A primer combination, kit for amplifying rabbit-derived single B cell antibody sequences and its application

Abstract

This invention discloses a primer combination, kit, and application for amplifying rabbit-derived single B cell antibody sequences. It belongs to the field of biotechnology. The purpose of this invention is to provide a method for amplifying, constructing, and validating antibody sequences. This invention provides a primer combination for amplifying antibody sequences. The primer combinations for amplifying the heavy chain variable region are as follows: SEQ ID NO.1, SEQ ID NO.10, and SEQ ID NO.17; the primer combinations for amplifying the light chain variable region are as follows: SEQ ID NO.24, SEQ ID NO.29, and SEQ ID NO.32; and the primer combination for reverse transcription is as follows: primer combination SEQ ID NO.35 and SEQ ID NO.36. This provides a reliable technical foundation for the efficient amplification and rapid development of rabbit-derived single B cell antibodies.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a primer combination, kit, and application for amplifying rabbit-derived single B cell antibody sequences. Background Technology

[0002] Monoclonal antibodies play an irreplaceable role in disease diagnosis, treatment, and basic research. Traditional hybridoma technology is the classic method for preparing monoclonal antibodies, but it suffers from drawbacks such as low fusion efficiency, long processing times, easy loss of antibodies, and large batch-to-batch variability. While display technology offers improved time efficiency compared to hybridoma, it is technically challenging and the heavy and light chains do not naturally pair. Recombinant rabbit antibodies produced using single-B cell technology exhibit good affinity and specificity while maintaining natural pairing. Furthermore, rabbit antibody light chains contain additional disulfide bonds, resulting in higher stability than antibodies from other species.

[0003] In recent years, single-B cell technology has rapidly gained widespread attention due to its ability to directly obtain naturally paired light and heavy chain variable region genes from B cells in vivo without the need for cell fusion. Flow cytometry is often an effective strategy for sorting B cells, which mainly relies on the binding of cell surface markers and fluorescently labeled antigens. However, there is a relative lack of commercially available antibodies and kits for sorting rabbit B cell surface markers, necessitating the use of customized staining reagent combinations and gating strategies for screening.

[0004] Most existing technologies use peripheral blood lymphocytes (PBMCs) for single B cell sorting. However, these technologies suffer from issues such as unclear clustering in flow cytometry, a low number of B cells sorted for the target antigen, and relatively low antibody affinity maturity. Furthermore, achieving efficient reverse transcription of low-abundance single-cell RNA is challenging. In addition, the entire procedure for amplifying antibody sequences from sorted single B cells usually requires eight or more primers, which may interfere with each other, resulting in relatively low amplification efficiency. This is largely related to reverse transcription efficiency, primer design, the characteristics of the PCR reaction principle, and the interactions between primers.

[0005] Currently, given the combined effects of low antigen-specific cell sorting yield and low reverse transcription and amplification efficiency, there is an urgent need for a simple, efficient, stable, and comprehensive technical solution suitable for rabbit single-chain / full-length antibody development, including single B cell screening, efficient reverse transcription and amplification, and construction. Summary of the Invention

[0006] The purpose of this invention is to provide a method for screening rabbit antigen-specific memory B cells and for rapidly preparing antibodies through efficient reverse transcription and amplification of the antibody variable region.

[0007] This invention provides a primer combination for efficient reverse transcription and amplification of antibody variable regions. The primer combinations for reverse transcription are as follows: SEQ ID NO.35 and SEQ ID NO.36; the primer combinations for amplifying the heavy chain are as follows: SEQ ID NO.1, SEQ ID NO.10, and SEQ ID NO.17; the primer combinations for amplifying the light chain are as follows: SEQ ID NO.24, SEQ ID NO.29, and SEQ ID NO.32; the primer combinations for amplifying the constant region of the heavy chain are as follows: SEQ ID NO.37 and SEQ ID NO.38; and the primer combinations for amplifying the constant region of the light chain are as follows: SEQ ID NO.39 and SEQ ID NO.40.

[0008] This invention provides a kit for efficient reverse transcription and amplification of antibody variable regions, the kit comprising the primer combination described above for amplifying antibody variable regions.

[0009] To further specify, the kit includes a combination of reagents for sample processing, cell lysis, reverse transcription, and amplification.

[0010] This invention provides an application of the above-described primer combination in amplifying monoclonal antibodies.

[0011] This invention provides an application of the above-described kit in the amplification of monoclonal antibodies.

[0012] This invention provides a method for amplifying the variable region of an antibody, the specific steps of which are as follows: Step 1: Using the primer combination described in claim 1, sample processing and sorting are performed to obtain individual B cells. After lysis, reverse transcription is performed using the reverse transcription primer combination to obtain cDNA. To further specify, the specific method for step 1 is as follows: After sorting and cleavage, antibody-specific reverse transcription primers SEQ ID NO.35 and SEQ ID NO.36 at the 3' end of the constant region were used in conjunction with Oligo(dT) targeted reverse transcription, with antibody-specific primers as the main component and Oligo(dT) as the auxiliary component, to perform efficient reverse transcription. Further specifying the reaction procedure: RNA denaturation at 65℃ for 5 min; 55℃ for 10 min, 85℃ for 5 min.

[0013] Further defining the reaction system: 4 μL of 5×HyperScript Supermix, 1 μL of rabbit IgG 3' end specific mixed primers (10 μM), 0.5 μL of Oligo(dT) (50 μM), and ddH2O to bring the reaction volume to 20 μL.

[0014] Step 2: Using the primer combination described above, perform semi-nested PCR to amplify the antibody variable region.

[0015] To further specify, the specific method for step 2 is as follows: First round of PCR: cDNA was collected, and the heavy chain variable region framework was amplified using SEQ ID NO.1 and SEQ ID NO.10; the light chain variable region framework was amplified using SEQ ID NO.24 and SEQ ID NO.29. Second round of PCR: Using the products from the first round, the heavy chain variable region was amplified using SEQ ID NO.1 and SEQ ID NO.17 as templates; the light chain variable region was amplified using SEQ ID NO.24 and SEQ ID NO.32 as templates.

[0016] Further defining the reaction procedure: denaturation at 98°C for 10 seconds; 68°C for 35 seconds, 35 cycles.

[0017] Further defining the reaction system: 25 μL of 2×PrimeSTAR Max Premix, 1 μL each of forward and reverse primers (10 μM), 1 μL of template, and ddH2O to bring the reaction volume to 50 μL.

[0018] The present invention also provides a method for amplifying the constant region of an antibody, using cDNA as a template, and the primer combinations for amplifying the heavy chain constant region are as follows: SEQ ID NO.37 and SEQ ID NO.38; the primer combinations for amplifying the light chain constant region are as follows: SEQ ID NO.39 and SEQ ID NO.40.

[0019] Beneficial Effects: This invention establishes a targeted reverse transcription method using antibody-specific 3' end reverse transcription primers combined with Oligo(dT). Using antibody-specific primers as the primary method and Oligo(dT) as a secondary method, the reverse transcription reaction is initiated simultaneously from multiple starting points, achieving multi-starting point cDNA synthesis. This method achieves precise capture of IgG heavy and light chain mRNA while ensuring efficient synthesis of full-length cDNA, significantly improving the reverse transcription efficiency of low-abundance antibody genes in single cells and the positive rate of subsequent PCR amplification. Furthermore, by comparing a large number of publicly available rabbit antibody sequences, primers are designed for relatively constant regions. A semi-nested PCR approach is used to share upstream primers, reducing interference between primers. Simultaneously, a spacing of more than 50 bp is maintained between downstream inner and outer primers to improve primer specificity. Only six primers are needed to achieve perfect amplification, resulting in high amplification efficiency. This invention also compared the sorting effects of peripheral blood lymphocytes and spleen lymphocytes. Spleen cells showed a greater sorting advantage. Through specially designed pre-screening sample processing and fluorescence gating schemes, the clustering effect and the screening rate of target cells were significantly improved. The present invention significantly improves the cell clustering and sorting effects, and achieves extremely high efficiency in reverse transcription and variable region amplification of rabbit-derived single B cell antibodies (amplification ratio 26 / 26). It also provides feasible examples of plasmid construction and expression verification. Attached Figure Description

[0020] Figure 1 This is a diagram of the antibody primer design for this invention (A and B represent the heavy chain and light chain, respectively). Figure 2 Image showing the results of flow cytometry sorting of a single B cell in rabbit peripheral blood. Figure 3 Image showing the results of flow cytometry sorting of a single B cell in a rabbit spleen; Figure 4 A diagram showing the amplification effect of the variable region of a single B cell; Figure 5 Amplification effect after redesigning primers for the variable region of a single B cell; Figure 6 A diagram showing the amplification effect of the constant region of B cells (A and B represent the heavy chain and light chain, respectively). Figure 7 This is a diagram showing the sequencing results of heavy chain positive clones; Figure 8 This is a graph showing the sequencing results of a light chain positive clone. Figure 9 Western blot identification of recombinant antibody expression in 293T cells; Figure 10 This represents the antibody binding activity ELISA value; Figure 11 SDS-PAGE was used to identify the antibody purification efficiency; Figure 12Western-Blot identification of antibody against BVDV-E2 protein. Detailed Implementation

[0021] Example 1 Primer Design Based on the alignment of the light and heavy chain gene sequences of rabbit immunoglobulin IgG collected in IMGT and GenBank, 59 sequences of the heavy chain leading chain were aligned, 68 sequences of the heavy chain constant region were aligned, 16 sequences of the light chain leading chain were aligned, and 20 sequences of the light chain constant region were aligned. Then, according to the primer design principle, specific reverse transcription primers for the 3' end of the antibody constant region and multiple pairs of specific amplification primers for the intact variable region of rabbit IgG and the antibody constant region were designed (see Table 1).

[0022] 3' end specific reverse transcription primers: 3' end gene-specific reverse transcription primers were designed targeting highly conserved regions of the IgG heavy and light chain constant regions, respectively, and at a certain distance from the polyA tail (see [link to primer design]). Figure 1 ).

[0023] First-round PCR primers: To obtain the complete variable region sequence, 5' primers targeting the leader peptide sequences (signal peptide regions) of the heavy chain (VH) and light chain (VL) variable regions were designed, along with a 3' primer targeting the constant region (see...). Figure 1 ).

[0024] Second-round (semi-nested) PCR primers: Considering the excessive use of primers and the short conserved region of the leader peptide sequence, a shared upstream primer was adopted. The downstream primer was designed within the first-round product, with a base spacing greater than 50 bp to improve specificity and yield (see...). Figure 1 ).

[0025] Constant region primers: The upstream primer for the constant region was designed using the portion that overlaps with the second-round PCR product by 18 bases. The downstream primer was selected from the end of the constant region (see...). Figure 1 ).

[0026] After multiple amplification tests and comparison of nucleic acid electrophoresis bands, the optimal primer combination was selected (see Table 2).

[0027] Table 1 Primers for rabbit IgG mRNA reverse transcription and complete sequence amplification assay

[0028] Table 2 Primers for rabbit IgG mRNA reverse transcription and complete sequence amplification

[0029] Example 2. Animal immunization and sample collection 1. Laboratory animals and immunogens Laboratory animals: New Zealand white rabbits, 6-8 weeks old, female, weighing 2.0-2.5 kg. All animal experiments were conducted in accordance with relevant animal welfare and ethical guidelines and were approved.

[0030] 2. Immunization schedule: For different experimental purposes, administer the corresponding antigen. Taking bovine viral diarrhea virus (BVDV) as an example, administer BVDV (10... 6 TCID 50 After thoroughly emulsifying the mixture with an equal volume of adjuvant, administer multiple subcutaneous injections on the back. On the 7th day after the last immunization, collect a small amount of blood from the marginal ear vein to test the serum antibody titer. Once the titer reaches the target level, euthanize the animal.

[0031] Table 3

[0032] 3. Preparation of peripheral blood single-cell suspension: Add 2 mL of ACK Lysis Working Buffer to every 100 μL of anticoagulated blood, mix well, and lyse at 4°C for 10 min. The blood sample will gradually become clear. Centrifuge at 350×g for 5 min, discard the supernatant, and obtain a grayish-white cell pellet. Add 2 mL of PBS, centrifuge at 350×g for 5 min (to wash away excess lysis buffer), discard the supernatant, and aspirate the supernatant with a pipette. Resuspend the cells in PBS, dilute, count, and adjust the concentration to 1×10⁻⁶. 8 cells / mL.

[0033] 4. Preparation of spleen single-cell suspension: The spleen was aseptically removed and placed in pre-chilled PBS. The spleen was ground on a 200-mesh cell sieve, and the cell suspension was collected in 50 ml centrifuge tubes. After treatment with erythrocyte lysis buffer, the suspension was resuspended in PBS and passed through a 70 μm cell sieve to obtain a single-cell suspension. The cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 8 Cells / mL, keep one vial, and freeze the rest.

[0034] Example 3: Flow cytometry sorting of antigen-specific single B cells 1. Pretreatment before sample loading Peripheral blood single-cell samples and spleen single-cell samples were collected and pre-stained with LIVE / DEAD dead cell staining agent (723). After staining, the cell pellet was resuspended in cell staining buffer, washed, and gently pipetted to form a single-cell suspension. Fc receptors were blocked using a blocking reagent, washed, and resuspended in cell staining buffer. Each 1×10-1 6Single-cell suspensions were prepared by adding 10 μL each of FITC-anti-rabbit CD4, FITC-anti-rabbit CD8, FITC-anti-rabbit T Lymphocytes, and 647-anti-rabbit IgG, along with 10 μL of PE-E2 protein (the product of PE fluorescent tagging of E2 protein against BVDV). The cells were incubated on ice for 1 hour. Blank tubes, single-stained tubes, negative controls, and isotype controls were also included.

[0035] 2. Flow sorting Samples were sorted using a flow cytometer. The gating strategy was as follows: FSC-A / SSC-A was used to screen lymphocyte populations, excluding cell debris and aggregates (FSC-H / FSC-A); 723-negative cells were used to screen live cells; FITC-negative cells were used to screen B cells; among B cells, 647-positive cells and PE-labeled antigen-positive cells were sorted, i.e., antigen-specific B cells. The instrument was set to single-cell sorting mode, and target cells were sorted one cell per well into the wells of a 96-well PCR plate (pre-filled with single-cell lysis buffer) for immediate use in subsequent experiments or storage at -80°C. Results: The sorting strategy of this invention, through flow cytometry gating, can clearly sort out single B cells with antigen specificity from both rabbit peripheral blood cells and rabbit spleen cells (see...). Figure 2 , 3 However, compared to the amount of target cells to be sorted and the maturity of antibody affinity of the cells themselves, it is more recommended to use the spleen for experiments.

[0036] Example 4. Reverse transcription and semi-nested expansion of VH and VL cells in a single B cell k Gene (1) The 96-well plate was immediately placed on ice after being lysed with single-cell lysis buffer. After lysis, the antibody constant region 3' end specific reverse transcription primers (SEQ ID NO.35 and SEQ ID NO.36) were used in combination with Oligo(dT) to target reverse transcription. The antibody specific primers were used as the main component and Oligo(dT) was used as the auxiliary component to perform efficient reverse transcription and finally obtain cDNA.

[0037] Reaction system: 4 μL of 5×HyperScript Supermix, 1 μL of rabbit IgG 3' end specific mixed primers (10 μM), 0.5 μL of Oligo(dT) (50 μM), and ddH2O to make up the reaction volume to 20 μL.

[0038] Reaction procedure: RNA denaturation at 65℃ for 5 min; 55℃ for 10 min, 85℃ for 5 min.

[0039] (2) Semi-nested PCR amplification of antibody variable region gene First round of PCR: Take an appropriate amount of cDNA obtained in step (1) and use the outer primers of heavy chain-F, heavy chain-R1, light chain-F and light chain-R1 in Table 2 to amplify the variable region framework simultaneously.

[0040] Reaction system: 12.5 μL of 2×PrimeSTAR Max Premix, 0.5 μL each of the outer and inner primers (10 μM stock concentration), 1 μL of cDNA, and ddH2O to make up the reaction volume to 25 μL.

[0041] Reaction program: denaturation at 98℃ for 10s, followed by denaturation at 68℃ for 35s, for 35 cycles.

[0042] Second round PCR: Use 1 μL of the first round product and use the heavy chain-F, heavy chain-R2, light chain-F, and light chain-R2 primers in Table 2 to amplify the heavy chain and light chain respectively. The reaction conditions are the same, only the number of cycles is reduced to reduce non-specific amplification.

[0043] Reaction system: 25 μL of 2×PrimeSTAR Max Premix, 1 μL each of forward and reverse primers (10 μM stock concentration), 1 μL of first-round product, and ddH2O to make up the reaction volume to 50 μL.

[0044] Reaction program: denaturation at 98℃ for 10s, followed by denaturation at 68℃ for 35s, for 35 cycles.

[0045] (3) Primer redesign If, based on the heavy chain-F, heavy chain-R2, light chain-F, and light chain-R2 primers in Table 2 of the second round, the required fragments are designed according to one's own experimental needs before primer synthesis, the preparation of single-chain / full-length antibodies can be achieved. After multiple tests, comparisons, and optimizations, when the added fragment is a short fragment, the second-round PCR reaction program only needs to change the annealing temperature according to the designed primers. If the added fragment is a long fragment, the second round of PCR reaction procedure should be changed to a two-step PCR amplification, as follows: Reaction system: 25 μL of 2×PrimeSTAR Max Premix, 1 μL each of forward and reverse primers (10 μM stock concentration), 1 μL of first-round product, and ddH2O to make up the reaction volume to 50 μL.

[0046] Reaction program: denaturation at 98℃ for 10s, followed by denaturation at 68℃ for 35s, for 35 cycles.

[0047] Results: Variable region amplification effect After the second round of PCR amplification, samples were spotted in the order of heavy and light chains for each cell. Agarose gel electrophoresis showed a clear target band at approximately 400 bp (see...). Figure 4 The results showed that the amplification success rate from the 26 antigen-specific B cells sorted was 26 / 26, and the pairing success rate of VH and VL genes reached the expected level, indicating that the experimental protocol was effective.

[0048] Example 5. Construction, expression, and validation of antibody plasmids Of the 26 cells from which the variable region could be successfully expanded, 16 cells (i.e., two columns) were selected for the construction experiment. The construction strategy is as follows: 5' end vector—Kozak sequence—rabbit wild-type signal peptide—rabbit variable region—rabbit constant region—3' end vector.

[0049] The second round of primers, designed and modified according to experimental requirements, was used for variable region amplification. Samples were spotted in the order of heavy and light chains for each cell. Agarose gel electrophoresis showed a clear target band at approximately 500 bp (see [link to sample]). Figure 5 The success rate was 16 / 16. Simultaneously, using cDNA as a template, the constant region was amplified. The reaction program was: 98℃ denaturation for 10 s; 68℃ for 40 s, 35 cycles. The reaction system consisted of 2×PrimeSTAR Max Premix 25 μL, 1 μL each of forward and reverse primers (10 μM), 1 μL of template, and ddH2O to bring the reaction volume to 50 μL. Agarose gel electrophoresis showed clear target bands at approximately 966 bp and 318 bp in the heavy and light chain constant regions, respectively (see [link to article]). Figure 6 ) Clear, paired bands of the same size were gel-cleaved and recovered, then seamlessly cloned and ligated with the corresponding constant region and vector. The clones were then transformed into competent cells, single colonies were picked, and colony PCR was performed using vector sequencing primers. Positive clones with the correct bands were selected for sequencing, and then used for full-length antibody expression and functional verification.

[0050] Sequencing results showed that the sequences were highly readable and of good quality. Analysis using tools such as Ig BLAST confirmed that all sequences were functional rabbit immunoglobulin VH and VL genes, with complete open reading frames and no obvious frameshifts or premature stop codons (see [link to analysis]). Figure 7 , 8 ).

[0051] Plasmids were extracted from 8 pairs of colonies with correct sequence alignment and co-transfected into HEK-293T cells for micro-expression in well plates. The expression supernatant was verified by Western blotting, showing antibody expression in 7 wells (see...). Figure 9The antibody heavy chain showed a band at approximately 55 kDa. Using BVDVE2 protein as the screening antigen, ELISA plates were coated with supernatant from 293T cells transfected with the antibody expression plasmid (sample) and supernatant from 293T cells transfected with the empty vector plasmid (control). The binding activity of each antibody to the antigen was detected by indirect ELISA. Results (see...) Figure 10 The results showed that the S / N values ​​of 5 antibodies were significantly greater than 2, indicating that these antibodies have certain binding activity with BVDV E2 protein.

[0052] Five antibody expression plasmids were transfected into ExpiCHO-S cells for low-level expression. The antibody expression supernatant was purified and concentrated using Protein A / G affinity chromatography resin. SDS-PAGE results (see [link to SDS-PAGE results]). Figure 11 The results showed that bands appeared at 55 kDa and 25 kDa for the antibody heavy and light chains, respectively. The diffusion of light chain bands after denaturation is normal. Five purified monoclonal antibodies were used as primary antibodies and validated by Western blotting of BVDV E2 protein. Clear bands of BVDV-E2 protein were visible, approximately 45-50 kDa (see...). Figure 12 This indicates that monoclonal antibodies against BVDV can be obtained through flow cytometry screening, amplification, construction, and expression.

[0053] The method established in this invention can efficiently and specifically sort single B cells from rabbit peripheral blood and spleen antigens. Using a designed primer combination significantly improves the efficiency of reverse transcription and amplification of the antibody variable region gene sequence. Furthermore, adding necessary homologous arms, restriction enzyme sites, and other fragments to the primers before amplification also yields high amplification efficiency. This invention also provides a feasible full-process example of constant region amplification, plasmid construction, and expression verification. Therefore, this patent provides a reliable technical platform for the rapid construction and development of high-performance rabbit monoclonal antibodies.

[0054] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer combination for amplifying an antibody sequence, characterized in that, The primer combinations used for reverse transcription are as follows: primer combinations SEQ ID NO.35 and SEQ ID NO.36; the primer combinations for amplifying the variable region of the heavy chain are as follows: SEQ ID NO.1, SEQ ID NO.10 and SEQ ID NO.17; the primer combinations for amplifying the variable region of the light chain are as follows: SEQ ID NO.24, SEQ ID NO.29 and SEQ ID NO.

32.

2. A kit for amplifying antibody sequences, characterized in that, The kit includes the primer combination for amplifying the antibody sequence as described in claim 1.

3. The reagent kit according to claim 1, characterized in that, The kit also includes primer combinations for amplifying the heavy chain constant region as follows: SEQ ID NO.37 and SEQ ID NO.38; and primer combinations for amplifying the light chain constant region as follows: SEQ ID NO.39 and SEQ ID NO.

40.

4. The application of the primer combination according to claim 1 in amplifying monoclonal antibodies.

5. The use of the kit according to claim 2 or 3 in amplifying monoclonal antibodies.

6. A method for amplifying the variable region of an antibody, characterized in that, The specific steps of the method are as follows: Step 1: Sample processing. Individual B cells are sorted, lysed, and then reverse transcribed using a reverse transcription primer combination to obtain cDNA. The reverse transcription primer combination is as follows: primer combination SEQ ID NO.35 and SEQ ID NO.

36. Step 2: Using the primer combination described in claim 1, perform semi-nested PCR to amplify the antibody variable region.

7. The method according to claim 6, characterized in that, The specific method for step 2 is as follows: Step 1: First round of PCR: Take cDNA and amplify the heavy chain variable region framework using SEQ ID NO.1 and SEQ ID NO.10; amplify the light chain variable region framework using SEQ ID NO.24 and SEQ ID NO.29; Step 2: Second round of PCR: Using the products from the first round, the heavy chain variable region was amplified using SEQ ID NO.1 and SEQ ID NO.17 as templates; the light chain variable region was amplified using SEQ ID NO.24 and SEQ ID NO.32 as templates.

8. The method according to claim 7, characterized in that, Reaction program: denaturation at 98℃ for 10 seconds; denaturation at 68℃ for 35 seconds, 35 cycles.

9. The method according to claim 7, characterized in that, Reaction system: 25 μL of 2×PrimeSTAR Max Premix, 1 μL each of forward and reverse primers, 1 μL of template, and ddH2O to make up the reaction volume to 50 μL.

10. A method for amplifying an antibody sequence, characterized in that, The antibody variable region is obtained using the method described in any one of claims 6-8. Using cDNA as a template, the primer combinations for amplifying the heavy chain constant region are as follows: SEQ ID NO.37 and SEQ ID NO.38; the primer combinations for amplifying the light chain constant region are as follows: SEQ ID NO.39 and SEQ ID NO.40.

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