CN1077801C - Carcinoembryonic antigen gene engineering antiboy CL-3-scFv - Google Patents

Abstract

The invention is a bioengineering product, which is a single-chain antibody expressed by the constructed antibody variable region fusion gene and capable of recognizing tumor cells in colorectal cancer and gastric cancer tissues. It can be used for auxiliary diagnosis and biological treatment of gastrointestinal malignant tumors. It can be expressed in the prokaryotic system, and the expression amount accounts for more than 35% of the total protein of the bacteria. It is conducive to mass production and can be recombined with the toxin gene to form a recombinant immunotoxin , to provide new targeted drugs for tumor biotherapy.

Description

Carcinoembryonic Antigen Genetic Engineering Antibody CL-3-scFv

The invention belongs to genetic engineering product. This product can specifically combine with carcinoembryonic antigen and recognize tumor cells in colorectal cancer and gastric cancer tissues. Combined to prepare targeted drugs for biological treatment of tumors.

Currently, cancer is still the number one killer of human beings. Therefore, the treatment of tumors is an important topic that people care about. The tumor biotherapy developed in recent years is the fourth major therapy after tumor chemotherapy, radiotherapy and surgery. Tumor targeting therapy using monoclonal antibody as carrier is an important part of tumor biotherapy. Its mechanism of action is that tumor cells have tumor-specific antigens and tumor-associated antigens that are different from normal cells. Carcinoembryonic antigen (CEA) is a tumor-associated antigen, and its chemical essence is an acidic glycoprotein with a molecular weight of 160-200kDa. This acidic glycoprotein is a normal component in tissues such as the fetal intestine, liver, and pancreas during early embryonic development. It gradually decreases in the late embryonic period, and the serum level is extremely low after birth. When a tumor occurs, cancer cells re-synthesize the antigen and express it on the surface of tumor cells. CEA was initially detected in colon cancer and rectal cancer tissues, and later found to exist in other malignant tumors derived from endoderm (such as esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, etc.). As an important marker on the cell surface of various malignant tumors, CEA has been widely used in basic research and clinical diagnosis and treatment of tumors.

There are many reports on the application of anti-CEA monoclonal antibodies and their conjugates in tumor diagnosis and treatment at home and abroad. Among them, the conjugated antibody CL-3 and ¹³¹ I shows high specificity and sensitivity in the localization diagnosis of colon cancer in vivo, and can inhibit the growth of colon cancer and alleviate the symptoms of tumor patients. However, the human anti-mouse antibody reaction after repeated administration and the large molecular weight of monoclonal antibodies make it difficult to enter solid tumor tissues and other problems, which limit its application.

Aiming at the above shortcomings, the present invention adopts the method of molecular biology to construct, clone and express the anti-CEA genetically engineered single-chain antibody. One of the innovations lies in the small molecular weight of the antibody. It is a single-chain antibody variable region fragment (singlechain Fv fragment, referred to as scFv) formed by linking the variable region of the heavy chain and the variable region of the light chain of the antibody through a small molecular peptide. The molecular weight is about 25kDa, which is one-sixth of the molecular weight of its parent monoclonal antibody. Compared with the parental monoclonal antibody, the advantages of CL-3-scFv are mainly manifested in its less immunogenicity and less likely to cause immune rejection in the human body; it has good permeability and can effectively penetrate the dense tumor barrier, which is beneficial to solid Diagnosis and treatment of tumors; Because the antibody CL-3-scFv does not contain Fc fragments, it avoids non-specific reactions with cells containing Fc receptors, so the background is low and the image is clear during in vivo imaging positioning inspection; Antibody CL -3-scFv does not require glycosylation, which is conducive to mass production with prokaryotic systems; the antibody CL-3-scFv is recombined with the toxin gene, and the recombinant immunotoxin produced can overcome the instability of traditional monoclonal antibody and toxin chemical conjugates To provide new targeted drugs for tumor biotherapy.

Innovation 2: Compared with hybridoma technology, its antibody preparation technology has the following advantages: It saves the time-consuming and laborious steps of cell fusion and immunization of humans and animals; it directly obtains and preserves antibody genes, avoiding repeated hybridoma The problem of gene loss after cryopreservation; through genetic modification of antibodies, the affinity of antibodies can be improved, and the effector function of antibodies can be improved; human antibodies can be developed; the method of phage screening antibodies is efficient, easy to operate, economical, and suitable for rapid and large-scale Production.

Innovation 3: The development of CEA monoclonal antibody has been reported, but CEA single-chain genetic engineering antibody has not been reported at home and abroad.

The specific technical scheme of the present invention is as follows: first, isolate and purify mRNA from mouse hybridoma cells secreting anti-CEA monoclonal antibody, and reverse transcribe it into the first strand of cDNA. The heavy chain variable region gene (V _H ) and the light chain variable region gene (V _L ) were respectively amplified by PCR using the first strand of cDNA as a template. The _VH and _VL are connected by a DNA sequence encoding (Gly4Ser)3 to form a _VH -Linker- _VL fusion gene. In order to insert the gene into an expression vector with Sfi1 and Not1 restriction sites, we added Sfi1 and Not1 restriction sites to both ends of the scFv gene fragment by PCR. The PCR amplified product was digested and recovered by endonucleases Sfi1 and Not1, and then ligated with the expression vector pCANTAB5E containing double restriction enzymes of Sfi1 and Not1 to transform Escherichia coli TG1 competent cells.

Because the single-chain antibody variable region gene is inserted into the phage coat protein g3p gene, it forms a fusion gene with the g3p gene. Therefore, the scFv and the coat protein g3p are expressed on the surface of the phage as a fusion protein. Using immunoaffinity screening method, human colon cancer CEA was used as antigen to screen recombinant phage antibody library. Those recombinant antibody phages that bind to CEA were isolated and used to infect TG1. These CEA recombinant phage antibodies were enriched by culture expansion. After three rounds of immunoaffinity-infection-amplification-rescreening, the CEA-specific recombinant phage antibody was finally obtained. This antibody can be used for in vitro immunodiagnosis of tumors.

In order to obtain a soluble single-chain antibody that can be used for tumor treatment, we selected an amber stop codon between the single-chain antibody gene and the g3p gene in the expression vector. The supE gene is contained in Escherichia coli TG1 cells, and the amber codon can be read through to produce scFv-g3p fusion protein. HB2151 cells do not contain the supE gene, so translation stops at the amber stop codon, scFv protein is produced and continuously transported to the periplasm, and gradually leaks into the culture medium, thereby obtaining soluble single-chain antibodies.

In order to detect the single-chain antibody conveniently, the present invention selects the pCANTAB5E expression vector. A gene encoding a small molecule peptide E-Tag is connected to the 3' end of the scFv gene. Therefore, the expressed scFv can be detected with an anti-E-Tag antibody. After screening by ELISA, 6 CEA single-chain antibody strains were obtained. Two strains were randomly selected for DNA sequence analysis, and the results showed that both were composed of 693 nucleotides, and their sequences were completely identical. We named it CL-3-scFv. Western Blot results showed that CL-3-scFv had the same specificity as the parent monoclonal antibody. It was able to recognize tumor cells in colorectal and gastric cancer tissues without reacting with normal gastrointestinal tissues.

In order to mass-produce antibody CL-3-scFv, the present invention cloned the antibody CL-3-scFv gene into expression vectors pJW2 and pET5a respectively, and constructed antibody recombinant plasmids pJW2-CL-3-scFv and pET5a-CL-3-scFv High-efficiency expression vector. Escherichia coli DH5α and BL21(DE3) were transformed respectively. Under induction with 42°C (pJW2-CL-3-scFv) or 1mM IPTG (pET5a-CL-3-scFv), the amount of expressed antibody was 35% of the total protein amount. After the purification and renaturation of the inclusion body protein, the antibody was purified by MonoQ and Q Sepharose FF ion exchange chromatography, and finally a single-chain antibody with a purity greater than 90% was obtained.

Example

(1) Cell culture and identification: Hybridoma cells secreting CL-3 antibody (Yuan Mei, Liu Chenggui, Li Li, etc., application of anti-colon cancer monoclonal antibody CL-3, CL-4 serological diagnosis. Chinese anal disease intestinal Journal, 1988, No. 4: 3-6) cultured in complete RPMI1640 medium containing 15% bovine serum. The incubator contains a gas mixture of 5% _CO2 and a humidity of 98%. The specificity and titer of monoclonal antibody in culture supernatant were identified by ELISA method.

(2) Isolation and purification of mRNA: mRNA was extracted and purified from approximately 2×10 ⁷ hybridoma cells with a rapid preparation and purification mRNA kit (Promega).

(3) Preparation of cDNA: Using the purified mRNA as a template, cDNA was synthesized by reverse transcription.

(4) Amplify the antibody variable region gene: use the PCR method to use cDNA as a template, add a set of light chain or heavy chain variable region primers to the PCR reaction system, 5 μl of 10×PCR buffer, and a final dNTP concentration of 2.5 Mm, after mixing, denature at 100°C for 5 minutes, and add 2 units of Taq DNA polymerase. The total reaction volume was 50 μl. After mixing, add mineral oil. Carry out 30 cycles of reaction, the conditions of each cycle are: denaturation at 94°C for 30s, annealing at 55°C for 90s, extension at 72°C for 90s, and keep warm at 72°C for 10min after the last cycle. After the reaction, 3 μl were taken out from the heavy and light chain variable region PCR reaction systems and subjected to 1.5% agarose gel electrophoresis, and the rest were recovered with Sephagles ^™ Bandprep Kit.

(5) Assembly and amplification of the single-chain antibody gene: the recovered _VH and _VL gene fragments and the connecting primers were equimolar or close to equimolar concentration, and 2.5mM dNTP and 5 units of Taq DNA polymerase were added, 10× 2.5 μl of PCR buffer, 2.5 μl of 25mM MgCl ₂ , the reaction volume is 25 μl, and seven annealing cycles are performed after blocking with liquid paraffin oil. The reaction conditions of each cycle are denaturation at 94°C for 30 seconds and annealing at 64°C for 4 minutes.

Using the scFv gene as a template, add a pair of primers containing Sfi I at the 5' end and Not I restriction site at the 3' end to the above reaction volume, and perform 30 PCR cycles, and the condition of each cycle is denaturation at 94°C 1 minute, anneal at 55°C for 2 minutes, extend at 72°C for 2 minutes, and hold at 72°C for 10 minutes after the last cycle. 3 μl was taken from the scFv gene PCR amplification reaction system and subjected to 1.2% agarose gel electrophoresis, and the rest was recovered with the above-mentioned recovery kit.

(6) Establishment of the single-chain antibody gene library: the recovered scFv genes were digested with endonucleases SfiI and NotI respectively, then ligated with the plasmid pCAMTNB5E double-digested with SfiI/NotI, and transformed into Escherichia coli TG1 competent cells. Cultivate transformed bacteria in culture medium 2×YT-G (1.7% Bacto-trypone, 1% Bacto-yeast extract, 0.5% NaCl, 2% glucose). A small portion (approximately 1/10 volume) of transformed bacteria was stored at -70°C with 13% glycerol.

(7) Expression of recombinant phage antibody and immunoaffinity screening: the remaining transformed bacteria were diluted with 10 ml of 2×YT-G, and cultured at 37° C. until the OD ₆₀₀ value was 0.5. Then add ampicillin to a final concentration of 100 μg/ml and M13K07 to a final concentration of 2×10 ⁹ pfu/ml, culture at 37°C for 1 hour, and centrifuge. The cell pellet was resuspended in 10ml 2×YT-AK (2×YT containing 100μg/ml ampicillin and 50μg/ml kanamycin) culture medium, cultured at 37°C overnight, and the supernatant containing recombinant phage was collected by centrifugation .

The supernatant containing the recombinant phage was incubated with the CEA antigen coated on the petri dish at 37°C for 1 hour. The plate was washed 20 times with PBST (PBS containing 0.05% Tween20), and the plate was washed 20 times with PBS. Add 1ml of 100mM triethylamine to elute the recombinant phage antibody bound on the plate, neutralize immediately with 1M Tris-HCl, pH9 and collect the eluted recombinant phage. The above infection-amplification-screening process was repeated for 2 rounds.

(8) Preparation and identification of recombinant phage antibody: Take 100 μl of recombinant phage antibody eluted in the third round and 200 μl of TG1 cells in the logarithmic growth phase, mix them and incubate them on a shaker at 37°C for 30 minutes, and infect the cells with 2 After doubling dilution of ×YT (1:10, 1:100, 1:1000), spread on SOBAG (2% Bacto-trypone, 0.5% Bacto-yeast extract, 0.05% NaCl, 10mMMgCl ₂ , 2% glucose, 100 μg/ml ampicillin, 1.5% Bacto-Agar) and cultured overnight at 30°C on a solid medium. Randomly pick 72 single colonies from the plate and inoculate them into 100μl 2×YT-

AG (2×YI containing 100 μg/ml ampicillin, 2% glucose) culture medium, cultured at 30° C. overnight. On the next day, transfer 20 μl of overnight bacteria to new 1.5ml Eppendorf centrifuge tubes containing 200 μl 2×YT-AG and 5×10 ⁸ pfu/ml M13K07. Incubate at 37°C for 2 hours, centrifuge, and wash with 200 μl 2×YT-AK (containing Ampicillin and kanamycin) culture medium to suspend the precipitated cells in each centrifuge tube, and culture overnight at 37°C. Centrifuge and collect the supernatant.

A 96-well enzyme-linked plate was coated with antigen CEA, the coating solution was 0.05M _{Na 2} CO ₃ (pH9.6), the antigen concentration was 10 μg/ml, 100 μl of coating solution was added to each well, and overnight at 4°C. Add blocking solution (1.5% BSA dissolved in PBS), and block at room temperature for 1 hour. Mix 100 μl of recombinant phage antibody supernatant with equal volumes of blocking solution, place at room temperature for 10 minutes, add to the coated enzyme-linked plate, and incubate at 37°C for 1 hour. M13 phage was used as negative control, 1% BSA, 2×YT, PBS was used as blank control, CL-3 monoclonal antibody was used as positive control, the plate was washed 3 times with PBST (PBS containing 0.05% Tween20), and 3 times with PBS Second-rate. Add 100 μl anti-M13 phage IgG-HRP labeled with horseradish peroxidase (1:5000), add 100 μl goat anti-mouse IgG-HRP to the positive control well, react at 37°C for 1 hour, wash the plate 3 times with PBST, wash with PBS Plate 3 times. Add 100 μl of substrate OPD-H ₂ O ₂ , react at room temperature for 20 minutes, add 50 μl 2M H ₂ SO ₄ to terminate the reaction, and detect the light absorbance of each well at 490 nm. Select several clones with higher activity, set up parallel wells to repeat the above results, and finally determine a positive clone with the strongest activity, named CL-3-scFv.

(9) Identification of recombinant plasmids of positive clones and DNA sequence analysis: Extract CL-3-scFv recombinant plasmids and digest them with SfiI and NotI restriction endonucleases, respectively. The digested products were analyzed by 1.2% agarose gel electrophoresis. The DNA sequence of the scFv gene on the recombinant plasmid was determined by T7DNASequence ^TM Kit. The results show that the CL-3-scFv gene is composed of 693 nucleotides, and its sequence is as follows: ATG GCC CAG GTC CAG CTG CAG GAG TCA GGG TAT ACT TTCACA ACC TAT GGA ATG AGCTGG GTG AAA CAG GCT CCA GGA AAG GGT TTA AAG TGG ATGGGC TGG ATA AAC ACC TACTCT GGA GTG CCA ACA TAT GCT GAT GAC TTC AAG GGA CGGTTI GCC TTC TCT TTG GAAACC TCT GTC AGC ACT GCC TAT TTG CAG ATC AAC AAC CTCAAA AAT GAG GAC ACG TCAACA TAT TTC TGT GCA AGA TAT GCC TTC GGC TCT TGG TACTTC GAT GTC AGG GGC CAAGGC ACC ACG GTC ACC GTC TCC TCA GGT GGA GGC GGT TCAGGC GGA GGT GGC TCT GGCGGT GGC GGA TCG GAC ATC GAG CTC ACT CAG TCT CCA ATGGCT TCT TTG GCT GTG TCTCTA GGG CAG AGG GCC ACC ATC TCC TGC AGA GCC AGC GAAAGT GTT GAT ACT TAT GCCGTT AGT TTT ATG AAC TGG TTC CAA CAG AAA CCA GGA CAGCCA CCC AAA CTC CTC ATCTAT ACT GCA TCC AAG CAA GGG TCC GGG GTC CCT GCC AGGTTT AGT GGC AGT GGG TCTGGG ACA GAC TTC AGC CTC AAC ATC CAT CCT ATG GAG GAGGAT GAT GCT GCA ATG TATTTC TGT CAA CAA AGT AAG GAG GTT CCG TGG ACG TTC GGTGGA GGG ACC AAG CTG GAAATA AAA CGG

(10) Preparation and identification of soluble single-chain antibody: Escherichia coli HB2151 was transformed with the identified recombinant plasmid. Pick 4 single colonies and culture them overnight in 2ml 2×YT-AG medium. After centrifugation, the cells were suspended and pelleted with 2ml of 2×YT culture medium containing 1mMIPTG, and cultured at 30°C for 20 hours. Centrifuge and collect the supernatants separately. Add an equal volume of acetone, mix well, and let stand in a refrigerator at 4°C for 4 hours. Centrifuge, remove supernatant, and air dry for 30 minutes. Add 50 μl PBS to dissolve the precipitate.

After 15 μl of the precipitate was subjected to 12% SDS-PAGE electrophoresis, the protein material on the gel was transferred to a nitrocellulose filter membrane by electroporation, and the soluble single-chain antibody was detected by anti-E-Tag antibody (Phamacia). Western Blot experiments proved that the molecular weight of the soluble scFv antibody fragment was about 26kDa (Figure 6). He is composed of 231 amino acids. The amino acid sequence deduced from the DNA sequence is as follows: M A Q V Q L Q E S G Y T F T T Y G M S W V K Q A P G K G L K WM G W I N T YS G V P T Y A D D F K G R F A F S L E T S V S T A Y L Q I N N LK N E D T ST Y F C A R Y A F G S W Y F D V R G Q G T T V T V S S G G G GS G G G G G S GG G G S D I E L T Q S P M A S L A V S L G Q R A T I S C R A S E SV D T Y A VS F M N W F Q Q K P G Q P P K L L I Y T A S K Q G S G V P A R FS G S G S G TD F S L N I H P M E E D D A A M Y F C Q Q S K E V P W T F G G GT K L E I K R

(11) Identification of the immunological activity of the soluble single-chain antibody: On ice, gradually add 58.2 g of ammonium sulfate to 200 ml of the supernatant to make the final concentration reach 50%, and keep stirring with a magnetic stirrer for 4 hours. Centrifuge, discard the supernatant, and dissolve the pellet in 2ml PBS buffer.

ELISA: Coat a 40-well enzyme-linked plate with antigen CEA, the coating solution is 0.05M Na ₂ CO ₃ (pH9.6), the antigen concentration is 10 μg/ml, add 100 μl of coating solution to each well, and overnight at 4°C. Add blocking solution (1% BSA, dissolved in PBS buffer) and block at room temperature for 1 hour. Set up parallel wells, dilute the concentrated immune supernatant (1:2, 1:4, 1:8, 1:16, 1:32, ...) with blocking solution, and add 100 μl of diluted immune supernatant to each well. 1% BSA and 2×YT were used as negative controls, PBS was used as blank control, and CL-3 monoclonal antibody was used as positive control, incubated at 37°C for 1 hour, and washed with PBST and PBS 3 times each. Add 100 μl of anti-E-Tag antibody diluted 1:2000. Add 100 μl of diluent to the positive control, incubate at 37°C for 1 hour, and wash the plate 3 times with PBST and PBS. Add 100 μl of goat anti-mouse IgG-HRP diluted 1:1000, and incubate at 37°C for 1 hour. Plates were washed 3 times each with PBST and PBS. Add 100 μl of substrate OPD-H ₂ O ₂ , react at room temperature for 20 minutes, add 50 μl 2M H ₂ SO ₄ to terminate the reaction, measure the OD490 value of each well at A490 and analyze the results.

Western Blot: Human colon cancer CEA was used as the antigen, and 2 μl of antigen dilution solution of different concentrations was used to spot on the nitrocellulose filter membrane, BSA was used as the negative control, and PBS buffer was used as the blank control. After drying in air for 20 minutes, add 5% skimmed milk powder/PBS, and block overnight at 4°C. The membranes were reacted with CL-3-scFv or CL-3 monoclonal antibody in the immune supernatant, and shaken gently at room temperature for 2 hours. Wash the membrane 3 times with PBS buffer, 10 minutes each time. Add 10 ml of anti-E-Tag antibody diluted 1:300 or goat anti-mouse IgG-HRP 10 ml diluted 1:500, and shake gently at room temperature for 1 hour. The membrane was then washed with phosphate-free buffer (150 mM Tris-HCl pH 7.4, 50 mM NaCl) for 10 minutes. Add 10ml of substrate (0.01M Tris-HCl pH7.4, 0.03% CoCl ₂ , 6mg diaminobenzidine, 15μl H ₂ O ₂ ), shake gently at room temperature for 2-3 minutes, rinse with distilled water, add 20ml of PBS buffer and store in 4°C. Dot Blot results showed that CL-3-scFv can specifically bind to CEA activity, which is similar to the activity of the positive control CL-3 monoclonal antibody. This shows that the soluble antibody fragment has the same affinity and specificity as the parent monoclonal antibody.

(12) Construction of CL-3-scFv high-efficiency expression vector: In order to produce CL-3-scFv in large quantities, we cloned the antibody CL-3-scFv gene into expression vectors pJW2 and pET5a respectively, and constructed recombinant plasmid pJW2-CL- 3-scFv and recombinant plasmid pET5a-CL-3-scFv high-efficiency expression vector. Escherichia coli DH5a and BL21(DE3) were transformed respectively. Randomly pick a single colony on the LB solid medium containing 100 μg/ml ampicillin, and culture it in 1000ml LB medium at 30°C until OD600=0.6, respectively with 42°C (pJW2-CL-3-scFv) or 1mM IPTG (pET5a -CL-3-scFv) induced culture for 5 hours. Centrifuge to collect cells.

(13) Extraction of inclusion body protein [5] and renaturation [6]: Resuspend the bacteria in 20ml of distilled water, and ultrasonically destroy the bacteria. Centrifuge at 12000×g, 4°C, 30min. The supernatant was discarded, and the precipitate was inclusion body. After washing once with 50mM TE, store at -20°C.

Dissolve the inclusion bodies with 8M urea (containing 10mM DTT), place at room temperature for 2 hours, centrifuge at 12000×g at 4°C for 20min, and discard the precipitate. The supernatant was added dropwise to refolding solution (0.1MTE, 0.5ML-arginine, 1mMGSSG, 0.2mM GSH, pH8.4) to make the final protein concentration about 30μg/ml. Stand at 10°C for more than 48 hours.

(14) Purification of antibody CL-3-scFv: Concentrate the refolded antibody with PEG. Antibodies were purified by MonoQ and Q SepharoseFF ion exchange chromatography. The column (2.6 x 20 cm) was equilibrated with 20 mMTE. The sample was loaded and eluted with 20mMTE at a flow rate of 60ml/h. After passing through the peak, elute with 20mMTE, 0.3M NaCl, and collect the target peak. The antibody was identified by SDS-PAGE and Western Blot, and the purity of the obtained single-chain antibody was greater than 90%.

Claims (3)

1. A biological preparation for the diagnosis or treatment of malignant tumors of the digestive tract, characterized in that it is a carcinoembryonic antigen single-chain antibody, which is composed of _VH -linker- _VL , wherein _VH and _VL They are the genes encoding the variable region of the heavy chain and the variable region of the light chain respectively, and the linker is a carcinoembryonic antigen single-chain antibody expressed by the fusion gene of the DNA sequence encoding (Gly4Ser)3. 2. The biological preparation according to claim 1, wherein the carcinoembryonic antigen single-chain antibody is composed of the following 231 amino acids: M A Q V Q L Q E S G Y T F T T Y G M S W V K Q A P G K G L K W M G W I N T YS G V P T Y A D D F K G R F A F S L E T S V S T A Y L Q I N N L K N E D T ST Y F C A R Y A F G S W Y F D V R G Q G T T V T V S S G G G G S G G G G S GG G G S D I E L T Q S P M A S L A V S L G Q R A T I S C R A S E S V D T YA V S F M N W F Q Q K P G Q P P K L L I Y T A S K Q G S G V P A R F S G SG S G T D F S L N I H P M E E D D A A M Y F C Q Q S K E V P W T F G G G TK L E I K R composition. 3. The biological preparation according to claim 1, wherein the fusion gene expressing the carcinoembryonic antigen single-chain antibody is composed of 693 nucleotides as follows: ATG GCC CAG GTC CAG CTG CAG GAG TCA GGG TAT ACT TTC ACA ACC TAT GGA ATG AGCTGG GTG AAA CAG GCT CCA GGA AAG GGT TTA AAG TGG ATG GGC TGG ATA AAC ACC TACTCT GGA GTG CCA ACA TAT GCT GAT GAC TTC AAG GGA CGG TTT GCC TTC TCT TTG GAAACC TCT GTC AGC ACT GCC TAT TTG CAG ATC AAC AAC CTC AAA AAT GAG GAC ACG TCAACA TAT TTC TGT GCA AGA TAT GCC TTC GGC TCT TGG TAC TTC GAT GTC AGG GGC CAAGGC ACC ACG GTC ACC GTC TCC TCA GGT GGA GGC GGT TCA GGC GGA GGT GGC TCT GGCGGT GGC GGA TCG GAC ATC GAG CTC ACT CAG TCT CCA ATG GCT TCT TTG GCT GTG TCTCTA GGG CAG AGG GCC ACC ATC TCC TGC AGA GCC AGC GAA AGT GTT GAT ACT TAT GCCGTT AGT TTT ATG AAC TGG TTC CAA CAG AAA CCA GGA CAG CCA CCC AAA CTC CTC ATCTAT ACT GCA TCC AAG CAA GGG TCC GGG GTC CCT GCC AGG TTT AGT GGC AGT GGG TCTGGG ACA GAC TTC AGC CTC AAC ATC CAT CCT ATG GAG GAG GAT GAT GCT GCA ATG TATTTC TGT CAA CAA AGT AAG GAG GTT CCG TGG ACG TTC GGT GGA GGG ACC AAG CTG GAAATA AAA CGG composition.