incubate in a 30°C water bath for 10 min, centrifuge at 12000 rpm for 10 min, transfer the supernatant to a new 1.5 mL EP tube, and take 10 μL of the eluted supernatant for gradient dilution for titer determination;

[0083] VI. Use 100 μL of the eluted phage display nanobody library for amplification and determine the titer of the amplified phage display library for the next round of panning.

[0084] Second round of selection

[0085] I. The library supernatant after the first round of amplification was blocked with 3% BSA blocking solution, and a total of 10 μg of 4WELP-NT-proBNP protein solution was added and mixed by inversion. A control group was added in this round, in which the panning protein as the target antigen was replaced with 1× PBS, and other conditions remained unchanged. The cells were incubated at 37°C on a shaker at 220 rpm for 30 minutes to fully react.

[0086] II. Add 5M NaCl solution to the tube to make the final concentration 0.5M, incubate in a 30℃ water bath for 10min, centrifuge at 30℃, 12000rpm for 20min, remove the supernatant with a pipette, and do not poke the precipitate at the bottom of the tube. Wash the precipitate 10 times with 0.5M NaCl / 0.5% PBST. After each wash, centrifuge at 30℃, 12000rpm for 2min to remove the supernatant, add 0.5% PBST solution to resuspend the precipitate, and bathe on ice for 30min to allow the protein to renature and become soluble.

[0087] III. Add 5M NaCl solution to the tube to make the final concentration 0.5M, incubate in 30℃ water bath for 10min, centrifuge at 12000rpm for 10min, discard the supernatant, wash 5 times with 0.5M NaCl / 0.5% PBST, resuspend the precipitate with 0.5% PBST solution, and incubate on ice for 30min to renature the protein and make it soluble;

[0088] IV. Add 5M NaCl solution again to a final concentration of 0.5M, incubate in a 30°C water bath for 10 min, centrifuge at 12000 rpm for 10 min, discard the supernatant, wash the precipitate at the bottom of the tube once with 0.5M NaCl / 1×PBS, add 400 μL of eluent (100 mM Gly-HCl) at pH = 2.2, and place at room temperature for 10 min on a rotating mixer at 20 rpm to fully elute the phage-displayed nanoantibodies bound to the target antigen;

[0089] V. Add 200 μL of neutralizing solution (1M Tris-HCl, pH=8.0) to adjust the pH, then add 5M NaCl solution to a final concentration of 0.5M, incubate in a 30°C water bath for 10 min, centrifuge at 12000 rpm for 10 min, transfer the supernatant to a new 1.5 mL EP tube, and take 10 μL of the eluted supernatant for gradient dilution for titer determination;

[0090] VI. Use 100 μL of the eluted phage display nanobody library for amplification and determine the titer of the amplified phage display library for the next round of panning.

[0091] The third round of selection

[0092] I. The library supernatant after the second round of amplification was blocked with 3% BSA blocking solution, and a total of 5 μg of 4WELP-NT-proBNP protein solution was added and mixed by inversion. A control group was also set up in this round, in which the panning protein as the target antigen was replaced with 1× PBS, and other conditions remained unchanged. The cells were incubated at 37°C on a shaker at 220 rpm for 30 minutes to fully react;

[0093] II. Add 5M NaCl solution to the tube to make the final concentration 0.5M, incubate in a 30℃ water bath for 10min, centrifuge at 30℃, 12000rpm for 20min, remove the supernatant with a pipette, and do not poke the precipitate at the bottom of the tube. Wash the precipitate 10 times with 0.5M NaCl / 1% PBST. After each wash, centrifuge at 30℃, 12000rpm for 2min to remove the supernatant, add 1% PBST solution to resuspend the precipitate, and bathe on ice for 30min to allow the protein to renature and become soluble.

[0094] III. Add 5M NaCl solution to the tube to make the final concentration 0.5M, incubate in 30℃ water bath for 10min, centrifuge at 12000rpm for 10min, discard the supernatant, wash 5 times with 0.5M NaCl / 1% PBST, resuspend the precipitate with 1% PBST solution, and incubate on ice for 30min to renature the protein and make it soluble;

[0095] IV. Add 5M NaCl solution again to a final concentration of 0.5M, incubate in a 30°C water bath for 10 min, centrifuge at 12000 rpm for 10 min, discard the supernatant, wash the precipitate at the bottom of the tube once with 0.5M NaCl / 1×PBS, add 400 μL of eluent (100 mM Gly-HCl) at pH = 2.2, and place at room temperature for 10 min on a rotating mixer at 20 rpm to fully elute the phage-displayed nanoantibodies bound to the target antigen;

[0096] V. Add 200 μL of neutralizing solution (1M Tris-HCl, PH=8.0) to adjust the pH, then add 5M NaCl solution to a final concentration of 0.5M, incubate in a 30°C water bath for 10 min, centrifuge at 12000 rpm for 10 min, transfer the supernatant to a new 1.5 mL EP tube, and take 10 μL of the eluted supernatant for gradient dilution for titer determination;

[0097] VI. Use 100 μL of the eluted phage display nanoantibody library for amplification, measure the titer of the amplified phage display library, and save it for later use; the results of the three rounds of selection are shown in Table 2.

[0098] Table 2 Results of three rounds of panning of phage display library

[0099]

[0100] Reverse screening was used for background subtraction, and phage-displayed nanoantibodies that specifically bind to NT-proBNP were enriched through three rounds of panning. The phage recovery rate in the third round of panning was about 30 times higher than that in the first round.

[0101] (3) Phage ELISA identification

[0102] Polyclonal Phage ELISA

[0103] I. Dilute 4WELP-NT-proBNP protein to 2 μg / mL and coat a 96-well plate, 100 μL / well, with 3 replicates. Seal the 96-well ELISA plate with a sealing film to prevent liquid evaporation and leave it at 4°C overnight;

[0104] II. The next day, pour off the antigen coating solution, wash the plate 5 times with 1×PBS, pat dry on absorbent paper to remove residual liquid in the wells, add 2% skim milk powder (dissolved in 1×PBS), 300 μL / well, and incubate at 37°C for 2 h.

[0105] III. Pour off the blocking solution, then wash the plate 5 times with 1×PBS, pat dry on absorbent paper to remove the residual liquid in the wells, and add a total of 1.0×10 11The phage display library solution (obtained by diluting the phage display library after each round of panning amplification described above with 1×PBS and blocking with 2% skimmed milk powder) was incubated in a 37°C incubator for 2 h;

[0106] IV. Pour off the liquid in the wells, wash the plate 5 times with 0.1% PBST, and then wash the plate 2 times with 1× PBS, and pat dry on absorbent paper to remove the residual liquid in the wells;

[0107] V. Dilute the Anti-M13-HRP antibody (M13 is a filamentous bacteriophage, Anti-M13-HRP is a secondary antibody) at 1:5000 in the blocking solution 1% skim milk powder (dissolved in 1×PBS), add 100 μL / well to the ELISA plate, and let it stand at room temperature for 1.5 hours;

[0108] VI. Pour off the liquid in the wells, wash the plate 5 times with 0.1% PBST solution, and then wash the plate 2 times with 1×PBS. Pat dry on absorbent paper to remove residual liquid in the wells;

[0109] VII. Add 100 μL TMB to each well, protect from light, incubate at 37°C for 10 min, add 50 μL 3M sulfuric acid to each well to terminate the reaction, and read the OD of each well using a microplate reader. 450 The results are shown in Table 3.

[0110] Table 3 Polyclonal Phage ELISA results

[0111]

[0112] Monoclonal Phage ELISA

[0113] I. Take out the phage display nanobody library eluted from the last round of panning from -80℃ and dilute it appropriately. Take 50μL of the phage display nanobody library to infect 100μL of Escherichia coli TG1 cultured to the logarithmic phase, let it stand at 37℃ for 30min, spread it on a 2YTAG (2YT solid medium containing 0.1% Amp and 1% glucose) solid plate, and culture it at 37℃ overnight;

[0114] II. Add 300 μL of 2YTAG (containing 0.1% Amp, 1% glucose) liquid culture medium to each well of a 96-well deep-well plate, inoculate each single colony independently in each well, and culture overnight at 37°C, 200 rpm;

[0115] III. On the second day, the culture solution in the well was pipetted and mixed, and then transferred to a new 96-well deep-well plate containing 300 μL fresh 2YTAG in each well at a ratio of 1:10. The culture was cultured at 37°C and 220 rpm until the logarithmic growth phase. Helper phage M13K07 (about 10 times the bacterial concentration) was added, and the culture was allowed to stand at 37°C for 30 min, and then placed in a 37°C shaker at 200 rpm for 30 min.

[0116] IV. Centrifuge at 4000 rpm for 20 min, discard the supernatant, resuspend the cells in 300 μL 2YTAK (Amp: 100 μg / mL, Kan: 50 μg / mL 2YT liquid medium) fresh medium, and culture at 220 rpm at 30°C overnight;

[0117] V. Dilute 4WELP-NT-proBNP protein to 2 μg / mL, add 100 μL / well to the ELISA plate, and incubate at 4°C overnight;

[0118] VI. On the third day, centrifuge the 96-well deep-well plate at 4000 rpm for 20 min, aspirate the supernatant for monoclonal ELISA, take out the 96-well ELISA plate coated the night before, discard the coating supernatant, pat dry on absorbent paper, and wash 3 times with 1× PBS, 1 min each time;

[0119] VII. Add 2% skim milk powder (dissolved in 1×PBS), 300 μL / well, incubate at 37°C for 1 h, discard the blocking solution, pat dry on absorbent paper, wash 3 times with 1×PBS, 1 min each time, and pat dry;

[0120] VIII. Dilute the monoclonal supernatant in VI by 1-fold with 1% skim milk powder (dissolved in 1×PBS), add 100 μL / well to the ELISA plate, and shake slowly at 37°C for 1 hour; discard the supernatant, pat dry, wash 5 times with 0.1% PBST, wash once with 1×PBS, and pat dry;

[0121] IX. Anti-M13-HRP antibody was diluted 1:5000 in blocking solution 1% skim milk powder (dissolved in 1×PBS), 100 μL / well was added to the ELISA plate, and allowed to stand at 37°C for 1 hour, the solution was discarded, and the plate was patted dry. The plate was washed 5 times with 0.1% PBST, washed once with 1×PBS, and patted dry.

[0122] X. Add 100 μL TMB to each well, incubate at 37°C in the dark for 10 min, add 50 μL 3M sulfuric acid to each well to terminate the reaction, and read the OD of each well using an ELISA reader. 450 The value, the result is Figure 4As shown, 14 phage-displayed nanobodies with good affinity were obtained by Phage ELISA detection of 40 monoclonal antibodies (numbered as: 5, 9, 11, 12, 14, 17, 18, 21, 25, 27, 28, 33, 34, 35).

[0123] The present invention designs a recombinant plasmid, expresses and purifies 4WELP-NT-proBNP protein as a panning protein, establishes a novel liquid phase panning method based on this, adopts reverse screening for background subtraction, enriches phage-displayed nano antibodies specifically binding to NT-proBNP through three rounds of panning, the phage recovery rate of the third round of panning is increased by about 30 times compared with that of the first round of panning, polyclonal Phage ELISA verifies the enrichment results of the three rounds of panning, 40 monoclonal Phage ELISA tests obtain 14 phage-displayed nano antibodies with good affinity, and the gene sequences obtained by sequencing will be used as nano antibody candidate genes, which can be used for the subsequent expression and evaluation of NT-proBNP nano antibodies.

[0124] The above-described embodiments are only preferred embodiments of the present invention, but not all feasible embodiments of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A recombinant negative sieve protein with elastic tag, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A recombinant panning protein with an elastic tag, characterized in that: The elastic-like tag in the elastic-like tag recombinant panning protein is connected to the antigen via a flexible chain, and the antigen includes the heart failure biomarker NT-proBNP.

3. The elastic tag-like recombinant panning protein according to claim 2, characterized in that: The amino acid sequence of the elastic tag-like recombinant panning protein is shown in SEQ ID NO.

3.

4. A recombinant expression plasmid into which is inserted a gene encoding the elastic-tagged recombinant negative screening protein of claim 1 or the elastic-tagged recombinant panning protein of claim 2 or 3.

5. An engineered bacterium carrying the recombinant expression plasmid according to claim 4.

6. The method for preparing the elastic-like recombinant negative screening protein of claim 1 or the elastic-like recombinant panning protein of claim 2 or 3, characterized in that: The recombinant expression plasmid described in claim 4 is transformed into Escherichia coli BLR (DE3) competent cells by heat shock method, positive single clones are screened by kanamycin, single clones are picked for expansion culture and induced to express recombinant protein, and after Escherichia coli BLR (DE3) is broken, reversible phase change cycle purification is performed to obtain elastic tag-like recombinant negative screening protein or elastic tag-like recombinant panning protein.

7. The preparation method according to claim 6, characterized in that: The induced expression conditions are automatic induction culture based on 25°C induction expression for 22 to 24 hours; the reversible phase change cycle purification process is: 0.5M NaCl, 30 to 32°C water bath for 15 to 25 minutes to denature the protein, 30 to 32°C, 10000 to 12000 rpm, centrifugation for 5 to 15 minutes to obtain a protein precipitate, and then use 0.2mol / L phosphate buffer to resuspend the precipitate, ice bath for 15 to 25 minutes to renature the protein, 4°C, 10000 to 12000 rpm centrifugation for 5 to 15 minutes to obtain a protein solution, and a total of 3 rounds of reversible phase change cycle purification are performed.

8. A method for panning a phage display library based on elastic tag-like recombinant protein, characterized in that: First, the background of the phage-displayed nanoantibody library constructed by immunization of alpaca is subtracted by using the recombinant negative screening protein of the elastic tag described in claim 1, and then the phage-displayed nanoantibody library after negative screening is mixed with the solution of the recombinant panning protein of the elastic tag described in claim 2 or 3 to allow the antigen and antibody to bind. During the reversible phase change cycle of the recombinant panning protein of the elastic tag, centrifugation and washing are repeated to remove unbound phage-displayed nanoantibodies and weakly bound or non-specifically bound products. The specifically bound phage-displayed nanoantibodies are eluted from the target antigen using an eluent, and the recombinant panning protein of the elastic tag is denatured and precipitated after adjusting the pH value to neutral. After separation, a phage-displayed nanoantibody solution is obtained. The obtained phage-displayed nanoantibody solution is mixed again with the solution of the recombinant panning protein of the elastic tag and the above panning process is repeated. A phage-displayed nanoantibody library that can specifically bind to the target antigen is obtained through multiple rounds of panning. After polyclonal and monoclonal Phage ELISA detection, a high-affinity phage-displayed nanoantibody that can specifically bind to the target antigen is obtained.

9. The phage display library panning method according to claim 8, characterized in that: The number of rounds of panning is 3; The parameters for the first round of selection were: antigen input 5-15 μg, total phage library input 2×10 11 ~9×10 11 , antigen-antibody binding time 40-60min, 0.5M NaCl, 30-32℃ water bath 5-15min denaturation, 30-32℃, 10000-12000rpm, centrifugation 5-15min, 0.5M NaCl / 0.1% PBST washing 9-11 times, 0.1% PBST resuspending precipitate, ice bath 20-40min renaturation; The parameters for the second round of selection were: antigen input 5-15 μg, total phage library input 2×10 11 ~9×10 11 , antigen-antibody binding time 20-40min, 0.5M NaCl, 30-32℃ water bath 5-15min denaturation, 30-32℃, 10000-12000rpm, centrifugation 5-15min, 0.5M NaCl / 0.5% PBST washing precipitate 14-16 times, 0.5% PBST resuspending precipitate, ice bath 20-40min renaturation; The parameters for the third round of selection were: antigen input 3-7 μg, total phage library input 2×10 11 ~9×10 11 , antigen-antibody binding time 20-40min, denaturation in 0.5M NaCl, 30-32℃ water bath for 5-15min, centrifugation at 30-32℃, 10000-12000rpm for 5-15min, wash the precipitate with 0.5M NaCl / 1% PBST 14-16 times, resuspend the precipitate with 1% PBST, and renature in ice bath for 20-40min.

10. Use of the elastic-tagged recombinant negative screening protein of claim 1, the elastic-tagged recombinant panning protein of claim 2 or 3, and the phage display library panning method of claim 8 or 9 in panning of phage display nanoantibody libraries.

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