Escherichia coli recombinant strain for producing nanobodies, construction method and application thereof

Abstract

The application relates to an E. coli recombinant strain for producing nanobody, a construction method and application thereof. The application compares three engineering bacteria E. coli BL21(DE3)pET28a-5-D8, E. coli BL21(DE3)pET28a-P T5 -5-D8 and E. coli ArcticExpress(DE3)pRARE2pET28a-P T5 -5-D8, finds that E. coli BL21(DE3)pET28a-5-D8 and E. coli BL21(DE3)pET28a-PT5-5-D8 are mainly expressed in a non-soluble manner, and E. coli ArcticExpress(DE3)pRARE2pET28a-PT5-5-D8 adopts a weak promoter to control nanobody 5-D8 and changes an expression host, and the specific process is as follows: the pET28a plasmid has BglII / XbaI enzyme cutting sites, a weak promoter (T5) is inserted, the transcription rate is down-regulated through promoter replacement, protein folding is promoted, and the soluble expression proportion is improved. In the E. coli ArcticExpress(DE3)pRARE2, the expression amount of the nanobody is significantly higher than that of E. coli(DE3), and the soluble protein proportion is further improved, so that the balance optimization of the expression amount and the solubility is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, in particular to a recombinant Escherichia coli strain for producing nanobodies and a construction method and application thereof. BACKGROUND

[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 easy engineering, they have shown great application prospects in disease diagnosis, treatment and biosensing. Currently, the traditional preparation methods of nanobodies mainly rely on animal immunization or mammalian cell culture, which have the disadvantages of long cycle, high cost and limited yield, and cannot meet the requirements of large-scale industrial production.

[0003] In recent years, the use of Escherichia coli recombinant expression system to construct nanobody production strains has become a new approach that attracts much attention. As a host bacterium, Escherichia coli has the advantages of fast growth, simple operation and low fermentation cost, and can realize efficient expression of nanobody genes through genetic engineering technology. However, due to the deposition of exogenous nanobodies in the form of inclusion bodies in Escherichia coli, the proportion of active nanobodies expressed is low, and complex folding and repair processes are required, which increases the production cost of nanobodies. Therefore, domestic and foreign scholars have carried out a lot of research on the optimization of protein expression in Escherichia coli, and have proposed various strategies such as low-temperature induction, molecular chaperone-assisted co-expression and codon optimization, which have effectively improved the solubility and activity of nanobodies in host cells. However, there are still problems such as poor solubility of nanobodies.

[0004] Therefore, the construction of a recombinant Escherichia coli strain for producing nanobodies is expected to overcome the problems of high cost, low efficiency and complex purification in traditional production methods, and provides a feasible technical route for the industrial production and wide application of nanobodies, which is of great significance to meet the market demand for high-quality and low-cost nanobody products. SUMMARY

[0005] OBJECTIVE

[0006] In view of the above problems, the present application provides a recombinant Escherichia coli strain and a construction method thereof, which realizes efficient and soluble expression of nanobodies by optimizing gene expression elements and host systems, simplifies the production process and reduces the cost.

[0007] TECHNICAL SCHEME

[0008] The present application first provides a recombinant bacterium for synthesizing nanobodies, wherein the recombinant bacterium is Escherichia coli as a host, and a plasmid free expression system for expressing nanobodies, and the plasmid free expression system is pET28a plasmid.

[0009] In an embodiment of the present application, the pET28a plasmid is used to express the nanobody.

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

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

[0012] In an embodiment of the present application, the P T5 The promoter nucleotide sequence is shown as SEQ ID NO: 7.

[0013] In an embodiment of the present application, the host is preferably E. coli ArcticExpress (DE3) pRARE2, which carries chaperone genes to assist the correct folding of proteins.

[0014] The present application also provides a method for producing a nanobody, which comprises culturing the recombinant E. coli.

[0015] In an embodiment of the present application, the culturing comprises culturing the recombinant E. coli in LB medium at 37℃ and 200 rpm for 8-10 h, then transferring to LB medium, culturing at 37℃ and 200 rpm until OD 600 = 0.6-1, adding IPTG, and inducing at 16-25℃ for 8 h.

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

[0017] Beneficial effects:

[0018] A nanobody (Nb) is an artificially designed antibody molecule, also known as a single-domain antibody (sdAb), VHH antibody or camelid antibody, which is a naturally heavy-chain antibody (HCAb) found in camelids such as llamas, alpacas, and sharks, and chondrichthyan fish, which lacks light chains. Although nanobodies are known for their high stability, they may still encounter solubility problems in practical applications. This is related to the following reasons:

[0019] (1) the difficulty of solubility caused by molecular structure characteristics, for example: (1) the CDR3 region is too hydrophobic; (2) the framework region (FR) mutation, which can affect the surface charge distribution and reduce the solubility; (3) the defect of disulfide bond, incomplete disulfide bond formation in the expression system will cause misfolding;

[0020] (2) expression system related problems, for example: the limitations of prokaryotic expression system: E. coli lacks eukaryotic post-translational modification system, and the inclusion body formation rate is as high as 30-70% (especially in high-density fermentation); the insufficient oxidation environment in the periplasmic space affects the formation of disulfide bonds.

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

[0022] The nanobody 5-D8 is a brand new antibody, and no literature report is found, which is an Her2 antigen, and its preparation also faces technical problems such as nanobody solubility, low expression amount and difficult separation and purification.

[0023] 1、The present application 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 engineering bacteria, it is found that E.coli BL21(DE3)pET28a-5-D8 and E.coli BL21(DE3)pET28a-PT5-5-D8 are mainly expressed in a non-soluble manner, and E.coli ArcticExpress(DE3)pRARE2 pET28a-PT5-5-D8 adopts a weak promoter to control the nanobody 5-D8 and changes the expression host, specifically as follows: the pET28a plasmid has BglII / XbaI enzyme cutting sites, and the weak promoter (T5) is inserted, the transcription rate is down-regulated by replacing the promoter, the protein folding is promoted, and the soluble expression proportion is improved. In E.coli ArcticExpress(DE3)pRARE2, the expression amount of the nanobody is significantly higher than that of E.coli(DE3), and the soluble protein proportion is further improved, so that the balance optimization of the expression amount and the solubility is realized.

[0024] 2、The present application adopts two-stage culture method: proliferation stage: 37℃, 200rpm culture to OD 600=0.6-0.8, fast accumulation of bacterial density. Induction stage: add 100 mg / L IPTG, low temperature induction at 16-25℃ for 8 hours, inhibit inclusion body formation, and directly obtain soluble nanobodies. Among them, by low temperature induction at 16-25℃, the misfolding of nanobodies in the cell is significantly reduced, the traditional inclusion body renaturation step is omitted, the downstream purification efficiency is improved, and the soluble expression is improved.

[0025] 3. The culture process only uses conventional LB medium without special ingredients or complex formula, which significantly reduces the fermentation cost while maintaining high expression and simplifies the production process. The process does not need complex feeding or induction agent gradient optimization, and the total culture period is ≤18 hours, which is suitable for industrial amplification. The whole process of the present application is simple, short production cycle, low cost and easy to amplify culture, which provides a reliable and economical technical platform for the industrial production of nanobodies.

[0026] 4. The expressed nanobodies (such as 5-D8) are verified by SPR detection, and have strong affinity (KD=2.40×10 -6 M) with antigens (Her2), which has practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Schematic diagram for adding a label to nanobody 5-D8.

[0028] Figure 2 Map of recombinant plasmid pET28a-5-D8.

[0029] Figure 3 Map of recombinant plasmid pET28a-P T5 -5-D8.

[0030] Figure 4 SDS identification diagram of nanobodies, wherein Marker is protein Marker, supernatant is E. coli BL21(DE3)pET28a-5-D8 broken liquid supernatant, and whole cell is E. coli BL21(DE3)pET28a-5-D8 broken liquid.

[0031] Figure 5 SDS identification diagram of nanobodies, wherein Marker is protein Marker, supernatant is E. coli BL21(DE3)pET28a-P T5 -5-D8 broken liquid supernatant, and whole cell is E. coli BL21(DE3)pET28a-P T5 -5-D8 broken liquid.

[0032] Figure 6SDS identification figure of nanobody, wherein Marker is protein Marker, supernatant is E. coli Arctic Express (DE3) pRARE2 pET28a-P T5 -5-D8 broken liquid supernatant, whole cell is E. coli Arctic Express (DE3) pRARE2 pET28a-P T5 -5-D8 broken liquid.

[0033] Figure 7 5-D8 affinity result of nanobody. DETAILED DESCRIPTION

[0034] The following examples are all adopted by conventional experimental methods, and the experimental materials are all available from commercial channels.

[0035] Sample pretreatment: take the recombinant E. coli bacteria after induction, ultrasonic broken, obtain whole cell broken liquid, centrifuge the whole cell broken liquid at 12000 rpm for 10 min to collect the supernatant, and detect the expression of nanobody by polyacrylamide gel electrophoresis.

[0036] Nanobody affinity detection conditions:

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

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

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

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

[0041] According to the amino acid sequence of nanobody 5-D8 (amino acid sequence as SEQ ID NO: 1), the nanobody 5-D8 gene is codon optimized (gene sequence as SEQ ID NO: 2), so that the gene sequence is more suitable for the expression system of E. coli. In order to facilitate the later purification, HisA (gene sequence as SEQ ID NO. 3) tag is added to the N terminal of nanobody 5-D8, and in order to facilitate the later further modification, FLAG (gene sequence as SEQ ID NO. 4) and HA (gene sequence as SEQ ID NO. 5) tags are added to the C terminal of nanobody 5-D8 in turn, and the final nucleotide sequence is shown as SEQ ID NO: 6, and the schematic diagram is shown as Figure 1 The artificially synthesized 5-D8 gene fragment is connected to the enzyme cut pET28a vector by homologous recombination to obtain the pET28a-5-D8 plasmid, as shown in Figure 2 .

[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 LB plates containing kanamycin, and the positive transformants obtained were identified by colony PCR to obtain the E. coli genetically engineered bacteria of the present application, designated 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, 200 rpm for 8-10 h. The culture was inoculated into LB medium containing 30 mg / L kanamycin at an inoculation amount of 2%, and the bacterial cells were cultured at 37°C, 200 rpm until the OD 600 = 0.6-0.8, 100 mg / L inducer IPTG was added, and the cells were induced at 16-25°C, 200 rpm for 8 h, and then centrifuged to collect the bacterial cells.

[0045] The bacterial cells collected above were washed with PBS for 2 times, and then disrupted by ultrasonication, and the expression effect of nanobody 5-D8 was analyzed by polyacrylamide gel electrophoresis.

[0046] Example 3: Construction of recombinant bacteria E. coli BL21(DE3)pET28a-P T5 -5-D8 for synthesizing nanobodies

[0047] According to the nucleotide sequence of P T5 , the primers P T5 -S and P T5 -A were synthesized, and the fragment PT was obtained by primer annealing. The fragment PT was phosphorylated by polyphosphatase to obtain the fragment PT-P. The fragment PT-P was ligated to the plasmid pET28a-5-D8 digested by BglII and XbaI to obtain pET28a-P T5 -5-D8, as shown in Figure 3 The recombinant plasmid was transformed into the expression host E. coli BL21(DE3), screened on LB plates containing kanamycin, and the positive transformants obtained were identified by colony PCR to obtain the E. coli genetically engineered bacteria of the present application, designated E. coli BL21(DE3)pET28a-PT5-5-D8. The primer sequences are shown in Table 1.

[0048] Table 1 Primer sequences for nanobody construction

[0049]

[0050] Example 4: Recombinant bacteria for synthesizing nanobodies: E. coli ArcticExpress(DE3)pRARE2 pET28a-P T5 Construction of -5-D8

[0051] The recombinant pET28a-5-D8 plasmid was transformed into the expression host *E. coli* ArcticExpress(DE3)pRARE2. Screening was performed on LB agar plates containing kanamycin and chloramphenicol. Positive transformants obtained by colony PCR identification were identified as the *E. coli* genetically engineered strain described in this invention, named *E. coli* ArcticExpress(DE3)pRARE2pET28a-P. T5 -5-D8.

[0052] Example 5: Detection of recombinant bacteria that synthesized 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 cultures were then inoculated at a rate of 2% into LB medium containing 30 mg / L kanamycin and 15 mg / L chloramphenicol, and cultured at 37°C and 200 rpm until the bacterial cells reached OD200. 600 =0.6-0.8, add 100 mg / L of IPTG as an inducer, incubate at 16-25℃ and 200 rpm for 8 h, then centrifuge to collect the bacterial cells. Sonicate to obtain whole-cell lysate, centrifuge at 12000 rpm for 10 min to collect the supernatant, and then analyze the expression of the 5-D8 nanobody using polyacrylamide gel electrophoresis.

[0054] like Figure 4 As shown, the nanobody 5-D8 showed obvious protein electrophoresis bands in the whole-cell lysate of E. coli BL21(DE3)pET28a-5-D8, while the bands were weak in the supernatant, indicating that the 5-D8 nanobody is mainly expressed in an insoluble manner in this engineered bacterium.

[0055] Similarly, such as Figure 5 As shown, the nanobody 5-D8 was synthesized in E. coli BL21(DE3)pET28a-PT5 There are obvious protein electrophoresis bands in the whole cell broken solution of 5-D8, but weak bands in the supernatant, which indicates that the expression of 5-D8 in the engineering bacteria is mainly in the form of non-soluble.

[0056] As shown in Fig. 2, the nanobody 5-D8 is expressed in the engineering bacteria E. coli ArcticExpress (DE3) pRARE2 pET28a-P Figure 6 T5 There are obvious protein electrophoresis bands in the whole cell broken solution and supernatant of 5-D8, which indicates that the expression of nanobody 5-D8 can be down-regulated by using weak promoter and changing the expression host, so that the nanobody can be expressed in soluble form.

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

[0058] The E. coli ArcticExpress (DE3) pRARE2 pET28a-P T5 5-D8 bacteria after induction were subjected to ultrasonic disruption until the solution was clear, and the supernatant was collected by centrifugation at 10000 rpm for 20 min at 4°C. The supernatant was filtered through a 0.22 μm filter membrane to remove bacteria, and then was subjected to loading. An AKTA was loaded with a 1 ml-Ni 2+ column, 20% alcohol, and deionized water, and the system parameters were set. The sample was loaded (loading volume 12 ml), and the elution gradient was 5%, 10%, 20%, 40%, and 100% to wash the column. The purification was completed, and nanobody 5-D8 was obtained.

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

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

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

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

[0063] Table 2: 5-D8 nanobody analyte loading concentration in turn 100 nM, 1 u, 5 u, 9 u, 1 :1 global fitting results as follows:

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

Claims

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

1.

2. An Escherichia coli recombinant bacterium producing a nanobody, characterized in that, E. coli Arctic Express (DE3) pRARE2 as the host bacteria, carrying recombinant plasmid pET28a, which contains the nucleotide sequence of SEQ ID NO: 2 Nanobody, the weak promoter T5 nucleotide sequence as shown in SEQ ID NO:

7.

3. The recombinant bacteria of claim 2, wherein, The N-terminal of the Nanobody is added with His tag, and the C-terminal is added with FLAG and HA tags.

4. The recombinant bacteria of claim 3, wherein, The nucleotide sequence of His is shown as SEQ ID NO. 3, the nucleotide sequence of FLAG is shown as SEQ ID NO. 4, and the nucleotide sequence of HA is shown as SEQ ID NO.

5.

5. The method for preparing nanobodies according to claim 1, characterized in that, The Nanobody is prepared by the recombinant bacteria according to any one of claims 2-4.

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

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