Anti-mouse IgG nano antibody and alkaline phosphatase fusion protein as well as preparation method and application thereof

Abstract

The invention relates to an anti-mouse IgG nano antibody and alkaline phosphatase fusion protein as well as a preparation method and application thereof. The anti-mouse IgG nano antibody and alkaline phosphatase fusion protein sequentially comprises an anti-mouse IgG nano antibody, a connecting peptide and shewanella alkaline phosphatase from an amino terminal to a carboxyl terminal. The fusion expression of the anti-mouse IgG nano antibody and shewanella alkaline phosphatase in escherichia coli is designed, so that various defects of a chemical labeling method can be avoided, a high-purity and high-specific activity enzyme-labeled antibody can be prepared, the preparation period of a second antibody can be shortened, the preparation process of the second antibody can be simplified, the stability of batches can be improved, and the dependence on experimental animals can be reduced. The prepared anti-mouse IgG nano antibody and alkaline phosphatase fusion protein can be applied to the field of immunodetection, such as a chemiluminescence immunoassay method, an enzyme-linked immunosorbent assay method, an enzymatic fluorescence immunoassay method, an immunoblotting method and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an anti-mouse IgG nano antibody and alkaline phosphatase fusion protein, and a preparation method and application thereof. Background Art

[0002] Immunoassays utilize antigen-antibody interactions to detect specific biomolecules such as proteins, hormones, and enzymes. They are widely used in disease diagnosis, treatment monitoring, and drug development. Common immunoassay techniques include chemiluminescent immunoassays, enzyme-linked immunosorbent assays (ELISAs), enzyme-catalyzed fluorescent immunoassays (ELISAs), and immunoblotting. These techniques all rely heavily on secondary antibodies that specifically recognize the primary antibody and enhance the signal. Rabbit and mouse antibodies are often used as the primary antibody (primary antibody). The secondary antibody (secondary antibody) is an antibody that binds to the primary antibody and primarily detects its presence and amplifies the detection signal. Traditional secondary antibodies are polyclonal antibodies, which are prepared by exploiting the immunogenicity of large proteins. These antibodies are then immunized in xenogeneic animals, where the immune system of the xenogeneic animal produces immunoglobulins specific to the antibody. This results in long production cycles, complex processes, and poor batch-to-batch controllability. Polyclonal antibodies also exhibit strong cross-reactivity with other antibodies, which can lead to nonspecific background staining. Furthermore, the production of polyclonal antibodies inevitably harms experimental animals, necessitating consideration of animal ethics issues.

[0003] Currently, the main method for preparing enzyme-labeled antibodies is chemical coupling labeling, such as using glutaraldehyde, periodate, SMCC reagent, 2-IT reagent, etc. to couple the antibody to the labeled enzyme. However, chemical coupling labeling is complex, has low coupling efficiency, unstable coupling sites, and harsh conditions that can easily lead to reduced antibody and enzyme activity. In addition, the antibody-enzyme conjugate is heterogeneous, and the removal of unbound antibody is crucial. This is because free antibody competes with the enzyme-labeled antibody for the corresponding antigen, reducing the amount of enzyme-labeled antibody bound to the solid phase and thus reducing the sensitivity of the test.

[0004] In summary, developing an efficient, highly stable and easy-to-operate method for preparing enzyme-labeled secondary antibodies while reducing dependence on experimental animals is of great significance in the field of enzyme-labeled secondary antibody preparation. Summary of the Invention

[0005] In response to the deficiencies in the existing technology and actual needs, the present invention provides an anti-mouse IgG nanobody and alkaline phosphatase fusion protein, as well as a preparation method and application thereof. The anti-mouse IgG nanobody and alkaline phosphatase fusion protein and an efficient preparation method are designed, which does not require the cumbersome enzyme-labeled secondary antibody process, optimizes the secondary antibody preparation strategy, thereby shortening the secondary antibody preparation cycle, simplifying the secondary antibody preparation process, improving the stability between batches, and reducing the dependence on experimental animals.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an anti-mouse IgG nanobody and alkaline phosphatase fusion protein, wherein the anti-mouse IgG nanobody and alkaline phosphatase fusion protein comprises an anti-mouse IgG nanobody, a connecting peptide and alkaline phosphatase from the amino terminus to the carboxyl terminus.

[0008] Preferably, the amino acid sequence of the anti-mouse IgG nanobody includes the sequence shown in SEQ ID NO.1.

[0009] Preferably, the connecting peptide comprises (GGGGS)n or A(EAAAK)nA, where n is selected from any one of 3-6.

[0010] Preferably, the alkaline phosphatase comprises Shewanella alkaline phosphatase.

[0011] Preferably, the amino acid sequence of the Shewanella alkaline phosphatase includes the sequence shown in SEQ ID NO.2.

[0012] Preferably, the Shewanella alkaline phosphatase is selected from any one of the carboxyl-terminal 25-443, 26-443, 27-443, 28-443, 29-443, 30-443, 31-443, 32-443, 33-443, 34-443, 35-443, 36-443, 37-443, 38-443, 39-443, 40-443, 41-443, 42-443, 43-443, 44-443, 45-443, 46-443, 47-443, 48-443, 49-443 or 50-443 fragments thereof.

[0013] The anti-mouse IgG nanobody and alkaline phosphatase fusion protein designed by the present invention is a fusion protein comprising an anti-mouse IgG nanobody and a Shewanella alkaline phosphatase carboxyl terminal 25-443, 26-443, 27-443, 28-443, 29-443, 30-443, 31-443, 32-443, 33-443, 34-443, 35-443, 36-443, 37-443, 38-4 The fusion of 43, 39-443, 40-443, 41-443, 42-443, 43-443, 44-443, 45-443, 46-443, 47-443, 48-443, 49-443 or 50-443 fragments can achieve the production of anti-mouse IgG nanoantibodies with alkaline phosphatase, while maintaining the high affinity of the fusion protein for mouse IgG and high enzymatic activity for alkaline phosphatase substrate.

[0014] It can be understood that based on the anti-mouse IgG nanobody and alkaline phosphatase fusion protein designed by the present invention, theoretically any anti-mouse IgG nanobody is applicable, and is not limited to a specific sequence.

[0015] SEQ ID NO.1: NbMIgG

[0016] QVQLVESGGGWVQPGGSLRLSCAASGFTFSDTAMMWVRQAPGKGRE WVAAIDTGGGYTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTARYYCA KTYSGNYYSNYTVANYGTTGRGTLVTVSS.

[0017] SEQ ID NO. 2: AP (1-443)

[0018] MSVTKTSLLLLLTIGLVFSASSKAAPELENGPMKPPSKPKNIVIMVGDGMGPSYTSAYRYFKDNPDTEEVEQTVFDRLLVGMASTYPASVSGYVTDSAAAATALATGVKSYN GAISVDTQKQHLPTMLEKAKALGLSTGVAVTSQINHATPAAFLAHNESRKNYDALALSYLDTNADVLLGGGQKYFSPELLEKFTAKGYQHISRFEDLATITQPKVIGLFAQ VQLPWALDEKNANRLSTMTQKALDLLSQNEQGFVLLVEGSLIDWAGHSNDIANTMGEMDEFANALEVVEQFVRQHPDTLMMVATADHNTGGLSIGAGGDYRWNPEILRNMSA STDTLALAALGGDQWQADLARGLGFELNADEVTQLSTARMQGLETMTEAIRKIIDKRTGTGWTTSGHTGTDVQVFAAGPAAELFNGHQDNTDIANKIFTLLPPKPKKAKTE.

[0019] It is understood that, based on the fusion protein designed by the present invention, fusion proteins with similar functions obtained by amino acid substitution, deletion or addition by genetic modification means in the art should be within the scope of protection of the present invention, and the number of amino acids substituted, deleted or added can be any value, such as 1, 5, 10, 15 and above, so that the sequence identity of the amino acid sequence after the change and its respective corresponding original sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or above. In the art, when conservative substitutions are made with amino acids with similar or similar properties, the function of the protein is generally not changed. For example, amino acids with similar properties are substituted in the CDR region and / or FR region. Amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code; therefore, antibodies obtained by conservative substitution with amino acids having similar or similar properties are also within the scope of protection of the present invention.

[0020] In a second aspect, the present invention provides a nucleic acid molecule encoding the anti-mouse IgG nanobody and alkaline phosphatase fusion protein described in the first aspect.

[0021] Preferably, a nucleic acid sequence of the nucleic acid molecule consists of an anti-mouse IgG nanobody coding sequence, a connecting peptide coding sequence and a Shewanella alkaline phosphatase carboxyl terminal 25-443 fragment coding sequence (NbMIgG-AP (25-443)), including SEQ ID The sequence shown in NO.3, the coding sequence of the fusion protein of the anti-mouse IgG nanobody and the carboxyl-terminal 26-443, 27-443, 28-443, 29-443, 30-443, 31-443, 32-443, 33-443, 34-443, 35-443, 36-443, 37-443, 38-443, 39-443, 40-443, 41-443, 42-443, 43-443, 44-443, 45-443, 46-443, 47-443, 48-443, 49-443 or 50-443 fragment is obtained by deleting 3 bases in sequence on the basis of the NbMIgG-AP (25-443) coding sequence.

[0022] SEQ ID NO.3: NbMIgG-AP(25-443)

[0023]

[0024] In the present invention, specific nucleic acid sequences are designed and optimized to adapt to host cell preferences, increase codon adaptation index, and reduce translation barriers, thereby significantly improving protein expression efficiency.

[0025] In a third aspect, the present invention provides a recombinant vector comprising the nucleic acid molecule described in the second aspect.

[0026] In one or more embodiments of the present invention, the starting vector of the recombinant vector may be a pET expression vector.

[0027] In a fourth aspect, the present invention provides a recombinant cell comprising the nucleic acid molecule described in the second aspect.

[0028] The recombinant cells of the present invention express the anti-mouse IgG Nanobody and alkaline phosphatase fusion protein described in the first aspect. They may comprise a nucleic acid molecule encoding the fusion protein or a recombinant vector containing the nucleic acid molecule. The host cell may be a prokaryotic cell, a lower eukaryotic cell, or a higher eukaryotic cell. Prokaryotic cells include bacterial cells, lower eukaryotic cells include yeast cells, and higher eukaryotic cells include mammalian cells. A representative example is Escherichia coli. In one or more embodiments, the E. coli cell may be E. coli Rosetta-gami2 (DE3) pLysS.

[0029] In a fifth aspect, the present invention provides a method for preparing the anti-mouse IgG nanobody and alkaline phosphatase fusion protein according to the first aspect, the preparation method comprising:

[0030] The recombinant vector described in the third aspect is introduced into host cells to obtain recombinant cells, which are cultured, and the cells are collected and mixed with a lysis buffer to obtain a lysis product; the lysis product is purified to obtain the anti-mouse IgG nanobody and alkaline phosphatase fusion protein.

[0031] The vector can be transformed into host cells using conventional methods well known to those skilled in the art. For example, the CaCl2 method, electroporation, calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. The obtained transformants can be cultured using conventional methods well known to those skilled in the art, and the culture medium can be a conventional culture medium. The fusion protein produced by the transformants can be isolated and purified using physical or chemical methods, such as salting out, centrifugation, cell disruption, chromatography, etc., using conventional methods well known to those skilled in the art.

[0032] In one or more embodiments, the culture is cultured using an autoinduction medium, which does not contain a chemical inducer such as IPTG; preferably, the autoinduction medium comprises: peptone, yeast extract, glycerol, lactose, glucose, Na2HPO4, NH4Cl, KH2PO4, Na2SO4 and MgSO4.

[0033] In one or more embodiments, the autoinduction medium includes: 10 g / L peptone, 5 g / L yeast extract, 5 g / L glycerol, 2 g / L lactose, 0.5 g / L glucose, 25 mmol / L Na2HPO4, 50 mmol / L NH4Cl, 25 mmol / L KH2PO4, 5 mmol / L Na2SO4, and 2 mmol / L MgSO4, wherein the amount of each component may fluctuate within 30%, 20%, 10%, or 5%.

[0034] Preferably, the culturing includes: culturing at 34-40°C (preferably 37±2°C, more preferably 37±1°C) for 4-8h (preferably 7±1h, more preferably 7±0.5h), and then adjusting to 17-23°C (preferably 20±2°C, more preferably 20±1°C) and culturing for 24-48h (preferably 40±4h, more preferably 40±3h).

[0035] In the present invention, by designing specific culture conditions and controlling specific temperature and culture time, the growth and metabolic efficiency of cells can be improved, the correct folding of proteins can be promoted, the biological activity and stability of recombinant proteins can be enhanced, and the expression level of recombinant proteins can be increased.

[0036] In one or more embodiments, the culture is performed on a shaking platform at 220±100 rpm, preferably 220±60 rpm, and more preferably 220±30 rpm.

[0037] Preferably, the lysis buffer contains phosphate, sodium chloride, glycerol and phenylmethylsulfonyl fluoride (PMSF).

[0038] In the present invention, a specific component lysis buffer is designed to improve protein extraction efficiency and protect protein structure and activity.

[0039] In one or more embodiments, the lysis buffer comprises:

[0040] Phosphate: 20±5 mM (preferably 20±3 mM, more preferably 20±2 mM);

[0041] Sodium chloride: 500±100 mM (preferably, 500±70 mM, more preferably 500±30 mM);

[0042] Glycerol: 5±2% (v / v) (preferably, 5±0.5%, more preferably 5±1% or 5±0.5%);

[0043] PMSF: 1±0.6 mM (preferably, 1±0.5 mM, more preferably 1±0.3 mM).

[0044] Preferably, the collecting cells and mixing with the lysis buffer further comprises performing ultrasonic treatment under ice bath conditions.

[0045] Preferably, the ultrasonic treatment includes an ultrasonic power of 150 to 250 W (for example, 160, 170, 180, 190, 200, 210, 220, 230 or 240 W, etc.), ultrasonication for 3 to 5 s (for example, 3.5, 4 or 4.5 s, etc.), pause for 5 to 7 s (for example, 5.5, 6, 6.5 s, etc.), and a total ultrasonication of 10 to 20 min (for example, 11, 12, 13, 14, 15, 16, 17, 18 or 19 min, etc.).

[0046] In the present invention, controlling specific ultrasonic treatment conditions can accelerate cell rupture, improve protein extraction efficiency, reduce protein degradation, improve protein quality, and enhance experimental repeatability.

[0047] In one or more embodiments, the steps of constructing the recombinant vector are as follows: (1) optimizing the gene sequence for expressing anti-mouse IgG nanobody and alkaline phosphatase fusion protein in Escherichia coli, and synthesizing the optimized gene fragment; (2) connecting the gene fragment to the expression vector; (3) transforming the ligation product into Escherichia coli DH5α competent cells, screening positive transformants, expanding the culture after Sanger sequencing verification, and extracting the recombinant plasmid.

[0048] In one or more embodiments, the preparation steps of the recombinant cells are as follows: (1) the recombinant vector is transformed into Escherichia coli competent cells; (2) at least 3 single colonies are picked and cultured in a small amount of LB culture medium, and then inoculated into a small amount of autoinduction culture medium for culture, and strains that efficiently express anti-mouse IgG nanoantibodies and alkaline phosphatase fusion proteins are screened; (3) the strains that efficiently express anti-mouse IgG nanoantibodies and alkaline phosphatase fusion proteins are inoculated into LB culture medium and cultured to the logarithmic growth phase, and then frozen after adding glycerol as seed bacteria.

[0049] In one or more embodiments, the steps of culturing the recombinant cells to express anti-mouse IgG nanoantibodies and alkaline phosphatase fusion proteins are as follows: (1) inoculating the recombinant bacterial seed bacteria into LB liquid culture medium and culturing for 8 to 20 hours; (2) inoculating the culture into an autoinduction culture medium at an inoculum size of 1‰ to 1%; (3) culturing in a constant temperature horizontal oscillator at 200 to 300 rpm, first at 37° C. for 4 to 8 hours, and then at 17 to 30° C. for 20 to 48 hours; (4) collecting the bacteria by filtration or centrifugation and removing the liquid culture medium.

[0050] In one or more embodiments, the purification process includes: (1) resuspending the recombinant cell culture in a lysis buffer, lysing the cells, and separating the supernatant and the precipitate; and (2) filtering the lysis supernatant and loading it onto a chromatography column for purification.

[0051] In one or more embodiments, nuclease and MgCl2 are further added to the lysate and incubated at 25±3°C (preferably, 25±2°C or 25±1°C).

[0052] Preferably, the purification method comprises affinity purification.

[0053] Preferably, the purification buffer for affinity purification contains phosphate, sodium chloride, glycerol and imidazole.

[0054] In the present invention, a specific affinity purification buffer component is designed to improve purification efficiency, protect protein activity, and reduce impurity interference.

[0055] In one or more embodiments, the purification buffer comprises: 20±6mM (preferably 20±4mM, more preferably 20±2mM) phosphate, 500±100mM (preferably 500±80mM, more preferably 500±400mM) sodium chloride, 5±1% (preferably 5±0.8%, more preferably 5±0.4%) glycerol, and 10±5mM (preferably 10±3mM, more preferably 10±2mM) imidazole.

[0056] In a sixth aspect, the present invention provides the use of the anti-mouse IgG nanobody and alkaline phosphatase fusion protein described in the first aspect in the preparation of a product for immunoassay.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] The present invention designs an anti-mouse IgG nanobody and a carboxyl-terminal fragment of Shewanella alkaline phosphatase for fusion expression in Escherichia coli, which can avoid various defects of chemical labeling methods and prepare high-purity and high-specific activity enzyme-labeled antibodies. It can also shorten the secondary antibody preparation cycle, simplify the secondary antibody preparation process, improve the stability between batches, and reduce the dependence on experimental animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Schematic diagram of the structure of anti-mouse IgG nanobody and alkaline phosphatase fusion protein;

[0060] Figure 2 Figure 2 is the result of SDS-PAGE detection of the expressed and purified anti-mouse IgG nanobody and alkaline phosphatase fusion protein;

[0061] Figure 3 This is the result of ELISA detection of mouse IgG using anti-mouse IgG nanobody, alkaline phosphatase fusion protein and commercially available secondary antibody;

[0062] Figure 4 The graph shows the results of ELISA detection of mouse IgG using a series of concentrations of anti-mouse IgG nanobody and alkaline phosphatase fusion protein and commercially available secondary antibodies;

[0063] Figure 5 The figure shows the results of immunoblotting detection using anti-mouse IgG nanobody, alkaline phosphatase fusion protein and commercially available secondary antibodies. DETAILED DESCRIPTION

[0064] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0065] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0066] Example 1

[0067] In this example, a recombinant plasmid expressing anti-mouse IgG nanobody and alkaline phosphatase fusion protein was constructed.

[0068] The anti-mouse IgG nanobody and alkaline phosphatase fusion protein (named NbMIgG-AP (25-443)) is composed of an anti-mouse IgG nanobody (NbMIgG), a linker polypeptide (Linker), and the carboxyl-terminal 25-443 fragment of Shewanella alkaline phosphatase (AP). The structure is as follows Figure 1 shown.

[0069] The nucleic acid sequence encoding the anti-mouse IgG nanobody and alkaline phosphatase fusion protein was codon optimized to achieve efficient expression in prokaryotic cells. The optimized gene fragment was synthesized, and Nco I / Xho I restriction site sequences were added at both ends. The anti-mouse IgG nanobody and alkaline phosphatase fusion protein encoding gene was approximately 1680 bp in size, and its nucleotide sequence is shown in SEQ ID NO.3.

[0070] SEQ ID NO.3:NbMIgG-AP(25-443)

[0071]

[0072] At the same time, the nucleic acid sequences of other anti-mouse IgG nanobodies and alkaline phosphatase fusion proteins were constructed. The coding sequences of the fusion proteins of the anti-mouse IgG nanobody and the carboxyl-terminal 26-443, 27-443, 28-443, 29-443, 30-443, 31-443, 32-443, 33-443, 34-443, 35-443, 36-443, 37-443, 38-443, 39-443, 40-443, 41-443, 42-443, 43-443, 44-443, 45-443, 46-443, 47-443, 48-443, 49-443 or 50-443 fragment of Shewanella alkaline phosphatase were obtained by deleting 3 bases in sequence on the basis of the NbMIgG-AP (25-443) coding sequence. The fusion proteins were named NbMIgG-AP(26-443), NbMIgG-AP(27-443) to NbMIgG-AP(50-443) in sequence.

[0073] The pET28b vector and the gene fragment encoding the synthetic anti-mouse IgG nanobody and alkaline phosphatase fusion protein were double-digested by Nco I / Xho I, gel electrophoresis, recovery and purification, and DNA ligase was used to connect the two gene fragments.

[0074] The ligation product was transformed into Escherichia coli DH5α competent cells and coated on LB plates. After inverted culture overnight, positive transformants were picked out. After sequencing to confirm the accuracy, the plasmid was extracted to obtain a recombinant plasmid expressing anti-mouse IgG nanobody and alkaline phosphatase fusion protein.

[0075] Example 2

[0076] In this example, a recombinant bacterium (Rosetta-gami2(DE3)pLysS / pET28b-NbMIgG-AP) was constructed.

[0077] The recombinant plasmids prepared in Example 1 were transformed into Escherichia coli Rosetta-gami2 (DE3) pLysS competent cells, and three single colonies of each were picked and cultured in a small amount of LB medium, and then inoculated into a small amount of autoinduction medium for culture to screen strains that efficiently expressed anti-mouse IgG nanoantibodies and alkaline phosphatase fusion proteins.

[0078] The strain highly expressing anti-mouse IgG nanobody and alkaline phosphatase fusion protein was inoculated into LB medium and cultured to the logarithmic growth phase, and then frozen after adding glycerol and stored as seed bacteria.

[0079] Example 3

[0080] In this example, anti-mouse IgG nanobody and alkaline phosphatase fusion protein were expressed.

[0081] Glycerol bacteria activation: 10 μL of glycerol-preserved recombinant bacteria (prepared in Example 2) was added to 2 mL of liquid LB medium, and Kana antibiotics were added at a final concentration of 100 μg / mL. The culture was incubated at 37° C. and 220 rpm overnight.

[0082] Auto-induced expression: inoculate 80 mL of auto-induction medium at 1% inoculum, culture at 37°C, 220 rpm, for 7 h, then transfer to 20°C, 220 rpm, and culture for 40 h.

[0083] The formula of the autoinduction medium is: peptone 10 g / L, yeast extract 5 g / L, glycerol 5 g / L, lactose 2 g / L, glucose 0.5 g / L, Na2HPO4 25 mmol / L, NH4Cl 50 mmol / L, KH2PO4 25 mmol / L, Na2SO4 5 mmol / L, MgSO4 2 mmol / L.

[0084] In addition, different auto-induced expression culture temperatures and times were adjusted for expression culture (other conditions were the same) as a control group, specifically including:

[0085] (1) Incubate at 37°C for 7 hours and then continue incubation for 17 hours;

[0086] (2) After culturing at 37°C for 7 h, transfer to 17°C, 25°C, or 30°C and continue culturing for 40 h;

[0087] (3) After culturing at 37°C for 7 h, transfer to 20°C and continue culturing for 48 h;

[0088] Most of the target protein expressed in the control group (1) was in the lysis precipitate, and the amount of target protein in the lysis supernatant of the control groups (2) and (3) was slightly lower than that in the control group (1). After culturing at 37°C for 7 hours, the cells were transferred to 20°C and cultured for 40 hours. This shows that the present invention controls the specific temperature and culture time to improve the growth and metabolic efficiency of cells, promote the correct folding of proteins, improve the biological activity and stability of recombinant proteins, and further improve the expression amount of recombinant proteins.

[0089] Example 4

[0090] This example involves bacterial cell collection, lysis, and protein purification.

[0091] The cultured cells of Example 3 were collected by centrifugation at 10000 g for 10 min, washed once with 10 mL of PBS, and resuspended in 4 mL of lysis buffer (containing 50 mM phosphate, 300 mM NaCl, 10 mM imidazole, 5% glycerol, 1 mM PMSF, pH 7.0).

[0092] Ultrasonic disruption was performed in an ice bath at a power of 150 W, for 4 seconds, followed by an 8-second pause, for a total of 18 minutes.

[0093] Add 30 U / mL nuclease and 3 mM MgCl2 to the lysate and incubate in a 25°C water bath for 10 min.

[0094] The supernatant and precipitate were separated by low-temperature centrifugation at 10000g for 10 min, and the supernatant was filtered through a 0.22μm filter and used for protein purification.

[0095] The filtered supernatant was loaded onto an equilibrated nickel column at a low flow rate, washed with 10 mM imidazole solution (containing 50 mM phosphate, 300 mM NaCl, 10 mM imidazole, 5% glycerol, 1 mM PMSF, pH 7.0) and the flow-through was collected. The nickel column was washed with 40 mM imidazole solution, and the target protein was eluted with 500 mM imidazole solution. The protein concentration was determined using the Bradford method, and each collected fraction was sampled for SDS-PAGE detection. After the protein concentration was determined, the fractions were aliquoted and stored at -80°C.

[0096] SDS-PAGE results are as follows Figure 2 As shown, the anti-mouse IgG nanobody and alkaline phosphatase fusion protein are mainly expressed in a soluble form. The NbMIgG-AP(33-443) protein obtained by nickel column affinity purification has high concentration and purity. The purity and total amount of the purified protein were calculated.

[0097] Protein purity = SDS-PAGE analysis using Imag J software to obtain the ratio of the grayscale of the corresponding protein band to the grayscale of the total protein bands in the lane.

[0098] Total protein amount = protein concentration measured by Bradford method × volume of protein solution.

[0099] The results showed that 10 mL of NbMIgG-AP (33-443) protein solution was purified, with a protein purity of over 95%. The concentration of NbMIgG-AP (33-443) protein was measured by the Bradford method to be 0.5 mg / mL. The total amount of purified NbMIgG-AP (33-443) protein was 5.0 mg, that is, 5.0 mg of NbMIgG-AP (33-443) protein was purified from 80 mL of culture product, with a high yield.

[0100] However, the recombinant bacteria were constructed and the antibody was expressed by using the nucleic acid sequence of anti-mouse IgG nanobody and other Shewanella alkaline phosphatase fragment fusion protein. The recombinant bacteria expressed a small amount of NbMIgG-AP (25-443) protein. The purified NbMIgG-AP (25-443) protein had a low concentration and purity of less than 50%, and the carboxyl-terminal truncated form of the protein ( Figure 2The protein band indicated by "#" on the right was detected by immunoblotting using an antibody against the carboxyl-terminal His tag. The signal intensity of this protein band is close to that of the full-length protein band. The amount of NbMIgG-AP(50-443) protein expressed by the recombinant bacteria is too low, and only a weak band can be detected by Western blotting. It is difficult to detect by Coomassie Brilliant Blue staining, making it difficult to obtain NbMIgG-AP(50-443) protein.

[0101] Example 5

[0102] In this example, the expressed and purified NbMIgG-AP was subjected to a phosphatase activity test.

[0103] Different concentration gradients of pNP were prepared using 0.97 M diethanolamine (pH 9.8) buffer, and the pNPOD values ​​at each concentration were measured using a microplate reader. 405nm To prepare a pNP standard curve, add the purified NbMIgG-AP protein and commercially available AP-labeled goat anti-mouse to a 10mM pNPP solution (prepared in 0.97M diethanolamine, containing 4.8mM MgCl2), mix well, and react at 37°C for 10 minutes. Then, add 1M NaOH to stop the reaction and measure the OD at this time. 405nm .

[0104] The amount of pNP produced was calculated according to the standard curve, and the specific activities of different protein phosphatases (U / mg) were calculated.

[0105] The specific activity of NbMIgG-AP (25-443) protein phosphatase was measured three times and was 2950, ​​3000 and 2980 U / mg respectively. The specific activity of NbMIgG-AP (33-443) protein phosphatase was measured three times and was 4950, 5000 and 4980 U / mg respectively. The specific activity of commercially available AP-labeled goat anti-mouse phosphatase was 5500, 5650 and 5560 U / mg respectively. The specific activity of the prepared NbMIgG-AP (33-443) protein phosphatase was close to that of commercially available AP-labeled goat anti-mouse phosphatase.

[0106] Example 6

[0107] This example uses enzyme-linked immunosorbent assay to compare the sensitivity of the NbMIgG-AP protein purified in Example 4 with commercially available secondary antibodies in detecting mouse IgG.

[0108] ELISA microplates were coated with a 3-fold serial dilution of mouse IgG (MAGEA4 M pAb) at 4°C overnight and blocked with 1% BSA at 37°C for 1 hour. The purified NbMIgG-AP protein from Example 4 and a commercially available AP-labeled goat anti-mouse antibody diluted to 0.3 μg / mL were added to the test wells and incubated at 37°C for 1 hour. The plates were washed five times with PBST and patted dry. 200 μL of AP substrate solution was added to each well and incubated for 4 minutes. The chemiluminescent signal was read using a microplate reader.

[0109] The results are as follows Figure 3 As shown, the signal value and signal-to-noise ratio (SNR) of NbMIgG-AP(33-443) protein in detecting mouse IgG (Group 1) were significantly higher than those measured by commercially available AP-labeled goat anti-mouse (Group 2). The SNR of NbMIgG-AP(33-443) protein in detecting mouse IgG at 1.3 ng / mL was still 20, while the SNR of commercially available AP-labeled goat anti-mouse was only 1.8. The sensitivity of NbMIgG-AP(33-443) protein in detecting mouse IgG was more than 10-fold higher than that of commercially available AP-labeled goat anti-mouse.

[0110] Example 7

[0111] In this example, the affinity activity of the NbMIgG-AP protein purified in Comparative Example 4 and a commercially available secondary antibody to mouse IgG was detected by enzyme-linked immunosorbent assay.

[0112] ELISA microplates were coated with 0.25 μg / mL mouse IgG overnight at 4°C and blocked with 1% BSA for 1 hour at 37°C. The purified NbMIgG-AP protein from Example 4 was diluted to 1000, 500, 100, 20, 4, 0.8, 0.4, and 0 ng / mL. Commercially available AP-labeled goat anti-mouse was diluted to 6000, 3000, 600, 120, 24, 4.8, 2.4, and 0 ng / mL, respectively, and added to the test wells. The plates were incubated at 37°C for 1 hour. The plates were washed five times with PBST and patted dry. 200 μL of AP substrate solution was added to each well and reacted for 4 minutes. The chemiluminescent signal was read using a microplate reader, and a four-parameter curve was plotted with the logarithm of the antibody concentration as the horizontal axis and the luminescent signal as the vertical axis.

[0113] The results are as follows Figure 4 As shown, the signal value of the detection reached the plateau phase after adding 500ng / mL NbMIgG-AP(33-443) protein (Group 1), and the signal value of the detection reached the plateau phase after adding 6000ng / mL commercially available AP-labeled goat anti-mouse (Group 2). According to the principle of ELISA saturation concentration method for determining affinity activity, EC 50 The value is the dissociation equilibrium constant (KD value), EC 50The smaller the value, the stronger the antibody's affinity for the antigen. EC obtained by detecting mouse IgG with NbMIgG-AP(33-443) protein 50 The value was 13.69 ng / mL, and the EC obtained by detecting mouse IgG with commercially available AP-labeled goat anti-mouse 50 The value was 243.8 ng / mL. The affinity activity of NbMIgG-AP(33-443) protein to mouse IgG was about 18 times higher than that of commercially available AP-labeled goat anti-mouse.

[0114] Example 8

[0115] In this example, immunoblotting was used to detect the effect of the NbMIgG-AP protein purified in Example 4 as a secondary antibody.

[0116] 5, 2.5, and 1.25 μg of MAGEA4 protein were loaded onto the wells of an SDS-PAGE gel. After electrophoresis separation, the membranes were transferred to PVDF membranes and blocked with 5% BSA. The membranes were then incubated with a mouse primary antibody against MAGEA4 and a rabbit primary antibody against MAGEA4 for 1 hour at 37°C. The membranes were washed five times with PBST. The membranes were then incubated with NbMIgG-AP protein, a commercially available AP-conjugated goat anti-mouse secondary antibody, and a commercially available AP-conjugated goat anti-rabbit secondary antibody at 37°C for 1 hour. The membranes were washed five times with PBST, and AP enzyme luminescent substrate solution was added for color development. Four antibody combinations were used for immunoblotting: combination 1: mouse primary antibody and NbMIgG-AP protein; combination 2: mouse primary antibody and commercially available AP-conjugated goat anti-mouse secondary antibody; combination 3: rabbit primary antibody and NbMIgG-AP protein; and combination 4: rabbit primary antibody and commercially available AP-conjugated goat anti-rabbit secondary antibody.

[0117] The results are as follows Figure 5 As shown, the signal intensity of the protein band detected with the NbMIgG-AP(33-443) protein secondary antibody was significantly higher than that detected with a commercially available AP-labeled goat anti-mouse secondary antibody. A band corresponding to the MAGEA4 multimer was clearly visible in the wells loaded with 1.25 μg of protein, whereas this protein band was not detected with the commercially available AP-labeled goat anti-mouse secondary antibody. The results from antibody combinations 3 and 4 demonstrate that the NbMIgG-AP(33-443) protein does not bind to the rabbit primary antibody, demonstrating high specificity.

[0118] In summary, the present invention designs the fusion expression of anti-mouse IgG nanobodies and Shewanella alkaline phosphatase in Escherichia coli, which can avoid the various defects of chemical labeling methods and prepare high-purity and high-specific activity enzyme-labeled antibodies, while also shortening the secondary antibody preparation cycle, simplifying the secondary antibody preparation process, improving the stability between batches, and reducing the dependence on experimental animals.

[0119] The applicant states that the present invention is intended to illustrate the detailed methods of the present invention through the above-described embodiments, but the present invention is not limited to the above-described detailed methods, that is, it does not mean that the present invention must rely on the above-described detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. An anti-mouse IgG nanobody and alkaline phosphatase fusion protein, characterized in that: The anti-mouse IgG nano antibody and alkaline phosphatase fusion protein comprises anti-mouse IgG nano antibody, connecting peptide and alkaline phosphatase from amino terminal to carboxyl terminal.

2. The anti-mouse IgG nanobody and alkaline phosphatase fusion protein according to claim 1, characterized in that The amino acid sequence of the anti-mouse IgG nanobody includes the sequence shown in SEQ ID NO.1; Preferably, the connecting peptide comprises (GGGGS)n or A(EAAAK)nA, where n is selected from any one of 3-6; Preferably, the alkaline phosphatase comprises Shewanella alkaline phosphatase; Preferably, the amino acid sequence of the Shewanella alkaline phosphatase comprises the sequence shown in SEQ ID NO.2; Preferably, the Shewanella alkaline phosphatase is selected from any one of the carboxyl-terminal 25-443, 26-443, 27-443, 28-443, 29-443, 30-443, 31-443, 32-443, 33-443, 34-443, 35-443, 36-443, 37-443, 38-443, 39-443, 40-443, 41-443, 42-443, 43-443, 44-443, 45-443, 46-443, 47-443, 48-443, 49-443 or 50-443 fragments thereof.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-mouse IgG nanobody and alkaline phosphatase fusion protein according to claim 1 or 2.

4. A recombinant vector, characterized in that The recombinant vector contains the nucleic acid molecule according to claim 3.

5. A recombinant cell, characterized in that The recombinant cell contains the nucleic acid molecule according to claim 3.

6. A method for preparing the anti-mouse IgG nanobody and alkaline phosphatase fusion protein according to claim 1 or 2, characterized in that: The preparation method comprises: The recombinant vector according to claim 4 is introduced into a host cell to obtain a recombinant cell, which is cultured, and the cells are collected and mixed with a lysis buffer to obtain a lysis product; the lysis product is purified to obtain the anti-mouse IgG nanobody and alkaline phosphatase fusion protein.

7. The method according to claim 6, characterized in that The culturing comprises culturing at 34-40° C. for 4-8 hours and then adjusting the temperature to 17-23° C. for culturing for 24-48 hours.

8. The method according to claim 6 or 7, characterized in that The lysis buffer contains phosphate, sodium chloride, glycerol and phenylmethylsulfonyl fluoride; Preferably, the collecting cells and mixing with the lysis buffer further comprises ultrasonic treatment under ice bath conditions; Preferably, the ultrasonic treatment includes ultrasonic power of 150-250W, ultrasonication for 3-5s, pause for 5-7s, and ultrasonication for a total of 10-20 minutes.

9. The method according to any one of claims 6 to 8, characterized in that The purification method includes affinity purification; Preferably, the purification buffer for affinity purification contains phosphate, sodium chloride, glycerol and imidazole.

10. Use of the anti-mouse IgG nanobody and alkaline phosphatase fusion protein according to claim 1 or 2 in the preparation of a product for immunoassay.

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