Molecular chaperone PDI for improving IgM recombinant expression efficiency and application of molecular chaperone PDI

Abstract

The invention relates to the technical field of recombinant antibody expression, in particular to a molecular chaperone PDI for improving IgM recombinant expression efficiency and application of the molecular chaperone PDI, and the molecular chaperone PDI comprises an amino acid sequence as shown in SEQ ID NO: 3. By means of molecular chaperone co-expression, the expression stability of IgM can be remarkably improved, meanwhile, the flow detection signal intensity of the recombinant expression IgM antibody is equivalent to that of a commercial IgM antibody from hybridoma, the expression condition of the recombinant antibody is stable, the batch-to-batch difference in the batch production process is small, and the production cost is low. The recombinant expression can better avoid the risk of antibody gene sequence loss (such as non-expression) generated in the continuous production process of the hybridoma, and also can avoid possible variation (such as glycosylation difference) of antibodies secreted in different batches, so that an optimization strategy is provided for recombinant expression of IgM.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of recombinant antibody expression, and in particular to a molecular chaperone PDI for improving IgM recombinant expression efficiency and application thereof. BACKGROUND

[0002] IgM antibody has wide application value in scientific research, diagnosis and treatment fields, especially in early immune response research, multivalent antigen binding, complement activation and disease intervention. Each IgM monomer includes two identical μ heavy chains and two identical light chains (κ / λ chain): μ heavy chain (about 70 kDa) is composed of variable region (VH) and four constant regions (CH1-CH4), and κ / λ chain is about 25 kDa, which is divided into variable region (VL) and constant region (CL). The most common natural form of IgM is connected by J chain, with a total molecular weight of about 900 kDa and 10 antigen binding sites.

[0003] The principle of IgM pentamer formation is that J chain connects IgM monomers through disulfide bond, which is a necessary condition for the formation of IgM pentamer. In addition, the molar ratio of IgM heavy chain (μ): light chain (κ / λ): J chain is also particularly important. Many documents suggest that the molar ratio of heavy chain: light chain: J chain is about 2:2:1, and too high J chain may lead to aggregation.

[0004] Due to the complex pentamer structure of IgM antibody and the strict requirement for functional maintenance, there are many challenges in the in vitro recombinant expression of IgM antibody. The most important challenge is: 1. Pentamer assembly problem. IgM is connected by disulfide bond and J chain to form 5 monomers, which requires the expression of heavy chain (μ), light chain (κ / λ) and J chain. Imbalance of each subunit ratio will lead to assembly failure (such as formation of hexamer or polymer when J chain is insufficient). Pentamer is easy to polymerize into larger complex (such as decamer) or degrade into monomer; 2. IgM contains a large number of intra / inter-chain disulfide bonds (such as 10 conserved cysteines in μ chain), and incorrect pairing leads to aggregation or degradation.

[0005] The main function of molecular chaperone in protein expression process is to assist the correct folding of new peptide chain, maintain protein stability, prevent aggregation, and participate in the repair or degradation of misfolded proteins. In mammalian cells, PDI (protein disulfide isomerase) can ensure the correct folding and disulfide bond formation of secreted proteins, especially monoclonal antibodies. Molecular chaperone is also a preferred key tool in the expression optimization and promotion of multi-structure proteins or membrane proteins. Some commercial CHO cell lines integrate specific molecular chaperones or folding cofactors, which are limited in use; some commercial vectors need authorization license in large-scale use.

[0006] At present, all mainstream technical solutions in the design level of IgM assembly carrier are concentrated on single-cistron carrier and multi-cistron carrier design, the ideas are concentrated in controlling the expression ratio of each subunit, and IgM can be expressed to a certain extent, but there are few reports on the assembly efficiency and expression stability of IgM pentamer, therefore, based on the basic idea of IgM recombinant expression plasmid structure, based on the problems of improving the stability and low assembly efficiency in the expression process of IgM, the co-expression of IgM element plasmid and PDI and its mutant is tried, and the commercial carrier element is also compared, which to a certain extent optimizes the problem of IgM recombinant expression. SUMMARY

[0007] In order to solve the above technical problems, the first aspect of the present application provides a molecular chaperone which can improve the efficiency of IgM recombinant expression, wherein the molecular chaperone PDI comprises an amino acid sequence as shown in SEQ ID NO: 3.

[0008] The second aspect of the present application provides a recombinant vector comprising the molecular chaperone PDI which can improve the efficiency of IgM recombinant expression.

[0009] Preferably, the recombinant vector comprises a recombinant vector expression element box and the molecular chaperone PDI. Preferably, the nucleotide sequence of the recombinant vector is as shown in SEQ ID NO: 1.

[0010] The third aspect of the present application provides an application of the molecular chaperone PDI which can improve the efficiency of IgM recombinant expression.

[0011] The present application has the following advantages and beneficial effects: The co-expression of the molecular chaperone of the present application can significantly improve the expression stability of IgM, and the flow cytometry detection signal intensity of the recombinant IgM antibody is equivalent to that of the commercial IgM antibody from hybridoma, the expression condition of the recombinant antibody is stable, the batch difference is small in the batch production process, and the recombinant expression can avoid the risk of antibody gene sequence loss (such as non-expression) in the continuous production process of hybridoma, and can also avoid the variation (such as glycosylation difference) of the antibodies secreted in different batches, which provides an optimization strategy for the recombinant expression of IgM.

[0012] The present application improves the folding, assembly and secretion efficiency of IgM antibody by introducing the co-expression strategy of molecular chaperone and its mutant. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 The construction process of the recombinant plasmid pDA1 in the embodiment of the present application is shown in the following figure: Figure 2 The construction process of the recombinant plasmid pDB2 in the embodiment of the present application is shown in the following figure: Figure 3 Figure 1 is a graph of SDS-PAGE detection of IgM expressed by pDA1 in an embodiment of the present application; Figure 4 Figure 2 is a graph of SDS-PAGE detection of IgM expressed by pDA1 co-expressed with pDB1 and pDB2 respectively in an embodiment of the present application; Figure 5 Figure 3 is a graph of SDS-PAGE detection of IgM expressed by pDA1 co-expressed with pDB3 in an embodiment of the present application; Figure 6 Figure 4 is a graph of flow cytometry detection of each sample of PAC-1 recombinant expression. DETAILED DESCRIPTION

[0014] The following examples are further illustrations of the present application, but the scope of the present application is not limited to the following examples.

[0015] CD41 / CD61 (GPIIb / IIIa complex) is also known as integrin allbβ3, which is expressed by megakaryocytes and platelets. Under the activation of substances such as ADP, thrombin and TXA2, it undergoes a conformational change, and then shows a ligand binding site specific to fibrinogen, von Willebrand factor (vWF), fibronectin and vitronectin. This conformational change is crucial for platelet adhesion. CD41 / CD61 monoclonal antibody (clone number PAC-1) is a monoclonal antibody that specifically binds to activated CD41 / CD61 (αIIbβ3), which has important value in clinical and scientific research as a detection index, and is mainly used for evaluating the degree of platelet activation and the risk of related diseases.

[0016] The present application uses the antibody sequence of CD41 / CD61 monoclonal antibody (clone number PAC-1) to perform IgM recombinant expression and functional verification using the above expression idea.

[0017] Example 1

[0018] The technical scheme of the present application for improving the efficiency of IgM recombinant expression using chaperones and their mutants is as follows: A method for improving the efficiency of IgM recombinant expression using chaperones, the structure of the IgM recombinant expression vector is pDA1: EF1α-VH-CH-CMV-VL-CL-P2A-J chain, pDB1: CMV-PDI1, pDB2: CMV-PDI2, pDB3: CMV-PDI3, wherein PDI1 (derived from the published sequence on www.uniprot.org, numbered P09103), PDI2 (SEQ ID No: 3 described in the present application), PDI3 (derived from the sequence in a commercialized vector).

[0019] Except for VH (SEQ ID NO: 4) and VL (SEQ ID NO: 5), all other elements in the recombinant vector pDA1 are disclosed in the prior art. Synthesizing this vector based on these elements is a conventional technique, and its sequence and synthesis method will not be described in detail here.

[0020] The sequences of the CMV promoters among the three molecular chaperones are also publicly available. The CMV promoter used in this embodiment is from the BioVector plasmid vector strain cell protein antibody gene depositation center, specifically the pTT5 vector. The sequence of pDB2 is shown in SEQ ID NO: 1, and the sequence of pDB3 is shown in SEQ ID NO: 2.

[0021] It should be noted that, except for the heavy chain variable region VH and the light chain variable region VL, the sequences of all other elements in the recombinant vector pDA1 were synthesized by genes. The chaperone plasmids: all elements of pDB1, including PDI1, were synthesized by genes; the PDI2 element in pDB2 was constructed by designing mutation primers using the synthesized PDI1 as a template; and all elements of pDB3, including PDI3, were synthesized by genes.

[0022] Specifically, the first step is to design and construct the basic plasmid framework for IgM expression. The specific approach is as follows: pDA: EF1α-HC-CMV-LC-P2A-J chain, such as Figure 1 As shown, EF1α and CMV in the component are commonly used promoters in the laboratory. The EF1α promoter expresses the heavy chain of the IgM antibody, while the CMV promoter expresses the light chain of the IgM antibody and the J chain. The P2A between the light chain and the J chain is a short sequence of 18-22 amino acids, belonging to the self-cleaving 2A peptide family, and is widely used in multi-gene co-expression systems. Its core feature is that it can induce ribosomes to "skip" peptide bond formation after translation, enabling a single mRNA to express multiple independent proteins, thereby achieving protein self-cleavage, and breaking the light chain and J chain. 2. The VH gene fragment of the antibody heavy chain variable region and the VL gene fragment of the light chain variable region of PAC-1 were obtained by PCR cloning; PCR reaction system: 10x PCR buffer, 5 μl; dNTP (2.5 mM), 4 μl; primer F (10 μM), 1 μl; primer R (10 μM), 1 μl; Pfu polymerase, 2 μl; template, 1 μl; add ddH2O to 50 μl. PCR amplification system: 95℃, 5 min; 95℃, 30 s, 55℃, 30 s, 72℃, 1 min, 30 cycles; 72℃, 5 min; 4℃, 10 min, the relevant amplification primer is shown in Table 1, and the obtained PCR product is recovered by 0.8% agarose gel; the recovery method refers to the specification of Century Gel Extraction Kit Agarose Gel DNA Recovery Kit Cat. No. CW2302; Table 1

[0023] The pDA empty carrier is first digested by restriction endonuclease EcoR I and Xba I, detected and recovered by agarose gel, and then homologously recombined with the recovered product of PAC-1-VL, and the product is directly transformed into E. coli Top10, and after verification by colony PCR and confirmation by sequencing, an intermediate plasmid with inserted light chain is obtained. The intermediate plasmid is then digested by Not I and BamH I, recovered by agarose gel, and then homologously recombined with the recovered product of PAC-1-VH, and the ligation product is again transformed into E. coli Top10, and after verification by colony PCR and confirmation by sequencing, the final plasmid pDA1 is obtained, and the construction process is shown in the attached Figure 1 The vector and enzyme digestion sites involved in the construction are shown in Table 2, wherein the enzyme digestion system is shown in Table 3: Table 2

[0024] Table 3

[0025] PDI2 in pDB2 was amplified by overlap extension PCR using PDI1 in pDB1 as template, the first segment was amplified using primers of SEQ ID NO: 10, 11, and the second segment was amplified using primers of SEQ ID NO: 12, 13, wherein SEQ ID NO: 11, SEQ ID NO: 12 were designed with mutation sites, after recovering the two segments, the first segment and the second segment were added to the PCR reaction system in a volume ratio of 2:1, and fusion PCR amplification was performed using primers of SEQ ID NO: 10, SEQ ID NO: 13 to obtain PDI2 containing mutation sites and perform 0.8% agarose gel electrophoresis detection, the PCR amplification system and the product recovery method are referred to 3, after pDB empty carrier was digested by restriction endonuclease EcoR I and BamH I and detected and recovered by agarose gel, it was subjected to homologous recombination with the recovered product of PDI2, the product was directly transformed into E. coli Top10, and after colony PCR verification and sequencing confirmation, the pDB2 plasmid was obtained; The pDA1, pDB1, pDB2 and pDB3 plasmids were extracted without endotoxin, and the operation steps were referred to the instructions of Kangweishiji EndoFree Plasmid Midi Kit Cat. No. CW2105S; Plasmid transfection 1) Host cell recovery and subculture: one tube of frozen CHO-K1 cells was recovered in a 37°C water bath, and after dissolution, the cells were transferred to a 50ml cell culture tube containing 10ml TransCHO medium for at least 3 times of subculture to restore the growth state to a viability of more than 95% for plasmid transfection preparation; 2) 24 hours in advance, CHO-K1 cells were inoculated in a 6-well plate at a density of 2-5x10 5 cells / well, using complete medium containing serum, and cultured at 37°C, 5% CO2 overnight; 3) PEI-plasmid complex preparation: Plasmid DNA dilution: take 200μg total amount of plasmid DNA into a sterile EP tube, dilute with 50-100 μL serum-free medium (such as Opti-MEM), and mix gently; PEI dilution: according to the PEI:DNA mass ratio of 3:1, take the corresponding amount of PEI (such as 1 μg DNA needs 3 μg PEI), dilute with equal volume of serum-free medium to 50-100 μL, and the dilution information of the expression plasmid and PEI is shown in Table 4; Mixing: slowly add the diluted PEI to the plasmid DNA solution, vortex immediately (or gently blow), and stand at room temperature for 15-20 minutes to form a complex.

[0026] Table 4

[0027] Transfection: The complex was added dropwise into 200 mL of complete medium, the culture bottle was gently shaken, and the culture was incubated at 37°C (the sugar content of the medium was monitored, and when the sugar content was less than 3 g / L, the sugar was supplemented to 6-8 g / L; 5% PFF05 feed was added every other day starting from the second day after transfection), and the cells or supernatant was collected after 7 days; Purification and quality inspection Purification: After centrifugation of the culture medium after expression of PAC-1-1#, PAC-1-2#, PAC-1-3#, and PAC-1-4#, the supernatant was purified using IgM Focurose HP (Huiyan Biology HQ060218001L), 0.8M (NH4)2SO4 was added to the culture medium, and after filtration, the filler was incubated at 4°C for 1 h, PBS, 0.8M (NH4)2SO4, pH 7.2 was washed, and PBS, pH 7.2 was eluted. The PAC-1-1#, PAC-1-2#, PAC-1-3#, and PAC-1-4# samples were detected by SDS-PAGE electrophoresis, as shown in the accompanying Figures 3-5

[0028] Figure 3 The SDS-PAGE detection of IgM expressed by pDA1 is shown, from left to right, 1, Protein Marker; 2, PAC-1-1# reduction; 3, PAC-1-1# non-reduction, wherein the IgM sample expressed by pDA1 without the addition of molecular chaperone elements is referred to as PAC-1-1#. As can be seen from the figure, the PAC-1-1# reduction detection in lane 2 shows that the heavy chain and light chain sizes are correct, and the PAC-1-1# non-reduction detection in lane 3 shows that the IgM pentamer size is correct. The IgM content of the PAC-1-1# sample without the addition of molecular chaperones is low.

[0029] Figure 4 ​Figure 6 shows the IgM expression results of pDA1 and pDB1 co-expression, from left to right, 1, protein Marker; 4, PAC-1-2# reduction; 5, PAC-1-3# reduction; 6, PAC-1-2# non-reduction; 7, PAC-1-3# non-reduction; wherein pDA1 and pDB1 chaperone element co-expression of IgM sample is referred to as PAC-1-2#, pDA1 and pDB2 chaperone element co-expression of IgM sample is referred to as PAC-1-3#, from the figure, it can be seen that 4 is the PAC-1-2# reduction test, which shows that the heavy chain and light chain size is correct, 6 is the PAC-1-2# non-reduction test, which shows that the IgM pentamer shows the correct size, 5 is the PAC-1-3# reduction test, which shows that the heavy chain and light chain size is correct, 7 is the PAC-1-3# non-reduction test, which shows that the IgM pentamer shows the correct size; comparison Figure 3 and Figure 4 It can be seen that the IgM assembly effect of PAC-1-2# and PAC-1-3# samples with added chaperone is better than that of PAC-1-1# sample without added chaperone element.

[0030] Figure 5 Figure 7 shows the IgM expression results of pDA1 and pDB3 co-expression, from left to right, 1, protein Marker; 2, PAC-1-4# reduction; 3, PAC-1-4# non-reduction; wherein pDA1 and pDB3 chaperone element co-expression of IgM sample is referred to as PAC-1-4#, from the figure, it can be seen that 2 is the PAC-1-4# reduction test, which shows that the heavy chain and light chain size is correct, 3 is the PAC-1-4# non-reduction test, which shows that the IgM pentamer shows the correct size, comparison Figure 3 and Figure 5 It can be seen that the IgM assembly effect of PAC-1-4# sample with added chaperone is better than that of PAC-1-1# sample without added chaperone element.

[0031] Flow cytometry: 1) 100ul of fresh human blood was aliquoted into a 2ml centrifuge tube, and 1.8ml of 1x lysis solution was added for lysis for 10min; 2) centrifugation, discard the supernatant, wash once with PBS, resuspend with 100ul PBS; 3) add corresponding primary antibody samples (PAC-1-1#, PAC-1-2#, PAC-1-3#, PAC-1-4#, PAC-1 commercial antibody) respectively, incubate for 30min, wash once with PBS, resuspend with 100ul PBS; 4) add corresponding secondary antibody, incubate for 30min, wash 2-3 times with PBS containing 5% mouse serum; 5) wash once with 100ul PBS containing 5% mouse serum, resuspend with 100ul PBS, and then load onto the machine; see Table 5 for flow cytometry sample preparation information.

[0032] Table 5

[0033] Figure 6 The flow cytometry detection results of PAC-1-1#, PAC-1-2#, PAC-1-3#, and PAC-1-4# samples are shown in the figure. As can be seen from the figure, under the condition of equal amount of antibody loading, the PAC-1 commercial antibody and the PAC-1-1#, PAC-1-2#, PAC-1-3#, and PAC-1-4# antibodies all have positive signals. PAC-1-1# represents the literature commonly reported without the addition of a molecular chaperone scheme, PAC-1-2# represents the wild-type molecular chaperone scheme, PAC-1-3# represents the present technical scheme, and PAC-1-4# represents the selected commercial molecular chaperone scheme. Comparison shows that the signal intensity PAC-1-3# > PAC-1-4# > PAC-1-2# > PAC-1-1#, and the PAC-1-3# antibody signal is comparable to that of the PAC-1 commercial hybridoma-derived antibody. Therefore, it can be concluded that the technical scheme of using the molecular chaperone PDI of the present application for recombinant expression of IgM is optimal, and the molecular chaperone PDI of the present application for recombinant expression of IgM is superior to the selected commercial molecular chaperone, superior to the wild-type molecular chaperone, and superior to the scheme without a molecular chaperone.

[0034] The application adopts a two-step strategy, the first step is a preliminary vector design aiming at simultaneously regulating the effective expression of heavy chain (mu), light chain (kappa / lambda) and J chain, two promoters respectively express the light and heavy chains of IgM, the light chain and J chain are connected by a self-cleavage peptide P2A, the expression of these elements provides necessary conditions for pentamer formation; the second step: in the design scheme obtained in the previous step, further improve the effect of correct folding and effective assembly of IgM, maintain the problems of antibody functional expression and secretion, etc., co-express the plasmid obtained in the first step with partner elements PDI (protein disulfide isomerase) and PDI mutants, and finally obtain a relatively stable co-expression combination strategy for efficient expression of IgM through expression, purification and quality inspection processes, at the same time, the application also has corresponding functions and reference significance for homodimeric protein, Fab antibody fragment and other related molecules which need to be folded and assembled into aggregates.

[0035] The above is the preferred embodiment of the application, of course, cannot limit the scope of the application, it should be noted that for those skilled in the art, without departing from the principles of the application, can make a number of improvements and changes, these improvements and changes are also considered to be within the scope of the application.

Claims

1. A molecular chaperone PDI for improving the efficiency of IgM recombinant expression, characterized in that: The molecular chaperone PDI comprises an amino acid sequence as shown in SEQ ID NO:

3.

2. A recombinant vector, characterized by: The nucleotide sequence encoding the molecular chaperone PDI for improving the efficiency of IgM recombinant expression according to claim 1.

3. The recombinant vector of claim 2, wherein: The recombinant vector further comprises a CMV promoter, which mediates the transcription and subsequent translation of the molecular chaperone PDI.

4. The recombinant vector of claim 2, wherein: The recombinant vector further comprises one or more expression elements such as enhancer, terminator.

5. The recombinant vector of claim 2, wherein: The nucleotide sequence comprises a nucleotide sequence as shown in SEQ ID No. 1.

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