CN117304329A - Method for improving expression quantity and sensitivity of 19-nor-testosterone nanobody - Google Patents

Abstract

The invention discloses a method for improving the expression quantity and sensitivity of a 19-nor-testosterone nanobody, which comprises the following steps: the expression vector is constructed by fusing 19-nortestosterone nanobody with thioredoxin A (TrxA), and the expression vector is transformed into an escherichia coli expression host for induced expression, insoluble components in thalli are nanobody inclusion body expression components, and the expression components of the inclusion bodies are denatured, renatured, purified and desalted by a one-step method, so that the expression quantity and the sensitivity of the nanobody can be improved. The invention can not only remarkably improve the expression level of the 19-nor-testosterone nano antibody, but also improve the expression level by 6 times; meanwhile, the sensitivity of the nano antibody can be obviously improved by 35.04 times by adopting a one-step method for renaturation, purification and desalination on the inclusion body column; and 19-nor-testosterone was detected by established ic-ELISA methods with a detection limit as low as 0.877pg/mL and a sensitivity of 43.67pg/mL.

Description

A method to improve the expression and sensitivity of 19-nortestosterone nanobodies

Technical field

The invention belongs to the technical field of food safety detection. More specifically, it relates to a method for improving the expression and sensitivity of 19-nortestosterone Nanobodies.

Background technique

Nandrolone phenylpropionate is one of the steroid hormones commonly used in animal-derived animals. It is widely used in animal husbandry to promote protein synthesis, reduce fat accumulation, and improve feed returns. However, the abuse and residue of nandrolone phenylpropionate will cause food safety problems. Therefore, it is necessary to strengthen the detection of nandrolone phenylpropionate in food. At present, its secondary metabolite 19-nortestosterone (19-NT) is mainly used as a marker for the detection of nandrolone phenylpropionate residues. Chromatography and mass spectrometry are often used to detect 19-nortestosterone. , immunoassay method; in the immunoassay method, the use of antibodies for detection has the advantages of rapidness, simplicity, and high sensitivity. In recent years, it has become a common method for food safety detection, and the preparation of core reagent antibodies is the key to immune detection technology.

Nanobody (Nb) is a new type of antibody molecule in camelids that naturally lacks the light chain and only has the variable region of the heavy chain. It has the advantages of small molecular weight, good solubility, high stability and easy expression, and has great potential in immune detection. great application potential. Chinese patent CN111269320A discloses a nanobody NT8 that specifically recognizes 19-nortestosterone and can be used to detect 19-nortestosterone. However, the expression level of the nanobody is not high and the sensitivity still needs to be improved, which limits the anti-19 - Application of nortestosterone Nanobodies.

Inclusion bodies are high-density, insoluble protein particles wrapped by membranes formed when foreign genes are expressed in prokaryotic cells, especially when they are highly expressed in E. coli. New creation of inclusion bodies: E. coli does not have the organelles and functional enzymes for post-translational modification of proteins carried in mammalian cells, resulting in a large accumulation of intermediates, prone to mismatching of inter-chain disulfide bonds, and aggravating the formation of inclusion bodies; The production is also closely related to the nature of the protein itself. Generally, the protein chain contains sulfur-containing disabilities such as multiple cysteine, which makes it easier to form disulfide bonds. The environment also has a great relationship. The electric potential and pH inside the cell are both It is possible to promote the formation of inclusion bodies, and an environment close to the isoelectric point of the protein will promote the formation of inclusion bodies; the structure of the target protein itself, the aggregation trend of the target protein itself, and its hydrophobicity are all related to the formation of inclusion bodies; the target protein may It is harmful to bacteria. Due to the self-protection mechanism of bacteria, the target protein is expressed in the form of inactive inclusion body precipitation.

The higher the protein expression and the faster the synthesis rate, the easier it is to form inclusion bodies. However, there are currently few studies on directly increasing the expression of active proteins by enhancing the expression of inclusion bodies, because the formation of inclusion bodies and the renaturation and purification of inclusion bodies are important in biology. , the medical field is a very important issue, and it is also a very difficult issue. Generally, the target protein content in inclusion bodies is high, about 50%, and the target protein can be obtained efficiently. However, its biological activity can only be restored after denaturation with urea or guanidine hydrochloride and then re-renatured, and the activity level is generally poor, so inclusion bodies The body is more difficult to use. If highly active proteins can be efficiently prepared from inclusion body components, the use of inclusion bodies will undoubtedly have important application significance.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the problems of low expression level of existing anti-19-nortestosterone nanobody NT8, low sensitivity and no biological activity of inclusion bodies, and provide a method to improve the expression level of 19-nortestosterone nanobody. and sensitivity methods.

The purpose of the present invention is to provide a method for improving the expression and sensitivity of 19-nortestosterone Nanobody.

Another object of the present invention is to provide an anti-19-nortestosterone Nanobody with high expression and high sensitivity.

Another object of the present invention is to provide a method for detecting 19-nortestosterone.

The above objects of the present invention are achieved through the following technical solutions:

The invention provides a method for improving the expression amount and sensitivity of 19-nortestosterone Nanobody, which includes the following steps:

S1. Fusion of the 19-nortestosterone Nanobody with thioredoxin A to construct an expression vector, and transform it into an E. coli expression host for induced expression. The insoluble component in the bacteria is the Nanobody inclusion body expression component;

S2. The Nanobody inclusion body expression components are denatured, renatured, purified and desalted through a one-step method to obtain 19-nortestosterone Nanobody with high expression and sensitivity.

Further, in step S1, the 19-nortestosterone Nanobody is Nanobody NT8, and its amino acid sequence is shown in SEQ ID NO.1.

The method provided by the invention can significantly improve the expression amount and sensitivity of 19-nortestosterone nanobody. By fusing the nanobody NT8 with thioredoxin A (TrxA), a recombinant expression vector pET-32a-NbNT8 is constructed, and by optimizing the expression in E. coli The host, inducer concentration, and induction temperature finally resulted in an expression system with the highest expression of nanobody inclusion bodies; and at the same time, the formula of the denaturation working solution of nanobody inclusion bodies was optimized to establish a one-step method for nanobody inclusion body renaturation, purification, and desalting on the column. , the expression level of the finally prepared nanobody TrxA-NbNT8 can reach 65 mg/L culture medium. Inclusion bodies are inactive target protein solids. Optimizing the formulas of inclusion body denaturation buffer and inclusion body renaturation buffer can improve the extraction efficiency of inclusion bodies and the activity level of the protein after renaturation. At the same time, the ic-ELISA method established by the present invention detects 19-nortestosterone, and the detection limit of nanobody TrxA-NbNT8 is as low as 0.877pg/mL, the linear range is 3.71-514.09pg/mL, and the sensitivity is 43.67pg/mL. The sensitivity (1.53ng/mL) of Nanobody NT8 expressed in the periplasmic space of the original pComb3xss vector is 35.04 times higher.

Furthermore, in step S1, the pET-32a expression vector carrying the strong promoter T7lac can be induced by the lactose analog IPTG to start the lactose operon with high efficiency and induce the rapid expression of the target protein; the vector is easy to clone using genetic engineering technology. Very suitable for constructing expression vectors.

Further, in step S1, the E. coli expression host is BL21DE3, OverExpress C43 (DE3), Rosetta DE3, Rosetta gamiB DE3 or Top 10F’.

Preferably, Escherichia coli (E.coli) expression host uses BL21DE3, and the constructed pET-32a-TrxA-NbNT8 is constructed into an expression engineering strain in E.coli BL21DE3.

Further, the conditions for inducing expression in step S1 are: inducing expression at 0.125-1mM IPTG and 20-37°C for 10-12 hours.

Preferably, medium induction expression conditions are: inducing expression at 1mM IPTG at 25°C for 12 hours.

Furthermore, the one-step method in step S2 is to perform inclusion body renaturation + purification + desalting in one step on the column. The purification is performed using AKTA pure and Ni Sepharose 6Fast Flow purification instruments and materials.

Further, the denaturation in step S2 is carried out using a denaturation buffer containing 20mM Tris-HCl, 0.5M NaCl, 6M guanidine hydrochloride, 5mM imidazole, 1mM β-mercaptoethanol, pH 8.0; and then using a denaturation buffer containing 20mM Tris, 0.5M NaCl, 6M Urea, 20mM imidazole, 1mM β-mercaptoethanol, pH 8.0 inclusion body denaturation enhancement solution were used for secondary denaturation treatment.

Preferably, the insoluble components are washed with inclusion body washing solution (20mM Tris, 0.5M urea, 0.5M NaCl, 2% Tween-20, pH 8.0) at room temperature for 5 minutes and then centrifuged twice to collect the precipitate, and the inclusion body denaturation buffer is used at room temperature. Resuspend the inclusion body, shake for 30 minutes, and centrifuge to collect the supernatant; use AKTA pure, Ni Sepharose 6Fast Flow and a desalting column to achieve refolding, purification and desalting treatment. First, use the inclusion body denaturation buffer for column equilibrium, with a constant flow rate of 0.5mL/ min to load the sample, and then switch the mobile phase to inclusion body denaturation enhancement solution with a flow rate of 1 mL/min.

Further, in step S2, the inclusion body refolding buffer containing 20mM Tris, 0.5M NaCl, 20mM imidazole, 1mM β-mercaptoethanol, 0.3M arginine, and pH 8.0 is mixed with the inclusion body denaturation enhancement solution.

Preferably, 30 CV of 0% to 100% inclusion body refolding buffer is used for on-column renaturation, with a flow rate of 0.5 mL/min; 10 CV of 100% inclusion body refolding buffer is used to pass through the column, with a flow rate of 1 mL/min.

Furthermore, in step S2, the inclusion body elution buffer (20mM Tris, 300mM NaCl, 500mM imidazole, pH 7.4) was used to elute the target protein at a flow rate of 1mL/min; then the antibody storage solution (10mM PBS, pH 7.4) Equilibrate the desalting column and load 4 mL of sample at a flow rate of 2 mL/min.

As an optimal implementation, the present invention provides a specific method:

S1. Based on genetic engineering technology, construct the recombinant expression vector pET-32a-NbNT8, perform PCR amplification of the target gene and vector pET-32a to achieve linearization, and clone and construct the fragment and vector through homologous recombination; and transform into E. coli Induced expression in the expression host, using BL21DE3, induced with 1mM IPTG at 25°C for 12 hours, collected the cells, extracted the inclusion body precipitate through high-pressure crushing, and obtained the insoluble component in the cells as the nanobody inclusion body expression component;

S2. After induced expression, the insoluble components (inclusion bodies) in the bacterial cells are washed with inclusion body washing solution (20mM Tris, 0.5M urea, 0.5M NaCl, 2% Tween-20, pH 8.0) for 5 minutes and then centrifuged again to remove the supernatant. . The precipitate obtained by the second centrifugation was resuspended in inclusion body denaturation buffer (20mM Tris-HCl, 0.5M NaCl, 6M guanidine hydrochloride, 5mM imidazole, 1mM β-mercaptoethanol, pH 8.0) for 30 minutes, and then centrifuged to collect the supernatant to obtain inclusion body denaturation. supernatant;

S3. Load the denatured inclusion body supernatant obtained in S2 above using the AKTA pure chromatography system. Combine the target protein with Cytiva nickel chromatography packing (Ni Sepharose 6Fast Flow), and use the inclusion body denaturation enhancement solution (20mM Tris, 0.5M NaCl , 6M urea, 20mM imidazole, 1mM β-mercaptoethanol, pH8.0), flow rate 1mL/min, perform secondary denaturation to wash away non-specific binding impurities;

S4. The target protein TrxA-NbNT8 bound on the column uses the gradient of the AKTA pure chromatography system to refold the inclusion body of the present invention (20mM Tris, 0.5M NaCl, 20mM imidazole, 1mM β-mercaptoethanol, 0.3M arginine, pH 8.0) was mixed with the inclusion body denaturation enhancement solution, and the ratio of the inclusion body refolding buffer was gradually increased to 100%, with a flow rate of 1mL/min to complete on-column refolding;

S5. Elute the renatured target protein TrxA-NbNT8 from the column, and use inclusion body elution buffer (20mM Tris, 300mM NaCl, 500mM imidazole, pH 7.4) to elute the target protein at a flow rate of 1mL/min; after elution Reload the sample and replace it into the storage solution (10mM PBS, pH 7.4) through the desalting column to obtain the 19-nortestosterone nanobody TrxA-NbNT8 with high expression and sensitivity.

The invention provides a high-expression, high-sensitivity anti-19-nortestosterone nanobody, which is processed by the above method. The anti-19-nortestosterone Nanobody treated with the method of the present invention has the smallest antibody fragment with complete antigen recognition ability (about 15kDa, TrxA-NbNT8 36kDa); compared with traditional antibodies, the method of the present invention maintains the small molecular weight of the Nanobody , has the advantages of high expression level and stability, strong affinity, and strong resistance to matrix interference. Moreover, the present invention further improves the expression level of Nanobodies in E. coli, and improves the sensitivity of Nanobodies by refolding recombinant antibodies with inclusion bodies.

The present invention also provides a method for detecting 19-nortestosterone, which uses high-expression, high-sensitivity anti-19-nortestosterone nanobody TrxA-NbNT8 for detection.

Preferably, the detection is an indirect competition detection, and the coating concentration of the coating agent is 0.25-1 μg/mL.

More preferably, the coating concentration of the coating agent is 1 μg/mL.

Preferably, the coating agent is 19-nortestosterone-OVA (obtained by coupling 19-nortestosterone hapten and ovalbumin (OVA) through an active ester method).

The invention has the following beneficial effects:

This invention expresses Nanobodies in the form of inclusion bodies for the first time, and processes them through one-step on-column renaturation, purification, and desalting, which provides a simple and efficient preparation method and greatly improves the expression of Nanobodies. The advantages of the method of the present invention are: 1) The prepared nanobody has high expression level, low cost and high sensitivity; 2) the one-step on-column inclusion body renaturation + purification + desalting operation is simple, fast and convenient, and is suitable for other proteins; 3 ) The high-quality anti-19-nortestosterone Nanobody prepared was used to establish an ic-ELISA method, which can be used for the detection of nandrolone phenylpropionate in food.

The present invention can not only significantly increase the expression of the nanobody TrxA-NbNT8 by 6 times, but also significantly improve the sensitivity of the nanobody by 35.04 times. It also establishes an ic-ELISA method to detect 19-nortestosterone with a low detection limit. to 0.877pg/mL, with a sensitivity of 43.67pg/mL and a detection range of 3.71-514.09pg/mL. It provides high-quality identification elements for the detection of nandrolone phenylpropionate in food and effectively reduces its production cost. In addition, the method provided by the present invention is suitable for other nanobodies or other proteins to effectively obtain biologically active substances from inclusion bodies, and provides a reference for the development of inclusion bodies in prokaryotic expression systems.

Description of the drawings

Figure 1 shows the expression results of different expression hosts.

Figure 2 shows the expression results at different induction temperatures.

Figure 3 shows the expression results of different inducer concentrations.

Figure 4 shows the inclusion body extraction effects of different inclusion body denaturation buffers (left) and the target protein purity identification results (right).

Figure 5 shows the effect of different 19-nortestosterone coating agent concentrations on the sensitivity of the ic-ELISA method.

Figure 6 is the standard curve for the detection of 19-nortestosterone by the ic-ELISA method of TrxA-NbNT8.

Detailed ways

The invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

Unless otherwise stated, the reagents and materials used in the following examples were all commercially available.

The 19-nortestosterone nanobody NT8 used in the embodiments of the present invention is derived from Chinese patent CN111269320A.

Example 1 Optimization of expression of inclusion bodies

In this example, the pET-32a expression vector carrying the strong promoter T7lac was used to construct the optimal recombinant expression system of NbNT8 through the preparation and expression optimization of nanobody inclusion bodies; the nanobody NT8 used can specifically recognize 19-nortestosterone. , whose amino acid sequence is shown in SEQ ID NO. 1, and fusion expression of thioredoxin (TrxA) is performed at the same time. TrxA, as a tag, has a solubilizing effect and prevents disulfide bond mismatching. Specifically, it includes the following steps:

S1. Design primers F1 and R1 based on the 5'- and 3'-sequences of nanobody NT8 to amplify the target fragment. Primers F2 and R2 are used to linearize the vector pET-32a. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. ;

Primer F1: 3’-gtaccgacgacgacgacaagGAGGTGCAGCTGGAGCAGTC-5’;

Primer R1: 3’-cagtggtggtggtggtggtgCTAAGAAGCGTAGTCCGGAACG-5’;

Primer F2: 3’-CACCACCACCACCACCACTG-5’;

Primer R2: 3’-CTTGTCGTCGTCGTCGGTACC-5’.

S2. Preparation of target fragment and vector: use Mut ⅡFast Mutagenesis Kit V2 is used for PCR amplification. The reaction system and conditions are shown in Table 1 and Table 2. Prepare the reaction system on ice. After preparation, mix gently, centrifuge briefly and then react on the PCR machine; the reaction product is processed for 1 % agarose gel electrophoresis for identification.

Table 1 Phanta Max DNA Polymerase reaction system

Note: The unit is μL; F/R: forward and reverse primers, concentration is 10 μM; dNTPs (concentration of each component is 10mM), template 1ng/μL.

Table 2 Phanta Max DNA Polymerase reaction conditions

S3. Use the homologous recombination system and procedures in Table 3 below to recombine the target fragment with the vector (50°C for 5 minutes), transform the clone into competent DH5α, and coat it with Amp-resistant solid incubator for overnight culture.

Table 3 Homologous recombination reaction system

S4. After sequencing verification, the successfully cloned target plasmid was transformed into five different expression competent states: BL21DE3, OverExpress C43(DE3), Rosetta DE3, Rosetta gamiB DE3, Top 10F', and small-scale expression of total bacterial protein Extract (extraction solution: Thermo B-PER Bacterial Protein Extraction Reagent) to verify its expression: 37°C, 1mM IPTG induced expression for 12 hours; select the optimal expression host.

The results are shown in Figure 1. From the changing trends of intracellular soluble components (↑) and inclusion body components (↓) of cells under different conditions, it can be seen that the position of the target protein band has soluble protein expression (↑) The process is accompanied by the production of a large number of inclusion bodies (↓), which means that at the same time as inclusion bodies are expressed, regular fusion expression is also induced.

After induced expression by different expression hosts BL21DE3, OverExpress C43 (DE3), Rosetta DE3, and RosettagamiB DE3, it can be found that the target protein band in the soluble expression (↑) lane is thin, and different expression hosts cannot improve the soluble expression of the target protein; comprehensively For the expression levels of soluble expression (↑) and inclusion body components (↓), engineering strain 1: BL21DE3 was selected for subsequent experiments.

S5. Optimize the induction temperature based on the engineering bacteria determined in S4: perform small-scale expression at 37°C, 25°C, 20°C, and 16°C and extract the total bacterial protein (extraction solution: Thermo B-PER Bacterial Protein Extraction Reagent) Verify its expression.

The results of induction temperature optimization are shown in Figure 2. Lowering the induction temperature can theoretically reduce the transcription and translation speed, reduce the accumulation of target protein intermediates, reduce disulfide bond mismatches, and reduce the formation of inclusion bodies. Although the target protein was induced at different induction temperatures, the expression test verified that there was no obvious difference in the soluble expression of the target protein band (↑) under the three conditions of 37°C, 25°C, and 20°C. The band was thinner at 16°C, indicating that the decrease in induction temperature did not promote the soluble expression of the target protein. The target protein band of the inclusion body component was the thickest and purer when induced at 25°C. Therefore, 25°C was selected for the next experiment based on comprehensive considerations.

S6. Further optimize the inducer concentration based on the engineering bacteria and induction temperature determined in S4 and S5: perform small-scale expression under the conditions of 1mM, 0.5mM, 0.25mM, 0.125mM and extract the total bacterial protein (extraction solution: Thermo B-PERBacterial Protein Extraction Reagent) to verify its expression.

The results of inducer concentration are shown in Figure 3. It shows that at the inducer concentration of 1mM, the soluble expression of the target protein band is the thickest (↑) and the inclusion body component is also the thickest (↓). Therefore, the inducer concentration of 1mM was selected. Carry out subsequent experiments.

In summary, it can be seen that under different induction conditions, there is no obvious difference in the soluble expression of the target protein, while the expression level of the target protein expressed in inclusion bodies is significantly improved. As a solubility-promoting tag, TrxA can reduce the formation of inclusion bodies and promote the soluble expression of the target protein in the protein cell, but it does not improve the overall expression of the target protein (the sum of soluble expression + inclusion bodies). conclusive. In this example, it can be found that after Nb was fused to express TrxA, after optimization of expression conditions, the target protein band expressed in the bacterial soluble component did not change significantly, so TrxA did not increase the expression level of Nb soluble expression in this experiment. . The optimal expression system finally selected was: pET-32a-TrxA-NbNT8 was constructed in E.coli BL21DE3 and induced by 1mM IPTG at 25°C for 12 hours. Under this system, the expression band of the target protein (34kDa) near marker 35kDa is the thickest, which can be used for the rapid and large-scale preparation of nanobody TrxA-NbNT8 in the next step.

Example 2 Renaturation, purification and desalting of inclusion bodies

Anti-19-nortestosterone Nanobodies were prepared according to the optimal expression system constructed in Example 1. After optimizing the inclusion body denaturation buffer and combining it with a high-efficiency refolding buffer, a one-step inclusion body method was established for on-column renaturation, purification and desalting. This method achieves the efficient preparation of anti-19-nortestosterone nanobodies. The specific method is as follows:

S1. Expression system determined according to Example 1: E.coli BL21DE3-pET-32a-TrxA-NbNT825°C, 1mM IPTG, induced for 12 hours and cultured in a shake flask;

S2. Collect the bacterial cells and crush them with high pressure (1000bar, 3 times). Collect the insoluble components of the crushed liquid and wash them with inclusion body washing solution (20mM Tris, 0.5M urea, 0.5M NaCl, 2% Tween-20, pH 8.0) at room temperature for 5 minutes. Collect the precipitate by centrifugation a second time, resuspend the inclusion bodies at room temperature using the following inclusion body denaturation buffers with different formulas, shake for 30 minutes, and centrifuge to collect the supernatant for the next step.

Different formulations of inclusion body denaturation buffers:

Inclusion body denaturation buffer 1: 20mM Tris-HCl, 0.5M NaCl, 6M urea, 5mM imidazole, 1mM β-mercaptoethanol, pH 8.0;

Inclusion body denaturation buffer 2: 20mM Tris-HCl, 0.5M NaCl, 6M guanidine hydrochloride, 5mM imidazole, 1mM β-mercaptoethanol, pH 8.0;

Inclusion body denaturation buffer 3: 0.2M PBS, 0.5M NaCl, 5mM β-mercaptoethanol, 8M urea, 0.5% triton, pH 8.0.

S3. Use AKTA pure, Ni Sepharose 6Fast Flow and desalting column (purchased from Tiandi Renhe) to achieve renaturation, purification and desalting:

1) Load the column and load the sample: Use inclusion body denaturation buffer for column equilibration, and load the sample at a constant flow rate of 0.5mL/min;

2) Inclusion body denaturation enhancement: After UV equilibrium, the mobile phase is switched to inclusion body denaturation enhancement solution (20mM Tris, 0.5M NaCl, 6M urea, 20mM imidazole, 1mM β-mercaptoethanol, pH 8.0), with a flow rate of 1mL/min;

3) Inclusion body refolding: After UV equilibrium, use 30CV of 0% to 100% inclusion body refolding buffer (20mM Tris, 0.5M NaCl, 20mM imidazole, 1mM β-mercaptoethanol, 0.3M arginine, pH 8.0 ) perform on-column renaturation at a flow rate of 0.5 mL/min; add 10 CV of 100% inclusion body renaturation buffer through the column at a flow rate of 1 mL/min;

4) Elution of target protein: Elute the target protein with inclusion body elution buffer (20mM Tris, 300mM NaCl, 500mM imidazole, pH7.4), with a flow rate of 1mL/min;

5) Load the column and load the sample: Use the antibody storage solution (10mM PBS, pH 7.4) to balance the desalting column, and load 4mL of sample at a constant flow rate of 2mL/min;

6) Peak collection: start collecting samples when UV≥5mAu, stop collecting samples when UV<5mAu;

7) After reducing SDS-PAGE to identify its purity, the sample can be directly used for subsequent antibody activity detection.

This example establishes a one-step inclusion body method that simultaneously performs on-column denaturation, renaturation, purification, and desalting. The renaturation method is combined with affinity purification, and the experiment is performed intelligently and controllably on the AKTA chromatography system, which frees up manpower and is fast. The protein is fixed on the affinity medium and remains permanently in the flow system without precipitation, resulting in small loss and high yield. However, the purification system established in this study does not require imidazole after the previous deformation enhancement solution impurity removal step. Concentration gradient elution can directly elute all proteins with high concentration of imidazole, which is fast, small in volume and high in concentration; the obtained affinity purification product can be directly desalted in a gel desalting column, and finally desalted to obtain a denaturant-free and imidazole-free protein solution. The concentration is high (this experiment can control the protein concentration at the mg/ml level) and can be directly tested, analyzed and stored.

The results are shown in Figure 4. By testing three different inclusion body denaturation buffers, the extraction effect of the target protein in the inclusion bodies was optimized, as shown in 20mM Tris-HCl, 0.5M NaCl, 6M guanidine hydrochloride, 5mM imidazole, 1mM β- Under the extraction of mercaptoethanol and pH 8.0 (inclusion body denaturation buffer 2), the target protein has the highest yield and the best purity.

Finally, the one-step method is used to perform inclusion body denaturation, on-column renaturation, purification and desalting. Compared with the conventional four-step method of denaturation, renaturation, purification and desalting, the operation is simpler, the time is shorter and the preparation efficiency is higher. . At the same time, it solves the problems of incomplete denaturation, time-consuming (several days), labor-consuming (manual), reagent-consuming (a large amount of renaturation buffer), cumbersome steps (possible addition of concentration steps), and large losses (refolding operation errors) existing in conventional methods. Problems such as precipitation, dialysis reverse salt dissolution precipitation, concentrated food precipitation), etc. This embodiment only takes about 6 hours to complete the steps of denaturation, renaturation, desalting, etc. at the same time. The product can be directly used for the next step of activity detection. The amount of harvested target protein is 65 mg/L culture medium, which greatly improves the efficiency of Nanobodies. The expression level effectively reduces the cost of antibody application.

Example 3 Detection of 19-nortestosterone

An ic-ELISA method for detecting 19-nortestosterone was established based on the anti-19-nortestosterone Nanobody prepared in Example 2. The specific method is as follows:

1) Determine the working concentration of antigen and antibody by the checkerboard method: dilute the coating antigen 19-nortestosterone-OVA (19-NT-OVA) to 1000ng/mL, 500ng/mL, 250ng/mL, and 125ng/mL and coat the plate for 12 hours ; Blocking: Take out the enzyme plate, wash it twice and pat dry the liquid, add 120 μL/well 3% skimmed milk powder (PBS), place it in a 37°C water bath for 3 hours, then dry the blocking solution, and dry it in an oven at 37°C for 30 minutes. ;Add the protein to be detected: double and dilute the antibody from 1 mg/mL, dilute 19-nortestosterone to 100ng/mL with PBST buffer, add 50 μL/well of PBST in odd-numbered columns, and add 50 μL/well of 19-nortestosterone in even-numbered columns. , then add 50 μL/well of the diluted antibody to each column to determine the antibody titer inhibition curve; wash the plate 3 times in a 37°C water bath for 40 minutes and pat the liquid dry; add anti-Nb-HRP Rab: dilute 5000 times with PBST, mix and add 100 μL/well, 37°C water bath for 30 minutes, wash the plate 3 times and pat dry; add chromogenic solution: 100 μL/well of chromogenic solution, 37°C water bath for 10 minutes, add 50 μL/well of stop solution to terminate the reaction; read: use a microplate reader Measure the absorbance at 450 nm, and select a working concentration of coating source and antibody with a reading of about 1.0 to further establish a competition inhibition curve.

2) Establish competitive inhibition curves under different working concentrations of coating agents and antibodies to determine the impact of different coating agent concentrations on the ic-ELISA method: Take an antigen-antibody concentration combination with an ABS value near 1.0, and configure a series of concentrations of 19- For methyltestosterone solution, add 50 μL of antibody protein and 50 μL of 19-nortestosterone solution of different concentrations to each well of the enzyme plate, and keep in a 37°C water bath for 40 minutes. Other experimental operations are the same as the previous step. The final results were obtained using the four-parameter fitting module of Origin 9.1 to establish the ic-ELISA standard curve. Calculate the IC50 value of the inhibition curve. Select the antigen and antibody concentration with a low IC50 value as the optimal working concentration.

The measurement results are shown in Figure 5, showing the impact of different 19-nortestosterone coating original concentrations on the sensitivity of the ic-ELISA method: the sensitivity of the standard curve established when the original coating concentration is low is poor, so 1000ng/mL was selected. For 19-NT-OVA, an antibody of 5 μg/mL is the optimal working concentration.

3) Using the above optimized conditions: 1000ng/mL 19-NT-OVA coating concentration, establish an ic-ELISA method based on TrxA-NbNT8 to detect 19-nortestosterone, and draw a detection standard curve.

The standard curve of the ic-ELISA method of TrxA-NbNT8 for detecting 19-nortestosterone is shown in Figure 6. Based on the preparation of nanobody TrxA-NbNT8, which highly expresses 19-nortestosterone, the ic-ELISA method was established and optimized. The original dosage is 1μg/mL, and the antibody dosage is 5μg/mL. The detection limit of this method for detecting 19-nortestosterone is as low as 0.877pg/mL, the linear range is 3.71-514.09pg/mL, and the sensitivity is 43.67pg/mL. The sensitivity (1.53ng/mL) of Nanobody NT8 expressed in the periplasmic space of the original pComb3xss vector is 35.04 times higher. In actual detection, it can be used to detect even trace amounts of residual 19-nortestosterone to achieve sensitive detection of nandrolone phenylpropionate.

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, etc. may be made without departing from the spirit and principles of the present invention. All simplifications should be equivalent substitutions, and are all included in the protection scope of the present invention.

Claims (10)

1. A method for improving the expression and sensitivity of 19-nortestosterone Nanobody, which is characterized by comprising the following steps: S1. Fusion of the 19-nortestosterone Nanobody with thioredoxin A to construct an expression vector, and transform it into an E. coli expression host for induced expression. The insoluble component in the bacteria is the Nanobody inclusion body expression component; S2. The Nanobody inclusion body expression components are denatured, renatured, purified and desalted through a one-step method to obtain 19-nortestosterone Nanobody with high expression and sensitivity. 2. The method according to claim 1, characterized in that in step S1, the 19-nortestosterone Nanobody is Nanobody NT8, and its amino acid sequence is as shown in SEQ ID NO.1. 3. The method according to claim 1, characterized in that, in step S1, a pET-32a expression vector carrying a strong promoter T7lac is used. 4. The method according to claim 1, characterized in that in step S1, the E. coli expression host is BL21DE3, OverExpress C43 (DE3), Rosetta DE3, Rosetta gamiB DE3 or Top 10F’. 5. The method according to claim 1, characterized in that the conditions for inducing expression in step S1 are: inducing expression at 0.125-1mMIPTG at 20-37°C for 10-12 hours. 6. The method according to claim 1, characterized in that the denaturation in step S2 is carried out by using a denaturation buffer containing 20mM Tris-HCl, 0.5M NaCl, 6M guanidine hydrochloride, 5mM imidazole, 1mM β-mercaptoethanol, pH 8.0; and then Use inclusion body denaturation enhancement solution containing 20mM Tris, 0.5M NaCl, 6M urea, 20mM imidazole, 1mM β-mercaptoethanol, pH 8.0 for secondary denaturation treatment. 7. The method according to claim 1, wherein the renaturation in step S2 uses an inclusion body renaturation buffer containing 20mM Tris, 0.5M NaCl, 20mM imidazole, 1mM β-mercaptoethanol, 0.3M arginine, and pH 8.0. The solution is mixed with the inclusion body denaturation enhancement solution. 8. A high-expression, high-sensitivity anti-19-nortestosterone Nanobody, characterized in that it is obtained by processing according to any one of claims 1 to 7. 9. A method for detecting 19-nortestosterone, characterized in that the anti-19-nortestosterone Nanobody according to claim 8 is used for detection. 10. The method according to claim 9, characterized in that, for indirect competition detection, the coating concentration of the coating agent is 0.25-1 μg/mL.