Escherichia coli recombinant bacterium capable of producing nano antibody as well as construction method and application of escherichia coli recombinant bacterium

Abstract

The invention discloses an escherichia coli recombinant strain for producing a nano antibody as well as a construction method and application of the escherichia coli recombinant strain. According to the present invention, three engineering bacteria such as E.coli BL21 (DE3) pET28a-5-D8, E.coli BL21 (DE3) pET28a-PT5-5-D8 and E.coli ArcticExpresses (DE3) pRARE2pET28a-PT5-5-D8 are compared, and it is found that the E.coli BL21 (DE3) pET28a-PT5-5-D8 and the E.coli BL21 (DE3) pET28a-PT5-5-D8 are mainly expressed in a non-soluble manner, while the E.coli ArcticExpresses (DE3) pRARE2pET28a-PT5-5-D8 uses a weak promoter to control the nanometer antibody 5-D8 and replace the expression host, specifically, the Protein folding is promoted, and the soluble expression proportion is increased. In E.coli ArcticExpresses (DE3) pRARE2, the expression quantity of the nano antibody is obviously higher than that of E.coli (DE3), the proportion of soluble protein is further increased, and balance optimization of the expression quantity and solubility is achieved.

Description

Technical field:

[0001] The present invention relates to the technical field of bioengineering, and in particular to a recombinant Escherichia coli strain producing nanoantibodies, a construction method thereof, and applications thereof. Background technology:

[0002] Nanobodies are a class of single-domain antibodies with unique biological activity. Due to their small molecular weight, high stability, strong binding affinity, and ease of engineering, they hold great promise for application in disease diagnosis, treatment, and biosensing. Currently, traditional methods for preparing nanobodies rely primarily on animal immunization or mammalian cell culture. These methods suffer from long production cycles, high costs, and limited yields, making them difficult to meet the requirements of large-scale industrial production.

[0003] In recent years, the use of Escherichia coli recombinant expression systems to construct nanoantibody production strains has become a new approach that has attracted much attention. As a host bacterium, Escherichia coli has the advantages of fast growth rate, simple operation and low fermentation cost, and can achieve efficient expression of nanoantibody genes through genetic engineering technology. However, since exogenous nanoantibodies are often deposited in the form of inclusion bodies in E. coli, the proportion of active nanoantibodies expressed is low, and it is necessary to rely on complex folding and repair processes, which increases the production cost of nanoantibodies. To this end, domestic and foreign scholars have conducted extensive research on the optimization of protein expression in E. coli, and proposed a variety of strategies such as low-temperature induction, molecular chaperone-assisted co-expression and codon optimization, which effectively improved the solubility and activity of nanoantibodies in host cells. However, there are still problems such as poor soluble expression of nanoantibodies.

[0004] Therefore, constructing a recombinant Escherichia coli that produces nanoantibodies is not only expected to overcome the problems of high cost, low efficiency and complex purification in traditional production methods, but also provides a practical technical route for the industrial production and wide application of nanoantibodies, which is of great significance to meeting the market demand for high-quality and low-cost nanoantibody products. Summary of the invention:

[0005] Purpose of the invention:

[0006] To address the above problems, the present invention provides a recombinant Escherichia coli strain and a method for constructing the same, which achieves efficient soluble expression of nanobodies by optimizing gene expression elements and host systems, thereby simplifying the production process and reducing costs.

[0007] Technical solution:

[0008] The present invention first provides a recombinant bacterium for synthesizing nanobodies. The recombinant bacterium uses Escherichia coli as a host, and a plasmid is used to express the nanobodies in an episomal manner. The plasmid-free expression system is a pET28a plasmid.

[0009] In one embodiment of the present invention, the pET28a plasmid is used to express Nanobodies.

[0010] In one embodiment of the present invention, preferably, the amino acid sequence of the Nanobody is as shown in SEQ ID NO: 1, and the nucleotide sequence after codon optimization is as shown in SEQ ID NO: 2.

[0011] In one embodiment of the present invention, a His (nucleotide sequence such as SEQ ID NO.3) tag is added to the N-terminus of the nanobody, and a FLAG (nucleotide sequence such as SEQ ID NO.4) and HA (nucleotide sequence such as SEQ ID NO.5) tag are added to the C-terminus, and the final nucleotide sequence is shown in SEQ ID NO:6.

[0012] In one embodiment of the present invention, the P T5 The promoter nucleotide sequence is shown in SEQ ID NO:7.

[0013] In one embodiment of the present invention, the host is preferably E. coli ArcticExpress (DE3) pRARE2, which carries a molecular chaperone gene to assist in the correct folding of proteins.

[0014] The present invention also provides a method for producing nanobodies, which comprises culturing the recombinant Escherichia coli.

[0015] In one embodiment of the present invention, the culture is to culture the recombinant E. coli in LB medium at 37°C, 200 rpm, for 8 to 10 hours, and then transfer to LB medium at 37°C, 200 rpm, and culture until the OD 600 =0.6-1, add IPTG, and induce at 16-25℃ for 8h.

[0016] In one embodiment of the present invention, the supernatant is purified directly by Ni column affinity chromatography (as in Example 6) without the need for inclusion body renaturation.

[0017] Beneficial effects:

[0018] Nanobodies (Nb) are artificially designed antibody molecules, also known as single-domain antibodies (sdAbs), VHH antibodies, or camelid antibodies. They are heavy-chain antibodies (HCAbs) that are naturally missing light chains and are found in camelids such as alpacas and dromedaries, as well as cartilaginous fish such as sharks and rays. Although nanobodies are known for their high stability, they may still encounter solubility issues in practical applications. This is related to the following reasons:

[0019] (1) Insolubility caused by molecular structural characteristics, such as: (1) excessive hydrophobicity of the CDR3 region; (2) framework region (FR) mutations, which may affect surface charge distribution and reduce solubility; (3) disulfide bond defects, incomplete disulfide bond formation in the expression system can lead to misfolding;

[0020] (2) Problems related to the expression system, such as the limitations of the prokaryotic expression system: Escherichia coli lacks a eukaryotic post-translational modification system, and the inclusion body formation rate is as high as 30-70% (especially during high-density fermentation); the insufficient oxidative environment in the periplasmic space affects the formation of disulfide bonds.

[0021] Therefore, solving the poor solubility of nanobodies has become a technical problem in this field.

[0022] Nanobody 5-D8 is a brand-new antibody that has not been reported in the literature. It is a Her2 antigen. At the same time, its preparation also faces technical problems such as poor solubility of nanoantibodies, low expression levels, and difficulty in separation and purification.

[0023] 1. The present invention compares E. coli BL21 (DE3) pET28a-5-D8, E. coli BL21 (DE3) pET28a-P T5 -5-D8 and E. coli ArcticExpress (DE3) pRARE2 pET28a-P T5 -5-D8 three kinds of engineered bacteria, found that E. coli BL21 (DE3) pET28a-5-D8 and E. coli BL21 (DE3) pET28a-PT5-5-D8 were mainly expressed in an insoluble manner, while E. coli Arctic Express (DE3) pRARE2 pET28a-PT5-5-D8 used a weak promoter to control the nanobody 5-D8 and replaced the expression host, as follows: pET28a plasmid has BglII / XbaI restriction sites, inserted a weak promoter (T5), through the promoter replacement, down-regulated the transcription rate, promoted protein folding, and increased the soluble expression ratio. In E. coli Arctic Express (DE3) pRARE2, the expression level of the nanobody was significantly higher than that of E. coli (DE3), and the proportion of soluble protein was further improved, achieving a balanced optimization of expression level and solubility.

[0024] 2. The present invention adopts a two-stage culture method: proliferation stage: culture at 37 ° C, 200 rpm until OD 600=0.6-0.8, rapidly accumulating bacterial cell density. Induction stage: Add 100mg / L IPTG and induce at 16-25°C for 8 hours to inhibit inclusion body formation and directly obtain soluble nanobodies. The low-temperature induction at 16-25°C significantly reduces the misfolding of nanobodies in cells, omitting the traditional inclusion body renaturation step, improving downstream purification efficiency, and enhancing soluble expression.

[0025] 3. The culture process only uses conventional LB medium, without the need for special ingredients or complex formulas, significantly reducing fermentation costs while maintaining efficient expression and simplifying the production process. This process does not require complex feeding or inducer gradient optimization, and the total culture cycle is ≤18 hours, making it suitable for industrial scale-up. The overall process of the present invention is simple, with a short production cycle, low cost and easy scale-up, providing a reliable and economical technical platform for the industrial production of nanobodies.

[0026] 4. The expressed nanoantibodies (such as 5-D8) were verified by SPR detection and showed strong affinity with antigen (Her2) (KD = 2.40 × 10 -6 M), has practical application value. Description of the drawings:

[0027] Figure 1 Schematic diagram of adding tags to nanobody 5-D8

[0028] Figure 2 This is a map of the recombinant plasmid pET28a-5-D8.

[0029] Figure 3 The recombinant plasmid pET28a-P T5 -5-D8 spectrum.

[0030] Figure 4 This is an SDS identification diagram of the nanobody, wherein Marker is a protein marker, the supernatant is the supernatant of the E. coli BL21 (DE3) pET28a-5-D8 disruption solution, and the whole cells are the E. coli BL21 (DE3) pET28a-5-D8 disruption solution.

[0031] Figure 5 This is the SDS identification diagram of the nanobody, where Marker is a protein marker and the supernatant is E.coli BL21 (DE3) pET28a-P T5 -5-D8 supernatant, whole cells are E. coli BL21 (DE3) pET28a-P T5 -5-D8 crushing liquid.

[0032] Figure 6This is the SDS identification diagram of the nanobody, where the Marker is a protein marker and the supernatant is E. coli Arctic Express (DE3) pRARE2 pET28a-P T5 -5-D8 supernatant, whole cells are E. coli Arctic Express (DE3) pRARE2 pET28a-P T5 -5-D8 crushing liquid.

[0033] Figure 7 The 5-D8 affinity results of the nanobody. Specific implementation method:

[0034] The following examples all use conventional experimental methods, and the experimental materials are all commercially available.

[0035] Sample pretreatment: The induced recombinant Escherichia coli cells were taken and ultrasonically disrupted to obtain whole cell disruption fluid. The whole cell disruption fluid was centrifuged at 12000 rpm for 10 min, and the supernatant was collected. The expression of nanoantibodies was detected by polyacrylamide gel electrophoresis.

[0036] Nanobody affinity detection conditions:

[0037] Antigen coupling conditions: 20 μg / ml, 5 μl / min, 20 minutes.

[0038] Antibody testing concentration gradient: 100nM, 1μM, 5μM, 9μM.

[0039] Regeneration solution: 7 mM NaOH, flow rate 30 μl / min, 1 minute.

[0040] Example 1: Construction of pET28a-5-D8 plasmid

[0041] According to the amino acid sequence of nanobody 5-D8 (amino acid sequence such as SEQ ID NO: 1), the nanobody 5-D8 gene was codon optimized (gene sequence such as SEQ ID NO: 2) to make the gene sequence more suitable for the large intestine expression system. In order to facilitate the subsequent purification, a HisA (gene sequence such as SEQ ID NO. 3) tag was added to the N-terminus of nanobody 5-D8. In order to facilitate further modification in the later stage, FLAG (gene sequence such as SEQ ID NO. 4) and HA (gene sequence such as SEQ ID NO. 5) tags were added to the C-terminus of nanobody 5-D8 in sequence. The final nucleotide sequence is shown in SEQ ID NO: 6, and the schematic diagram is shown in FIG. Figure 1 The artificially synthesized 5-D8 gene fragment was connected to the pET28a vector after enzyme digestion by homologous recombination to obtain the pET28a-5-D8 plasmid, see Figure 2 shown.

[0042] Example 2: Construction of recombinant bacteria BL21 (DE3) pET28a-5-D8 for synthesizing nanobodies

[0043] The recombinant pET28a-5-D8 plasmid was transformed into the expression host E. coli BL21 (DE3), screened on an LB plate containing kanamycin, and the positive transformants obtained by colony PCR identification were the genetically engineered E. coli bacteria of the present invention, named BL21 (DE3) pET28a-5-D8.

[0044] The recombinant bacteria containing the recombinant plasmid obtained above were inoculated into LB liquid medium containing 30 mg / L kanamycin and cultured at 37°C and 200 rpm for 8 to 10 h. The above culture was inoculated into LB medium containing 30 mg / L kanamycin at a 2% inoculum size and cultured at 37°C and 200 rpm until the OD 600 =0.6-0.8, add 100 mg / L inducer IPTG, 16-25°C, 200 rpm, induce for 8 h, and collect the bacteria by centrifugation.

[0045] The collected bacteria were washed twice with PBS, disrupted by ultrasound, and the expression effect of nanobody 5-D8 was analyzed by polyacrylamide gel electrophoresis.

[0046] Example 3: Synthesis of Nanobodies by Recombinant E. coli BL21 (DE3) pET28a-P T5 -5-D8 Construction

[0047] According to P T5 The nucleotide sequence of SEQ ID NO: 7 was synthesized by primer P T5 -S and P T5 -A, primer annealing was used to obtain fragment PT. Using polyphosphate kinase, fragment PT was phosphorylated to obtain fragment PT-P. Fragment PT-P was ligated into plasmid pET28a-5-D8 double-digested with BglII and XbaI to obtain pET28a-P T5 -5-D8, such as Figure 3 The recombinant plasmid was transformed into the expression host E. coli BL21(DE3), screened on LB plates containing kanamycin, and identified by colony PCR. The positive transformants obtained were the genetically engineered E. coli strain of the present invention and were designated E. coli BL21(DE3)pET28a-PT5-5-D8. Primer sequences are shown in Table 1.

[0048] Table 1 Primer sequences used for nanobody construction

[0049]

[0050] Example 4: Synthesis of Nanobodies by Recombinant E. coli ArcticExpress (DE3) pRARE2 pET28a-P T5 -5-D8 Construction

[0051] The recombinant pET28a-5-D8 plasmid was transformed into the expression host E. coli ArcticExpress (DE3) pRARE2), and the positive transformants obtained by colony PCR identification were the genetically engineered E. coli bacteria of the present invention, and were named E. coli ArcticExpress (DE3) pRARE2pET28a-P T5 -5-D8.

[0052] Example 5: Detection of recombinant bacteria synthesizing nanobody 5-D8

[0053] Combine E.coli BL21(DE3)pET28a-5-D8, E.coli BL21(DE3)pET28a-P T5 -5-D8 and E. coli Arctic Express (DE3) pRARE2 pET28a-P T5 -5-D8 recombinant bacteria were inoculated into LB liquid medium containing 30 mg / L kanamycin and 15 mg / L chloramphenicol, and cultured at 37°C and 200 rpm for 8-10 h. The above culture was inoculated into LB medium containing 30 mg / L kanamycin and 15 mg / L chloramphenicol at a 2% inoculum size, and cultured at 37°C and 200 rpm until the bacteria reached OD 600 =0.6-0.8, add 100 mg / L of the inducer IPTG, induce at 16-25°C, 200 rpm, and induce for 8 hours. Collect the cells by centrifugation. Ultrasonic disruption is performed to obtain a whole-cell lysate, which is then centrifuged at 12,000 rpm for 10 minutes to collect the supernatant. The whole-cell lysate and supernatant are then combined and analyzed by polyacrylamide gel electrophoresis to detect the expression of the nanobody 5-D8.

[0054] like Figure 4 As shown, the nanobody 5-D8 has obvious protein electrophoresis bands in the whole cell disrupted liquid of E. coli BL21 (DE3) pET28a-5-D8, while the bands in the supernatant are weak, indicating that it is mainly expressed in an insoluble manner in the engineered bacteria 5-D8.

[0055] Similarly, if Figure 5 As shown, Nanobody 5-D8 was expressed in E. coli BL21 (DE3) pET28a-PT5 -There are obvious protein electrophoresis bands in the whole cell disrupted liquid of 5-D8, while the bands in the supernatant are weak, indicating that the protein is mainly expressed in an insoluble manner in the engineered bacteria 5-D8.

[0056] And as Figure 6 As shown, nanobody 5-D8 was expressed in the engineering bacteria E. coli ArcticExpress (DE3) pRARE2pET28a-P T5 Obvious protein electrophoresis bands appeared in the whole cell lysate and supernatant of -5-D8, indicating that the use of a weak promoter to control the nanobody 5-D8 and the replacement of the expression host can downregulate its expression level and make the nanobody expressed in a soluble manner.

[0057] Example 6: Purification of Nanobody 5-D8

[0058] The induced E. coli ArcticExpress (DE3) pRARE2 pET28a-P T5 -5-D8 bacteria were ultrasonically disrupted until the solution was clear, and the supernatant was collected by centrifugation at 10000 rpm at 4°C for 20 min. The supernatant was sterilized by filtration through a 0.22 μm filter membrane before loading. 2+ The column was washed with 20% alcohol and deionized water, the system parameters were set, the sample was loaded (the sample volume was 12 ml), and the elution gradient was 5%, 10%, 20%, 40%, and 100%. The column was washed to complete the purification and obtain the nanobody 5-D8.

[0059] Example 7: Affinity detection of Nanobody 5-D8

[0060] The CM5 chip channel (Fc2-Fc1) was cleaned with a 50mM NaOH solution and an NHS-EDC mixture was injected (flow rate 10μl / min, continued for 15 minutes) to activate the carboxyl groups on the chip surface. The antigen (her2, purchased from Yiqiao Shenzhou) solution (20μg / ml) was injected into the Fc1 channel at a flow rate of 5μl / min for 20 minutes to covalently immobilize the antigen on the chip surface. Unreacted active sites were blocked using an ethanolamine solution (flow rate 10μl / min, continued for 10 minutes), and the antigen coupling amount was confirmed to be 2304 milliseconds (mRU) by SPR signal.

[0061] Different concentrations of 5-D8 nanoantibodies (100 nM to 9 μM) were sequentially injected into the chip (Fc2-Fc1 channel) at a flow rate of 30 μl / min, with an association time of 2 minutes and a dissociation time of 300 seconds. The binding signal was monitored in real time. After each test, the chip was regenerated with 7 mM NaOH (flow rate of 30 μl / min for 1 minute) to remove bound antibodies and restore chip activity.

[0062] SPR Analysis software was used to perform a 1:1 global fitting of the binding curve to calculate the association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD). The experimental results showed that the KD value of the 5-D8 nanobody was 2.40×10 -6 M, indicating that it has a higher affinity with the antigen.

[0063] Table 2: The loading concentrations of 5-D8 nanobody analytes are 100nM, 1u, 5u, and 9u, respectively. The 1:1 global fitting results are as follows:

[0064] curve Rmax ka(1 / (M*s)) kd(1 / s) KD(M) RI Chi2 Curve 2(1e-07) 57.7459 2.13E+03 5.10E-03 2.40E-06 0.3417 2.4 Curve 3(1e-06) 57.7459 2.13E+03 5.10E-03 2.40E-06 0.963838 2.4 Curve 4(5e-06) 57.7459 2.13E+03 5.10E-03 2.40E-06 4.08542 2.4 Curve 5(9e-06) 57.7459 2.13E+03 5.10E-03 2.40E-06 1.86842 2.4

Claims

1. A nanobody, characterized in that Its amino acid sequence is shown in SEQ ID NO:

1.

2. A recombinant Escherichia coli producing nanobodies, characterized in that: E. coli Arctic Express (DE3) pRARE2 was used as the host bacteria, carrying the recombinant plasmid pET28a, which contained the nanobody shown in the nucleotide sequence of SEQ ID NO: 2 and the weak promoter T5 shown in the nucleotide sequence of SEQ ID NO:

7.

3. The recombinant bacterium according to claim 2, characterized in that A His tag is added to the N-terminus of the nanobody, and a FLAG and HA tag are added to the C-terminus.

4. The recombinant bacterium according to claim 3, characterized in that The nucleotide sequence of His is shown in SEQ ID NO.3, the nucleotide sequence of FLAG is shown in SEQ ID NO.4, and the nucleotide sequence of HA is shown in SEQ ID NO.

5.

5. The method for preparing a nanobody according to claim 1, wherein The nanobody is prepared by the recombinant bacteria according to any one of claims 2 to 4.

6. Use of the recombinant bacteria according to any one of claims 2 to 4 in the preparation of nanobodies.

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