Mutant of Taq enzyme, nucleic acid molecule and application thereof

Abstract

The invention relates to the field of bioengineering, in particular to a Taq enzyme mutant, a nucleic acid molecule and application of the Taq enzyme mutant and the nucleic acid molecule. The invention provides a mutant of Taq DNA (deoxyribonucleic acid) polymerase, which has one or more amino acid site mutations on the basis of wild Taq DNA polymerase: the 507th site, the 543th site, the 551th site, the 742th site and the 664th site, and the amino acid sequence of the wild Taq DNA polymerase is as shown in SEQ ID NO: 75. The invention provides a novel mutant DNA (deoxyribonucleic acid) polymerase. Compared with a wild type DNA polymerase, the novel mutant DNA polymerase has higher inhibition resistance or higher dUTP (deoxyuridine triphosphate) doping capacity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, in particular to a mutant of Taq enzyme, a nucleic acid molecule and application thereof. BACKGROUND

[0002] DNA polymerase is a very useful biological reagent, which has a wide range of applications in genetic engineering, sequencing, nucleic acid amplification detection and other directions. It is also the most important reagent in PCR nucleic acid detection, and PCR method is one of the most important methods in molecular in vitro diagnosis field.

[0003] DNA polymerase can be divided into 7 families (A, B, C, D, E, X, and Y) according to its nucleic acid sequence similarity, and the enzymes mainly used in actual clinical application are mainly from A family (also known as Pol-I type polymerase) and B family (also known as α-type polymerase).

[0004] The most widely used DNA polymerase in A family is Taq DNA polymerase from Thermus aquaticus species, and many polymerases used in PCR kits at present stage adopt Taq DNA polymerase. In addition, Pol-I type DNA polymerase from Thermus thermophilus species (also known as Tth DNA polymerase), Pol-I type DNA polymerase from Geobacillus stearothermophilus species (also known as Bst DNA polymerase) and other polymerases also belong to A family.

[0005] PCR (polymerase chain reaction) is a technique for rapidly amplifying DNA fragments in vitro, which is based on the replication mechanism of DNA and the catalytic effect of enzymes. The elements required for PCR technology are as follows: 1. DNA template: PCR reaction needs a target DNA fragment as the template for amplification. This DNA can come from any source, such as bacteria, plants, animals or humans. 2. Primer: Two primers are needed in PCR reaction, one is forward primer and the other is reverse primer. Primer is a short DNA sequence, which will bind to the two ends of the target DNA fragment. 3. DNA polymerase: PCR reaction needs a heat-stable DNA polymerase (for example, Taq polymerase). This enzyme can remain stable at high temperature, and can catalyze the synthesis of DNA in each PCR cycle.

[0006] The steps of PCR reaction are as follows: 1. Preparation of reaction system: mix the reagents required for PCR reaction (such as DNA template, primers, polymerase, buffer and dNTPs, etc.) together according to the specific proportion to form the PCR reaction system. 2. Denaturation: heat the PCR reaction system to 94-98℃ to separate the double-stranded DNA into two single-stranded DNAs. 3. Annealing: cool the PCR reaction system to 50-65℃ to allow the primers to bind to the complementary DNA single strand. The selection of primers is determined according to the sequence of the target DNA fragment. 4. Extension: warm the PCR reaction system to 72℃, at which point the DNA polymerase starts to catalyze the synthesis of DNA. It will extend along the DNA template strand to synthesize new DNA strands complementary to the primers. 5. Repeat the cycle: the above three steps (denaturation, primer binding and extension) constitute a PCR cycle. Each PCR cycle doubles the amount of target DNA fragment. Through multiple cycles, a large amount of target DNA fragment can be amplified in a short time. The number of PCR cycles depends on the amount of DNA amplification required. Generally, the number of PCR cycles is usually between 20-40 times.

[0007] The advantages of PCR technology include high specificity, high sensitivity and rapid amplification speed. It is widely used in genomics research, disease diagnosis, genetic analysis, forensic science and bioengineering fields.

[0008] The overall folding shape of polymerase is similar to the right hand of human, which is divided into palm, fingers, thumb and other domains. The amino acid sequence of the main active site of polymerase is highly conserved, and polymerases of different species perform their functions through similar mechanisms. In addition, some amino acid sites of polymerase may not affect its main function after mutation. Through changes in amino acid sequence, including point mutation, insertion, deletion, recombination and other ways, the enzyme activity function can be changed as desired.

[0009] Although PCR technology has many advantages, it is also easy to cause contamination of PCR amplified products and lead to false positive results. The template amount of PCR amplified products is very large, and if the PCR reaction container is not tightly sealed, it is easy to escape into the air. Once these amplified products nucleic acids fall into the next round of PCR reagent preparation or negative samples with aerosol, it will cause the PCR result to present false positive, thus leading to experimental error.

[0010] In order to avoid the contamination of PCR reaction products, people usually separate the space of PCR reaction from the space of reagent preparation, such as into several rooms. But the flow of personnel and materials cannot completely avoid the occurrence of pollution.

[0011] One solution widely used in the industry is to use dUTP and UNG enzyme combination to prevent contamination of PCR amplification products. The principle is as follows:

[0012] The use of dUTP in combination with UNG enzyme can effectively prevent contamination of PCR products. dUTP is a deoxyuracil nucleotide triphosphate, which can replace the commonly used dTTP (deoxythymine nucleotide triphosphate) in PCR reactions. In PCR reactions, dUTP will be introduced into the amplified DNA strand. UNG enzyme (uracil nucleotidase) is a specific enzyme that can recognize and remove uracil groups in DNA strands. After PCR reaction, if foreign DNA is contaminated into the reaction system, which may contain uracil groups. When using dUTP and UNG enzyme, the uracil groups in the foreign DNA will be removed by UNG enzyme, thereby avoiding the amplification of these DNA contaminants in the PCR reaction.

[0013] Although the combination of dUTP and UNG enzyme can prevent contamination of PCR amplification products, because dUTP is not the preferred substrate of Taq DNA polymerase, its amplification efficiency will decrease with the incorporation of dUTP. This is more obvious in multiplex amplification.

[0014] In order to overcome this difficulty and improve the amplification efficiency of PCR when using dUTP as a substrate, it is necessary to genetically engineer Taq DNA polymerase. SUMMARY

[0015] Therefore, the present application provides a mutant of Taq enzyme, a nucleic acid molecule and its application. The present application provides a new mutant DNA polymerase, which has stronger inhibition resistance or stronger dUTP incorporation ability compared with wild-type DNA polymerase.

[0016] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0017] The present application provides a mutant of Taq DNA polymerase, which has one or more amino acid site mutations at positions 507, 543, 551, 742 and 664 based on the wild-type Taq DNA polymerase, and the amino acid sequence of the wild-type Taq DNA polymerase is shown in SEQ ID NO: 75.

[0018] The mutation at position 507 includes E507K or E507Q.

[0019] The mutation at position 543 includes S543N.

[0020] The mutation at the 551st site includes any one of D551K, D551R and D551Q.

[0021] The mutation at the 742nd site includes any one of E742Q, E742N and E742K.

[0022] The mutation at the 664th site includes T664Q or T664N.

[0023] In some embodiments of the present application, the above-mentioned mutant further includes one or more mutations at the following mutation sites: E117Q, D119N, D142N, D144N.

[0024] In some embodiments of the present application, the above-mentioned mutant has:

[0025] (1) an amino acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 35; or

[0026] (2) a sequence in which one or more amino acids in the amino acid sequence as shown in (1) are substituted, deleted, added and / or replaced; or

[0027] (3) a sequence having more than 90% homology with the amino acid sequence as shown in (1) or (2).

[0028] MRGMLPLFEP KGRVLLVD GHHLAYRTFHA LKGLTTSRGEP VQAVYGFAKS LLKALKEDGDA VIVVFDAKAPS FRHEAYGG YKAGRAPTPED FPRQLALIK ELVDLLGLAR LEVPGYEADD VLASLAKKAE KEGYEVRILT AAKDLYQLLSD RIHVLHPEGY LITPAWLWE KYGLRPDQWAD YRALTGDESD NLPGVKGIGE KTARKLLEEW GSLEALLKN LDRLKPAIRE KILAHMDDDL KLSWDLAKVR TDPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLES PKALEEAPWPP PEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARG LLAKDLSVLA R EGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSE RLFA NLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLE AEVFRLAGHP FNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVE KILQYRELTK LKSTYIDPLP DLIHPRTGRL HTRFNQTATAT GRLSSSDPN LQNIPVRTPL GQRIRRAFIA EEGWLLVALD YSQIELRVL A HLSGDENLIR VFQEGRDIHT ETASWMFGVP REAVDPLMRRA AKQINFGVL YGMSAHRLSQ ELAIPYEEAQ AFIERYFQSF PKVRAWIEKT LEEGRRRGYV ETLFGRRRYV PDLEARVKSV RNAAERMAFN MPVQGTAADL MKLAMVKLF PRLEEMGARML LQVHDELVLE APKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0029] MRGMLPLFEP KGRVLLVD GHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPED FPRQLALIK ELVDLLGLAR LEVPGYEADD VLASLAKKAE KEGYEVRILT ANKDLYQLL SDRIHVLHPE GYLITPAWLW E KYGLRPDQW ADYRALTGDE SDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIRE KILAHMDD LKLSWDLAKV RTRDLPLEVD FAK RREPDRERLR AFLERLEFGS LLHEFGLLE SPKALEEAPW PPPEGAFVGF VLSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEAR GLLLAKDLSV LALREGLGLP PG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSE RLFA NLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLE AEVFRLAGHP FNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVE KILQYRELTK LKSTYIDPLP KLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPL GQRIRRAFI AEEGWLLVALD YSQIELRVL A HLSGDENLIR VFQEGRDIHT ETASWMFGVP REAVDPLMRRAAKQINFGVL YGMSAHRLSQ ELAIPYEEAQ AFIERYFQSF PKVRAWIEKT LEEGRRRGYV ETLFGRRRYV PDLEARVKSV REAAERMAFN MPVQGTAADL MKLAMVKLF PRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0030] In some embodiments of the application, the sequence of SEQ ID NO: 3 is: MRGMLPLFEP KGRVLLVD GHHLAYRTFH AL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPED FPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ADKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTEKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKNTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKTINFGVL YGMSAHRLSQ ELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV RKAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0031] MRGMLPLFEP KGRVLLVD GHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPED FPRQLALIK ELVDLLGLAR LEVPGYEADD VLASLAKKAE KEGYEVRILT ADKDLYQLL SDRIHVLHPE GYLITPAWLW E KYGLRPDQW ADYRALTGDE SDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIRE KILAHMDD LKLSWDLAKV RTRDLPLEVD FAK RREPDRERLR AFLERLEFGS LLHEFGLLE SPKALEEAPW PPPEGAFVGF VLSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEAR GLLLAKDLSV LALREGLGLP PG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSE RLFA NLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLE AEVFRLAGHP FNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVE KILQYRELTK LKNTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPL GQRIRRAFI AEEGWLLVALD YSQIELRVL A HLSGDENLIR VFQEGRDIHT ETASWMFGVP REAVDPLMRRAAKQINFGVL YGMSAHRLSQ ELAIPYEEAQ AFIERYFQSF PKVRAWIEKT LEEGRRRGYV ETLFGRRRYV PDLEARVKSV REAAERMAFN MPVQGTAADL MKLAMVKLF PRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0032] MRGMLPLFEP KGRVLLVD GHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPED FPRQLALIK ELVDLLGLAR LEVPGYQAND VLASLAKKAE KEGYEVRILT ANKNLYQLL SDRIHVLHPEG YLITPAWLWE KYGLRPDQW ADYRALTGDE SDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIRE KILAHMDDDL KLSWDLAKVR TDLPLEVDFA KRREPDRERL RAFLERLEFG SLLHEFGLLE SPKALEEAPW PPPEGAFVGF VLSRKEPMWA DLLALAAAR GG RVHRAPEPY K ALRDLKEAR GLLAKDLSVL ALREGLGLPP G DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSE RLFA NLWGRLEGE ERLLWLYREV ERPLSAVLAH MEATGVR LDVAYLRALS LEVAEEIARL EAEVFRLAGH PFNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVE KILQYRELTK LKSTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPL GQRIRRAFIA EEGWLLVALD YSQIELRVL A HLSGDENLI RVFQEGRDIH TETASWMFGV PREAVDPLMR RA AKQINFGVL YGMSAHRLSQ ELAIPYEEAQ AFIERYFQSF PKVRAWIEKT LEEGRRRGYV ETLFGRRRYV PDLEARVKSV REAAERMAFN MPVQGTAADL MKLAMVKLF PRLEEMGARML LQVHDELVLE APKERAEAVAR LAK E VMEGV YPLAVPLEV E VGIGEDWLSA KE.

[0033] MRGMLPLFEP KGRVLLVD GHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPED FPRQLALIK ELVDLLGLAR LEVPGYEADD VLASLAKKAE KEGYEVRILT ANKDLYQLL SDRIHVLHPE GYLITPAWLW E KYGLRPDQW ADYRALTGDE SDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIRE KILAHMDD LKLSWDLAKV RTRDLPLEVD FAK RREPDRERLR AFLERLEFGS LLHEFGLLE SPKALEEAPW PPPEGAFVGF VLSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEAR GLLLAKDLSV LALREGLGLP PG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSE RLFA NLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLE AEVFRLAGHP FNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVE KILQYRELTK LKSTYIDPLP QLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPL GQRIRRAFI AEEGWLLVALD YSQIELRVL A HLSGDENLIR VFQEGRDIHT ETASWMFGVP REAVDPLMRRAAKQINFGVL YGMSAHRLSQ ELAIPYEEAQ AFIERYFQSF PKVRAWIEKT LEEGRRRGYV ETLFGRRRYV PDLEARVKSV REAAERMAFN MPVQGTAADL MKLAMVKLF PRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0034] MRGMLPLFEP KGRVLLVD GHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPED FPRQLALIK ELVDLLGLAR LEVPGYEADD VLASLAKKAE KEGYEVRILT ANKDLYQLL SDRIHVLHPE GYLITPAWLW E KYGLRPDQW ADYRALTGDE SDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIRE KILAHMDD LKLSWDLAKV RTRDLPLEVD FAK RREPDRERLR AFLERLEFGS LLHEFGLLE SPKALEEAPW PPPEGAFVGF VLSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEAR GLLLAKDLSV LALREGLGLP PG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSE RLFA NLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLE AEVFRLAGHP FNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVE KILQYRELTK LKSTYIDPLP RLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPL GQRIRRAFI AEEGWLLVALD YSQIELRVL A HLSGDENLIR VFQEGRDIHT ETASWMFGVP REAVDPLMRRAAKQINFGVL YGMSAHRLSQ ELAIPYEEAQ AFIERYFQSF PKVRAWIEKT LEEGRRRGYV ETLFGRRRYV PDLEARVKSV REAAERMAFN MPVQGTAADL MKLAMVKLF PRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0035] MRGMLPLFEP KGRVLLVD GHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPED FPRQLALIK ELVDLLGLAR LEVPGYEADD VLASLAKKAE KEGYEVRILT ANKDLYQLL SDRIHVLHPE GYLITPAWLW E KYGLRPDQW ADYRALTGDE SDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIRE KILAHMDD LKLSWDLAKV RTRDLPLEVD FAK RREPDRERLR AFLERLEFGS LLHEFGLLE SPKALEEAPW PPPEGAFVGF VLSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEAR GLLLAKDLSV LALREGLGLP PG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSE RLFA NLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLE AEVFRLAGHP FNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVE KILQYRELTK LKNTYIDPLP KLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPL GQRIRRAFI AEEGWLLVALD YSQIELRVL A HLSGDENLIR VFQEGRDIHT ETASWMFGVP REAVDPLMRRA AKTINFGVL YGMSAHRLSQ ELAIPYEEAQ AFIERYFQSF PKVRAWIEKT LEEGRRRGYV ETLFGRRRYV PDLEARVKSV REAAERMAFN MPVQGTAADL MKLAMVKLF PRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0036] MRGMLPLFEP KGRVLLVD GHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPED FPRQLALIK ELVDLLGLAR LEVPGYEADD VLASLAKKAE KEGYEVRILT ADKDLYQLL SDRIHVLHPE GYLITPAWLW E KYGLRPDQW ADYRALTGDE SDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIRE KILAHMDD LKLSWDLAKV RTRDLPLEVD FAK RREPDRERLR AFLERLEFGS LLHEFGLLE SPKALEEAPW PPPEGAFVGF VLSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEAR GLLLAKDLSV LALREGLGLP PG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSE RLFA NLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLE AEVFRLAGHP FNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVE KILQYRELTK LKNTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPL GQRIRRAFI AEEGWLLVALD YSQIELRVL A HLSGDENLIR VFQEGRDIHT ETASWMFGVP REAVDPLMRRAAKQINFGVL YGMSAHRLSQ ELAIPYEEAQ AFIERYFQSF PKVRAWIEKT LEEGRRRGYV ETLFGRRRYV PDLEARVKSV RQAAERMAFN MPVQGTAADL MKLAMVKLF PRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0037] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ADKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKSTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKNINFGVL YGMSAHRLSQELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV RNAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0038] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ANKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTEKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKNTYIDPLP KLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKTINFGVL YGMSAHRLSQ ELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV REAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0039] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ADKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKNTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKTINFGVL YGMSAHRLSQ ELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV RQAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0040] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT AAKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKSTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKNINFGVL YGMSAHRLSQELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV RQAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0041] In some embodiments of the application, the sequence of SEQ ID NO: 14 is: MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ADKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKSTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKTINFGVL YGMSAHRLSQ ELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV RQAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0042] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ADKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTEKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKSTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKQINFGVL YGMSAHRLSQELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV RQAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0043] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ADKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKSTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKQINFGVL YGMSAHRLSQELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV REAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0044] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ANKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKSTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKQINFGVL YGMSAHRLSQELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV REAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0045] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT AAKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKSTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKTINFGVL YGMSAHRLSQ ELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV RQAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0046] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ANKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKNTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKQINFGVL YGMSAHRLSQELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV RQAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0047] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT AAKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTEKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKSTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKQINFGVL YGMSAHRLSQELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV RQAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0048] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT AAKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKNTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKQINFGVL YGMSAHRLSQELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV RQAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0049] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ANKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTEKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKSTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKQINFGVL YGMSAHRLSQELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV RQAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0050] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ADKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKSTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKQINFGVL YGMSAHRLSQELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV RQAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0051] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ADKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTEKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKNTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKQINFGVL YGMSAHRLSQELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV RQAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0052] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ADKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKNTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKTINFGVL YGMSAHRLSQ ELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV RQAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0053] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT AAKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKSTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKQINFGVL YGMSAHRLSQELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV REAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0054] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT AAKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTEKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKNTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKQINFGVL YGMSAHRLSQELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV RQAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0055] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT AAKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKNTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKQINFGVL YGMSAHRLSQELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV REAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0056] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT AAKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKSTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKQINFGVL YGMSAHRLSQELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV RQAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0057] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ANKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKSTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKQINFGVL YGMSAHRLSQELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV REAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0058] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ANKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTEKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKSTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKTINFGVL YGMSAHRLSQ ELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV RKAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0059] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ADKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKSTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKTINFGVL YGMSAHRLSQ ELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV REAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0060] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ADKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKNTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKTINFGVL YGMSAHRLSQ ELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV REAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0061] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ADKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTKKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKSTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKTINFGVL YGMSAHRLSQ ELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV REAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0062] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ANKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTQKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKNTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKQINFGVL YGMSAHRLSQELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV REAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0063] MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ANKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTEKTG KRSTSAAVLE ALREAHPIVEKILQYRELTKLKSTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKTINFGVL YGMSAHRLSQ ELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV REAAERMAFN MPVKGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0064] In some embodiments of the application, the sequence of SEQ ID NO: 37 is: MRGMLPLFEP KGRVLLV DGHHLAYRTFHAL KGLTTSRGEP VQAVYGFAKS LLKALKEDGDAVIVVFDAKA PSFRHEAYGG YKAGRAPTPEDFPRQLALIK ELVDLLGLAR LEVPGYEADDVLASLAKKAE KEGYEVRILT ADKDLYQLLSDRIHVLHPEGYLITPAWLWE KYGLRPDQWADYRALTGDESDNLPGVKGIG EKTARKLLEE WGSLEALLKN LDRLKPAIREKILAHMDDLKLSWDLAKVRT DLPLEVDFAK RREPDRERLR AFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFV LSRKEPMWAD LLALAAARGG RVHRAPEPYK ALRDLKEARGLLAKDLSVLALREGLGLPPG DDPMLLAYLL DPSNTTPEGV ARRYGGEWTE EAGERAALSERLFANLWGRLEGEERLLWLY REVERPLSAV LAHMEATGVR LDVAYLRALS LEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGL PAIGKTEKTG KRSTSAAVLQ ALREAHPIVEKILQYRELTKLKSTYIDPLP DLIHPRTGRL HTRFNQTATA TGRLSSSDPN LQNIPVRTPLGQRIRRAFIAEEGWLLVALD YSQIELRVLA HLSGDENLIR VFQEGRDIHT ETASWMFGVPREAVDPLMRRAAKTINFGVL YGMSAHRLSQ ELAIPYEEAQ AFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYV PDLEARVKSV REAAERMAFN MPVQGTAADL MKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVA RLAKEVMEGV YPLAVPLEVE VGIGEDWLSA KE.

[0065] The present application also provides nucleic acid molecules encoding the above mutants.

[0066] In some embodiments of the present application, the above-mentioned nucleic acid molecule has:

[0067] (4) a nucleotide sequence represented by any one of SEQ ID NO: 38 to SEQ ID NO: 72; or

[0068] (5) a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence represented by (4), and a nucleotide sequence which is functionally identical or similar to the nucleotide sequence represented by (4); or

[0069] (6) a nucleotide sequence which has at least 90% identity to the nucleotide sequence represented by (4) or (5).

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[0107] The present application also provides an expression vector comprising the nucleic acid molecule and acceptable genetic elements.

[0108] The present application also provides a host transformed and / or transfected with the expression vector.

[0109] The present application also provides a method for preparing the mutant, which comprises culturing the host, collecting the bacterial cells, and purifying the mutant.

[0110] The present application also provides a product comprising the mutant, the nucleic acid molecule, the expression vector, the host, and / or the mutant prepared by the method.

[0111] The present application also provides the use of the mutant, the nucleic acid molecule, the expression vector, the host, the mutant prepared by the method, and / or the product in any of the following:

[0112] (I) polymerase chain reaction;

[0113] (II) nucleic acid detection for non-diagnostic purposes;

[0114] (III) reducing contamination of products of polymerase chain reaction;

[0115] (IV) improving amplification efficiency of polymerase chain reaction;

[0116] (V) improving tolerance to dUTP incorporation in polymerase chain reaction.

[0117] The present application also provides a method for polymerase chain reaction, which comprises mixing the mutant and / or the mutant prepared by the method with a template and raw materials, amplifying, and detecting.

[0118] In some embodiments of the present application, the method, the pretreatment comprises a hot start step.

[0119] In some embodiments of the present application, the method, the raw materials comprise dUTP.

[0120] The present application provides a mutant of Taq DNA polymerase, which has one or more amino acid site mutations at positions 507, 543, 551, 742, and 664 based on a wild-type Taq DNA polymerase, and the amino acid sequence of the wild-type Taq DNA polymerase is shown in SEQ ID NO: 75.

[0121] The present application provides a novel mutant DNA polymerase, which has stronger anti-inhibition ability or stronger dUTP incorporation ability compared with wild-type DNA polymerase. BRIEF DESCRIPTION OF DRAWINGS

[0122] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows.

[0123] Figure 1 SDS-PAGE electrophoretogram of Taq enzyme protein, MK represents protein molecular weight marker, and Taq represents Taq enzyme (taking TQ1068 as an example);

[0124] Figure 2 Multiple PCR capillary electrophoretogram of wild-type Taq and different mutant Taqs, in which the Taq enzyme uses dUTP to replace dTTP as a raw material to amplify 0.5 ng human genomic DNA;

[0125] Figure 3 Multiple PCR capillary electrophoretogram of wild-type Taq and different mutant Taqs, in which the Taq enzyme uses dUTP to replace dTTP as a raw material to amplify 0.5 ng human genomic DNA; DETAILED DESCRIPTION

[0126] The present application discloses a mutant of Taq enzyme, a nucleic acid molecule and application thereof.

[0127] It should be understood that the expression "one or more of" includes each of the objects recited after the expression and various different combinations of two or more of the recited objects, individually, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0128] The terms "comprising", "having" or "including", including their grammatical variations, should generally be understood to be open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0129] It should be understood that the order of steps or order of performing certain actions is immaterial so long as the application remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0130] The use of any and all examples, or exemplary language herein, for example, "for instance" or "such as", is intended merely to better illustrate the application and does not indicate that any non-recited element is essential to the practice of the application unless claimed accordingly. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0131] In addition, numerical ranges and parameters anywhere in this specification are intended to be approximate, unless otherwise indicated. Where particular values are given in specific examples, they are understood to be approximations unless otherwise indicated. Any numerical value, however, inherently contain certain errors necessarily resulting from the standard deviation found in the respective testing measurements.

[0132] The DNA polymerase nucleic acid sequence of the present application can be obtained by whole gene synthesis, or can be obtained by mutation based on the existing wild-type DNA polymerase nucleic acid sequence.

[0133] The DNA polymerase of the present application can be expressed and produced by conventional molecular cloning techniques. Mainly including the following three steps:

[0134] (1) The DNA polymerase nucleic acid sequence of the present application is recombined into a suitable expression vector by molecular cloning techniques, and the recombinant expression vector is transformed into a host cell.

[0135] (2) The host cell is cultured in a culture medium, and the host cell is allowed to express the DNA polymerase of the present application.

[0136] (3) The DNA polymerase of the present application is isolated and purified in the culture medium or the cell.

[0137] Among them, in step 1, there are various commonly used molecular cloning techniques for recombining the DNA polymerase nucleic acid sequence of the present application into a suitable expression vector. These methods include homologous recombination technology, enzyme digestion and ligation technology, nucleic acid synthesis technology, etc. The expression vector can use commercially available mature expression vectors, such as the pET series vectors produced by Novagen company. These vectors contain complete expression elements, and can express the protein produced by the target nucleic acid sequence in a large amount after transforming the host cell under suitable conditions.

[0138] The host cell can be a prokaryotic cell, such as an E. coli cell. It can also be a eukaryotic cell, such as a yeast cell, or it can also be a eukaryotic cell, such as an animal cell or a plant cell. The technology of transforming the recombinant expression vector into the host cell is also a technology known to the skilled person, which can use CaCl2 method or electroporation method.

[0139] After obtaining the engineered cells containing the recombinant vector by the above method, the engineered cells are cultured in a suitable culture medium. The culture and induction conditions of the engineered cells are within the skills known to those skilled in the art. For example, the Escherichia coli is cultured at a temperature of 37°C and a rotation speed of 200 rpm, and when the OD value reaches about 0.8, the corresponding inducer is added to induce the expression of the target protein, and then the culture is continued for 4 hours or the temperature is lowered to 16°C for 4-16 hours. It should be noted that the skilled person can adjust the fermentation time and temperature according to the actual situation to obtain higher expression or more convenient production process.

[0140] If it is a secretory expression, the fermentation broth needs to be collected. If it is a non-secretory expression, the cells after expression need to be collected by centrifugation or the like. Then the cells are broken by high pressure, enzymatic hydrolysis, mechanical shearing force or the like to release the target protein. The lysate of the host cells needs to be purified to obtain the target protein with high purity and activity.

[0141] Protein purification is a common biochemical technique, including but not limited to affinity chromatography, ion chromatography, hydrophobic chromatography, gel exclusion chromatography, ammonium sulfate precipitation and the like. The target protein with high purity and activity can be obtained by using various methods properly.

[0142] In the embodiments 1-5 of the present application, the raw materials and reagents used can be purchased from the market.

[0143] The present application is further described below in combination with the embodiments:

[0144] Example 1: Construction of the expression vector of the mutant Taq enzyme

[0145] Through rational design, we synthesize the corresponding nucleic acid sequence by GenScript. The target fragment of Taq polymerase is cleaved by NcoI and XbaI enzyme sites and connected to the commercial vector pTrc99A. The restriction endonuclease used for cleavage and T4 ligase used for connection are purchased from NEB company.

[0146] Example 2: Expression and purification of the mutant Taq enzyme

[0147] 1) The successfully constructed expression vector is transformed into host cells, liquid medium is cultured, and when the OD600 value is 0.9-1.2, 1 mM of IPTG is added, and the expression is induced at 37°C overnight, and the bacterial cells are collected.

[0148] 2) Cell disruption: 1 g of cells was added to 4 mL of bacterial disruption buffer (potassium chloride 4.5 g, 200 mM sodium phosphate solution 150 g, EDTA 0.4 g, concentrated phosphoric acid solution to adjust pH to 7.0, H2O to 1 L), 37 °C water bath, 0.5 mg / mL final concentration of lysozyme was added, stirring for 15 min, then centrifuged, centrifugal force 45000 G, 20 min, the supernatant was heated in a 75 °C water bath for 30 min, then centrifuged at 12000 rpm for 20 min, about 4 mL of supernatant was collected, 1.2 mL of 4 M ammonium sulfate was added, and then column purification was performed,

[0149] 3) Butyl column purification: the Butyl column was equilibrated with 2-3 column volumes of lysis buffer (20 mM phosphate buffer + 1 M ammonium sulfate, pH 7.6); the lysis buffer was transferred to the Butyl column (incubated for 1 h) and the flow-through was collected; the column was equilibrated with 4 column volumes of lysis buffer, and the equilibration buffer was collected; elution was performed with 60% B / 95% B / 100% buffer B (20 mM phosphate buffer, pH 7.6) for 3-4 column volumes each, and the eluate was collected separately; the above samples were run on a gel to identify the position of the target protein;

[0150] 4) Q column purification: 3-5 column volumes of buffer F (20 mM Tris-HCl + 100 mM NaCl, pH 8.0) were used to equilibrate the column until the baseline was stable; the previous sample was dialyzed into buffer F and then loaded and incubated for 0.5 h; after loading was completed, 3-5 column volumes of buffer F were used for equilibration; 12% / 17% / 100% buffer G (20 mM Tris-HCl + 1000 mM NaCl, pH 8.0) was used for washing, and the eluate was run on a gel to determine the position of the target band;

[0151] 5) Sample post-treatment: according to the electrophoresis results, the target protein was collected, and protein concentration was performed using an ultrafiltration tube to obtain purified mutant Taq DNA polymerase, which was diluted with a storage solution (20 mM Tris-HCl, 120 mM kCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Tween 20, 50% glycerol, pH 8.0) after standard activity measurement, and stored at -20 °C

[0152] Example 3 Obtaining a hot-start mutant Taq enzyme (antibody modification)

[0153] The protein concentration of Taq and the antibody (specific Taq enzyme antibody purchased from Baiying Biotechnology) were mixed in equal volumes according to a ratio of 1:4 (Taq was 1 mg / mL, and the antibody required 4 mg / mL), an EP tube was sealed with a sealing mold, and placed in a 37 °C incubator, and after two hours, it was taken out and stored at -20 °C.

[0154] Obtain the hot start Taq enzyme.

[0155] Example 4 Mutant Taq enzyme uses dUTP to replace dTTP as raw material for PCR amplification

[0156] 1) The modified Taq is diluted to 4U / μL with 1* Taq storage buffer, and the DNA template is diluted to 0.5ng with 1* Low-TE.

[0157] Mix preparation: PCR amplification system.

[0158] Table 1

[0159]

[0160] PCR program:

[0161] 95℃ 1min; 95℃ 10s, 60℃ 1min 30s (28 cycles); 60℃ 10min, 4℃ Hold.

[0162] 2) 3500 electrophoresis:

[0163] Formamide and internal standard (Xinhai internal standard NS600) are prepared at a ratio of 100:2, 9μL is added to each reaction, and 1μL of amplified product is added.

[0164] Experimental results:

[0165] This round of multiplex amplification should have 42 peaks, and in the hybrid, one allele is amplified and the other is not amplified, which is recorded as 0.5. The more complete the peak number is, the better the amplification is. In the case of dUTP incorporation, wild type Taq enzyme amplification appears incomplete.

[0166] Table 2

[0167]

[0168]

[0169] The nucleotide sequence of the wild type is shown in SEQ ID NO: 83; the nucleotide sequence of TQ1083 is shown in SEQ ID NO: 84; the nucleotide sequence of TQ1081 is shown in SEQ ID NO: 85; the nucleotide sequence of TQ1082 is shown in SEQ ID NO: 86; the nucleotide sequence of TQ1032 is shown in SEQ ID NO: 87; the nucleotide sequence of TQ1078 is shown in SEQ ID NO: 88; the nucleotide sequence of TQ1079 is shown in SEQ ID NO: 89; and the nucleotide sequence of TQ1043 is shown in SEQ ID NO: 90.

[0170] Electrophoresis (partial) map as shown Figure 2 ;

[0171] The above experimental results show that TQ1068 (D142A, T664Q, E507Q, E742N), TQ1058 (E507Q, T664Q, D142N, D551K) have better amplification under dUTP compared with the wild type control.

[0172] Example 5 Mutant Taq PCR amplification of templates with inhibitors

[0173] 1) The modified Taq was diluted to 4U / μL with 1* Taq storage buffer, and the DNA template was diluted to 0.5ng with 1* Low-TE.

[0174] Mix preparation: PCR amplification system.

[0175] Table 3

[0176]

[0177] PCR program:

[0178] 95℃ 1min; 95℃ 10s, 60℃ 1min 30s (28 cycles); 60℃ 10min, 4℃ Hold.

[0179] 2) 3500 electrophoresis:

[0180] Formamide and internal standard (Xinhai internal standard NS600) were prepared at a ratio of 100:2, 9μL was added to each reaction, and 1μL of amplified product was added.

[0181] 3) Experimental results:

[0182] Table 4

[0183]

[0184] Electrophoresis (partial) map as shown Figure 3 ;

[0185] The above experimental results show that TQ1059 (E507Q, T664Q, D142N, D551R), TQ1058 (E507Q, T664Q, D142N, D551K) have better amplification in the presence of inhibitors.

[0186] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A mutant of Taq DNA polymerase, characterized in that, Based on the wild-type Taq DNA polymerase, it has one or more of the following amino acid site mutations: site 507, site 543, site 551, site 742 and site 664, and the amino acid sequence of the wild-type Taq DNA polymerase is shown in SEQ ID NO:75; Wherein: the mutation at position 507 includes: E507K or E507Q; The mutation at site 543 includes: S543N; The mutation at site 551 includes any one of D551K, D551R, and D551Q. The mutation at position 742 includes any one of E742Q, E742N, and E742K; The mutation at position 664 includes either T664Q or T664N.

2. The mutant as described in claim 1, characterized in that, It also includes one or more mutations at the following mutation sites: E117Q, D119N, D142N, and D144N.

3. The mutant as described in claim 1 or 2, characterized in that, It has the following characteristics: (1) Any amino acid sequence as shown in SEQ ID NO:1 to SEQ ID NO:35; or (2) A sequence based on the amino acid sequence shown in (1) by substitution, deletion, addition and / or replacement of one or more amino acids; or (3) A sequence that is more than 90% homologous to the amino acid sequence shown in (1) or (2).

4. A nucleic acid molecule encoding the mutant as described in any one of claims 1 to 3.

5. The nucleic acid molecule as described in claim 4, characterized in that, It has the following characteristics: (4) Any nucleotide sequence as shown in SEQ ID NO:38 to SEQ ID NO:72; or (5) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (4), and which has the same or similar function as the nucleotide sequence shown in (4); or (6) A nucleotide sequence that is at least 90% identical to the nucleotide sequence shown in (4) or (5).

6. An expression carrier, characterized in that, include: The nucleic acid molecule as described in claim 4 or 5, and the acceptable gene element.

7. The host, characterized in that, Transformation and / or transfection of the expression vector as described in claim 6.

8. The method for preparing the mutant according to any one of claims 1 to 3, characterized in that, After culturing the host cell as described in claim 7, the bacterial cells are collected, purified, and the mutant is obtained.

9. The product, characterized in that, include: The mutant as described in any one of claims 1 to 3, the nucleic acid molecule as described in claim 4 or 5, the expression vector as described in claim 6, the host as described in claim 7, and / or the mutant obtained by the preparation method as described in claim 8.

10. The use of the mutant as described in any one of claims 1 to 3, the nucleic acid molecule as described in claim 4 or 5, the expression vector as described in claim 6, the host as described in claim 7, the mutant obtained by the preparation method as described in claim 8, and / or the product as described in claim 9 in any of the following: (I) Polymerase chain reaction; (II) Nucleic acid testing for non-diagnostic purposes; (III) Reduce contamination from polymerase chain reaction products; (IV) Improve the amplification efficiency of polymerase chain reaction; (V) Improve the tolerance of dUTP incorporation in polymerase chain reaction.

11. A method for polymerase chain reaction, characterized in that, The pretreated mutant as described in any one of claims 1 to 3 and / or the mutant obtained by the preparation method as described in claim 8 are mixed with the template and raw materials, amplified, and detected.

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