Helicobacter pylori antigen fusion construction method and application thereof

Abstract

The invention discloses a helicobacter pylori antigen fusion construction method and application thereof. A TrxA tag, a D3 sequence and a sumo tag are added to the N end of helicobacter pylori antigen-urease Ureb for fusion and dissolution promotion, and after purification, ulp1 protease can be used for enzyme digestion removal; a PA tag is added to the C end of helicobacter pylori antigen-urease Ureb, so that the Ureb can be anchored on GEM particles, finally, a fusion antigen which can be expressed in escherichia coli broken supernatant and can be successfully anchored on the GEM particles is obtained, the fusion antigen is used for immune effect evaluation, and compared with the prior art, soluble expression of Ureb is achieved, Ureb can be anchored on GEM, and immune prototypes are increased.

Description

Technical Field

[0001] The present invention relates to an antigen fusion construction method and application thereof, and more specifically, to a Helicobacter pylori antigen fusion construction method and application thereof, and belongs to the field of biological genetic engineering. Background Art

[0002] Helicobacter pylori (HP) is the causative factor of various gastrointestinal diseases such as gastritis, gastroduodenal ulcer, gastric mucosa-associated lymphoma and gastric cancer, and is recognized as a Class I carcinogen worldwide. Drug treatment has serious toxic side effects and is prone to recurrence and reinfection. Therefore, the development of a safe and effective vaccine is the best way to prevent Helicobacter pylori infection and reduce the incidence of related gastrointestinal diseases.

[0003] Urease (Ureb) is a commonly used antigen in the development of Helicobacter pylori (HP) vaccines. It is distributed on the surface of bacteria and accounts for 5-10% of bacterial proteins. It decomposes urea and neutralizes gastric acid during the colonization and infection process. It has a conservative sequence and strong immunogenicity. Heat shock protein (HspA) is a stress protein molecule with a unique nickel binding region. It has a synergistic effect with urease (Ureb). The fusion of the two can enhance the immunogenicity of the antigen.

[0004] Thioredoxins are a series of oxidoreductases widely present in organisms, which can reduce the binding of disulfide bonds by replacing thiodisulfide. Thioredoxin A (TrxA), which is often used as a fusion expression tag, has a weight of 11.6kDa and is derived from Escherichia coli. The most unique advantage of the TrxA tag is its thermal stability. TrxA itself is not used as an affinity tag to optimize recombinant proteins, but the recombinant protein is preserved by denaturing and removing impurities under high temperature conditions. TrxA also has excellent solubilization efficiency. When used as a solubilization tag, it can increase the soluble expression of recombinant proteins from 12% to 95%.

[0005] D3 is a domain sequence of Protein A. Relevant experiments in the laboratory of this applicant have proved that it has the ability to be secreted outside the cell.

[0006] The GEM-PA surface display system is composed of a Grampositive Enhancer Matrix (GEM) and an anchor protein (PA). GEM particles are the display core, and foreign proteins are displayed on the surface of GEM particles through fusion expression with PA anchor proteins. GEM particles have the advantages of high biosafety, strong ability to bind foreign proteins, and autologous adjuvant effect.

[0007] GEM particles are inactive particles formed by Gram-positive bacteria after being treated with heat and acid. The current mainstream GEM particles are obtained by processing Lactococcus lactis. The main component of the cell wall of Lactococcus lactis is a peptidoglycan skeleton, which is thick and has a closed network structure. Peptidoglycan is one of the ligands of TLR2, and TLR2 is a member of the Toll-like receptor family. It can recognize different pathogen-associated molecular patterns and plays an important role in the key link between innate immunity and specific immunity. This characteristic makes GEM particles have a self-adjuvant effect. Because GEM particles are small, about 1-2μm, they are well matched to the size requirements of the M cells on the mucosal surface for uptake of antigens. Therefore, as an adjuvant, they also have a good effect in activating mucosal immunity.

[0008] Protein Anchor PA is the C-terminal cell wall binding active center of the peptidoglycan hydrolase AcmA expressed by Lactococcus lactis. It is composed of repeated autolysin motifs (Lysine motif, LysM). LysM consists of 45 amino acid residues and is a very common amino acid sequence in prokaryotes and eukaryotes, existing in at least more than 4,000 proteins. In prokaryotes, LysM has evolved into a universal peptidoglycan binding motif. Summary of the invention

[0009] The purpose of the present invention is to provide a Helicobacter pylori antigen fusion construction method with the technical characteristics of being able to achieve soluble expression of Ureb, anchoring on GEM, increasing immune prototypes, and the like.

[0010] Another object of the present invention is to provide an application of a fusion antigen in the preparation of Helicobacter pylori vaccines and medicines.

[0011] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0012] The invention discloses a method for constructing a Helicobacter pylori antigen fusion, which comprises: fusing a lytic tag to the N-terminus of the Helicobacter pylori antigen-urease Ureb, and fusing a heat shock protein HspA and a PA sequence (PA tag) to the C-terminus of the Helicobacter pylori antigen-urease Ureb, so as to obtain a fusion antigen that can be expressed in Escherichia coli supernatant and can be successfully anchored on GEM particles.

[0013] Preferably, fusing the N-terminal lysis-promoting tag of Helicobacter pylori antigen-urease Ureb specifically includes: adding TrxA tag, D3 sequence, and SUMO tag to the N-terminal of Helicobacter pylori antigen-urease Ureb for lysis-promoting, and removing the N-terminal fusion sequence by ulp1 protease after purification.

[0014] Preferably, pET32a-TrxA-his-pA_D3-SUMO-Ureb is obtained by using the Helicobacter pylori antigen fusion construction method.

[0015] Preferably, the TrxA sequence is:

[0016] ATGAGCGATAAAATTATTCACCTGACTGACGACAGTTTTGACACGGATGTACTCAAAGCGGACGGGGCGATCCTCGTCGATTTCTGGGCAGAGTGGTGCGGTCCGTGCAAAATGATCGCCCCGATTCTGGATGAAATCGCTGACGAATATCAGGGCAAACTGAC CGTTGCAAAACTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGCAACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCC.

[0017] Preferably, the D3 sequence is:

[0018] GCAGATAATAAGTTCAATAAGGAACAACAGAACGCCTTTTATGAAATCCTGCATCTGCCTAATCTGACCGAAGAACAGCGTAATGGCTTTATTCAGTCACTGAAAGATGACCCGAGTGTGAGCAAAGAAATTCTGGCCGAAGCGAAAAAAACTGAACGATGCACAGGCACCGAAG.

[0019] Preferably, the SUMO sequence:

[0020] GATAGCGAAGTTAATCAGGAAGCAAAACCGGAAGTTAAACCGGAAGTGAAACCGGAAACCCATATTAATCTGAAAGTGAGTGATGGTAGCAGTGAAATTTTCTTTAAAATCAAGAAGACCACCCCGCTGCGTCGTCTGATGGAA GCATTTGCCAAACGCCAGGGTAAAGAAATGGATAGCCTGCGTTTTCTGTATGATGGTATTCGTATTCAGGCCGATCAGACCCCGGAAGATCTGGATATGGAAGATAATGATATTATCGAAGCCCATCGCGAACAGATTGGTGGC.

[0021] The invention discloses application of a fusion antigen in preparing Helicobacter pylori vaccine and medicine.

[0022] Beneficial effects: The invented method can be used to express fusion antigens in the supernatant of E. coli fragments and successfully anchor them on GEM particles, thereby increasing immune prototypes for immune effect evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The graph shows the expression of the fusion antigen Ureb-HspA at 16°C and 28°C without the addition of TrxA and D3 sequences.

[0024] Figure 2 The graph shows the expression of the fusion antigen Ureb-HspA at 37°C without the addition of TrxA and D3 sequences.

[0025] Figure 3 The graph shows the expression of the fusion antigen Ureb-HspA with TrxA and D3 sequences added at 16°C, 28°C and 37°C.

[0026] Figure 4 This is a graph showing the expression status of BL21(DE3)-pET32a-TrxA-his-pA_D3-SUMO-Ureb after 7 hours of fermentation culture.

[0027] Figure 5 This is a diagram of the incubation of recombinant antigen and GEM particles.

[0028] in, Figure 5 : ①Uncooked lactic acid bacteria; ②Lactic acid bacteria cooked in 0.1M HCl (GEM particles); ③Supernatant after Ureb expression; ④Supernatant after crushing passed 0.45um membrane; ⑤Supernatant after passing through membrane and incubating with GEM particles and then centrifuging the supernatant; ⑥Supernatant after passing through membrane and incubating with GEM particles and then centrifuging and precipitating. DETAILED DESCRIPTION

[0029] The following is in conjunction with the instruction manual Figure 1-5 , the present invention is further illustrated, but the present invention is not limited to the following embodiments.

[0030] 1. Test whether TrxA and D3 sequences can effectively promote the expression of fusion antigen in the supernatant

[0031] Embodiment 1:

[0032] The existing BL21(DE3)-pET32a-SUMO-UrB strain was induced to express in a small amount (5 ml) at 220 rpm, 16°C, 28°C and 37°C, and 1 mM IPTG (induced overnight at 16°C, and induced for 4 h at 28°C and 37°C). The results are as follows: Figure 1 , Figure 2 As shown, most of the target proteins are expressed in the precipitate.

[0033] Embodiment 2:

[0034] The plasmid was reconstructed based on the existing plasmid, and the lytic tag TrxA and D3 sequences were added to obtain the strain BL21(DE3)-pET32a-TrxA-his-pA_D3-SUMO-Ureb. The recombinant expression was carried out under the same expression conditions as before the construction. The results are as follows Figure 3 As shown, it indicates that the expression level of the target protein in the supernatant increased significantly under 16°C and 28°C conditions.

[0035] Embodiment 3:

[0036] The recombinant BL21(DE3)-pET32a-TrxA-his-pA_D3-SUMO-Ureb strain was expressed in a fermenter under the induction conditions of 37°C and 0.1 mM IPTG. After 7 hours of induction, the results were as follows: Figure 4 As shown, obvious expression can be seen in the supernatant.

[0037] 2. Test whether the PA tag can be well anchored on the GEM particles

[0038] Embodiment 4:

[0039] 1) Preparation of GEM particles

[0040] Take 10μl of frozen strains of Lactococcus lactis MG1363 and add them to 10ml GM17 medium, culture at 37℃ overnight, and transfer to 500ml GM17 medium, and continue to culture at 37℃ for 12-16 hours. Collect the cells by centrifugation at 6000rpm for 5 minutes, wash once with sterile water, resuspend the cells with 0.1N hydrochloric acid, and boil at 100℃ for 60 minutes. Centrifuge at 6000rpm for 5 minutes to remove the acid solution, and wash 3 times with phosphate buffered saline (PBS) to collect the cells.

[0041] 2) Incubation of GEM particles with antigen

[0042] The obtained GEM particles were mixed with the supernatant obtained by E. coli expression, incubated at room temperature for 30 minutes, centrifuged at 6000 rpm for 5 minutes, washed 3 times with PBS, and the precipitate was collected. Figure 5 As shown, after incubation, the target protein has obvious bands in the precipitate, indicating that the PA tag is successfully bound to the GEM particles.

[0043] The sequence of pET32a-TrxA-his-pA_D3-SUMO-Ureb in the present application example is as follows:

[0044] TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGA

[0045] CCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTC

[0046] GCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGGCA

[0047] CCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTT

[0048] TTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTC

[0049] AACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAA

[0050] TGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCA

[0051] CTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCG

[0052] CTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACA

[0053] TTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGC

[0054] TGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAAC

[0055] AGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCT

[0056] GCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATT

[0057] CTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGA

[0058] GAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGG

[0059] AGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGG

[0060] AACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACA

[0061] ACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGAT

[0062] GGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATA

[0063] AATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCC

[0064] CGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGA

[0065] GATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTG

[0066] ATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAA

[0067] ATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTG

[0068] AGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTT

[0069] GTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCA

[0070] AATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATA

[0071] CCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGG

[0072] ACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCC

[0073] AGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCT

[0074] TCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGG

[0075] AGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGT

[0076] CGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACG

[0077] GTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATA

[0078] ACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCA

[0079] GTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACA

[0080] CCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCG

[0081] CTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACG

[0082] GGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGA

[0083] GGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGC

[0084] GATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTG

[0085] GCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAG

[0086] GGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACT

[0087] GATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGAGCGGCGGGA

[0088] CCAGAGAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCACAGGGTA

[0089] GCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGA

[0090] CTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCA

[0091] GTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTA

[0092] GCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCC

[0093] TGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCC

[0094] GAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCC

[0095] AGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATA

[0096] GTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCC

[0097] CGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAA

[0098] ACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGC

[0099] CAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAG

[0100] AGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAAC

[0101] GGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATGTCCGCACCAACGCG

[0102] CAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAG

[0103] TGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCT

[0104] TCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACG

[0105] CGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCT

[0106] CCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACA

[0107] TCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGG

[0108] ATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGA

[0109] CGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCC

[0110] GCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTT

[0111] GCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTT

[0112] CCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCG

[0113] GCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGG

[0114] GCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCC

[0115] TTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAG

[0116] GAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGC

[0117] CGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAG

[0118] GCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGAT

[0119] CGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTC

[0120] TAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAGCGATAAAATTATTCACCTGACTG

[0121] ACGACAGTTTTGACACGGATGTACTCAAAGCGGACGGGGCGATCCTCGTCGATTTCTGGGCAGAGTGG

[0122] TGCGGTCCGTGCAAAATGATCGCCCCGATTCTGGATGAAATCGCTGACGAATATCAGGGCAAACTGAC

[0123] CGTTGCAAAACTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATCCGTGGTATCCCGA

[0124] CTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGCAACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTG

[0125] AAAGAGTTCCTCGACGCTAACCTGGCCGGTTCTGGTTCTGGCCATATGCACCATCATCATCATCATTC

[0126] TTCTGGTGCAGATAATAAGTTCAATAAGGAACAACAGAACGCCTTTTATGAAATCCTGCATCTGCCTA

[0127] ATCTGACCGAAGAACAGCGTAATGGCTTTATTCAGTCACTGAAAGATGACCCGAGTGTGAGCAAAGAA

[0128] ATTCTGGCCGAAGCGAAAAAACTGAACGATGCACAGGCACCGAAGTCTTCTGGTGATAGCGAAGTTAA

[0129] TCAGGAAGCAAAACCGGAAGTTAAACCGGAAGTGAAACCGGAAACCCATATTAATCTGAAAGTGAGTG

[0130] ATGGTAGCAGTGAAATTTTCTTTAAAATCAAGAAGACCACCCCGCTGCGTCGTCTGATGGAAGCATTT

[0131] GCCAAACGCCAGGGTAAAGAAATGGATAGCCTGCGTTTTCTGTATGATGGTATTCGTATTCAGGCCGA

[0132] TCAGACCCCGGAAGATCTGGATATGGAAGATAATGATATTATCGAAGCCCATCGCGAACAGATTGGTG

[0133] GCaagaaaatatcaaggaaagaatatgtaagtatgtatggtccgaccaccggtgacaaggtacgtctg

[0134] ggggacacggatctgatagcggaggtcgagcacgattacaccatttacggcgaggagctgaagttcgg

[0135] cggtggtaaaaccctgcgtgagggcatgagccaaagcaataacccgagcaaagaggagcttgatctga

[0136] tcattaccaacgccctaattgtggactacaccggcatctataaagcggacatcggcattaaagacggc

[0137] aagattgccggcatcggaaagggcggtaataaggacatgcaggatggcgtcaaaaacaacctgagcgt

[0138] tggtccggctactgaggccttagcgggcgaaggcctgattgttaccgcgggtggtattgatacgcata

[0139] tccactttatctctccgcaacagatcccgacgacgtttgcgagtggtgttaccaccatgatcggtggt

[0140] ggtactggcccagcggacggcacgaacgctaccaccatcacgccgggtcgccgtaacttgaagtggat

[0141] gctgcgcgcagctgaagaatatagcatgaatcttggcttcctggcgaaaggcaacacctccaatgatg

[0142] caagcttggcggaccagattgaagctggcgccatcggtttcaaaatccacgaagattggggtacgacc

[0143] ccaagcgcaattaaccacgctctcgacgttgctgacaaatacgacgtgcaggtcgcgattcacaccga

[0144] caccttgaatgaagccggctgcgttgaagatacgatggcagcgatcgctggcagaaccatgcatacct

[0145] tccacaccgaaggtgcaggcggtggtcatgcaccggatatcatcaaggtggcgggtgagcacaacatt

[0146] ctgccggcgtccaccaatccaactatcccgtttaccgttaataccgaagcggaacacctggatatgct

[0147] gatggtgtgccatcatctggataagagcatcaaggaggacgtgcagtttgcggactctcgcattcgtc

[0148] cgcagacgattgctgcggaggacactctgcatgatatgggtattttctccattacctctagcgatagc

[0149] caagccatgggtcgtgtgggtgaggttattacccgtacctggcaaaccgcggacaagaacaagaaaga

[0150] gttcggtcgcttgaaagaagagaaaggtgataacgacaactttcgtattaaacgctatctgagcaaat

[0151] acaccatcaacccggcgatcgcgcatggtatcagcgaatacgtgggttctgttgaggttggtaaggtc

[0152] gctgatttggttctgtggtcgccggcgttttttggcgtgaagccgaatatgattatcaagggcggatt

[0153] catcgcgctgtcccaaatgggcgatgccaacgcatcgatcccgacgccacaaccggtttattatcgtg

[0154] aaatgtttgcacaccacggcaaggcgaaatacgacgccaatattaccttcgttagccaagcagcctac

[0155] gaaaaggttatcaaaagagagctgggtttggaacgtcaggttctgccggtgaaaaactgtcgtaacat

[0156] cactaaaaaggacatgcagttcaacgataccacggctcacattgaagttaatccgggaaacctatcatg

[0157] tgttcgtggatggtaaggaggtgacttccaagccggcaaacaaagtgagcttggcgcagctgtttagc

[0158] atcttcggtggcggcggttccaaattccagccgctgggtgaacgcgtgctcgtcgagcgtctgggaga

[0159] agacaacaagacctcttccggcattatcattccggataatgcgaaagagaaaccgctgatgggtgttg

[0160] ttaaggcagtgagccacaagatctccgaagggtgcaaatgcgtgaaggagggtgacgttattgcgttt

[0161] ggtaaatacaaaggtgccgagattgtgctggacggcaccgagtacatggtattggagctggaagatat

[0162] cttgggtatcgtgggtagcggtagttgctgccacaccggtaatcatgaccacaagcacgctaaagagc

[0163] acgaagcatgttgccatgatcacaagaaacatggtggcagctcgggcggtgcgagctctgctggcaac

[0164] acaaacagcggcggcagcaccacgacgaacaccaacaacaactccggtacgaactcctctagcactac

[0165] atataccgtcaagagcggcgacactctgtggggtatcagccagcgttatggtatttccgtggcgcaaa

[0166] tccagagcgctaataacctgaagagcacgatcatctacatcggtcagaagctggtcttaaccggttct

[0167] gccagctcaaccaacagcggcggttctaacaattcagcgagcacgactccgaccaccagcgtcacgcc

[0168] agcgaaaccggcttcccaaaccacggttaaggtgaagagcggcgacactctgtgggcactgtccgtga

[0169] agtacaagacctctattgctcagctgaaaagctggaaccacttgagctctgataccatatacattggc

[0170] caaaacctgatcgttagccagtccgcggcaagcagcaatccgagcactggttcgggctcgaccgcgac

[0171] caacaactccaacagcaccagctctaacagcaacagttctatccataaggtggttaagggcgacacgt

[0172] tatggggcctgtcgcaaaagtctggttcgccgattgcgagcatcaaggcgtggaatcacttgtccagc

[0173] gataccatccttatcggccagtatctgcgtattaaataaCTCGAGCACCACCACCACCACCACTGAGA

[0174] TCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCAT

[0175] AACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT.

[0176] Finally, it should be noted that the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

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