Nano antibody fusion expression system based on escherichia coli and preparation method thereof

Abstract

The invention relates to the technical field of biological pharmacy, in particular to an escherichia coli-based nano antibody fusion expression system and a preparation method thereof. The nano-antibody sequence VHH is an expression sequence SEQ ID NO.1, pET39b is used as a carrier to construct and express the nano-antibody, and the C terminal of the nano-antibody is respectively connected with a His tag. Soluble high expression of the nano-antibody can be realized, the finally obtained nano-antibody has the characteristics of uniform structure, no residual tag sequence and high expression quantity, and the expression quantity of 5.6 mg / g thalli can be finally realized based on the 581 nano-antibody. The expression system provides a platform technology for the establishment of an early screening system of the nano-antibody and the industrialization of the nano-antibody in the future.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and more specifically, to an Escherichia coli-based nano-antibody fusion expression system and a preparation method thereof. Background Art

[0002] Since the birth of the first monoclonal antibody in 1975, more and more antibodies (such as chimeric antibodies, humanized antibodies, and fully human antibodies) have been used in the diagnosis and treatment of various diseases, showing an expanding application prospect. Camels and sharks naturally have a unique antibody lacking light chains, called variable heavy chain domains, also known as nanobodies (VHH). Compared with the complex processing of traditional antibodies, VHH has the characteristics of smaller size (12-15kDa) and simpler structure, and has better research and development application value in laboratory environments. For example, VHH can be used as an immune recognition antibody, molecular probe, or molecular imaging tool.

[0003] At present, there are many ways to express and produce nanobodies in vitro: such as Escherichia coli, Pichia pastoris, fungi, mammalian cells, plant cells, etc. The yeast expression system is carried out by secretory expression, but yeast has serious problems of protease-mediated degradation and post-translational modification, which is not conducive to downstream processes. Mammalian cells such as HEK293, CHO, etc. are one of the suitable expression systems for nanobodies, but their expression systems are complicated to operate and costly, and are not suitable for early process development. Escherichia coli mainly expresses soluble expression through extracellular periplasmic secretion expression. There are literature reports that the expression amount can be increased by optimizing the signal peptide, but the periplasmic space of Escherichia coli is small and the expression amount is limited. Escherichia coli gene operation is simple and inexpensive. However, Escherichia coli lacks the necessary post-translational folding and disulfide bond formation mechanisms, and most exogenous proteins are expressed in the form of inclusion bodies. Although the expression product can obtain active proteins through downstream processes such as denaturation, the denaturation process is still not applicable to early screening.

[0004] Although nanobodies have a simple structure, using the E. coli expression system and introducing signal peptides to localize the expression products to the extracellular periplasm for soluble secretion is a common strategy at present. However, due to the lack of chaperone proteins, inefficient translocation of the inner membrane, and the small extracellular periplasm space, the upper limit of its production is far lower than expected. Establishing a nanobodies expression system with simple process, high purity and high expression is an urgent problem to be solved. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, one aspect of the present invention is to provide a nanobody fusion expression system based on Escherichia coli, wherein the nanobody sequence VHH is the expression sequence SEQ ID NO.1, and pET39b is used as a vector to construct and express the nanobody, and His tags are respectively connected to the C-termini of the nanobody.

[0006] Preferably, the nanobody fusion is to add a protein tag and / or an enzyme cleavage site to the expression sequence SEQ ID NO.1.

[0007] Preferably, the restriction site is DDDDK.

[0008] Preferably, the restriction sites include EK enzyme and TEV protease.

[0009] Another aspect of the present invention is to provide a method for preparing a nanobody fusion expression system based on Escherichia coli, wherein the specific steps of the preparation method are:

[0010] S1. After the bacterial culture and induction expression are completed, the E. coli cells are collected, and the soluble expressed protein is crushed and the supernatant is collected and directly purified;

[0011] S2. The collected supernatant solution is purified by affinity chromatography, desalted preparation and enzymatic cleavage, and affinity purification of the enzymatic cleavage product, and finally a nano-antibody with good uniformity and purity is obtained.

[0012] Preferably, the bacterial culture in S1 is to inoculate the recombinant expression vector into 2YT+G medium (containing Kana + ) and cultured at 37°C and 220 rpm; the induced expression was that during the bacterial culture process, when the OD600nm value was detected to reach 0.6-0.8, IPTG with a final concentration of 1 mM / mL was added to induce the expression of the recombinant nanoantibody, and the induction conditions were: 20°C, 16 hours.

[0013] Preferably, the E. coli cells are collected in S1 by taking 500 μL of the cultured cells and centrifuging at 4° C. and 12,000 rpm for 1 minute after the cells are cultured and induced to express, discarding the supernatant, disrupting the cells and detecting the protein expression by SDS PAGE.

[0014] Preferably, the affinity chromatography purification in S2 is to dilute the collected supernatant sample solution, filter and load it, first rinse it with an equilibrium buffer, then elute it with an elution buffer, collect the elution peak, and obtain a purified nanobody fusion protein with an affinity tag.

[0015] Preferably, the equilibration buffer has a pH value of 8.5, and is composed of a solvent and a solute, wherein the solvent is water, and the solute and its concentration are: 20 mM Tris-HCl, 300 mM NaCl; the elution buffer has a pH value of 8.5, and is composed of a solvent and a solute, wherein the solvent is water, and the solute and its concentration are: 20 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole.

[0016] Preferably, the desalting preparation and enzyme digestion in S2 are to desalt the affinity purified sample into an enzyme digestion buffer, detect the sample concentration by ultraviolet spectrophotometer A280nm, and use EK enzyme for enzyme digestion, the enzyme digestion ratio of EK enzyme: protein = 1:50 to 1:500 (mg:mg), and the enzyme digestion is performed at 20°C and 37°C for 14-18 hours to obtain the recombinant nanobody after enzyme digestion; the pH value of the enzyme digestion buffer is 8.0, and it is composed of a solvent and a solute; the solvent is water, and the solute and its concentration are: 20mM Tris-HCl, 50mMNaCl, 2mM CaCl 2 .

[0017] The beneficial effects of the present invention are as follows:

[0018] The nanobody expression system based on Escherichia coli of the present invention can achieve high soluble expression of nanobodies, and the nanobodies finally obtained have the characteristics of uniform structure, no residual tag sequence, and high expression amount. Based on 581 nanobodies, the expression amount of 5.6 mg / g of bacteria can be finally achieved. The expression system of the present invention provides a platform technology for the establishment of an early screening system for nanobodies and the future industrialization of nanobodies.

[0019] Additional aspects and advantages of the invention will become apparent from the following description, or may be learned by practice of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0021] Figure 1 This is the SDS-PAGE electrophoresis pattern of the recombinant nanobody connected to the His tag in Example 1 of the present invention;

[0022] Figure 2 This is the SDS-PAGE electrophoresis pattern of the recombinant nanobody connected to His+SUMO in Example 1 of the present invention;

[0023] Figure 3 This is the SDS-PAGE electrophoresis pattern of SUMO-581 affinity purification in Example 2 of the present invention;

[0024] Figure 4This is the SDS-PAGE electrophoresis pattern of the SUMO-581 affinity purification elution sample digestion in Example 2 of the present invention;

[0025] Figure 5 This is the SDS-PAGE electrophoresis pattern of affinity purification of DsbA-EK-581 in Example 2 of the present invention;

[0026] Figure 6 This is the SDS-PAGE electrophoresis pattern of the affinity purification elution sample of DsbA-EK-581 in Example 2 of the present invention;

[0027] Figure 7 This is the SDS-PAGE electrophoresis pattern of affinity purification of DsbA-EK-581-Flag in Example 3 of the present invention;

[0028] Figure 8 This is the SDS-PAGE electrophoresis pattern of the DsbA-EK-581-Flag affinity purification elution sample digestion in Example 3 of the present invention;

[0029] Fig. 9 This is the SDS-PAGE electrophoresis pattern of affinity purification after DsbA-EK-581-Flag digestion in Example 3 of the present invention;

[0030] Fig.10 This is the affinity purification chromatogram after DsbA-EK-581-Flag digestion in Example 3 of the present invention;

[0031] Fig.11 This is the mass spectrum of the affinity purified sample after DsbA-EK-581-Flag digestion in Example 3 of the present invention;

[0032] Fig.12 This is the ELISA activity determination of the affinity purified sample after DsbA-EK-581-Flag digestion in Example 3 of the present invention. DETAILED DESCRIPTION

[0033] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0034] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from the description. Therefore, the protection scope of the present invention is not limited to the specific embodiments disclosed below.

[0035] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0037] IPTG is a product of MacLean, catalog number 367-93-1;

[0038] The pET28b vector in the following examples is a product of Novagen, with a product catalog number of 69865-3.

[0039] The pET39b vector in the following examples is a product of Novagen, with a product catalog number of 70090-3.

[0040] The relevant culture medium and solution in the following examples are as follows:

[0041] LB medium: Yeast extract 5 g, Tryptone 10 g, NaCl 10 g, pH 7.0, dilute to 1 L, sterilize at 121°C for 20 min.

[0042] 2YT+G medium: Yeast extract 30 g, Tryptone 10 g, NaCl 5 g, Glycerol 10 g, pH 7.0, dilute to 1 L, sterilize at 121°C for 20 min.

[0043] 2YT+G medium (containing Kana + ): Yeast extract 30g, Tryptone 10g, NaCl 5g, Glycerol 10g, pH 7.0, dilute to 1L, sterilize at 121℃ for 20min, add kanamycin with a final concentration of 50ug / mL after sterilization and cooling.

[0044] The pH value of the equilibrium buffer is 8.5, and it is composed of a solvent and a solute. The solvent is water, and the solute and its concentration are: 20mM Tris-HCl, 300mM NaCl.

[0045] The elution buffer has a pH value of 8.5 and is composed of a solvent and a solute. The solvent is water, and the solute and its concentration are: 20 mM Tris-HCl, 300 mM NaCl, and 500 mM imidazole.

[0046] The pH value of the digestion buffer is 8.0, and it consists of a solvent and a solute. The solvent is water, and the solutes and their concentrations are: 20mM Tris-HCl, 50mM NaCl, 2mM CaCl 2 .

[0047] Example 1. Nanobody linked to His tag and His+SUMO tag

[0048] The present invention uses phage display technology to screen the nano antibody sequence VHH obtained from the constructed nano antibody library by immunizing alpacas, uses pET28b as a vector to construct and express nano antibodies, and connects His tags to the N-terminal of the nano antibodies to obtain sequences shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4. At the same time, the recombinant protein is expressed in Escherichia coli.

[0049] The His-tagged recombinant nanobody was cultured in 5 mL LB medium in the E. coli BL21 (DE3) expression strain at 37°C and 220 rpm. When the OD600nm value reached 0.6-0.8, IPTG with a final concentration of 1 mM / mL was added to induce the expression of the recombinant nanobody. The induction conditions were: 20°C for 16 hours. After the culture was completed, 500 μL was taken and centrifuged at 4°C and 12,000 rpm for 1 minute, the supernatant was discarded, the bacteria were broken and the protein expression was detected by SDS PAGE.

[0050] The nanobody was connected to a His+SUMO tag and expressed in E. coli BL21 (DE3) using pET SUMO as a vector. The sequences shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 were obtained by connecting the His+SUMO tag. At the same time, the recombinant protein was expressed in E. coli.

[0051] The His+SUMO-linked recombinant nanobody expression strain was cultured in 5 mL LB medium at 37°C and 220 rpm. When the OD600nm value reached 0.6-0.8, IPTG with a final concentration of 1 mM / mL was added to induce the expression of the recombinant nanobody. The induction conditions were: 20°C for 16 hours. After the culture was completed, 500 μL was taken and centrifuged at 4°C and 12,000 rpm for 1 minute, the supernatant was discarded, the bacteria were broken and the protein expression was detected by SDS PAGE.

[0052] The results showed that the SDS-PAGE assay showed that Figure 1 and Figure 2 shown.

[0053] Figure 1It is the SDS-PAGE electrophoresis profile of the recombinant nanobody connected to the His tag. Lane 1-BI is the whole bacterial protein of AnHSA before induction, lane 1-Sup is the supernatant of ultrasonic crushing and centrifugation of AnHSA after induction, lane 1-IBs is the precipitate of ultrasonic crushing and centrifugation of AnHSA after induction, lane Marker is the protein molecular weight standard with a molecular weight of 10-180kD, lane 2-BI is the whole bacterial protein of 156 before induction, lane 2-Sup is the supernatant of ultrasonic crushing and centrifugation of 156 after induction, lane 2-IBs is the precipitate of ultrasonic crushing and centrifugation of 156 after induction, lane 3-BI is the whole bacterial protein of 581 before induction, lane 3-Sup is the supernatant of ultrasonic crushing and centrifugation of 581 after induction, and lane 3-IBs is the precipitate of ultrasonic crushing and centrifugation of 581 after induction.

[0054] Figure 2 The figure is the SDS-PAGE electrophoresis of the recombinant nanoantibody connected to His+SUMO. The lane marker is a protein molecular weight standard with a molecular weight of 10-180kD, the lane 1-BI is the whole bacterial protein of sumo-581 before induction, the lane 1-Sup is the supernatant of the ultrasonic crushing and centrifugation of sumo-581 after induction, the lane 1-IBs is the precipitate of the ultrasonic crushing and centrifugation of sumo-581 after induction, the lane 2-BI is the whole bacterial protein of sumo-156 before induction, the lane 2-Sup is the supernatant of the ultrasonic crushing and centrifugation of sumo-156 after induction, the lane 2-IBs is the precipitate of the ultrasonic crushing and centrifugation of sumo-156 after induction, the lane 3-BI is the whole bacterial protein of sumo-AnHSA before induction, the lane 3-Sup is the supernatant of the ultrasonic crushing and centrifugation of sumo-AnHSA after induction, and the lane 3-IBs is the precipitate of the ultrasonic crushing and centrifugation of sumo-AnHSA after induction.

[0055] Compared with the His+SUMO recombinant nanoantibody, the expression level of the target protein His-tagged recombinant nanoantibody produced by the His-tagged recombinant nanoantibody is significantly lower than that of the Sumo fusion expression method as shown in Table 1 below, and some target proteins are expressed in inclusion bodies.

[0056] Table 1. Summary and comparison of 581 nanobody expression levels

[0057]

[0058] Example 2. Nanobody linked to DsbA+His tag

[0059] The present invention uses phage display technology to screen and obtain nano antibody sequence VHH from a constructed nano antibody library by immunizing alpacas, uses pET39b as a vector to construct and express nano antibodies, connects a DsbA tag to the N-terminus of the nano antibody, adds an EK restriction site between the tag and the nano antibody, and connects a His tag to the C-terminus, thereby obtaining a sequence as shown in SEQ ID NO. 8. At the same time, the recombinant protein is expressed in Escherichia coli.

[0060] The DsbA+His recombinant nanobody expression strain was cultured in 5 mL LB medium at 37°C and 220 rpm. When the OD600nm value reached 0.6-0.8, IPTG with a final concentration of 1 mM / mL was added to induce the expression of the recombinant nanobody. The induction conditions were: 20°C, 16 hours. After the culture was completed, 500 μL was taken and centrifuged at 4°C and 12,000 rpm for 1 minute, the supernatant was discarded, the bacteria were broken and the protein expression was detected by SDS PAGE.

[0061] The DsbA+His recombinant nanobody expression strain was cultured in 2L 2YT+G medium at 37°C and 220 rpm. When the OD600nm value reached 0.6-0.8, IPTG was added at a final concentration of 1mM / mL to induce the expression of the recombinant nanobody. The induction conditions were: 20°C, 16 hours. After the bacterial culture and induced expression were completed, the Escherichia coli cells were collected, and the soluble expressed protein was directly purified after being crushed and the supernatant was collected. The collected supernatant solution was subjected to the purification steps of affinity chromatography (for example, using a GE HisTrap FF chromatography column), desalting enzyme cleavage (for example, using a Sephadex G-25Fine chromatography column), and enzyme penetration (for example, using a GE HisTrapFF chromatography column).

[0062] 1) In affinity chromatography purification, the collected supernatant sample solution is diluted, filtered and loaded, first washed with an equilibrium buffer, then eluted with an elution buffer, and the elution peak is collected to obtain a purified recombinant protein with an affinity tag.

[0063] 2) Desalting the affinity purified sample into the enzyme digestion buffer, detecting the sample concentration by UV spectrophotometer A280nm, and digesting with EK enzyme, EK enzyme: protein = 1:500-1:2000 (mg:mg) digestion ratio, digesting at 4°C, 20°C, and 30°C for 14-18 hours to obtain the recombinant nanoantibody after digestion, and selecting the best way for digestion.

[0064] The nanobody was connected to a polycistronic DsbA+His tag to obtain a sequence as shown in SEQ ID NO.9. pET39b was used as a vector for expression in Escherichia coli BL21 (DE3). The recombinant nanobody expression strain connected to the polycistronic DsbA+His tag was cultured in 5 mL LB medium at 37°C and 220 rpm. When the OD600nm value reached 0.6-0.8, IPTG was added at a final concentration of 1 mM / mL to induce the expression of the recombinant nanobody, and the induction conditions were: 20°C for 16 hours. After the culture was completed, 500 μL was taken and centrifuged at 4°C and 12,000 rpm for 1 minute, the supernatant was discarded, the bacteria were broken and the protein expression was detected by SDS PAGE.

[0065] The recombinant nanobody expression strain connected to the polycistronic DsbA+His tag is cultured in 2L 2YT+G medium at 37°C and 220 rpm. When the OD600nm value reaches 0.6-0.8, IPTG with a final concentration of 1mM / mL is added to induce the expression of the recombinant nanobody, and the induction conditions are: 20°C, 16 hours. After the bacterial culture and induced expression are completed, the Escherichia coli cells are collected, and the soluble expressed protein is directly purified after being crushed and the supernatant is collected. The collected supernatant solution is subjected to the purification steps of affinity chromatography (for example, using a GE HisTrap FF chromatography column), desalting enzyme digestion (for example, using a Sephadex G-25Fine chromatography column), and enzyme penetration (for example, using a GE HisTrap FF chromatography column).

[0066] 1) In affinity chromatography purification, the collected supernatant sample solution is diluted, filtered and loaded, first washed with an equilibrium buffer, then eluted with an elution buffer, and the elution peak is collected to obtain a purified recombinant protein with an affinity tag.

[0067] 2) The affinity purified sample was desalted into enzyme cleavage buffer, the sample concentration was detected by UV spectrophotometer A280nm, and EK enzyme was used for enzyme cleavage. Different enzyme cleavage conditions, different temperatures, enzyme amounts, buffer systems and denaturation conditions were tried. The construction was successfully cleaved by EK enzyme at an enzyme cleavage ratio of EK enzyme: protein = 1:500 (mg:mg) and 20°C, and the tag was successfully cleaved from the nanoantibody.

[0068] The results showed that the SDS-PAGE detection results were as follows Figure 3-6 shown.

[0069] Figure 3The SDS-PAGE electrophoresis pattern of sumo-581 affinity purification. Lane 581-2 is the sumo-581 supernatant washing stock solution, lane FT is the sumo-581 affinity purification flow-through sample, lane M is the protein molecular weight standard with a molecular weight of 10-180 kD, lane EL1 is the sumo-581 affinity purification 0-10% B solution elution sample, lane EL2 is the sumo-581 affinity purification 11%-35% B solution elution sample, lane EL3 is the sumo-581 affinity purification 36%-70% B solution elution sample, and lane EL4 is the sumo-581 affinity purification 71%-100% B solution elution sample.

[0070] Figure 4 The SDS-PAGE electrophoresis of the eluted sample of SUMO-581 affinity purification was shown in Figure 1. Lane 4℃, 1:500 is the eluted sample of SUMO-581 purified by 500-fold dilution at 4℃, lane 4℃, 1:1000 is the eluted sample of SUMO-581 purified by 1000-fold dilution at 4℃, lane 4℃, 1:2000 is the eluted sample of SUMO-581 purified by 2000-fold dilution at 4℃, lane M is the protein molecular weight standard with a molecular weight of 10-180kD, lane 20℃, 1:500 is the eluted sample of SUMO-581 purified by 500-fold dilution at 20℃, lane 20℃, 1:1000 is the The purified elution sample of SUMO-581 was digested with 1000-fold dilution of Sumo enzyme at 20℃, the purified elution sample of SUMO-581 was digested with 2000-fold dilution of Sumo enzyme at 20℃, the purified elution sample of SUMO-581 was digested with 500-fold dilution of Sumo enzyme at 30℃, the purified elution sample of SUMO-581 was digested with 1000-fold dilution of Sumo enzyme at 30℃, the purified elution sample of SUMO-581 was digested with 1000-fold dilution of Sumo enzyme at 30℃, and the purified elution sample of SUMO-581 was digested with 1:2000-fold dilution of Sumo enzyme at 30℃.

[0071] Figure 5This is the SDS-PAGE electrophoresis pattern of affinity purification of DsbA-EK-581. Lane 581-4 is the DsbA-EK-581 supernatant washing stock solution, lane FT is the DsbA-EK-581 affinity purification flow-through sample, lane M is the protein molecular weight standard with a molecular weight of 10-180kD, lane EL1 is the DsbA-EK-581 affinity purification 0-20% B solution elution sample, lane EL2 is the DsbA-EK-581 affinity purification 21%-45% B solution elution sample, lane EL3 is the DsbA-EK-581 affinity purification 46%-65% B solution elution sample, lane EL4 is the DsbA-EK-581 affinity purification 66%-85% B solution elution sample, and lane EL5 is the DsbA-EK-581 affinity purification 86%-100% B solution elution sample.

[0072] Figure 6 The figure is the SDS-PAGE electrophoresis of the DsbA-EK-581 affinity purified elution sample digested with enzyme. The lane 4℃, 1:500 is the purified elution sample of DsbA-EK-581 digested with EK enzyme 500 times diluted at 4℃, the lane 4℃, 1:1000 is the purified elution sample of DsbA-EK-581 digested with EK enzyme 1000 times diluted at 4℃, the lane 4℃, 1:2000 is the purified elution sample of DsbA-EK-581 digested with EK enzyme 2000 times diluted at 4℃, the lane M is the protein molecular weight standard with molecular weight of 10-180kD, the lane 20℃, 1:500 is the purified elution sample of DsbA-EK-581 digested with EK enzyme 500 times diluted at 20℃, the lane 20℃, 1:1000 is the purified elution sample of DsbA-EK-581 digested with EK enzyme The enzyme was diluted 1000 times and the purified elution sample of DsbA-EK-581 was digested at 20℃, the lane 20℃, 1:2000 is the purified elution sample of DsbA-EK-581 digested at 20℃ by EK enzyme at 2000 times dilution, the lane 30℃, 1:500 is the purified elution sample of DsbA-EK-581 digested at 30℃ by EK enzyme at 500 times dilution, the lane 30℃, 1:1000 is the purified elution sample of DsbA-EK-581 digested at 30℃ by EK enzyme at 1000 times dilution, the lane 30℃, 1:2000 is the purified elution sample of DsbA-EK-581 digested at 30℃ by EK enzyme at 2000 times dilution.

[0073] Compared with the recombinant nanoantibody with polycistronic DsbA+His tags, the recombinant nanoantibody with polycistronic DsbA+His tags successfully cut the tag from the nanoantibody at an enzyme cleavage ratio of EK enzyme: protein = 1:500 (mg:mg) and 20°C, while the recombinant nanoantibody with DsbA+His tags was not cut under various enzyme cleavage conditions.

[0074] Example 3. Nanobody linked to DsbA+His+Flag tag

[0075] The present invention uses phage display technology to screen and obtain nano antibody sequence VHH from a constructed nano antibody library by immunizing alpacas, uses pET39b as a vector to construct and express nano antibodies, inserts DsbA and EK restriction sites at the N-terminus of the nano antibody, and connects His and Flag tags at the C-terminus, respectively, to obtain a sequence as shown in SEQ ID NO. 1. At the same time, the recombinant protein is expressed in Escherichia coli.

[0076] The DsbA+His+Flag recombinant nanobody expression strain was cultured in 5 mL LB medium at 37°C and 220 rpm. When the OD600nm value reached 0.6-0.8, IPTG with a final concentration of 1 mM / mL was added to induce the expression of the recombinant nanobody. The induction conditions were: 20°C, 16 hours. After the culture was completed, 500 μL was taken and centrifuged at 4°C and 12,000 rpm for 1 minute, the supernatant was discarded, the bacteria were broken and the protein expression was detected by SDS PAGE.

[0077] The DsbA+His+Flag recombinant nanobody expression strain was cultured in 2L 2YT+G medium at 37°C and 220 rpm. When the OD600nm value reached 0.6-0.8, IPTG with a final concentration of 1mM / mL was added to induce the expression of the recombinant nanobody. The induction conditions were: 20°C, 16 hours. After the bacterial culture and induced expression were completed, the Escherichia coli cells were collected, and the soluble expressed protein was directly purified after being crushed and the supernatant was collected.

[0078] The collected supernatant solution is subjected to the purification steps of affinity chromatography (eg, using a GE HisTrap FF chromatography column), desalting enzyme cleavage (eg, using a Sephadex G-25Fine chromatography column), and enzyme penetration (eg, using a GE HisTrap FF chromatography column).

[0079] 1) In affinity chromatography purification, the collected supernatant sample solution is diluted, filtered and loaded, first washed with an equilibrium buffer, then eluted with an elution buffer, and the elution peak is collected to obtain a purified recombinant protein with an affinity tag.

[0080] 2) Desalting the affinity purified sample into the enzyme digestion buffer, detecting the sample concentration by UV spectrophotometer A280nm, and digesting with EK enzyme, EK enzyme: protein = 1:50 to 1:500 (mg:mg), digesting at 20°C and 30°C for 14-18 hours to obtain the recombinant nanobody after digestion, and selecting the best way for digestion (e.g., EK enzyme: protein = 1:250, 20°C).

[0081] 3) After the digested sample is filtered and loaded, it is first washed with an equilibrium buffer, and the eluted sample is collected, which is the recombinant nanobody after digestion, and the result is detected by SDS PAGE.

[0082] 4) The antibody concentration of the sample after enzyme digestion and purification was determined by BCA, and the molecular weight of the nanobody was analyzed by mass spectrometry. The molecular weight of the nanobody was analyzed by high-resolution mass spectrometry, ultra-performance liquid chromatography and deconvolution software, and was 17206.5 Da, which was consistent with expectations.

[0083] 5) Coat the target antigen at an appropriate concentration on the ELISA plate, block the non-specific wells with 1% BSA, add the nanoantibodies after gradient serial dilutions, and use the secondary antibody labeled with rabbit anti-Flag peroxidase to analyze the binding activity of the nanoantibody by indirect ELISA.

[0084] The results showed that the SDS-PAGE assay showed that Figure 7-10 shown.

[0085] Figure 7 The SDS-PAGE electrophoresis pattern of DsbA-EK-581-Flag affinity purification. Lane M is a protein molecular weight standard with a molecular weight of 10-180 kD, lanes 581-5 are DsbA-EK-581-Flag supernatant wash stock solution, lane FT is DsbA-EK-581-Flag affinity purification flow-through sample, lane EL1 is DsbA-EK-581-Flag affinity purification 0-20% B solution elution sample, lane EL2 is DsbA-EK-581-Flag affinity purification 21%-50% B solution elution sample, lane EL3 is DsbA-EK-581-Flag affinity purification 51%-75% B solution elution sample, and lane EL4 is DsbA-EK-581-Flag affinity purification 76%-100% B solution elution sample.

[0086] Figure 8The SDS-PAGE electrophoresis of the DsbA-EK-581-Flag affinity purified elution sample digested. Lane M (left) is a protein molecular weight standard with a molecular weight of 10-180kD, the lane before digestion (left) is the DsbA-EK-581-Flag affinity purified elution sample, the lane 20℃, 1:50 is the EK enzyme 50 times diluted and digested the DsbA-EK-581-Flag purified elution sample at 20℃, the lane 20℃, 1:150 is the EK enzyme 150 times diluted and digested the DsbA-EK-581-Flag purified sample at 20℃ Elution sample, lane 20℃, 1:250 is the purified elution sample of DsbA-EK-581-Flag digested with EK enzyme at 20℃ with 250 times dilution, lane 20℃, 1:350 is the purified elution sample of DsbA-EK-581-Flag digested with EK enzyme at 20℃ with 350 times dilution, lane 20℃, 1:500 is the purified elution sample of DsbA-EK-581-Flag digested with EK enzyme at 20℃ with 500 times dilution, Lane M (right) is a protein molecular weight standard with a molecular weight of 10-180 kD. Lane M before enzyme digestion (right) is the elution sample of DsbA-EK-581-Flag affinity purification. Lane 37℃, 1:50 is the elution sample of DsbA-EK-581-Flag purified by EK enzyme 50 times diluted at 37℃. Lane 37℃, 1:150 is the purification of DsbA-EK-581-Flag by EK enzyme 150 times diluted at 37℃. Elution sample, lane 37℃, 1:250 is the purified elution sample of DsbA-EK-581-Flag digested with EK enzyme at 250 times dilution at 37℃, lane 37℃, 1:350 is the purified elution sample of DsbA-EK-581-Flag digested with EK enzyme at 350 times dilution at 37℃, lane 37℃, 1:500 is the purified elution sample of DsbA-EK-581-Flag digested with EK enzyme at 500 times dilution at 37℃.

[0087] Fig. 9The SDS-PAGE electrophoresis of affinity purification after DsbA-EK-581-Flag digestion. Lane M is a protein molecular weight standard with a molecular weight of 10-180kD, lane before digestion is the purified elution sample before DsbA-EK-581-Flag digestion, lane digestion is the EK digestion sample after DsbA-EK-581-Flag purification, lane FT1 is the affinity purification flow-through sample 1 after DsbA-EK-581-Flag digestion, lane FT2 is the affinity purification flow-through sample 2 after DsbA-EK-581-Flag digestion, and lane EL1 is Lane EL2 is the sample eluted from B solution with 0%-21% affinity purification after DsbA-EK-581-Flag digestion, lane EL3 is the sample eluted from B solution with 48%-70% affinity purification after DsbA-EK-581-Flag digestion, lane EL4 is the sample eluted from B solution with 71%-100% affinity purification after DsbA-EK-581-Flag digestion.

[0088] Fig.10 The left ordinate is the absorbance value (A280nm), the right ordinate is the percentage (%) of elution buffer B, and the abscissa is the affinity purification sample volume (mL).

[0089] The recombinant nanobody connected to the DsbA+His+Flag tag was successfully cleaved from the tag by EK enzyme: protein = 1:250 (mg:mg) at 20°C. The results of mass spectrometry analysis are as follows Fig.11 shown.

[0090] Fig.11 This is the mass spectrum of the affinity purified sample after DsbA-EK-581-Flag digestion. The left ordinate is the mass spectrum intensity value, and the abscissa is the molecular mass (Da).

[0091] The molecular weight of the recombinantly expressed nanobody is 17206.5Da, and the specific binding capacity of the nanobody ELISA analysis is 234ng / mL. Fig.12 shown.

[0092] Fig.12 The left ordinate is the absorbance value (OD450nm), and the abscissa is the concentration (ng / mL) of the affinity purified sample after DsbA-EK-581-Flag digestion.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may also have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A nanobody fusion expression system based on Escherichia coli, characterized in that: The nanobody sequence VHH is the expression sequence SEQ ID NO.1, pET39b is used as a vector to construct and express the nanobody, and a His tag is connected to the C-terminus of the nanobody.

2. The nanobody fusion expression system based on Escherichia coli according to claim 1, characterized in that: The nanobody fusion is to add a protein tag and / or an enzyme cleavage site to the expression sequence SEQ ID NO.

1.

3. The nanobody fusion expression system based on Escherichia coli according to claim 2, characterized in that: The restriction site is DDDDK.

4. The nanobody fusion expression system based on Escherichia coli according to claim 2, characterized in that: The enzyme cleavage sites include EK enzyme and TEV protease.

5. The method for preparing a nanobody fusion expression system based on Escherichia coli according to claim 1, characterized in that: The specific steps of the preparation method are: S1. After the bacterial culture and induction expression are completed, the E. coli cells are collected, and the soluble expressed protein is crushed and the supernatant is collected and directly purified; S2. The collected supernatant solution is purified by affinity chromatography, desalted preparation and enzymatic cleavage, and affinity purification of the enzymatic cleavage product, and finally a nano-antibody with good uniformity and purity is obtained.

6. The method for preparing a nanobody fusion expression system based on Escherichia coli according to claim 5, characterized in that: The bacterial culture in S1 is to inoculate the recombinant expression vector into 2YT+G culture medium at a ratio of 1:1000, and culture at 37°C and 220 rpm; the induced expression is to add IPTG with a final concentration of 1 mM / mL to induce the expression of the recombinant nanoantibody when the OD600nm value reaches 0.6-0.8 during the bacterial culture process, and the induction conditions are: 20°C, 16 hours.

7. The method for preparing a nanobody fusion expression system based on Escherichia coli according to claim 5, characterized in that: The E. coli cells collected in S1 are collected after the cell culture and induction expression are completed, and 500 μL is centrifuged at 4° C. and 12,000 rpm for 1 minute, the supernatant is discarded, the cells are broken, and the protein expression amount is detected by SDS PAGE.

8. The method for preparing a nanobody fusion expression system based on Escherichia coli according to claim 5, characterized in that: The affinity chromatography purification in S2 is to dilute the collected supernatant sample solution, filter and load it, first rinse it with an equilibrium buffer, then elute it with an elution buffer, collect the elution peak, and obtain a purified nanobody fusion protein with an affinity tag.

9. The method for preparing a nanobody fusion expression system based on Escherichia coli according to claim 8, characterized in that: The pH value of the equilibrium buffer is 8.5, and the solvent is water, and the solute and its concentration are: 20mM Tris-HCl, 300mM NaCl; the pH value of the elution buffer is 8.5, and the solvent is water, and the solute and its concentration are: 20mM Tris-HCl, 300mM NaCl, 500mM imidazole.

10. The method for preparing a nanobody fusion expression system based on Escherichia coli according to claim 5, characterized in that: The desalting preparation and enzyme digestion in S2 are to desalt the affinity purified sample into an enzyme digestion buffer, detect the sample concentration by ultraviolet spectrophotometer A280nm, and use EK enzyme for digestion, the enzyme digestion ratio of EK enzyme: protein = 1:50 to 1:500 (mg:mg), and digestion is carried out at 20°C and 37°C for 14-18 hours to obtain the recombinant nanoantibody after digestion; the pH value of the enzyme digestion buffer is 8.0, and it is composed of a solvent and a solute; the solvent is water, and the solute and its concentration are: 20mM Tris-HCl, 50mM NaCl, 2mM CaCl2.

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