TWI321567B - Antifreeze protein analogue and nucleic acid coding sequence thereof, and production and use of the same - Google Patents

Description

1321567 Patent No. 095118228 Revision of Amendment Page of Patent Application No.: October 1, 1998. Illustrated: Technical Field of C Invention Y Field of the Invention The present invention relates to the reduction of water; the freezing point 5 novel antifreeze peptide analogues, which can be used in industries such as food, pharmaceutical, biotechnology and genetic engineering, such as 'as water containing various types of water to be refrigerated or frozen Resistance to water-containing products (including pharmaceuticals, food, animal and plant cells, microorganisms, blood, etc.); anti-freezing agent or 10 cryoprotectant. The invention also relates to a nucleic acid coding sequence of the antifreeze peptide analog, and the use of recombinant DNA technology to produce a recombinant anti-beam protein analog. C Previous 3 Background 15 Antifreeze proteins (AFPs) are proteins that are unique to certain organisms that live in polar or cold regions. Such proteins present in such organisms have the effect of reducing the freezing point of body fluids of such organisms, thereby rendering such organisms free from death by freezing. Current research shows that in addition to fish in the polar waters, some organisms that survive in the frigid zone, such as plants (M a/.

Plant Physiol., 100:593-596; M. Urrutia et al. (1992),

Biochim. Biophys., Acta. 1121:199-206, ^^ (Duman. JG and Olsen, TM (1993), Cryobiology, 30: 322-328) Gra good gas (LA Graham, et al. (1997) , Nature, 388: 727-728), in the latter also 5 1321567 No. 095118228 Patent Application Manual Amendment Page Correction Period: October 1st, 1998 contains antifreeze protein. However, the amino acid composition and structure of the antifreeze proteins possessed by these organisms are very different. In today's research on antifreeze proteins, the antifreeze proteins of fish have been studied more clearly (Fee«少, 兄五.yeA, Z, di/v. 5 Chem. (1978), 32:191-282, (Technology Technol. (1993), 47: 82-90, and Trends in Food Sci. and Technol. (1998), 9:102-106; AL DeVries, Annu. Rev. Physiol. (1983), 45:245-260 Davies, PL and Hew, CL·, FASEB J. (1990), 4: 2460-2468). Antifreeze egg yolk can be divided into two categories according to the presence or absence of glycosyl group: one is 10 with glycosylation; the antifreeze glycoproteins (AFGPs), and the other is glycosyl-free; Antifreeze proteins (AFPs). In addition, antifreeze proteins can be classified into type I (type I), type II (type II), type III (type III), and type IV (type IV) according to the difference in amino acid composition and structure. ) and so on. 15 Type I antifreeze proteins are alanine-riched a-helical polypeptides found in right-eye flounders (P/ewrowechi/ae) With the sculpins. Type II anti-globin is a cyste-rich protein of 11 to 24 kDa, which was found in the sea 20 raven 'smeit' and squid ( Herring). Nuclear Magnetic Resonance (NMR) structural measurements showed that Type II anti-Kang protein is a yS-structured protein with some disulfide bridges. Type III anti-image proteins are 7 kDa and 14 kDa globular proteins, which were found in cotton [fish carp] 6 1321567 No. 095118228 Patent Application Revision Revision Date: October 1, 1998 Zoarcoid fish, including eelpout and wolffish. Type IV antifreeze protein was found in longhorn sculpin (see G. Deng et al. (1997), FEBS Letters, 402: 17-20), 矣 12.3k 〇 2i 5 and was predicted to have A helix bundle structure. Although the above-mentioned various types of antifreeze proteins are structurally distinct, they have a common ability to inhibit ice crystal growth by binding to an ice surface. Table 1 below lists the type I anti-beam proteins reported to date, which have a molecular weight of about 3 to 10 kDa, which is characterized by alanine-rich (alanine content > 60%) and in the primary structure ( The primary structure mainly contains a 11-residue repeating unit. In particular, the amino acid sequence of the repeating unit of the type I antifreeze protein of Winter flounder is "Thr-X2-Asx-X7", wherein X is more than 15 is alanine or the other is beneficial. The α-helix forms an amino acid, and Asx represents asparagine (Asn) or aspartic acid (Asp). 7 1321567 Revision No. 095118228 Patent Application Revision Date: October 1st, 1st step «^^^-emperature of thermal hysteresis); ^s plus *--3 Deduction Shen 0 Per* protection 0 1 mg /mL^薄3筚3渰*萆"谇: its-^tss^atM^*^^柯务,-ijuoit^s-^Νδ^2•Patch+ 扣蜗6?* 阵护7 mg/mL Infertile*|Sheep S splashing sea berry 谇·· 4»"Ice 3 worm 卟1?3 屮±^^妒9.7kDa· '"Preface^奇53*^3"谇抹谇硌冲和蜗卧洚谇洚谇洚谇10 39/彐|_+5渔^3 x=>^Bss{asparagine)'58ssuthreonine)iaaaB{-anine): {Longhorn sculpin) detail>-m.#. 舛令 &quot ;•ta.·* sss+> (Dusky Sn3il'-sh) Α»^*·^.·* {Atlantic wna two fiwh) -*^5ζ.Α -*^50 (Arctic sculpin> 逭^谇茁片.·* ^^^^50 (GrubCTy sculpin) 茚尨佑妗'*, 茚syou 5C .·* {Shorthorn sculpin) 3^5Λ0δΑ {Aimskmn plaice) {Yellowtail flounder) 详爷-B.·* ^ -^-0^ Detailed order tta'*, (winterflouncjer) $

IssAFP

SssAFP2

WfsAFP3 IAFP2

Lg.AFP2

La-AFP AS3 AS1 GS8

Gs sw8 SS3

AP

YTAFP 5a-滓申证珀{5'm'-ike protein) AFP9 HPLC8 HPLC6 morphine*/ itchitch* 泠;12! 7.* 0.30 {skin-type) $ $ 玮5! (liver-type) 3.5 3·4 3.4 9,6 9.3 2.9 3.4 3.4 3.0 4.0 2.9 •5 4.0 16·7 3 3.3 3.2 Cloud tkpa) 81 CM· 83 79 73 s 00°1 Code 1 (./0) P15 0.1°1 0.6® 0.67

PS 0.56 0.60 0.68 SDAPAKAAAK TAADAKAAAAK TAADALAAANK ΤΑΑΑΑΚΑΑΑΚ5-3διί " 19 > MAAAAKAAEAAAMAAANAAEAAATKAADAAASAAAAAIAAIAEAAEAAEAAATKSANVAAAAAATSAAAAAKATA NAAAAASAAAAAAAAVA {present study Su enamel hull cen: 1s) _ SDAPAKAAAATAAAAKAAAEATAAAAAKAAADTKAKAAR {4 * 238143-- MDAPAKAAAA TAAAAKAAAEA TAAAAAKAAAA TKAGAAR5238 waves%: 16) SDAPA ^ AAAA TAAAAKAAAmA TKAAAAKAAAA TKAAAH {4 -35 * 51: 1- MAAATPAQ ^ AAATATAAAAAAASAAAAAAATTASTAAKVSAGAAATAAAAVVAAKNAATAVAPNTG AITAATAASATAAAAAKAAQATADAAATKAAAAAVTSKAAAALPf ^ l (^ '- ^' - race Optional '-1-_ MAAATPAQRAAATATAAAAAAASAAAAAAATTASTAAK < SAGAAATAAAA < VAAKNAATA < APNTG AITAATAASATAAAAAKAAQATADAAATKAAAAAVTSKAAAAALAAl (fist Chicago | 3'-race ^: 13) _ MDAPARAAAK. TAADALAAANK TMDAAAAAAAA (本-迮珐*5%:-) SDGETPAGifRIAAAAAlAAK TAADAAAKAAAIAAAAA523珐麻"11) SDGETPAQKAA RLAAAAAALAAK TAADAAAKAAA1AAAAA c^-355^-3 SDAPA1AAAK TAADALAAAKK TAADAAAAAAKP5-38*"3 MNGETPAQKAA^LAAAAALAAK TAADAAAKAAAKAAAIAAAAASA (Stained 355" 2 MNAPARAAAK TAADALAAAKK TAADAAAAAAA (^-3s6s:7) D TASDAAAAAAA TA AAAKAAAEK ΤΑ knife DAAAAAAA TAAAA335-3ssst6) D TASDAAAAAAATAAAAAKAAAD TAAAAAKAAAD TAAAAAEAAAA TAR {heap 23853:- X a D PAAFIA AAA Ai * {本-35*5^:- D TASDAAAAAAA TAATAAAAAAA TAVTAAKAAAL TAANAAAAAAA TAAAA^G AA^G {^2asss:3 D TASDAAAAAAL TAANAKAAAKL TADNAAAAAAATAX {^-3^^51:2) D TASDAAAAAAL TAANAKAAAEL TAANAAAAAAA TAf^/ffJ^^^itlKSEQ ID NO:1) 8 1321567 Patent Application No. 095118228 Revision Date Amendment Date: October 1998 1. The results shown in Table 1 above are extracted by reference to the following literature and the database of the NCBI website: 1. RP Evans and GL Fletcher, Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular 5 Enzymology , 11 June 2001, 1547 (2): 235-244; 2. Information on 5a-like proteins is a reference; 8 Marshall et al. (2005), "/. and · 〇 / CTzew., May 6, 2005, 280 (18): 17920-17929; 3. For the amino acid sequence of La-AFP, see NCBI Accession No. 10 AAR22528 (NCBI accession number: AAR22528); 4. About Lg-AFP2 Amino acid sequence See NCBI Accession No. AAR22529; and 5. For information on wfsAFP3 and Longhorn sculpin, refer to WK Low ei α/. (2001),··.〇/. CT^m·,276 (15 ): 15 11582-11589. Previous studies have shown that threonine (Thr) residues in type I antifreeze proteins play an important role in the binding of this type I antifreeze protein to ice crystals (JCC·Chou (1992),丄Mol. Biol., 223:509-517·, Margaret M. Harding et al. (1999), Eur. J. Biochem., 264, 20 Chou Proposal: Thr Residue on the same side of the Type I antifreeze protein The hydroxyl group on the base will bind to the oxygen atom in the [0112] direction of the ice lattice, thereby inhibiting or delaying the growth of the ice crystal. Since the type I antifreeze protein combines with the ice crystal to form a hydrogen bond, the type is similar to a zipper. The binding mode is called a "zipper-like model," (foot _C. C/zom, (7PP2), the same as above.) This 9 1321567 patent application specification revision page is amended: On October 1st, 1998, H. Chao et al. found that the greater the number of repeating amino acid units in type I antifreeze proteins, the greater the ability of the type I antifreeze protein to lower the freezing point of water. (H. Chao et al. (1996), Protein Science, 5: 1150-1156). Antifreeze proteins slow down the formation of ice crystals, and use this The application of anti-frozen protein is extremely wide. For example, in the preservation of tissue culture or cell strain, the addition of antifreeze protein can improve the survival rate after freezing, reduce the damage of the organelle, and make the membrane permeability. Preservation is good (and α/ (1991), Biochem. Biophys. Res. Comm·, 180:566-571, 氙, epiprotein is also available for egg and sperm preservation, and by surgery 10 by ice crystal The "knife type" replaces the role of the laser knife (//ew, CZ. am/汾 "Private DSC (1992), Journal of Biochemistry, 203: 33-42, also, in the food industry, Adding antifreeze protein to the manufacture of ice cream can make the palatability and quality of the ice cream better (F from No. 5, Yeh, Y. (1998), Trends in Food Sci. and Technol, 15 The addition of antifreeze protein can improve the quality of frozen meat (S.R_ Payne et a丨(J994), Meat Science, 37·42Ρ-43§). In addition, many products need to be refrigerated and sold. The use of anti-/east protein can avoid the quality of these products during the transportation process due to temperature changes. Getting worse. 20 At present, the antifreeze protein is obtained by separating and purifying the antifreeze protein from the fish body. This must be compatible with the fishery and the source is unstable. The price depends on the purity of the protein. The price per gram can reach about 5 US dollars. The price is not cheap, and even if it is using existing synthetic technology, it can even reach 1 times the price (httP://www.preparedf〇〇ds_C〇m). Fletche^ Person Evaluation: Nature 10 1321567 Revision Date: October 1998 I曰 No. 095118228 Patent Application Specification Amendment Page The fish blood antifreeze protein is about 2g/L plasma, therefore, 1 metric tons The fish body (based on plasma 〇·5% body weight) can only obtain 抗Kg antifreeze protein. The estimated amount of antifreeze protein used for tumor resection is 5~1〇mg/mN. Calculate the annual requirement of antifreeze protein. It is necessary to have 5〇〇~1〇〇〇 metric tons of fish to fully supply the fish. need. As for the ice cream industry, the amount of antifreeze protein used in ice cream manufacturing is estimated to be 0.1 mg/L, and the annual demand for antifreeze protein is calculated accordingly, and 150,000 metric tons of fish is required to fully supply the required amount. In addition, related applications of antifreeze proteins are still under development. Obviously, fish-free antifreeze proteins from natural sources are not sufficient for future needs (GZ to 10 al. (1999), Chemtech 30: 17-28. Another way to supply antifreeze proteins is to use peptide synthesizers. (peptide synthesizer) to synthesize, but the cost of this method is too high. Therefore, the most ideal way to supply antifreeze protein should be genetic engineering technology, which saves time, saves money and has no seasonal and geographical restrictions. Regarding genetic engineering research on antifreeze proteins, there are many transgenic animals, for example, fish that have been successfully genetically transferred, and their cold tolerance is improved, for example, goldfish (goldfish) (Brothers et al. (1995), Mol. Mar. Biol. Biotechnol., Mar. 1995, Cui, Salmon (CHew ei a/· (79 5 5J, 〇 / 20 Fish Biology. 47:1) -19, special. JM Logsdon, et al. (1997), Proc. ΑΓαί/. jcac?· Further explore the molecular evolution of antifreeze protein genes. However, in the genetic engineering of anti-bed protein, there are also Related reports on genetically transgenic plants, see, for example, from Holmberg Et al., Gene, September 5, 2001, 275(1): 115-24. 11 i 1321567 Patent Revision No. 095118228 Revision Date: January 1st, 1998, wants to mass produce antifreeze proteins, It can be considered to be produced by cuhured cells or genetically engineered microorganisms. With regard to cultured cells, it is difficult to establish cell cultures from various parts of the fish body, and various antibiotics 5 The regulation of frozen protein expression in eukaryotic cells is quite complex, and stimulations such as hormones are often needed to increase yield. In addition, the protein processing mechanism of antifreeze proteins is complex and requires Multi-organelles are processed and transported through the cell membrane and yield is not high. 10 For genetically engineered microorganisms, ID Peters et al. constructed a semi-synthetic winter flounder resistance; semiSynthetic winter flounder antifreeze proptein , pro-AFP) coding region, and inserting the coding region into an iacZ representation Inside, then formed recombinant expression vector Transformation to E. coli cells. The thus obtained transformed E. coli cells (transformed cells of quinolol) produce a fusion protein with a pro-AFP and a -gal domain (-gal domain). That is, cold-gal-pro-AFP (/·Ζλ Peier·?, ei α/· (1989), Protein Engineering, 3: 145-15Γ). H丄.Jing et al. constructed a expression vector with a chimeric AFP/CAT (or 20 pr〇AFP/CAT) cDNA in which the AFP (or proAFP) cDNA was structurally fused to the 5'-end of the CAT reporter gene. (was fused, in frame, to the 5'-end of the CAT reporter gene), and the E. coli was transformed with the expression vector. However, the transduced Escherichia coli thus obtained was measured only in the form of a fusion form of AFP (or proAFP). Table 13 1321567 Patent Application Publication No. 095118228 Revision Date: October 1998 1 low level expression 'and the resulting fusion protein is located in the bacteria, not easy to recover and purify (/ / force ·% with α / ·), 57π ·

Yan Sheng Wu Xue Βαο· 1995 Μακ,28(1):77-83). RG Solomon and R. Appels used an inducible 77 polymerase transcription expression system to successfully display a recombinant variant of the i-type antifreeze protein in E. coli ( Recombinant variant), rAFP. The recombinant protein is in the form of inclusion bodies and accumulates in the bacteria to a high level, and a simple and rapid purification protocol is designed for the recombinant type I to freeze. Purification of protein »So/omo« irnd Λ. 々τρΑ (7PP9", iVoiezw Εχρκ Purif·, 16:53-62. Also attempts to transform the antifreeze protein encoding gene into yeast cells. R. Mckown and GJ Warren found that a 15-chimeric antifreeze protein in yeast cells doubled the survival rate of yeast cells after rapid freezing (R. Mckown and GJ Warren, (J99J), Cryobio! 28: 474-482) In addition, Dr. Leondonk et al. used the 37 amino acids of East Protein 20 AFP6 isolated from the polar fish (Piewdop/ewrowec/M·? americrawers) The template is used, and the coding gene of the type I anti-combin protein is chemically synthesized using the preferred codons of the yeast, and the coding gene is implanted into an episomal expression vector and a plurality of Repeat this Inserted into the plastid (multicopy integration vector) and induced by the inducible promoter GA67 13 1321567 Patent No. 095118228 Patent Description Amendment Page Revision Period: October 1 曰 (induce) performance. In the obtained transf〇rmant, the expression level can reach about 100 mg/L. However, since the proteolytic processing of the performance product is incomplete, the obtained performance product is a chimeric protein or AFP oligomers (7M W α/., 5 Yeast, July, 1995, II (9): 849-864). Synthetic anti-East peptides are disclosed in US 5,925,540 and US 5,932,697 to Thomas Caceci et al. It has the amino acid sequence shown in Figure 4 attached to the specification of the two U.S. patents, or the specific repeating unit having π amino acid residues shown in Figure 4. Two of the 10 U.S. patents also disclose synthetic genes for use in bacteria to express the antifreeze peptide. However, from the examples provided in the specification of the two U.S. patents, Thomas Caceci et al. Synthetic antifreeze peptide and coding base Because there is still room for improvement. The isolated sculpin-type intracellular AFPs derived from sh〇rthorn sculpin and their corresponding are disclosed in US 6,429,293 to Choy L. Hew. Nucleic acid. Although the above-mentioned literature reports and patents are not stable and expensive in the supply of fish blood antifreeze proteins from natural sources today, and there are insufficient problems to supply the future. The art still needs to be developed. It can be used to mass produce new technologies for antifreeze proteins with excellent antifreeze activity. To this end, genetic engineering technology is considered an ideal tool because it saves time, money, and no seasonal or geographical constraints. C SUMMARY OF THE INVENTION 3 SUMMARY OF THE INVENTION 14 1321567 Patent Application Publication No. 095118228 Revision Date: October 1998 1曰I * In the present invention, the applicant designs the antifreeze effect using the existing knowledge and technology in the field of biotechnology. Novel antifreeze protein analogues that can be compared to natural antifreeze proteins, which can be produced in high yield and are easy to purify, while having high pH stability. And high thermal stability. The antifreeze protein analog of the present invention comprises an amino acid sequence represented by the formula (I): YP-[AR] aWZ (1) wherein: 10 a is an integer from 1 to 20; in each -[AR]- The R inside means independently a direct bond or a spacer sequence containing 3 to 15 amino acid residues. A in each -[AR]- independently represents a meeting. Forming an α-helical structure of 15-Qm-, wherein: m is an integer from 3 to 20; Q is an amino acid repeating unit comprising 11 amino acid residues and represented by formula (II): Xl-X2-X3-X4-X5-X6-X7-X8-X9-Xl0_Xn_ (II) 20 where: Χι in each Q is a threonine (Thronine, Thr), or at least one Q within Q Can be replaced by valine (Val); X4 in each Q is an amino acid residue selected from asparagine (Asn) and aspartic acid (Asp) 15 1321567 Patent Publication No. 095118228, Revision of Amendment Page: October 1st, 1998, or X4 with at least one Q can be replaced with an amino acid selected from the following group Threonine, valine and alanine; having at least one quinone in X and X1() is a group of amino acid residues which form a salt bridge in the amino acid sequence to stabilize the 〇:-helical structure. 5 One of the middle 6 and Χίο is one selected from the group consisting of lysine (IySine,

Lys) with amino acid residues of arginine (Arg), and the other is glutamic acid (Glu); X丨丨 in each Q is a leucine, Leu ), or having at least one Q in the " can be replaced with an amino acid selected from the group consisting of alanine and 10 aspartic acid; and the remaining amino acid residues in each Q are alanine, The alanine provides the resistance; the van der Waals force required for the combination of the eastern protein analog with the ice crystals, or the X3 of at least one Q can be replaced by a Amino acids in the following 15 groups: serine (Ser), proline, aspartic acid, and arginine; Y is absent or represents a protein marker (pr〇 Tein tag); P represents one or several amino acid residues, wherein the amino acid residue directly adjacent to _[A_R]a_ is an acidic amino acid selected from aspartic acid and glutamic acid 20 (acidic amino acid); W represents one or several amino acid residues, wherein at least one amino acid residue is selected from the group consisting of arginine, histidine (His) and Acidic basic amino acid; and Z is absent or represents a protein standard; 16 1321567 Patent Application Publication No. 095118228 Revision Date: October 1st, 1998, but conditional The antifreeze protein analog is not a protein having an amino acid sequence as shown in SEQ ID NO: 1. In a preferred embodiment of the invention, the antifreeze protein analog comprises an amino acid sequence represented by the formula: 5 YPA-[RA]nWZ (in) wherein: η is 0 or one An integer from 1 to 15; R' is, at each occurrence, independently a spacer sequence comprising from 3 to 15 amino acid residues; 10 Α each occurrence is independently an alpha-helical structure -Q m -, wherein: m is an integer from 3 to 20; Q is an amino acid repeating unit comprising 11 amino acid residues and represented by formula (II): 15 -Xi_X2_X3_X4_X5_X6_X7_X8_X9_Xi_Xi_Xi1- (II) wherein: X in each Q is a sulphonic acid, or X at least one Q can be replaced with valine; X4 in each Q is selected from aspartame The amino acid residue of the acid and aspartic acid 20, or X4 in at least one Q, may be replaced by an amino acid selected from the group consisting of sulphonic acid, proline and alanine; X6 and X10 in at least one Q are a group of amino acid residues which form a salt bridge in the amino acid sequence to stabilize the α-helical structure, 17 1321567 095H8228 Patent Application Specification Revision w Revision Date: October 1998! One of X6 and XlO g in the day is an amino acid residue selected from the group consisting of lysine and arginine, and the other is glutamic acid; Χιι in each Q is a leucine, or Xn in at least one q may be replaced with an amine 5-based acid selected from the group consisting of alanine and aspartic acid; and the remaining amino acid residues in each Q are alanine, the alanine providing the The vanguard force required for the combination of the antifreeze protein analog with the ice crystal, or the at least one Q in the Q can be replaced by an amino acid selected from the group consisting of: serine, Resveric acid, aspartic acid, and arginine; Y is absent or represents a protein marker; p represents one or several amino acid residues, which are directly adjacent to the amine of _A_[R_A]n_ The acid residue is an acidic amino acid selected from the group consisting of aspartic acid and glutamic acid; W represents one or several amino acid residues, wherein at least one of the amino acid residues 15 is selected from a basic amino acid of arginine, histidine and lysine; and Z is absent or represents a protein weave; but conditionally the antifreeze The white analog is not a protein having an amino acid sequence as shown in Sequence Identification No.: 1 (SEQ ID NO: 1). In order to achieve the goal of stably providing a large amount of inexpensive antifreeze protein, the applicant further derives and prepares a selected host cell based on the amino acid sequence of the antifreeze protein analog. A corresponding nucleic acid coding sequence of a preferred codons, followed by preparation of a recombinant expression vector carrying the nucleic acid coding sequence (recombinant 18 1321567 095118228 patent application revision page ^正曰Period: October 1 曰 expression vector), and the resulting recombinant expression vector is transformed into the selected host cell, and the nucleic acid coding sequence is expressed in the transformed host cell to cause desired Generation of antifreeze protein analogs. The practice of the present invention is not intended to be limited to the use of specific host cells, but can be applied to a variety of prokaryotic and eukaryotic cells, including bacteria, yeast ( Yeasts, fungi, plant and animal cells, etc. and can be used to generate various forms of proteins, including proteins located in the cytoplasmic (four) (four) or periplasmic space, on the membrane 10 or Extracellular protein. In a preferred embodiment, the transformed microbial host cell obtained by the present invention secretes the expressed antifreeze protein analog to the outside of the cell, which is not reported otherwise. The above and other objects, features, and advantages of the present invention will be apparent from the description of the accompanying claims appended claims 1 shows an amino acid sequence of an antifreeze protein analog 1 (SEQ ID NO: 20) and an antifreeze protein analog Γ (SEQ ID NO: 21) designed according to the present invention, wherein an antifreeze protein analog 1 , X1 = Gly, 2〇X2 = Ser ; and in terms of resistance; East protein analog 1 ''Xj = Asp, X2 = Pro; Figure 2 schematically illustrates the use of overlap extension polymerase chain reaction method ( Overlapping-extension polymerase chain reaction, OEPCR) s use of four primers that are complementary between their sequences. 19 1321567 Revision No. 095118228 Patent Description Amendment Page Revision: October 1st, 1998 Generating a PCR product encoding the antifreeze protein analog 1 shown in Figure 1, wherein the label 2 and the label 3 respectively represent a template primer (t:empiat;eprimer), the two template primers at their 3, end Complement each other; the number 1 indicates An amplification primer or a 5 forward primer for the template primer of the amplification label 2; and 4 indicates an amplification primer or a forward primer for amplifying the template primer of the label 3. When the PCR reaction begins, the template primer will first bind and extend (3, to 5, the DNA as a template and extend from 5, to 3'), thus generating a full-length DNA. After that, the amplification primer starts to mass-amplify the DNA fragment with the full-length DNA as a template; 10 Figure 3 shows the construction process of the plastid vector pRP, wherein: orM7, E. coli Ab co/ί) plaque of the plastid pACYC177 Starting point; ori-pAM α 1 'D. faecalis (iSVrepiococcM·? /aeca/h) DS-5 plastid ρΑΜ α 1 replication initiation point; Apr, ampicillin resistant gene; Tcr , tetracycline 15 resistant gene; P, promoter; SD, SD sequence (Shine-Dalgarno sequence); pre, the DNA coding sequence of the message (signal peptide); pro, pre-sequence (prosequence) DNA coding sequence, which is involved in the folding and secretion of proteases; mature, DNA coding sequence of mature protease; MCS, multiple selection sites (multiple Cloning site); α/eC, a downstream DNA sequence, which may contain a sequence of transcription terminators; Figure 4 shows the use of primers in Table 2 and the use of overlap extension polymerase chain reaction (overlapping-extensio) n polymerase chain reaction, 20 1321567 No. 095118228 Patent Application Specification Revision Page Revision Date: October 2015 曰 OEPCR) Agarose gel electrophoresis results of PCR products obtained, wherein Μ: 100 bp DNA molecular marker ; Path 1: PCR products obtained by OEPC using primers AFP·1/1/, AFP-SS, AFP1 and AFP2; and diameter 2: OEPCR using primers AFP-valence wffl, AFP-SS, AFP1 and AFP2 5 obtained PCR product; Figure 5 shows the construction process of a plastid vector PRP-AFP; Figure 6 shows the construction process of a plastid vector PQE30-AFP, wherein: /acO, /ac promoter; ori-pBR322, pBR322 The origin of replication; the pQE30 terminator, which contains the Lambda transcriptional termination region and the rmS T1 transcriptional termination region; Figure 7 shows the plastid vector pET12a -AsAFP construction process, wherein the message peptide is the message peptide of E. coli outer membrane protease 〇 mpT; 15 Figure 8 shows the construction process of the plastid vector pET12a-OsAFP; Figure 9 shows the colon Tricine-SDS-PAGE electrophoresis results of recombinant antifreeze protein analogs produced by the transformed strains (f. co" transformants), in which the diameter Μ: low molecular weight protein marker; diameter 1: from E. coli M15 ( Protein purified from cytoplasm 2〇 fractionation of pREP4, pQE30-AFP); diameter 2: protein purified from the medium fraction of E. coli BL21 (DE3) (pET12a-AsAFP); Protein purified from the intracellular soluble fraction of E. coli BL21 (DE3) (pET 12a-AsAFP); PATH 4: Amendment from the amendment page of the E. coli 21 1321567 Patent No. 095118228 patent application: Protein purified from the intracellular insoluble fraction of BL21(DE3)(pET12a-AsAFP) in October 1998; Path 5: intracellular from Escherichia coli BL21(DE3)(pET12a-OsAFP) a protein partially purified; and a protein purified from the intracellular insoluble portion of Escherichia coli BL21 (DE3) (pET12a-5 OsAFP); and Figure 10 shows a recombinant antifreeze protein according to the present invention. The effect of reducing ice crystal formation, where area A (panel A): water; area B (panel B): lysis buffer containing 8 Μ urea (urea); area C (panel C) : Protein purified from the cytoplasmic portion of Escherichia coli M15 (pREP4, pQE30-AFP); D region (panel D): a protein partially purified from the medium of Escherichia coli BL21 (DE3) (pET12a-AsAFP); Panel (panel E): a protein purified from the intracellular soluble fraction of E. coli BL21 (DE3) (pET12a-AsAFP); F region (panel F): from Escherichia coli BL21 (DE3) (pET12a-AsAFP) 15 insoluble protein in the insoluble fraction of the cells; G-zone (panel G): a protein purified from the intracellular soluble fraction of E. coli BL21 (DE3) (pET12a-OsAFP); and n-region (panei Η): from E. coli 81^21 (0£3) (卩 卩 丁 123-〇3 into? ? Figure 7 is a schematic diagram showing three genes for the expression vectors pSP1Atag, pSP2Atag and pSP3Atag of the anti-beam protein 20 (AFP) analogs Α, 2Α and 3Α according to the present invention. Composition, wherein: SD, SD sequence; $, message peptide encoding DNA; alpha force, anti-; East protein gene; and, (His) 6 DNA; Figure 12 schematically illustrates the use of polymerase chain reaction (PCR) Using the primers described in Table 5 of Example 7 below, and the amendments to the specification of the patent application No. 22 1321567, No. 095,118,228, date of revision: October, October 1曰pET-12a-AsAFP or p-RP- The AFPI gene in AFP is used as a template to generate a PCR product encoding three antifreeze protein analogs 2, 2A and 3A having different numbers of repeating amino acid units according to the present invention, wherein: π, the message peptide encodes DNA; <3 force 7, anti-; East protein gene; and, (His) 6 5 DNA; Figure 13 shows Escherichia coli BL21 (DE3) (pSP1Atag), Escherichia coli BL21 (DE3) (pSP2Atag) and Escherichia coli BL21 (DE3) (pSP3Atag) induced by 1 mM IPTG for 3 hours The results of Tricine-SDS 10 PAGE analysis of the nutrient fraction and the protein collected by total cell lysate; Figure 14 shows Escherichia coli BL21(DE3)(pSPlAtag), Escherichia coli BL21(DE3)(pSP2Atag) and Escherichia coli BL21 (DE3) (pSP3Atag) was induced with 1 mM IPTG for 3 hours, and the results were analyzed by Tricine-SDS PAGE of recombinant antifreeze protein analog ΙΑ, 2A and 3A purified from the medium fraction, wherein the diameter was 1: Recombinant antifreeze protein analog 1A from the medium portion of E. coli BL21 (DE3) (pSP1Atag); diametric 2: recombinant antifreeze protein analog 2A from the medium portion of E. coli BL21 (DE3) (pSP2Atag); Path 3: Recombinant antifreeze protein analog 3A from the medium portion of Escherichia coli BL21(DE3) (pSP3Atag); 20 Figure 15 shows the expression vectors pSP1Atag, pSP2Atag with antifreeze protein (AFP) analog ΙΑ, 2A and 3A Cell growth of Escherichia coli BL21 (DE3) transformed with pSP3Atag and secretion production of recombinant antifreeze protein analogs 1A, 2A and 3A; Figure 16 shows recombinant antifreeze protein analogues 1 a, 2A and 3A inhibit ice crystals 23 1321567第09 Revision No. 5118228 Patent Application Revision Date: The analysis of the ability of recrystallization in October 1998, in which the concentrations of recombinant antifreeze protein analogues 1A, 2A and 3A were both 400 pg/pL; C was the control group. (PBS buffer, 50 mM NaH2P04, 300 mM NaCl, pH 8.0); and CAFPI is a commercially available type I antifreeze protein (A/F protein Canada Inc.), which is from the cold ocean fish (cold ocean fish) The purity of the blood is between 90 and 99%; Figure 17 shows the results of a single ice crystal configuration of the recombinant antifreeze protein analogs 1A, 2A and 3A, wherein the recombinant anti-east protein analogue 1A, The concentrations of 2A and 3 A were both 400 pg/pL; C was the control group (PBS buffer, 50 mM 10 NaH2P04, 300 mM NaCl, pH 8.0); and CAFPI was a commercially available type I anti-beam protein (A/ F protein Canada Inc.), which is isolated from the blood of cold ocean fish, with a purity between 90 and 99%; Figure 18 shows the pH of recombinant antifreeze protein analogues ία, 2A and 3A Value 15 stability, in which the concentrations of recombinant antifreeze protein analogs 2, 2A and 3A are both 400 pg/p L; and C are the control group (PBS buffer, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0); Figure 19 shows the thermal stability of the recombinant antifreeze protein analogs 丨a, 2A and 3A, wherein the recombinant antifreeze protein is similar The concentrations of 1A, 2A, and 3A were both 400 20 叩/叫; and C was the control group (PB S buffer, 50 mM NaH 2 P 〇 4, 300 mM NaCl, pH 8.0); and Figure 20 shows recombinant resistance. The minimum inhibitory concentration (MIC) of frozen protein analogs 2, 2A and 3A. t: Embodiment; 1 24 1321567 Patent Application No. 095118228 Revision page Revision period: October 1998 1 Detailed description of the preferred embodiment (detailed description of the invention) Type I antibody; The blood or epidermis of right-eye flounders, corpses, sculpins, snailfish, etc., and the 5-amino acid sequence of the j-type antifreeze protein known today is listed. It is shown in Table 1 as described above. It is analyzed that the known Type I antifreeze proteins have the following structural and functional properties: the configuration is an α-helical structure, and the molecular weight is between 3 and 10 kDa. It is mainly composed of a repeating unit "Thr-X2-Asx-X7" having 11 amino acid residues, wherein X is mostly Ala ' and Asx is Asp or Asn, Thr residue in the repeating unit of amino acid Base 1 plays an important role in the binding of type I antifreeze proteins to ice crystals, and the greater the number of amino acid repeat units, the better the ability of type I antifreeze proteins to lower freezing points. Retaining the α-helical configuration of type I antifreeze proteins and repeating the amino acid The important function-related Thr residue in the element should be able to construct 15 protein analogs with antifreeze properties. In addition, increase the number of amino acid repeating units in the antifreeze protein' or stabilize the antifreeze protein α. The spiral configuration should achieve the purpose of improving the ability of antifreeze proteins to inhibit the growth of ice crystals. Therefore, the applicant developed a novel method based on the structural and functional properties of type I antifreeze protein. Antifreeze protein analog. 20 According to the technical idea of the present invention, the antifreeze protein analog is designed in the primary structure to be a repeating unit "Thr-Xz-Asx-X7" having 11 amino acid residues. The basis of the invention is to retain the essential amino acid sequence with antifreeze characteristics and to correct the n-terminus of the synthetic antifreeze protein analog and the specification of the patent application No. 095118228 Page correction period: 98 years and 1 month! The amino acid residue of the 疋 疋 螺 螺 螺 螺 螺 螺 螺 螺 螺 螺 螺 螺 螺 螺 螺 螺 螺 胺 胺 胺 胺 胺 胺 胺 胺 胺 胺 胺 胺 胺 胺 胺 胺 胺 胺The acid residue, and the number of repeating single 7L of the amino acid is increased', whereby the anti-mystery analog is more stable than the natural antifreeze protein and the anti-freezing effect is comparable or better. Thus, the present invention provides an anti-freezing effect. a frozen protein analog comprising an amino acid sequence represented by formula (1): YP-[AR]a_W-Z (1) wherein: a is an integer from 1 to 20; 10 is in each -[AR]- R independently represents a direct bond or a spacer sequence containing 3 to 15 amino acid residues; A in each -[AR]- independently represents a Forming a helical structure of -Qm-, wherein: 15 m is an integer from 3 to 20; Q is an amino acid repeating unit comprising 11 amino acid residues and represented by formula (11):

Xl-X2-X3-X4-X5~X6-X7-X8-X9~Xl〇-Xll- (II) where: X丨 in each Q is a threonine (Threonine, Thr), or at least one The Χι in Q can be replaced by valine (Val); X4 in each Q is selected from asparagine (Asn) and aspartic acid (Asp). The amino acid residue, or the X4 in at least one Q, may be replaced by an amino acid selected from the following group: 26 1321567 Patent No. 095,118,228, Patent Application Revision Date: October 1, 1998 : sulphate, valine and alanine; having at least one Q and 6 and 1 () are a group of amino acid residues which form a salt bridge in the amino acid sequence to stabilize the helical structure, wherein One of Χ6 and Χίο is an amino acid residue selected from the group consisting of lysine (5 Lys) and arginine (Arg), and the other is glutamic acid (Glu). Χι丨 in each Q is leucine (Leu)' or Xn in at least one Q can be replaced with an amino acid selected from alanine and aspartic acid; Within each Q The amino acid residue is alanine, which provides the vanderWaalsforce required for the binding of the anti-bedin analog to ice crystals, or the X3 of at least one Q can be Replaced with an amino acid selected from the group consisting of serine (Ser), imine 15 acid, aspartic acid, and arginine; Y is absent or represents a protein A protein tag; P represents one or several amino acid residues, wherein the amino acid residue directly adjacent to -[AR]a- is an acid selected from aspartic acid and glutamic acid. Acidic acid; 20 w represents one or several amino acid residues, wherein at least one of the amino acid residues is selected from the group consisting of arginine, histidine (His) and A basic acid of amino acid; and Z is absent or represents a protein standard; the condition of the protein is 疋; the east protein analogue is not one having the sequence identification 27 1321567 095118228 Amendment date of the patent application specification revision date: October 1st, 1998: Amino acid sequence shown in 1: Protein. Preferably, a is an integer of from 1 to 12, more preferably from 1 to 8, still more preferably from 1 to 4, in accordance with the present invention. In a preferred embodiment of the present invention, the antifreeze protein analog package 5 comprises an amino acid sequence represented by the formula (III): YPA-[RA]nWZ (in) wherein: η is 0 or a An integer from 1 to 15; R/ is independently a spacer sequence containing from 3 to 15 amino acid residues 10 at each occurrence; 独立 is independently formed at each occurrence to form &lt;2 - -Qm- of the helical structure, wherein: m is an integer from 3 to 20; Q is an amine 15 acid repeating unit comprising 11 amino acid residues and represented by formula (II): -Χΐ-Χ2_Χ3_Χ4 ·Χ5_Χ6_Χ7-Χ8_Χ9_Χΐ0_Χΐ1_ (II) where: X in each Q is a threonine, or there is at least one X in Q, which can be replaced by proline; 20 X4 in each Q is one selected from The amino acid residue of aspartic acid and aspartic acid, or X4 in at least one Q, may be replaced by an amino acid selected from the group consisting of sulphonic acid and proline. And alanine; having at least one of X6 and X10 in a Q is a group of amino acid sequence 28 1321567 Patent Application No. 095118228 Authentic date: 98 years 1 month] The formation of a salt bridge to stabilize the helical amino acid residue, wherein one of Χ6 and Χ1〇 is an amino acid residue selected from the group consisting of lysine and arginine. And the other is glutamic acid; Χιι in each Q is a leucine, or at least one quinone in Q can be replaced by an amino acid selected from alanine and aspartic acid And the remaining amino acid residues in each Q are alanine, which provide the van der Waals required for the binding of the anti-bedin analog to ice crystals, or at least one X of Q Replaced with 10 amino acids selected from the group consisting of: serine, valine, aspartic acid, and arginine; Y is absent or represents a protein standard; Represents one or several amino acid residues, wherein the amino acid residue directly adjacent to the amino acid residue is an acidic amino acid selected from the group consisting of aspartic acid and glutamic acid; 15 w represents one or several amino acids a residue wherein at least one amino acid residue is a basic amino acid selected from the group consisting of arginine, histidine, and lysine; And Z is absent, or represents a protein flag; however, that the conditions for the non-antifreeze protein analog having a sequence as Identification Number: amino acid sequence of the protein shown in Figure 1. According to the invention, preferably, η is an integer from 〇 to 11, more preferably from 〇 to 7 ′ and even more preferably from 〇 to 3. According to the present invention, the larger the value of m, the better the antifreeze activity of the antifreeze protein analog. Preferably, m is a number of turns of 4 to 15. More preferably, m is an integer from 4 to 12. In order to make the anti-; East-protein analog 〇; - Helical knot 29 1321567 No. 095118228 patent application revised page revised period: 98 years 1 month month, I曰 structure can maintain good, when m ^ 4, it is recommended The ruler or ruler should be present and preferably comprise from 5 to 7 amino acids, which may comprise proline. In a preferred embodiment of the invention, πι=4. According to the present invention, the "_Qm_," having an -propylamine 5 acid content of the antifreeze protein analog falls within the range of 5% to 85%, preferably 55% to 80%. More preferably, the antifreeze protein The "_Qnr" of the analog has a monoalanine content of greater than 60% 'more preferably greater than 65%. According to the invention, X6 in at least one Q in each -Qm- is an lysine (Lys), while χ10 It is glutamic acid (Glu). 10 According to the present invention, the amino acid residue which must be maintained as alanine in -Qm-, for example, m=4, may be the sixth, ninth of _Qm• 1〇, 14, 17, 21 ' 25 ' 28, 29, 32, 36, 39, 40, 43 and 44 amino acid residue positions, as they are roughly on the surface where antifreeze proteins bind to ice crystals The amino acid residue 15 group at positions 17 and 21 and positions 28 and 32 may be replaced with an amino acid group capable of forming a salt bridge to enhance the stability of the α_helical structure. There are at least one salt bridge in each _(^_, preferably two or more. When more than two salt bridges are present in each, they may be present adjacent or alternately in the amine The acid repeats in unit q. *° According to the present invention, the protein label at the Y or Z position available as the anti-Kang protein analog is preferably a protein-friendly purification and does not affect the resistance; Protein markers, and suitable examples include, but are not limited to: H1S-tag, GST-tag, Strep.tag, Arg_tag, FLAG-tag, c-myc tag, S-tag, HAT-tag, Camodulin_tag, cellulose binding </ RTI> </ RTI> </ RTI> </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Tag (maltose-binding tag), etc. In a preferred embodiment of the invention, the protein marker is a 5 His6-tag which allows the antifreeze protein analog to be used after being generated - Purified by afnnity c〇iUIIm chromatography of nickel chelating nitrilotriacetic acid (Ni-NTA). In a better example, one Antifreeze egg according to the invention The white analogue has a His6-tag at its purine position. 10 According to the present invention, preferably 1 to 15 of the anti-frozen protein analog has a P position of formula (I) or formula (III). The amino acid residue is more preferably 3 to 10 amino acid residues, still more preferably 5 to 7 amino acid residues. According to the present invention, preferably 1 to 15 amino acid residues, more preferably 3_1〇15 amino acid residues, are present at the W position of the antifreeze protein analog of formula (1) or formula (m). More preferably, the radical '5-7 amino acid residues. According to the present invention, the antifreeze protein analog preferably has 3-1 amino acid residues, more preferably 5-7, at the r position of the formula (1) or the R/position of the formula (III). Amino acid residues. According to the present invention, the antifreeze protein analog may have an aspartic acid (Asp) or glutamic acid residue at the p position of the formula (I), and may have a arginine residue at the w position. Base, hydrazine to enhance the stability of the helical structure of the antifreeze protein analog (cf. G. Merutka, et al. (1991), Biochem, 30: 4245-4248). According to the present invention, the applicant has designed Amino acid 31 1321567 shown in 1 Revision No. 095118228 Patent revision page Correction period: October 1 98 sequence of antifreeze protein analog 1 (where X1 == Gly, X2 = Ser) (sequence Identification number: 20) with antifreeze protein analog r (where Xl = Asp, X2 = Pro) (SEQ ID NO: 21), which have 4 amino acid repeating units at positions 20 and 31 amino acid Is an amino acid, at the 24th and 35th amino acid positions, 5 is glutamic acid' such that it forms between the 20th and 24th amino acids and between the 31st and 35th amino acids. Salt bridge, thus having the function of a stable "·helical structure. Again" has an aspartic acid (Asp) residue set in the first amine of the antifreeze protein analogues 1 and I The N-termininal upstream of the acid repeat unit and a arginine (Arg) residue are placed downstream of the C-terminus of the last amino acid repeat unit of the antifreeze protein 10 and 1 Here, it is advantageous to stabilize the α-helical structure. Furthermore, the antifreeze protein analogues 1 and 1 are designed to have a six-member histidine tag (C-terminial) at the C-terminial position. The antifreeze protein analogs 1 and r are allowed to be purified by affinity column chromatography using a Ni-NTA resin. 15 Alternatively, the antifreeze protein analog 1 and the first amine of the hydrazine can be used. A threonine (Glu) residue is placed upstream of the N-termininal of the repeating unit of the acid, which also contributes to the stable:-helical structure. As used herein, "polypeptide", "peptide" And the terms "protein" and the like may be used interchangeably and mean a polymeric 2 quinone consisting of amino acid residues, wherein one or more amino acid residues are naturally occurring amino acids (naturally Occurrence amino acids) or artificial chemical mimics. In this paper, The amino acid can be represented by its full name, a three-letter abbreviation, and a single letter symbol, as is well known in the art. In addition, the revision page of the patent application specification of No. 32 1321 567 In October 1st, the convention of the singularity is that the left end of the protein is the N-terminus (amine-based end) and the right end is the C-terminus (carboxyl end). In the antifreeze protein analog of the present invention, the C-terminus is usually a carboxylic acid (-COOH) or carboxylate (-COO-) group, but the C-terminus may also be a guanamine 5 (_CONH2) or a slow mutated ester ( a -COOR) group, wherein R can be: a Ck alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl and the like; a CS-8 cycloalkyl group such as a cyclopentyl group and a ring Hexyl and analogs; c6_12 aryl, such as phenyl, a-naphthyl and the like; and C7_i4 aralkyl, such as benzyl, phenethyl 10 (Phenethyl), α-naphthylmethyl and the like. When the antifreeze protein analog of the present invention has a monocarboxylic acid or a carboxylate group at a position other than the C terminal, a protein containing a slow group or a glycosylation is also included in the present invention. The ester groups herein may be those described above for the C terminal. 15 Optionally, the amine group of the amino acid residue at the N-terminus of the antifreeze protein analog of the present invention may be protected by a protecting group which may be a C丨·6醯Acyl group, a Cukanyl group such as foryl group, acetyl group or the like. The antifreeze protein analogs according to the present invention may be formed in the form of physiologically acceptable salts, including those having a mineral acid such as hydrogen chloride, hydrogen bromide, sulfuric acid, Salts of phosphoric acid; those salts with organic acids such as acetic acid 'maleic acid, tartaric acid, saprotic acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.; Those with an amino acid (such as arginine, aspartic acid, amylin, etc.) 33 1321567 Patent Application Publication No. 095118228 Revision Date: October 1st, 1998 Salts According to the invention Frozen protein analogs can be made by conventional chemical synthesis, for example, in a homogeneous solution or on a solid phase. In this regard, reference is made to, for example, 'C/jaAraZjctri/y 5 Hent (1991), Eur. 丄 Biochem., 202: 1057-1063. In order to be able to provide a large number of inexpensive antifreeze proteins more economically, it may be a good choice to use genetic engineering techniques to produce antifreeze protein analogs according to the present invention. Therefore, according to the known knowledge and technology of genetic engineering technology, when the amino acid 10 sequence of the antifreeze protein analog according to the present invention is extracted, it can be based on the genetic codon table (genetic codon table). , in particular, a preferred host cell preferred codon to generate a corresponding nucleic acid coding sequence for the anti-linguistic analog, and then the nucleic acid coding sequence is inserted into a suitable host cell and the nucleic acid is encoded The sequence is expressed in the host cell, followed by purification of the expressed antifreeze protein 15 analog. As used herein, the terms "nucleic acid" or "nucleic acid sequence" and the like mean a deoxyribonucleotide sequence or ribonucleotide sequence in the form of a single strand or a double strand and is known to have Naturally occuiring nucleotides or aritificial 20 chemical mimics. As used herein, "nucleic acid," the term can be used with "gene", "cDNA", "mRNA", "Oligonucleotide 4 acid" and "polynucleic acid" are used interchangeably. Unless otherwise indicated, 'a nucleic acid sequence other than the particular sequence shown herein' also encompasses its complementary sequence (c〇mplementary seqUences) to 34 1321567 Revision No. 095,118,228, the revised page of the specification of the patent application: October 1st, 1998 and conservative analogs, such as homologous sequences with degenerative codon substitutions (homologous) In particular, degenerate codon substitutions can be made, for example, by replacing the third position of one or more selected codons 5 in a nucleic acid sequence with other Nucleotide residues are produced. Techniques for manipulating nucleic acids, such as, for example, for generating mutations, subcloning, labelling, probing, and Sequencing, hybridization, etc., are described in detail in the scientific and patent literature. See, for example, Sambrook 10 J, Russell DW (2001) Molecular Cloning: a Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, New York; Current Protocols in Molecular Biology, Ausubel ed., John Wiley &amp; Sons, Inc., New York (1997); Laboratory Techniques in Biochemistry and Molecular Biology: 15 Hybridization With Nucleic Acid Probes, Part I, Theory and Nucleic Acid Preparation, Tijssen ed" Elsevier, NY (1993) o To obtain a nucleic acid coding sequence for an antifreeze protein analog according to the present invention, it can be produced by a conventional chemical synthesis method, for example, by synthesizing a partial sequence ( Partial sequences) and then connected. Thereafter, 20 can be amplified by a polymerase chain reaction (PCR).

In a preferred embodiment of the present invention, the applicant utilizes an overlap-extension polymerase chain reaction (OEPCR) to synthesize a DNA encoding the above-described antifreeze protein analog. 35 1321567 Patent Application No. 095118228 Manual revision page revision period: October 1st, 1998 sequence. OEPCR is a method for preparing a chimeric DNA sequence by a polymerization-free linkage reaction method, in which the specificity of a pCR primer and the control of an anneaiing temperature can be skillfully controlled. Two or more 5 or more DNA fragments originally separated are spliced into one chimeric DNA sequence. In addition, by using the specificity of the end of the PCR primer and the control of the binding temperature, it is also possible to introduce a DNA fragment to be added, such as a restriction enzyme cutting site, a ribosome binding site (RBS). , promoter sequences, and the like. 10 Further, the 'OEPCR was carried out by performing amplification of DNA using a conventional sputum DNA polymerase in combination with P/w DNA polymerase. DNA aggregation has a proof-reading function that reduces the chance of errors in DNA synthesis. This method has been utilized to synthesize a number of DNA coding sequences which are limited to synthetic lengths. In a preferred embodiment of the invention, the Applicant uses a preferred codon of Bacillus subtilis to generate a DNA fragment encoding the above-described anti-binding protein analog 1 and purine, using the primers shown in Table 2 below. It was synthesized by performing 0EPCR as shown in FIG. 2. Referring to Figure 2, four primers having complementarity between their sequences can be used to generate a PCR product encoding the antifreeze proteins 20 and I shown in Figure 1. Reference numerals 2 and 3 denote template primers, respectively, which are complementary to each other at their 3' ends. For example, reference numeral 2 may be the reference AFP1 (sequence identification number: 25), and reference numeral 3 may be the reference AFP2 (sequence identification number: 26). Reference numeral 1 denotes an amplification initiator or a forward modification of a template primer for amplifying the reference numeral 2. Amendment date: October 1, 1998, a forward primer. For example, the reference numeral 1 may be the primer AFP-BamHI (sequence identification number: 22) or the primer AFP-SphI (sequence identification number: 23) for amplifying the primer AFP1. Reference numeral 4 denotes an extension primer or a forward primer for amplifying the template primer of the numeral 3. For example, reference numeral 4 may be the primer AFP-SS (sequence identification number: 24) for amplifying the primer AFP25. These amplification primers can be designed to have restriction enzyme cutting sites. When the PCR reaction begins, the two-template primers (labels 2 and 3) will first stick and extend (using 3' to 5' DNA as a template and extending from 5' to 3), thus generating full-length DNA. Thereafter, the amplification primers (reference numerals 1 and 4) start to mass-amplify the DNA fragment with the full-length 10 DNA as a template. Table 2. Primer name for the synthesis of the AFPI gene. Nucleotide sequence (5^3·) AFP-BamHI BamHl 5'-caatcag£ai££gatacagcgtcagatgcggcagcg-3' (SEQ ID NO: 22) AFP-SphI Sphl 5 '-aatttag£ai^£aagatcctgatacagcgtcagatgcggca-3' (SEQ ID NO: 23) AFP-SS Sail Spel 5'-atttag This £g^£ta£itattagtggtggtggtggtggtgtcttgcg-3·(Serial Identification Number: 24) AFPI 5'-gatcctgatacagcgtcagatgcggcagcggctgcggctcttactgcagctactgcggctcttactgcagctaacgcaaaag Ccgcagctgaactgacggctgccaatgcgaaggcg-3·(SEQ ID NO: 25) AFP2 5'-ttagtggtggtggtggtggtgtcttgcggtggcagcggctgcagcagcagcgttagctgctg tgagctcggccgccgccttcgcattggcagccgt-3·(Sequence ID: 26) Note: The restriction enzyme cleavage site located in the primer is underlined or framed, where SWI and The cutting address has been re-examined. Thus, the present invention synthesizes PCR amplification products which each substantially correspond to the DNA sequence encoding the above-mentioned anti-frozen protein analog 1 and the plant, which have the sequence identification number: 27 and the sequence identification number: 28, respectively. The nucleic acid sequence. A nucleic acid coding sequence according to the invention can be carried in an expression cassette. "Performance" is a synthetically produced or recombinantly produced nucleic acid construct. The term "synthetically or recombinantly produced nucleic acid construct" is disclosed in the specification of the modified version of the specification of the patent application No. 095118228. , with a series of nucleic acid elements to allow transcription and translation of a particular nucleic acid carried in the target sputum within a target cell. The expression 依据 according to the invention comprises a promoter sequence operably linked upstream of the nucleic acid coding sequence. "Operably linked" means that a first sequence is placed sufficiently close to a second sequence ' such that the first sequence can affect the region controlled by the second sequence 10 or the second sequence. For example, a promoter sequence is operably linked to a gene sequence 'and is typically at position 5 of the gene sequence, The gene sequence is rendered under the control of the promoter sequence. In addition, a regulatory sequence can be operably linked to a a subsequence that enhances the ability of the promoter sequence to promote transcription. In this case, the regulatory sequence is often located at position 5 of the promoter sequence. The term "promoter sequence" refers to a DNA sequence. , which is usually located in front of a gene present in a DNA polymer, and provides a site for initiating transcription of the gene to generate mRNA (site f〇r initiati〇n of the transcription of said gene Into mRNA) The promoter sequence suitable for use in the present invention may only be adapted to initiate the expression of a nucleic acid coding sequence according to the invention in a selected host cell, which may be derived from any of the following: Virus, bacteriophage, bacterial cell, yeast cell, fungal cell, algae cell, plant cell, insect cell, animal cell, and human cell, and may be a constitutive promoter 38 1321567 Patent No. 095118228 Patent Application Revision Revision Date : October 1 曰 I * (constitutive promoter) or an inducible promoter (inducible promoter). The promoter can also be modified A synthetic promoter of any native promoter sequence. For example, when the host is an Escherichia bacterium, the promoters available for 5 include, but are not limited to: T5 promoter, T7 promoter, iac promoter, T7A1 Promoter, /ac promoter, irp promoter, ire promoter, recA promoter, lpp promoter, ara Fan 4Z) promoter and promoter; etc.; when the host is Bacillus (10)) bacteria, can be used Promoters include, but are not limited to, SPO1 promoter, SP02 promoter, penP 10 promoter, etc.; and when the host is a yeast, available promoters include, but are not limited to: PH05 promoter, PGK promoter, GAP Promoter and ADH promoter, etc. When the host is an insect cell, useful promoters include, but are not limited to, a polyhedrin promoter and a P10 promoter. When the 15 host is an animal cell, useful promoters include, but are not limited to, the SRa promoter, the SV40 early promoter, the RSV-promoter, the HIV LTR promoter, the HSV-TK promoter, the CMV promoter, and CMV- HSV thymidine kinase promoter and the like. When the host is a plant cell, a usable promoter includes, for example, a 35S 2〇 CaMV promoter, an actin promoter, a ubiquitin promoter, and the like. According to the invention, the promoter sequence is preferably selected from the group consisting of T5, T7, Pjpac, P/flC, Pw, Pw, Pveg, P..., Pw, Pw, Pflra, Pw and Pw And the promoters derived from them. 39 1321567. 904118228 Patent Application Revision Revision Date: October 1, 1998 According to the present invention, a representation of a nucleic acid coding sequence according to the present invention can be contained in a vector to form a recombinant Performance carrier. Vectors suitable for use in the present invention include vectors used in general genetic engineering techniques, such as: bacteriophages, such as sputum bacteria; plastids 5 such as plastids from coliforms (eg, pBR322, pBR325) , pUC12, pUC13, pQE-30, pET12, pET30), a shuttle vector from Bacillus subtilis (such as pUBllO, pTP5, pC194), Escherichia coli (five co/z·) and Bacillus subtilis (shuttle) Vector) pHY300PLK 'plasty from yeast (eg pSH19, pSH15); adhesion. 10 masos (cosmids); viruses (viruses), such as vaccinia viruses, retroviruses, or such as An animal virus of the baculovirus and the like. Vectors suitable for use in the present invention may contain other expression control elements, such as a transcription starting site, a transcription termination site, a ribosome binding site, An RNA splicing site, a polyadenylation site, and a translation termination site. Vectors suitable for use in the present invention may further comprise additional regulatory elements, such as transcription/translating enhancer sequences, enhancer sequences, and at least one suitable for sorting the vector under appropriate conditions. Marker gene or reporter gene. Marker genes useful in the present invention include, for example, a dihydrofolate reductase gene for use in eukaryotic cell cultures, and a G418 or neomycin resistance gene, and the specification of the patent application No. 40 1321567, No. 095118228 Amendment page correction period: October 1st, 1998, ampicillin, streptomycine, tetracycline or kanamycine resistance genes in E. coli and other bacterial cultures . Furthermore, a vector suitable for use in the present invention may further comprise a nucleic acid sequence encoding a secretion signal 5 located at the 5' end of the nucleic acid coding sequence according to the present invention such that a secretion message sequence is present in the expressed recombinant form. The N-terminal side of the protein. For example, when the host is a bacterium belonging to the genus Escherichia, the PhoA message sequence, the OmpT message sequence, and the 〇mpA message sequence can be used; when the host is a bacterium belonging to the genus Bacillus, the α-amylase message sequence and 10 Bacillus subtilis can be utilized. The protease (subtilicin) message sequence, etc.; when the host is a yeast, the MFa message sequence, the SUC2 message sequence, etc.; and when the host is an animal cell, the insulin message sequence, the a-interferon message sequence, the antibody molecule message can be utilized. Sequence, etc. The sequences described above are known to those skilled in the art. As used herein, the term "expression carrier" means any recombinant expression system that can be constructed in vitro (/« Wiro) or in vivo (ζ·« Wvo) in any host cell ( The nucleic acid coding sequence according to the invention is expressed constitutively or inducibly. The expression vector can be a linear or circular expression system and encompasses expression systems that maintain the free gene form or are integrated into the genome of the host cell. The expression system may or may not have the ability to self-replicate, it may only drive the transient performance of the host cell. As used herein, "cell", "host cell,", "transformed host cell" and "recombinant host cell" (reconibinant 41 1321567 patent application specification revision page amended period) : "98 October 1 host cell)" and the like can be used interchangeably, and not only refers to specific individual cells but also sub-cultured offsprings or potential progeny (potential) Offsprings). The progeny cells may undergo specific genetic modification in subsequent generations due to mutation or environmental influences, and the daughter cells may in fact not coincide with the mother cells, but the daughter cells are still covered. Within the scope of the term used herein. The host cell suitable for use in the present invention may be a prokaryotic cell or an eukaryotic cell and is a non-transformed/transfected cell or has been At least one other recombinant nucleic acid sequence transformed/transfected cells ° Prokaryotic cells suitable for use in the present invention include, but are not limited to, cells derived from bacteria such as E. coli (£. , Bacillus subtilis 15 (5 &lt; 3 ^ 7 / Lactobacillus species, genus genus (Strepiomyces 57?) and Salmonella typhi (Salmonella typhi), only green f (Cyanobacteria), so gas Bacteria, etc. Eukaryotic cells suitable for use in the present invention include, for example, fungal cells, protozoa cells, plant cells, insect cells, animal cells, and human cells. Representative examples of suitable fungal cells are yeast cells. For example, baker's yeast cereWs/ae) or methanolophilic yeast. Suitable plant cells are derived from gymnosperms or angiosperms, with a single Leaf plant 42 1321567 095118228 Patent application specification revision page Revision date: October 1 曰 (monocots) and dicots (dic〇ts) are preferred, especially crops (crops), and are taken from these plants Roots, stems, leaves or meristems are specialized in 'protoplasts or callus' forms. Suitable insect cell representatives are Drosophila 82 cells and derived from Sf21 cells and Sf9 cells of the autumn army insects. Suitable animal cells may be cultured cells or cells in vivo, and preferably vertebrate cells, more preferably mammalian cells, and are derived from animals, liver, liver, Organs or tissues such as lungs, ovaries, breasts, skin, bones, blood, etc., such as CHO, COS, BHK, 10 HEK-293, Hela, NIH3T3, VERO, MDCK, MOLT-4, Jurkat, K562, HepG2, etc. Suitable media and culture conditions suitable for use in host cells for DNA recombination techniques are well known in the art of biotechnology. For example, host cells can be cultured in the art. Fermentation bioreactor with shaking burning 15 flasks, test tubes, microtiter dishes with flat shallow dish, and the culture may be suitable for recombinant host cell growth temperature, pH, and lower oxygen content. In accordance with the present invention, the term "transfection" can be used interchangeably with the term "transformation" and generally refers to the manner in which a nucleic acid molecule is introduced into a selected host cell. According to techniques known in the art, a nucleic acid molecule (e.g., a recombinant DNA construct or a recombinant vector) can be introduced into a selected host cell by a variety of techniques, such as filling an acid bow or Transfection, electroporation, microinjection, particle bombardment, transfection of liposome mediators (lip〇s〇rne- 43 1321567) • Revision No. 095,118,228, the date of revision of the patent application specification: 98 years of mediated transfection), transfection with bacterial bacillus, use of retrovirus or other viruses [eg, vaccinia virus (eg, vaccinia virus) Vacnation (transduction) of vaccinia virus or baculovirus of insect cells, transformation of protoplasts (pr〇t〇plast fusion), transformation of agrobacterium 5 strains or other methods. In order to transform a Bacillus bacterium, it can be referred to, for example, 'Using in Molecular &amp; General Genetics (1979), Vol. 168 77/, etc.; when transducing yeast, reference can be made. For example, 10 notes are performed by Methods in Enzymology (1991), Vol. 194, 182-187, Proc. Natl. Acad. Sci. USA, Vol. 75, 1929 (1978). For the transformation of insect cells or insects, refer to, for example, the method described in (5, 47_55 &quot; illusion, etc.. When you want to transform animal cells, you can refer to it, for example, Vir〇1〇gy, v〇 1 52, 456 15 (1973), etc. The recombinant protein produced by the present invention may be one according to the vector system used and the position of the nucleic acid coding sequence according to the present invention inserted into the vector. a form of a separate protein or a fusion protein (fusi〇n pr〇tein). For example, when a nucleic acid coding sequence according to the present invention is inserted into a 20-containing gene sequence encoding a further protein (for example, a reporter gene) If the position of the insertion is such that the nucleic acid coding sequence according to the present invention is linked to the gene sequence or partially overlaps at the end, a recombinant protein in the form of a fusion protein may be expressed. The activity X of the protein is adversely affected. The 44-square correction date of the spacer can be designed as needed: October 1st, 1998. Insertion is well known to those skilled in the art. Techniques for use. Depending on the vector used and the host system, the recombinant protein produced by the present invention can be maintained in a recombinant host cell, secreted into the medium, and crazed to a periplasmic space or It is retained on the outer surface of the fine membrane 5. The recombinant protein produced by the method of the invention can be purified using a variety of standard protein purification techniques. Primarily, after the transformed host cell has been cultured for a period of time, it is known The method collects the cells and then floats them in an appropriate buffer, and then breaks the 10 cells by means of ultrasound, lysozyme and/or freeze-dissolution. Immediately thereafter, by centrifugation and / Or filter to obtain a crude protein extract. The buffer may also contain a protein modifier such as urea and guanidine hydrochloride, and a surfactant such as Triton®. When the recombinant protein is secreted into the medium, After the termination of the culture, the cells and the supernatant are separated by a conventional method, and the supernatant is collected. For example, the protein contained in the culture supernatant obtained is 15 The purification treatment can be carried out by a combination of traditional protein separation and purification techniques. The conventional separation and purification methods include, but are not limited to, salting out, solvent precipitation, Dialysis, ultrafiltration, gel filtration, SDS-polyacrylamide 20 guanamine gel electrophoresis, isoelectric point electrophoresis, reverse phase chromatography, anion exchange chromatography Method, affinity chromatography, chromatofocusing, etc. Preferably, the recombinant protein produced by the present invention is recovered "in a substantially pure form". As used herein, the amendments to the specification of the patent application No. 095,118, 228, filed on November 1, 1998, "substantially pure" refers to the purity of a purified protein. The protein can be effectively used as a commercial product. In another preferred embodiment of the present invention, the applicant uses the DNA sequence having the sequence identification number: 27 obtained above as a template, and uses the table of the fifth embodiment. The primers shown in 5 are Ndelsp-AFP (sequence identification number: 29), Kpnl-AFP (sequence identification number: 30), BamHI-AFP (sequence identification number: 31), KpnITAR-AFP (sequence identification number: 32), BamHIGDP-AFP (SEQ ID NO: 33) and SpeSalHtagTAA-AFP (SEQ ID NO: 34), and further obtain 10 to 3 DNAs with sequence identification number: 35, sequence identification number: 36 and sequence identification number: 37, respectively. Sequences, which respectively encode an antifreeze protein analog having a different number of repeating units of amino acid, ie, antifreeze protein (AFP) analog 1A (SEQ ID NO: 38 ' contains 4 antifreeze units), Protein analog 2A (SEQ ID NO: 39, containing 8 antifreeze units) and antifreeze protein 15 analog 3A (SEQ ID NO: 40, containing 12 antifreeze units). According to the present invention, by recombination The antifreeze protein obtained by DNA technology has been confirmed to have the ability to inhibit the formation of ice crystals, and can be genetically produced in high yield, and thus has potential for supply in medicine, food, various frozen and frozen products, and other related biotechnology fields. Including: 20 (1) preservation of tissue culture or cell strain: addition of AFP can improve the survival rate of cryopreservation; (2) cryopreservation of eggs and sperm; (3) preservation of tissue and organ transplantation, increase the success rate of surgery; (4) Preservation of cord blood; 46 1321567 Revision No. 095118228 Patent application revision page Revision period: October 1998 i曰(5) In the surgical operation, the role of the laser knife is replaced by the "knife type" of ice crystal; (6) Applied to foods, such as ice cream, to make the taste and quality better; (7) Applied to the aquatic products and meat industry to improve the quality of frozen and frozen meat; (8) Other needs to be frozen Refrigerated way to save or transport products, in order to avoid the formation of ice crystals 5 and affect the quality; and (9) anti-ice crystals form a variety of cold τ regional products. The invention will be further described in the following examples, but it should be understood that the examples are merely illustrative and are not to be construed as limiting. 10 EXAMPLES General Experimental Methods and Materials: The experimental methods employed in the present invention, as well as related techniques for DNA cloning, are referenced to textbooks well known in the art: Sambrook J, Russell DW (2001) Molecular Cloning: A 15 Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, New York, for example, DNA cleavage reaction by restriction enzymes, DNA adhesion using T4 DNA ligase DNA ligation with T4 DNA ligase, polymerase chain reaction (PCR), 20 agarose gel electrophoresis, sodium dodecanoate-polyacrylamide gel electrophoresis (sodium) Dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting and plasmid transformation, etc., and these techniques are all familiar to those skilled in the art, and can be used as a patent of No. 47 1321567 No. 095118228. The amendment date of the application manual revision date: October 1, 1998, according to its professional quality to implement. For example, the plastid transformation method used in the following examples is carried out by electrotransformation. In addition, the cell density was measured using a spectrophotometer (V530, Jasco) with a measurement wavelength of 600 nm, and the resulting absorbance value of 5 was recorded as OD600. The protein concentration analysis was performed by using a protein analysis reagent (Protein Assay Reagent, BioRad Co.) and analyzing the gel electrophoresed protein by an image analyzer (GAS9000, UVItec). Furthermore, the present invention uses an overlap-extension polymerase chain reaction (OEPCR) 10 to synthesize a DNA sequence encoding an antifreeze protein analog of the present invention, wherein the materials used include: nucleotides ( dNTPs) (Protech Technology. Enterprise CO., Ltd., Taiwan), P/w DNA polymerase 10X reaction buffer [i ° / w DNA polymerase 1 OX reaction buffer, containing 200 mM Tris-HCl (pH 8.8, 25 〇 C ), 100 mM KC1, 100 mM (NH4)2S04, 15 20 mM MgS04, 1.0% Triton® X-100 with 1 mg/mL nuclease-free BSA (nuclease-free BSA)] and cadaveric/m DNA polymerase It was purchased from Promega, Madison, USA, and the primers were supplied by GENSET Corp., La Jolla, CA, USA, and the instrument used was perkin-Elmer GeneAmp PCR System 2400 thermocycler (Perkin-Elmer Corp., 20 Wellesley, MA). ,USA). Anti-Freeze Protein Activity Assays Anti-bed proteins have the ability to reduce solution seizures, inhibit ice recrystallization inhibition, and modify ice crystal morphology. In the present case, the Applicant determines the anti-freezing egg 48 1321567 according to the present invention. Amendment page of the specification of the patent application No. 095118228. Date of revision: October 1998, the white analogue inhibits ice recrystallization inhibition and The ability of the ice crystal morphology to modify the activity of the anti-canon analog is evaluated. In the experiment, the cooling and temperature control of the sample to be tested was performed using an LNP cooling system and a temperature controller TMS94 (temperature programmer 5 TMS94). The LNP cooling system used here is a combination of a LNP94 liquid nitrogen pump and a 2 liter liquid nitrogen drum (2 Litre dewar). The liquid nitrogen is charged into a liquid nitrogen drum before use. The method for testing the antifreeze protein to inhibit recrystallization of ice crystals is as follows: First, 10 μL of antifreeze protein solution or buffer droplets are placed on a glass slide, and then covered with a coverslip. The sample to be tested was placed in a Linkam BCS196 freezing stage, and the temperature control was performed using the LNP cooling system and the temperature controller TMS94. The temperature of the sample in the freezing platform was first lowered to _4 以 at a rate of 90 ° C / min. (:, and maintain this temperature for 1 minute and 15 minutes. After that, heat up to -7eC at 90 °C / minute and maintain it at this temperature for 60 minutes. Use 〇lympus BX41 microscope with Roper Scientific (USA The digital camera observes the size of the ice crystals formed in the test sample at 1 〇χ magnification. The method for testing the anti-Kein modified ice crystal configuration is as follows: first take 2 2 抗 antifreeze protein solution or buffer Drop on glass slides, then cover with coverslips. Place the sample to be tested into the Linkarn BCS196 freezing platform and drop the temperature of the sample to _4〇°c at 90 C/min and maintain At this temperature, it lasts for a few minutes. After that, it is heated to a temperature of 9 〇 〇 / min to a single ice crystal opening temperature, and then continuously cooled to grow ice crystals. Lee 49 Revision date: October 1, 1998 No. 095,118,228 patent application revision page with Olympus BX41 microscope with R〇per stealing (10) digital camera 'observation test sample (4) ice crystal configuration at 1GX magnification. Analysis method for anti-freeze protein thermal stability 10 Prepare 100 anti-Kang protein solution (concentration 0.4 mg/mL 'pH 8 · 〇), and heat it at HKTC. After adding hour or 2 hours, 'take out 2G μί of anti-; East protein solution and give Placed on ice. Analyze the ability to modify the configuration of ice crystals by the above method. When the configuration of ice crystals is double cone, 'represents resistance; East protein activity is not affected by heating; when the configuration of ice crystals is In the case of hexagons, it indicates a decrease in anti-whitening activity; when the configuration of ice crystals is circular, it indicates loss of antifreeze protein activity. The analytical method for pH stability of anti-proteins utilizes Microcon YM-3 centrifuge tubes (Millip〇re, USA) to replace the components of the anti-conne protein solution so that the ?11 value of the antifreeze protein solution reaches the required 2, 4, 6, 8, 10, and 12 in the experiment. The treatment is as follows: take the resistance of 400 15 Frozen protein solution (concentration 0.4〇^/11^呷118.0) placed in a

In a Microcon®-3 centrifuge tube, centrifuge at i4〇〇〇Xg, substituting to concentrate the solution to a volume of 50 pL, then add 350 μί of a specific pH buffer, continue centrifugation, and concentrate. To a volume of 5 inches. The buffer addition and centrifugation were repeated twice. Finally, the concentrated liquid was mixed with 350 2 Torr of buffer and placed on ice. The ability of the antifreeze protein to modify the ice crystal configuration was analyzed as described above. Analytical method for minimal inhibition concentration of anti-linguistic protein

The analysis of the minimum inhibitory concentration of the antifreeze protein is in accordance with the specification of the GM 50 1321567 Patent Application No. 095118228. Revision date: October 1, 1998

Mueller et αί· (1991), J. Biol. Chem., 266: 7339-7344, and modified. The antifreeze protein solution was appropriately diluted with a buffer to a concentration of 400 肫 / 吣 ~ 1 〇 pg / pL, and the ability of the antifreeze protein to inhibit recrystallization of ice crystals was analyzed by the aforementioned method. When the ice crystals in the sample are 5 μ fine, the antifreeze protein at this concentration has antifreeze activity; on the contrary, if the ice crystals grow significantly, the antifreeze protein at this concentration does not have antifreeze activity. The concentration at which ice crystals begin to increase significantly is used as the minimum inhibitory concentration of this antifreeze protein. Example 1. Construction of plastid vector PRP_AFP 10 plastid vector PRP-AFP was inserted into a plastid vector pRP by encoding a DNA sequence encoding the antifreeze protein analog 1 of the present invention (SEQ ID NO: 27). It is formed, and its construction process is explained as follows. I. Construction of plastid vector pRP: The plastid vector pRP is a shuttle vector, and its construction flow is shown in Fig. 153, in which the vector pEX600A and the vector pEX500A are used. The vector PEX600A and the vector pEX500A were constructed by taking two from the basophilic Bacillus subtilis YaB (alkalophilic 5" to 7/milk YaB) (KC.

Tsai et al. (J983), Biochem. Int., 7:577-583) (alkaline 20 elastase gene, ale) (Ryuta Kaneko et al., generously provided by Professor Cha Yingjie from Yangming University) (1989), J. Bacteriol. 777..523U236, NCBI accession number M28537 (NCBI accession no. M28537)] different DNA fragments inserted into E. coli co//) and Bacillus subtilis like a shuttle of '/(4) The shuttle vector pHY300PLK (Takara Shuzo Co., Tokyo, 51 1321567, revised date: October 2014, No. 095118228, patent application specification page, Japan) was formed. Primarily, a plastid pEX301 carrying the a/e gene was established to add deductions from the "Finance, as described above". The gene can be divided into a region (a structural gene), an upstream region 5 domain, and a downstream region). The initiation codon of the β/e gene on the plastid PEX301 is then genetically engineered from "ttg" to "atg", while the three nucleotides preceding the start codon are " Gaa,, was changed to "cat" and a cleavage site for gene selection was introduced there to obtain a derivative plasto ρΕΧ3〇1Α. 10 Then, the plastid was treated with the selected restriction enzyme. The o/eC-a/eE fragment and a fragment were obtained from the or/e gene on pEX301A, and then the two DNA fragments were separately inserted into the vector pHY300PLK to obtain the vector pEX500A and the vector pEX600A, wherein the vector pEX500A was compared to pEX600A. An α/eC fragment is added downstream of the α/eE fragment (refer to the master's thesis written by Li Yaocheng from the National Institute of Biochemistry, Yangming University, 1994, entitled “Experimental Elastase Research on the Analytic YaB (1) Enzyme matrix-specific protein engineering (Π) in the gene expression of 仰如仏 DB104”, directed professor Professor Cai Yingjie. The function of the fl/eC fragment is unknown, and may have a transcription termination site (transcrip) Tion 20 terminatin site) ° Amplify the sequence containing the α/eE promoter-SD sequence by PCR and using the vector PEX600A as a temperte (aleE promoter-SD sequence-signal peptide coding sequence) a DNA sequence in which the primer set used to amplify the DNA sequence is modified as disclosed in the specification of the specification of the patent application No. 095,118, 228, filed on Jan. 1, 1998, and the PCR operating conditions are shown in Table 3. And wherein in the design of the PCR primer set, 'a five-cleavage site CEcoAI cutting site is added upstream of the MeE promoter, and a multiple selection site is added downstream of the message peptide coding sequence (multiple) Cloning site, MCS), which contains 5 restriction enzyme cleavage sites for selection: 5 tears 1«-5&gt;7/21-5^1-melon?1-printed eI (5'-3 '), where &amp;ci overlaps with the cleavage site of the 〇i. Table 3 The nucleotide sequence of the primer name (5'·3·) The forward primer pRP-£coi?I £coRI 5'-tagactgaMl£gtatatttcccaatagctaaaaaggtttcc -3丨 (Serial Identification Number: 41) Reverse Primer pRP-MCS Spel Sad Sphl BamHl 5*-taaatcactaet|ctceaektca2catect2eatccctc22ctPcttptpr,patr-V ^ year 5ii Identification Xhol Identification Number: 42) PCR conditions: 2 minutes at 94 ° C, 1 cycle (CyCle); 30 seconds at 94 ° C, 1 at 56 ° C minutes and 30 seconds, 1 minute, 35 cycles at 72 ° C; and at 72 ° C 7 min 1 cycle "PCR product taeacteaattcetatatttcr.rafltapctaaaaapgmr.rraatarraAiingagttQ ^^ flii ^ gi ^ attttagattaccagctgcgcaggttggaacattttgaggaggtataaccatatgaataagaaaatgg ggaaaattgttgccggaacagcactaattatatcagtagcatttagttcatcgatcecacaaecagc cgag ££ M ^ aacatpcteadctceap | Actaetgattta i 辨识 辨识 辨识 辨识 注 Note: The restriction enzyme cleavage site located in the primer is indicated by the bottom line or the framed frame, where the ^^ and ΧΑοΙ cleavage sites overlap. 10 The PCR product obtained above (sequence identification) ID: 43) encodes a message peptide derived from the basophilic Bacillus subtilis YaB alkaline elastase at its nts 123-203, and an SD sequence at its nts 108-113. The restriction enzyme cMIASpel was used to excise the α/eE gene in the vector pEX500A, and the resulting cleavage product was ligated with the 15 amplification product obtained above. Thereafter, the formed ligated product was transformed to be cultured to contain 20 pg/mL ampicillin

E. coli DH10B 53 1321567 in LB medium (LB broth) of (ampicillin, Ap) Amendment date of patent application No. 095118228 Revision date: October 1st, 1998 (five DH10B, available from Gibco-BRL, Within Rockville, MD), it was then incubated with LB containing 20 pg/mL ampicillin (apicillin, Ap). A transformant was randomly selected and a plasmid vector 5 was extracted. A plastid vector confirmed by restriction enzyme cleavage and sequence obtained after DNA sequencing is named pRP, which carries a DNA sequence containing a nucleotide sequence which corresponds substantially to the above The amplification product is of the kind, and there is an α/eC fragment downstream of the DNA sequence that has an unclear function and may have a transcription termination 10 site. Retention of the α/eC fragment facilitates the following recombinant DNA technology manipulations. According to our earlier studies, the plastid vector pRP can elicit a secretion of a heterologous protein in at least the following host cells: Bacillus subtilis, including the bacillus subtilis YaB (alkalophilic YaB) ); and the bio-source collection and research center (BCRC), Hsinchu, which is available from the Food Industry Research and Development (FIRDI). Food Road No. 331].

20 Π·Synthesis of the DNA sequence encoding the antifreeze protein analog 1 of the present invention (that is, the AFP I gene): Applicants based on the structural and functional properties of the cold winter flounder type I antifreeze protein HPLC6, and the protein The antifreeze protein analogs of the invention are designed hierarchically. 54 1321567 • Revision No. 095118228 Patent Revision Revision Date: October 1st, 1998 曰 HPLC6 is mainly composed of 3 residues with 1 amino acid and with the formula “Thr-X^Asx-X7” Represented by the repeating unit. Referring to the applicant, the amino acid sequence of the antifreeze protein analogs 1 and 1 of the present invention is designed to have 4 repeating units, and 5 is provided at the c-terminus of the antifreeze protein analog 丨 and 1, Six histidine acids facilitate the purification of pr〇tein purification. Further, an aspartic acid-proline sequence was designed to be located at the N-terminus of the amino acid sequence of the antifreeze protein analog I of the present invention. Thus, if the antifreeze protein analog of the present invention is expressed in the form of a fusion protein, formic acid can be used to excise the N-terminus of the anti-bundle egg 10 white analog Γ. Fusion partner. Then, based on the amino acid sequence designed by the antifreeze protein analog 1 and hydrazine, the preferred codons of Bacillus subtilis can be used to derive their nucleic acid coding sequence' and then according to the derived nucleic acid coding sequence. Design and synthesize primers for OEPCR, and then use these primers to perform oepcr, and 15 to obtain an amplification product containing a nucleotide sequence encoding antifreeze protein analogs 1 and 1, encoding the antifreeze of the present invention. The DNA fragment of the protein analog 1 and hydrazine was subjected to the use of the primer shown in the above Table 2 to carry out the amplification as shown in Fig. 2 (: 11 and was synthesized) 20 〇 EPCR reaction conditions are as shown in Table 4 below. 55 1321567 No. 095118228 Patent Application Specification Revision Date: October 10, 1998 Table 4. OEPCR Operating Conditions for Synthesis of AFP I Gene

Content volume &amp;L) Final reaction concentration water 36.6 One-to-one DNTPs (2.5 mM each) 4 200 μΜ 10X Reaction Buffer 5 IX AFP-5a/«HI or AFP-^j/jI Introduction (50 pmole^L 1 1 μΜ AFP-SS primer (50 pmole^L) 1 1 μΜ AFP1 primer (10.66 ng/pL) 1 10.66 ng AFP2 primer (10.35 ng/pL) 1 10.35 ng P/m DNA polymerase 0.4 1.2 U Reaction conditions 1 minute at 94 ° C, 1 cycle; 30 seconds at 94 ° C, 30 seconds at 70 ° C, 72 ° (: 35 cycles at 2 minutes; and 5 minutes at 72 ° C, 1 cycle). The reaction mixture used for PCR was 50 μL of reaction buffer [20 mM Tris-HCl, 10 mM KC1, 10 mM (NH4) 2S04, 2 mM MgS04j 0.1% Triton® X-l00, ol mg/mL nuclease-free The nuclease-free Bovine serum albumin contains 200 μM of d ATP, dTTP, dGTP and (1(:ΤΡ, 1μΜ amplification primer AFP-5 (10) HI (or AFP- SpAI), 1 μΜ amplification primer AFP-SS, 10.66 ng AFP1 primer, 10.35 ng AFP2 primer and 1.2 UP/m DNA polymerase. After the reaction is completed, the obtained PCR product is sequentially phenol/chloroform/isoamyl alcohol (phenol/chl Oroform/isoamylalcohol, v/v/v = 25:24:1) and extract once with chloroform/isoamylalcohol (v/v = 24:1). After that, take the solution containing DNA and add 2 A volume of absolute alcohol and 0.1 volume of sodium citrate (pH 5.2) were placed at -70 ° C for 1 hour to precipitate the DNA. Washed with 70% alcohol to remove salt [desalt] and vacuum 15 evaporation ( After vacuum evaporation, the DNA is dissolved back into an appropriate amount of sterile deionized water and stored at -20 ° C for use. 56 1321567 Patent Application No. 095118228 Revision of the amended page: 98 years On October 1st, the purity and concentration of the PCR product were observed by electrophoresis. The procedure for electrophoresis was as follows: First, a 2% agarose gel was prepared. The DNA sample was mixed uniformly with 6X loading buffer in a ratio of 5:1 and then applied to the sample well of the 2% agarose gel 5. Electrophoresis was carried out at 1 〇〇 v for 30 minutes. After the electrophoresis element was completed, the gel was dyed with ethidium bromide (EtBr) and observed under an ultraviolet lamp. The DNA concentration was determined by using a Kodak D C26 5 zoom digital camera with a dedicated UV light source filter for gel photography and using the analysis software Gel-Pro AnalyzerTM version 3.0 1〇 to analyze the DNA concentration. Figure 4 shows the results of agarose gel electrophoresis of two PCR products, wherein the diameter 1 is an amplification product (amplicon) obtained by OEPCR using the primers AFP-5^/, AFP-SS, AFP 1 and AFP2. Identification number: 28), and diameter 2 is a 15 amplification product (SEQ ID NO: 27) obtained by OEPCR using primers AFP-5i and HI, AFP-SS, AFP1 and AFP2. The PCR product of diaphoresis 1 can be incorporated into a plastid PEX500A which is cleaved by ZzIASpd to obtain a derivative plastid pAFP with a nucleic acid which expresses a basic elastase (partial)-AFP fusion protein. Encoded fragments (data not shown). 20 E. Construction of plastid vector pRP-AFP: Figure 5 shows the construction of recombinant vector PRP-AFP, in which the primers AFP-5 &lt;awHI, AFP-SS, AFP1 and AFP2 were used for OEPCR in the above procedure. The resulting amplification product (SEQ ID NO: 27) was used in this experiment as the AFPI gene. 57 1321567 Patent No. 095118228 Revision of Amendment Page of the Patent Application: October 1st, 1998 First, 'restore the amplified product stored at _2〇 °C, then take out the appropriate amount for Ba/nHIASpel restriction enzyme cleavage (restriction enzyme cleavage). Further, the plastid vector pRP obtained in the above Procedure I was cleaved with the restriction enzyme 5awHI/printed eI and the cleavage product was recovered. With respect to the obtained two cleavage products, the vector DNA and the insert DNA were ligated at a molar ratio of 1.1 Å. Thereafter, the thus obtained ligation product was fed into the cells of Escherichia coli strain DH10B by electrotransformation. Transformants were randomly selected and 10 plasmids were extracted. A plastid vector confirmed by restriction enzyme cleavage and sequence obtained after DNA sequencing was named pRP-AFP 'it can be used in Bacillus subtilis (10) such as ///?) The expression of the AFP I gene was revealed in the cells of the bacillus Yab (alkalophilic YaB) and the sub-drug lactobacillus (data not shown), and was used to construct the Escherichia coli described in Examples 2 to 4 below. The source of the antifreeze protein gene carried in the expression vector. Example 2. Construction of plastid vector pQE30-AFP and its expression in Escherichia coli strain M15 (pREP4) 20 I Construction of plastid vector pQE30-AFP: Fig. 6 shows the construction procedure of plastid vector pQE30-AFP. The plastid vector pQE-30 (purchased from Qiagen) is a T5 expression vector featuring: (1) a T5 promoter (pT5), and an RNA capable of E. coli 58 1321567 . Patent No. 095118228 Revision of Amendment Page of the Patent Application No. #月: October 1st, 1998 1 identified by the enzyme; (2) Contains 2 /ac operons (/ac 〇perat〇r, /ac〇), It can strictly regulate the transcription of a target gene. The /ac inhibitory protein gene repressor 5 geneW) present on another vector pREP4 can express a large amount of lactose repressor, and the expression of the target gene can be inversely suppressed (ira/w-repression); (3) The protein product expressed by this vector will carry an amino acid sequence of MRGSHHHHHH (SEQ ID NO: 44) at the n-terminus, which will be present in the cytoplasm of the transgenic host cell, and This protein product can be purified by affinity column chromatography using a Ni-NTA resin. The plastid vector pQE-30 was cleaved with the restriction enzyme 5amHIASa/I, and the cleavage product was recovered. Further, the recombinant vector pRP-AFP obtained in Example 1 was cleaved using a restriction enzyme to obtain a DNA fragment containing AFP-a/eC' and the DNA fragment was recovered. For the two cleavage products obtained, the vector DNA and the insert DNA were ligated at a molar ratio of 1:5. Thereafter, the thus obtained ligation product was fed into cells of Escherichia coli strain DH10B by electrotransformation. The transgenic plants were selected with 20 machines and the plastid carriers were extracted. A plastid vector confirmed by restriction enzyme cleavage and sequenced after DNA sequencing was named PQE30-AFP, which carries a DNA fragment encoding a recombinant antifreeze protein analog at the N-terminus. There is an amino acid sequence of MRSSHHHHHH (Procedure 59 1^21567 No. 095118228 Patent Application Revision Revision Date: October 1998! 辨识列号: 44). Π·Recombinant antifreeze protein analog was produced by E. coli transform strain M15 (pREP4, PQE30-AFP): The known plastid vector PQE30-AFP was transformed into Escherichia coli 5 strain M15 (pREP4) [ The plastid PREP4 has a repressor that can strictly regulate the expression of E. coli] and E. coli strain Ml5 (the two E. coli strains are purchased from Qiagen), and the transformed strain M15 (pREP4, pQE30) is obtained. -AFP) (experimental group) and M15 (pQE30-AFP) (control group). Thereafter, the obtained transformant strain was treated with 1 mM IPTG to induce production of a recombinant antifreeze protein analog. After 4 hours of induction of the transformant strain Ml5 (pREP4, pQE30-AFP) by IPTG, the obtained recombinant antifreeze protein analog was the most abundant, mainly in the soluble fraction of the host cell. After 6 hours of IPTG induction, the number of recombinant antifreeze protein analogs obtained was decreased by 15 , which may be because the recombinant antifreeze protein analogs expressed were in the form of peptides and were easily hosted. Intracellular protease or peptide peptidase is decomposed. In addition, the reduced amount of recombinant antifreeze protein analogs expressed may also be due to insufficient dose of inducer due to consumption of IPTG (data not shown). 20 Example 3. Construction of plastid vector pET-12a-AsAFP and its expression in Escherichia coli strain BL21(DE3) I. Construction of plastid vector pET-12a-AsAFP: Figure 7 shows plastid vector pET-12a -AsAFP construction process. The plastid vector pET-12a (purchased from Novagen) has a size of approximately 4.7 60 1321567. Date of revision: October 2014, pp. 095118228, patent application specification page kb, which is a T7 expression vector with an amine The ampicillin resistance gene (Apr) and the message peptide coding sequence. This plastid vector can be utilized to express a large number of target genes placed downstream of the Τ7 promoter. 5 To facilitate the manipulation of the plastid, the plastid vector pET-12a was first cleaved with the restriction enzyme 5amHI, and then Fill_in and ligati〇n were added to make the BamHI site on the plastid vector pET-12a. The ligated product formed by the disappearance of β is then cleaved with a restriction enzyme, and the cleavage product is recovered. Further, the recombinant vector 10 PRP_AFP ' obtained in Example 切割 was cleaved using restriction enzyme 5 (10) HIAWI to obtain a DNA fragment containing AFP-a/eC, and the DNA fragment was recovered. With respect to the obtained two cleavage products, the carrier dNA and the insert DNA were ligated with a molar ratio of 1:5, and the thus obtained ligated product was fed to Escherichia coli strain DH10B by electroporation. Within the cell. The transgenic plants were randomly selected and the plastid vector was extracted. 15 A sequence-corrected T7 plastid vector obtained after restriction enzyme cleavage confirmation and DN Α sequencing was named pET-La-AsAFP, which was able to initiate downstream of the T7 promoter under the induction of 0.4 mM IPTG. The expression of the AFP gene is represented by a recombinant antifreeze protein analog with a message peptide derived from the basic elastase of the albileophilic 2〇YaB at the N-terminus. . Π. Recombinant antifreeze protein analog was generated by E. coli transform strain BL21 (DE3) (pET-12a-AsAFP): The obtained plasmid vector pET-12a-AsAFP was transformed into a T7 expression system. Escherichia coli strain BL21 (DE3) (purchased from Novagen), 61 1321567 No. 095118228 patent application revised page revised period: 98 years 1 month 1曰 and obtained transformed strain BL21 (DE3) (pET-12a -AsAFP). After 2 hours from the obtained transgenic strain induced by 〇4 mM IPTG, a recombinant plastic antifreeze protein analog was started to be produced. By cell fractionation analysis, it was found that the expressed recombinant antifreeze protein 5 analog was mainly secreted into the periplasmic space or extracellular. This shows that the secretion-associated protein in the E. coli host cell recognizes the message peptide of the alkaline elastase carried by the recombinant antifreeze protein analog, and the recombinant antifreeze protein analog is carried to the cell membrane. The recombinant antifreeze protein analog is delivered to the cell wall space via the translocation unit (transl〇cati〇n 10 machinery). Example 4. Construction of the plasmid vector PET-12a-OsAFP and its expression in E. coli strain BL21 (DE3) I. Construction of the plastid vector pET-12a-OsAFP: Figure 8 shows the plastid carrier PET-12a- OsAFP build process. 15 The plastid vector pET-12a-AsAFP was cleaved with a restriction enzyme to obtain a DNA fragment containing AFP-a/eC, and the DNA fragment was recovered. Further, the plastid vector pET-12a' was cleaved using restriction enzyme B (10) HIAEC0RI and the cleavage product was recovered. For the two cleavage products obtained, the vector DNA was ligated with the insert DNA at a molar ratio of 丨:5. Thereafter, the thus obtained conjugated product was fed into cells of Escherichia coli strain DH10B by electroporation. The transgenic plants were randomly selected and the plastid vector was extracted. A T7 plastid vector with the correct sequence obtained after restriction enzyme cleavage confirmation and DN A sequencing was named pET-12a-OsAFP with - 62 1321567. The revised page of the patent application No. 095118228 was amended. : In 1998, a DNA fragment encoding a recombinant anti-Kang protein analog with a message peptide at the N-terminus. Π. Recombinant antifreeze protein analog was generated by E. coli transform strain BL21(DE3)(pET-12a-〇sAFP): 5 The obtained plasmid vector pET-12a-OsAFP was transformed into Escherichia coli strain BL21 (DE3) in the 'and get the transformed strain BL21 (DE3) (pET12a_

OsAFP). After the resulting transgenic strain was induced with 0.4 mM IPTG for 2 hours, a recombinant antifreeze protein analog was started to be produced. By cell division analysis, it was found that the recombinant antifreeze protein analog 10 exhibited was mainly present in the intracellular insoluble fraction. Analysis by transmission electron microscopy revealed the formation of an inclusion body in the cells of the E. coli transformant. Although the N-terminus of the recombinant antifreeze protein analog obtained in this example carries the 〇 mp τ message peptide, 15 but failed to efficiently secrete the recombinant anti-beam protein analog into the cell wall space. This may be because the rate of protein synthesis is too fast, and it is too late to allow the expressed recombinant antifreeze protein analog to be recognized by the secretory-related protein in the E. coli host cell, so that the recombinant antifreeze is expressed. The protein analog forms insoluble inclusion bodies in the cells of the E. coli transforming strain. Example 5. Expression of the antifreeze protein encoding gene in E. coli transformants and recombinant antifreeze protein analogs Purification I. Expression of anti-bundle-encoding gene in E. coli transgenic strain: For the modified page containing the recombinant plastid vector obtained in the above Examples 2 to 4, the amendment page of the patent application No. 095118228 is amended: In October 1998, a strain of E. coli was selected, and a single colony (c〇l〇ny) was selected and inoculated into an antibiotic-containing LB liquid medium (LB broth) for preculture overnight. Then '1% of the overnight preculture was inoculated into 100 mL of fresh 5 fresh LB liquid medium containing antibiotics (the total culture volume can be adjusted as needed), with regard to the pQE performance system, in the medium Add 25 pg/mL connamycin

(kanamycine) with 100 pg/mL ampicillin (ampicillin); for the pET expression system, 50 pg/mL ampicillin was added to the medium. The thus-formed initial culture thus formed was cultured overnight at 37 ° C, 10 150 rpm. 1% of the overnight culture was added to 100 mL of fresh LB liquid medium containing antibiotics. After incubation at 37 ° C, 180 rpm to 〇d600 and 0.6~0.8, add appropriate amount of iptg (for pQE performance) In the system, 1 mM IPTG was added to the medium; for the pET expression system, 0.4 mM or 0.05 mM IPTG was added to the medium to induce the expression of the antifreeze protein-encoding gene, and then the culture was continued at 37 ° C and 180 rpm. . II. Detection of Recombinant Antifreeze Protein Analogs in E. coli Transgenic Plants: Refer to the Novagen handbook method to distinguish the site of the protein produced by E. coli transformant 20 into an extracellular fraction. Four parts of the cell wall space (peripiasmic space fracti〇n) and intracellular soluble fracti〇n and insoluble fraction, and protein electrophoresis to detect the expressed recombinant form The location of the anti-beam protein analog. Experimental procedure 64 1321567. Revision No. 095118228 Patent Application Revision Date: October 1, 1998 as follows: According to the above procedure I, the E. coli transformation strain is cultured, and when cultured until OD600 = 0_6~0.8, it is added. Appropriate amount 11) Ding in order to induce the expression of the antifreeze protein encoding gene. 5 After 2 hours and 4 hours of 1PTG induction, 40 mL of the culture was taken out and centrifuged at 1000 x g and 4 ° C for 1 minute. The supernatant was collected, filtered through a 0.22 μηη filter membrane, and the filtrate was collected as a protein sample of the extracellular portion. The precipitate obtained after the above centrifugation was again suspended in 30 μL of T S 10 buffer. Then, 60 pL of EDTA was added, followed by slowly stirring at room temperature for 10 minutes. The supernatant was decanted by centrifugation at 10 °C for 10 minutes at 4 °C. The precipitate was again suspended in 30 mL of ice-cold MgS04 solution (5 mM MgS04, 10% TCA) and slowly stirred for 10 minutes in an ice bath. The supernatant was obtained by centrifugation at 10,000 x g and 4 ° C for 10 minutes to obtain a protein sample of 15 as a part of the cell wall space. The precipitate left after the above centrifugation was precipitated and concentrated by trichloroacetic ccid (TCA), and the precipitate was again dispersed in 4 mL of ice-cold Tris-HCl (pH 7.5), and lysozyme was added. , purchased from Sigma, final concentration of 100 pg/mL) and allowed to act at 30 ° C for 20 15 minutes. Thereafter, the cells were disrupted by ultrasonic treatment (XL Sonicator ultrasonic processor, Misonix Inc., Farmingdale, NY, USA). The resulting product was taken out in 1.5 mL and placed in a microtube, and centrifuged at 10,000 x g and 4 ° C for 10 minutes. Thereafter, the supernatant of 200 μΙ&gt; was taken out as the intracellular soluble fraction 65 1321567. The revised version of the specification of the patent application No. 095118228 was amended: the protein sample of October 1, 1998, and the precipitate was a cell. Insoluble part. The pellet was washed twice with 2 mM Tns-HCl (pH 7.5) to avoid possible residual soluble proteins to interfere with experimental judgment. Thereafter, 丨5 mL &amp;i% SDS was added to reconstitute the insoluble protein, which was then heated and vigorously mixed, until the precipitate was completely dispersed in the solution, thereby serving as a protein sample of the insoluble portion of the cell. . Protein samples of different bacterial fractions were mixed with 5X sample buffer (or 2X sample buffer) and heated at 10 ° C for 5 minutes. 'Tricine_SDs PAGE for egg 10 white matter Electrophoresis. The operation of Tricine-SDS PAGE is described in accordance with the method of H. Schagger and G. von Jagow (1987), Anal. Biochem" 166: 368-379, and is modified. First, a polypropylene decylamine is prepared. Film, which is divided into 3 layers, respectively, is a stacking gel (4% T, 3% 〇, spacer gel (10% T, 3% 〇, and separation gel (separati〇n) Gel) (16.5/i T, 3/» C) 'The protein sample is then loaded (i〇a(jing) into the sample well with 0.2 M Tris-base (pH 8.9) as the positive buffer and Electrophoresis was carried out at a voltage of 80 V using 0.1 M Tris-base (containing oi μ Tricine, 0.1% SDS, pH 8.25) as a negative electrode buffer, and stopped when the tracking dye ran to the bottom of the gel sheet. Electrophoresis: The coagulation piece was then stained with a 10% acetic acid solution containing 0.025% masal blue R-250 (c〇〇massive blue R-250) for an hour and a half. After that, it was treated with 1〇0/〇 acetic acid solution. Dyeing. Purification of the dish-recombinant antifreeze protein analog: 66 Revision No. 095H8228 Patent Application Revision Date: October 10, 1998 Cardiac treatment (10 μg, 4 ° C, 1 〇 minutes) to harvest the transfected cells cultured according to the above procedure I and induced by IPTG, thereby obtaining the supernatant fraction (ie, extracellular Part of the performance) first filter the membrane with 〇22^ claws; consider 'collecting the liquid' and then concentrate it by ultrafiltration. 5 Add Tris, NaH2P04 and urea to the final concentration of the obtained concentrate. The pH of the resulting mixture was adjusted to 8.0 at 10 mM, 100 mM, and 8 Torr, and allowed to slowly mix for 1/2 hour at room temperature. In addition, the cells &gt; cell pellete were resuspended (re -suspend) in a sonication buffer, followed by ultrasonic treatment with a Sonicator 10 (Ultrasonic processor model XL; Heat Systems, Farmingdale, NY) to break the cells. Thereafter, centrifugation (10000 x g '10 minutes) And a soluble portion and an insoluble portion are obtained. The insoluble portion is resuspended in a lysis buffer, and the soluble portion is added to a final concentration of 8 M. Leave at room temperature slowly 15 Stir for 1 hour. Purification of the protein was carried out according to the method of The QIAexpressionistTM (fourth edition, Qiagen), in which the histidine acid (Histidine) was purified in a batch manner in a densuring environment (in a batch manner). ) protein. The purification procedure was as follows: 4 ml of the 20 protein sample was uniformly mixed with 1 mL of Ni-NTA resin, and subjected to rotating shaking for 2 hours at room temperature. The resin and protein sample mixture was packed in a column and rinsed with 4 mL Wash Buffer C1 [wash buffer C1, containing 100 mM NaH2P04, 10 mM Tris-Cl, 8 尿素 urea (urea), pH 7.0] at room temperature. Resin twice, then 4 mL of washing buffer C2 67 1321567 No. 095118228 patent application revision page correction period: October 1st, 1998 (containing 100 mM NaH2P〇4, lOmM Tris-Cl, 8 Μ urea, pH 6.3) The resin was rinsed twice to separate the protein that could not be bound to the resin. Finally, 2 mL of elution buffer D (containing 100 mM NaH2P04, 10 mM Tris-Cl, 8 Μ urea, pH 5.9) and 2 mL of Washing 5 rinsing E (containing 100 mM NaH2P04, lOmM Tris) -Cl, 8 M urea, pH 4.5) The protein adsorbed on the resin was flushed out. At the time of elution, every 5 5 mL of eluate was collected in a test tube. Figure 9 shows the results of 10 Tricine-SDS-PAGE electrophoresis of recombinant antifreeze protein analogs produced by the E. coli transformed strains obtained in the foregoing Examples 2 to 4, wherein the diameter M: low molecular weight protein label; Path 1 : Protein purified from the cytoplasmic portion of E. coli M15 (pREP4, pQE30-AFP); diascript 2: protein partially purified from the medium of E. coli BL21 (DE3) (pET12a-AsAFP); run 3: from the large intestine Protein purified from the intracellular 11 soluble fraction of Bacillus sp. BL21 (DE3) (pET12a-AsAFP); PATH 4: Protein purified from the insoluble fraction of cells in E. coli BL21 (DE3) (pET12a-AsAFP); : a protein purified from the intracellular soluble fraction of Escherichia coli BL21 (DE3) (pET12a_〇SAFP); and a diameter 6: purified from the intracellular insoluble fraction of Escherichia coli BL21 (DE3) (pET12a-OsAFP) 20 proteins. According to the experimental results, the recombinant antifreeze protein analog produced by the E. coli transformed strain obtained in Example 2-3 is located in the host cell, and can be roughly summarized as follows: 1. Escherichia coli M15 (pREP4, Recombination generated by pQE30-AFP) 68 1321567 Revision No. 095118228 Patent Revision Date: 98 years of the first month of the antifreeze protein analogue is mainly present in the soluble part of the cell, with a molecular weight of approximately 6.48 kDa; 2. The rAFP produced by E. coli BL21 (DE3, pET-12a-AsAFP) is mainly present in the extracellular (supernatant) fraction, while the soluble and insoluble fractions in the cell can also be purified to protein. The recombinant antifreeze protein analog purified from the insoluble portion of the cell has a large molecular weight (about 8.14) and is presumed to have a message peptide (path 4). Recombination from the extracellular and intracellular soluble fractions

The antifreeze protein analog has a small molecular weight (approximately 5.42 kDa). The N-terminal sequencing results showed that the informative peptide derived from the experimental elastase of the albileophilic YaB (alkalophilic YaB) can be recognized and excised by the E. coli BL21 (DE3) host cell (diameter 2, diameter 3 15 20 3. The recombinant antifreeze protein analog produced by E. coli BL21 (DE3, pET-12a-0sAFP) is mainly present in the insoluble part of the cell, and the soluble part in the cell can also be purified. protein. Self-celling; the trough fraction can be purified to two proteins of different molecular weights (estimated molecular weights of approximately 7.53 and 5.41 kDa). We hypothesized that the smaller molecular weight may be a recombinant resistance in which the N-terminal message peptide is cleaved; the East Egg ^ analog's position is close to the expected one. The numerator = larger electric material is likely to move faster because it has more hydrophobic amine groups, so it moves faster than the expected position. 6. Recombinant anti-resistant strain produced by E. coli transformant; analysis of the ability of the eastern protein analog to reduce the formation of ice crystals 69 1321567 Patent Application No. 095118228 Revision of the revised page: October 1st, 1998 The recombinant antifreeze protein analog produced by the E. coli transformed strain obtained in the previous examples was tested for its antifreeze ability, and the specific anti-freezing resistance was determined by using a Linkam HFS91 cold stage in combination with a photomicrography system. The ability of the merlin analog to reduce ice crystal formation and modify the ice crystal 5 configuration. Different concentrations of 2 pL of recombinant antifreeze protein analog solution [with water or buffer (1 mM NaH2P〇4) according to the "Anti-freeze protein activity analysis method described in the above "General Experimental Methods and Materials" , 10 mM Tds-HCl, pH 8.0) was placed on a glass slide and covered with a coverslip, 10 and then placed in a Linkam HFS91 cold stage, the temperature was rapidly reduced to -40 ° C ' and observed The form of ice crystal formation. Figure 10 is a graph showing the effect of reducing the formation of ice crystals by the recombinant antifreeze protein analog according to the present invention, wherein the A region shows ice crystals formed by water and the particles are extremely large; and the B region is formed by lysing buffer containing 8 guanidine urea. Ice 15 crystals, smaller particles; C region is a protein purified from the cytoplasmic portion of E. coli M15 (pREP4, pQE30-AFP), and D region is a medium from Escherichia coli BL21 (DE3) (PET 12a-AsAFP) Part of the purified protein, the E region is a protein purified from the soluble portion of E. coli BL21 (DE3) (pET12a-AsAFP), and the F region is not from E. coli 20 BL21 (DE3) (PET12a-AsAFP). The protein purified from the soluble fraction; the G region is a protein purified from the soluble fraction of Escherichia coli BL21 (DE3) (pET12a_〇sAFp) and the sputum region is from Escherichia coli BL21(DE3) (pET12a-OsAFP) The protein purified from the insoluble fraction. The results of the C~Η region are shown in the presence of recombinant antifreeze protein analogues. 70 1321567 Patent No. 95118228 Patent Application Specification Revision Date: October 1998! The ice crystals formed on the day were significantly smaller, and it was confirmed that the recombinant antifreeze protein analogs according to the present invention have the ability to reduce ice crystal formation. Example 7. Nucleic acid coding sequences of antifreeze protein analogs having different number of repeating units of amino acid and their carrier construction 5 This example further constructs three different antifreeze protein analog expression vectors, each of which carries There is a nucleic acid fragment encoding an anti-conne analog having a number of repeating units of different amino acids, ie, an anti-; Eastin (AFp) analog 1A (containing 4 antifreeze units), an antifreeze protein analog 2A ( Containing 8 anti-; East units) and anti-Kang analogs 3A (containing 12 anti-bed 10 units) 'their expression vectors are shown schematically in Figure 11. Figure 12 is a schematic illustration of the use of the polymerase chain reaction (PCR), using the primers shown in Table 5 below, and using the AFPI gene in the plastid vector pET-12a-AsAFP or p-RP-AFP as a template. The PCR products encoding the antifreeze protein analogs ΙΑ, 2A and 3A were amplified in large amounts, respectively. 15 Table 5. Primer Names of Nucleic Acid Coding Sequences for Synthesis of Antifreeze Protein Analogs 2, 2A and 3A Nucleotide Sequences (5·-3·) Ndelsp-AFP Ndel 5'-gatata£^iai£aataagaaaatggggaaaattgttgc- 3' (SEQ ID NO: 29) KpnI-AFP Kpnl 5'-caatat^ftiacc-patacagcgtcagatgcggc-3, (Serial Identification Number: 30) BamHI-AFP BamHl 5'-catattg1gai££tgatacagcgtcagatgcgg-3, (sequence Identification number: 31) KpnITAR-AFP Kpnl S'-tapataggtacctcttac2stggcapcggct-3* (sequence resolution: 32) BamHIGDP-AFP Bamm V-ttactteeatcccctctteceeteecaeceect-V i bed identification number: 33) SpeSalHtagTAA-AFP Sail Spe\ 5*-tagataktcgadia£itattagtggtggtggtest2ststci&gt;3' (sequence identification number: 34) Note: The restriction enzyme cleavage site located in the primer is underlined or framed to indicate 'where and ^1 and

Spel's cutting addresses overlap.

The reaction content of the PCR is 100 μm of the reaction buffer [20 mM 71 1321567 No. 095118228 patent application revision page Revision period: October 1st, 1998

Tris-HCl, 10 mM KC1, 1 mM mM (NH4) 2S04, 2 mM MgS04, 0.1% Triton® X-100, 0.1 mg/mL nuclease-free BSA], containing 400 μM each of dATP, dTTP , dGTP and dCTP, 2 μΜ amplification primers '50 ng template (pET-12a-AsAFP or p-RP-AFP) and 2.4 U /?/« 5 DNA polymerase. The reaction conditions for PCR were: 2 minutes at 94 ° C, 1 cycle; 30 seconds at 94 ° C, 30 seconds at 59 ° C, 1 minute at 72 ° C, 35 cycles; 7 minutes at 72 ° C, 1 Cycles). After the reaction was completed, the obtained PCR product was extracted once with phenol/form/isoamyl alcohol (v/v/v = 25:24:1) and chloroform/isoamyl alcohol (v/v = 24:1). . After 10, the DNA-containing solution was taken, and 2 volumes of anhydrous alcohol and 0.1 volume of sodium citrate (pH 5.2) were added and allowed to stand at -70 ° C for 1 hour to precipitate DNA. After desalt and vacuum evaporation with 70% alcohol, the DNA was dissolved back in an appropriate amount of sterile deionized water and stored at -20 ° C until use. 15 The PCR products encoding the antifreeze protein analogs 1A, 2A and 3A obtained above and the plastid pET29a (Novagen, USA) were cleaved with appropriate restriction enzymes, respectively. Then the Gene-SpinTM Extraction kit was used to recover the insert DNA. And carrier DNA. For the two cleavage products obtained, the carrier DNA and the insert DNA were ligated at a molar ratio of 1:5. After 20, the thus obtained ligation product was fed into the cells of Escherichia coli strain DH10B by an electrotransformation method. The transgenic plants were randomly selected and the plastid vector was extracted&apos; followed by restriction enzyme cleavage confirmation and DNA sequencing. The PCR fragment amplified using the primer Ndelsp-AFP and the primer SpeSalHtagTAA-AFP was cleaved with iW/el and restriction enzymes, and then the plasmid was cleaved. 72 1321567 Patent No. 095118228 Revision of the patent application Revision date: October 1, 1998 The substance was inserted into the plastid pET29a, and a plastid was constructed and named pSP1Atag. The PCR fragment amplified using the primers Ndelsp-AFP and KpnlTAR-AFP was cleaved with the //mI restriction enzyme, and then the cleavage product was inserted into the plastid pET29a to form plastid pNK. Thereafter, the PCR fragment amplified by the primers KpnIAFP and SpeSalHtagTAA-AFP and the restriction enzyme cleavage were used, and then the cleavage product was inserted into the plastid pNK, and a plastid was constructed and designated as pSP2Atag. A PCR fragment of 10 amplified by the primers BamHI-AFP and SpeSalHtagTAA-AFP and restriction enzyme cleavage were used, and then the cleavage product was inserted into the plastid pET29a to construct a plastid PBS. Thereafter, the PCR fragment amplified using the primers KpnIAFP and BamHIGDP-AFP was cleaved with the parental "I and restriction enzymes, and then the cleavage product was inserted into the plastid pBS to construct a plastid pKBS. Finally, the PCR fragment amplified by Ndelsp-AFP and 15 KpnITAR-AFP was cleaved with the paw/eI and your restriction enzyme, and then the cleavage product was inserted into the plastid pKBS, and a plastid was constructed and named pSP3Atag. The above recombinant antifreeze protein analog expression vector was individually transformed into a protein shell expressing Escherichia coli BL21 (DE3), and the resulting transformed 20 strain cells were cultured in 13/connamycin medium. Then, 0.4 mM IPTG was added for protein induction. The medium fraction and total cell inclusions were collected and analyzed for protein performance by Tricine_SDS_PAGE. Further, a recombinant anti-whitening analog in the medium portion was purified using a Ni-NTA affinity resin. The results obtained are shown in Figures 13 and 14. Purification of the weight 73 1321567 Revision No. 095,118,228 Patent Revision Revision Date: October, 1998 1 The antifreeze protein analog was further analyzed for antifreeze activity and the results are shown in Figures 16-20. 13 and 14, after induction with 4 mM IPTG, Escherichia coli BL21 (DE3) (pSP1Atag), Escherichia coli BL21 (DE3) (pSP2Atag) was 5 and E. coli BL21 (DE3) (pSP3Atag) was intracellular. The expression of antifreeze protein was observed in the part and the medium part, which showed that the recombinant antifreeze protein analog containing the basic elastase message peptide of Bacillus subtilis YaB (alkalophilic 5_7 heart YaB) can be Performance can be secreted or leaked extracellularly' and the amount of expression is at least 1 〇 mg / L or more. 10 See Figure 15 'E. coli BL21 (DE3) (pSP2Atag) can reach more than 90 mg / L; E. coli BL2i (DE3) (pSPlAtag) performance of at least 19 mg / L; five. eo &quot; BL21 (DE3) (pSP3Atag) can perform at least 10 mg/L. L. Tong et al. have embedded the synthetic anti-beam protein gene into the expression vector containing tac initiation 15 mobilization, and fused the E. coli 〇mpA message peptide coding sequence at the gene level. After induction for 32 hours, The extracellular production of anti-depressant protein was 16 mg/L (Z. deduction α/· (&quot;2000), corpse coffee and ^· hn/, (8)). In the present invention, the expression of 20 antifreeze proteins of Escherichia coli BL21 (DE3) (pSP2Atag) and Escherichia coli BL21 (DE3) (pSP 1 Atag) is higher than that reported by Tong et al., and the production cost is The time is also short. Referring to Figures 16-20, the recombinant antifreeze protein analogs 1A, 2A and 3A according to the present invention have the ability to inhibit the recrystallization of ice crystals, and at the same time, they have excellent pH stability and have a pH ranging from 2 to 12 Antifreeze activity. In the heat 74 1321567 No. 095118228 patent application revision page revision date: October 1st, 1998, 1st, the recombinant antifreeze protein analog 3A is the best, and the recombinant anti-bed protein analogues 1A and 2A are also Have a good thermal stability performance. As for the minimum inhibitory concentration (MIC) of antifreeze protein inhibition of ice crystal formation, the measured values of recombinant antifreeze protein analogs 2, 2A and 3A were 3〇网/叫, 35 5 pg/pL and 35 μδ/μί, respectively. . Based on the above, the present invention is expected to have potential for development as an antifreeze for use in industries such as food, pharmaceutical, biotechnology and genetic engineering. • All documents and patents cited in this specification are incorporated by reference into this document as a reference. In the event of a conflict, the detailed description of the case (including definitions) will prevail. While the invention has been described with reference to the specific embodiments of the invention, it will be understood that many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that the present invention be limited only by the scope of the patent application 15 as set forth in the accompanying application. 75 1321567 ji / 095118228 Patent Application Specification Revision Date: October 1998 1曰I: Figure BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows an antifreeze protein analog 1 (SEQ ID NO: 20) and an antifreeze protein analog 丨 designed according to the present invention, (the sequence identification number of the amino acid sequence ' For protein analog 1, X1 = Gly, 5 X2 - Ser, and for antifreeze protein analog 1 ', X丨 = Asp, X2 = Pro; Figure 2 schematically illustrates the use of overlap extension polymerase chain reaction Overlapping-extension polymerase chain reaction (OEPCR) &gt; Using four primers 10 with complementarity between their sequences to generate a PCR encoding the antifreeze protein analog 1 shown in Figure 1. Product, wherein: reference numerals 2 and 3 respectively denote template primers, which are complementary to each other at their 3' ends; and reference numeral 1 denotes an amplification primer for amplifying the labeling primer of the label 2 (amplifying prime r) or a forward primer; and reference numeral 4 denotes an amplification primer or a forward primer for amplifying the template primer of reference numeral 3 15. When the PCR reaction starts, the template primer will first adhere and extend (to 3 The DNA of 'to 5' is a template and extends from the 5th to the 3' direction.] Thus, the full-length DNA is generated. Then, the amplification primer starts to amplify the DNA fragment in a large amount using the full-length DNA as a template; FIG. 3 shows The construction process of a plastid vector pRP, wherein: ori-17, E. coli co/z·) plastid pACYC 177 replication origin; ρΑΜα 1 replication initiation point; A〆, ampicillin resistance Gene (ampicillin resistant gene); Tcr, tetracycline resistant gene; P, promoter; SD, SD sequence 76 1321567 Patent 095118228 Patent application revision page Revision date: October 1, 1998 Shine-Dalgarno sequence; pre, the DNA coding sequence of the signal peptide; pro, the DNA coding sequence of the prosequence, which is folded and secreted by the protease. related Mature, mature protease DNA encoding 5 sequence, MCS 'multiple cloning site; β/eC, α/eE downstream DNA sequence, which may contain a transcription terminator Figure 4 shows the results of agarose gel electrophoresis of PCR products obtained using the primers in Table 2 and using the overlap-extension polymerase chain reaction (10 OEPCR), wherein the diameter is 100 Bp DNA molecular marker; PATH 1: PCR product obtained by OEPCR using primers AFP-, AFP-SS, AFP1 and AFP2; and diameter 2: using primers AFP-5i?mHI, AFP-SS, AFP1 and AFP2 to perform PCR products obtained by OEPCR; 15 Figure 5 shows the construction process of the plastid vector PRP-AFP; Figure 6 shows the construction process of the plastid vector PQE30-AFP, wherein: /acO ' /ac promoter; ori -pBR322, the origin of replication of pBR322; the PQE30 terminator, which contains the Lambda transcription termination region and the T1 transcription termination region. Ion region); Figure 7 shows the construction of a plastid vector pET12a-AsAFP, in which the message peptide is the message peptide of E. coli outer membrane protease 〇 mpT; Figure 8 shows the construction process of the plastid vector pET12a-OsAFP; 1321567, j Revision No. 095118228 Patent Application Revision Date: October 1, 1998 Figure 9 shows the recombinant form produced by the large intestine rod-shaped transgenic plants (five co/z·transf〇nnants) and purified. Results of Tricine_SDs_PAGE electrophoresis of antifreeze protein analogs, in which the diameter Μ: low molecular weight protein marker; 丨: protein purified from the cytoplasm 5 fraction of E. coli M15 (pREP4, PQE30-AFP); Protein purified from the medium portion of Escherichia coli BL21(DE3)(pET12a-AsAFP)*(me£jium fracti〇n); diameter 3: soluble in cells from Escherichia coli BL21 (DE3) (pET 12a-As AFP) Protein purified by intracellular soluble fraction; diameter 4: protein purified from intracellular insoluble fraction of Escherichia coli 10 BL21(DE3)(pET12a-AsAFP); Path 5: from Escherichia coli BL21 a protein purified from the intracellular soluble fraction of (DE3) (pET12a-OsAFP); and a protein purified from the intracellular insoluble portion of E. coli BL21 (DE3) (pET12a_OsAFP); and 15 Figure 10 shows The effect of reducing ice crystal formation by the recombinant antifreeze protein analog of the present invention, wherein panel A: water; panel B: lysate buffer containing 8 Μ urea (urea) Ysis buffer) ; c region (panel C): protein purified from the cytoplasmic portion of E. coli M15 (PREP4, PQE30-AFP); D region (panel D): from E. coli 20 BL21 (DE3) (PETl2a-AsAFP) Part of the medium purified from the protein; £ area coffee 1^1 ugly): from E. coli muscle 21 (〇 ugly 3) (? ugly Ding 12 &amp; - eight 8 eight?细胞) a protein purified from the intracellular soluble fraction; F-region (panelF): a protein purified from the intracellular insoluble fraction of Escherichia coli muscle 21 (0£3) 1'123-八3 八卩卩) ;G-zone (panel G): purification of intracellular soluble fractions from E. coli BL21 (DE3) (pET12a-78 1321567, 095,118, 228, pp. 095, 218, 228, pp. Protein; and Η(panel Η): a protein purified from the insoluble portion of the cell of Escherichia coli BL21(DE3) (pET12a-OsAFP); Figure 11 is a schematic diagram showing three antibodies according to the invention Frozen protein 5 (AFP) analog ΙΑ, 2A and 3A expression vectors pSP1Atag, pSP2Atag and pSP3Atag gene composition, wherein: Sd, SD sequence; ίρ, message skin coding DNA, α力 7 'anti; East protein gene; And sigh, (His)6 DNA; Figure 12 schematically illustrates the use of the polymerase chain reaction (PCR), using the primers described in Table 5 below, Example 7, and with 10 pET-12a - AFPI gene in AsAFP or p-RP_AFP as a template to generate three types of coding according to the present invention There are PCR products of the antifreeze protein analogs 2, 2A and 3A with different numbers of repeating amino acid units, where: π, the message wins the DNA, and? 'Anti; East Protein Gene; and Rushen, (His)6 DNA; 15 Figure I3 shows Escherichia coli BL21 (DE3) (pSP1Atag), Escherichia coli BL21 (DE3) (pSP2Atag) and Escherichia coli BL21 (DE3) (pSP3Atag) Results of Tricine-SDS PAGE analysis of proteins collected from the medium fraction and total cell lysate after induction with 1 mM IPTG; 20 Figure 14 shows Escherichia coli BL21(DE3)(pSPlAtag), large intestine Bacillus BL21 (DE3) (pSP2Atag) and Escherichia coli BL21 (DE3) (pSP3Atag) Tricine-SDS PAGE of recombinant antifreeze protein analog ΙΑ, 2A and 3A purified from the medium fraction after induction with 1 mM IPTG for 3 hours The results of the analysis, wherein the diameter 1 is: from the Escherichia coli BL21 (DE3) (pSP1Atag) 79 1321567 No. 095118228 patent application revised page revision date: October 1998 1 曰 medium part of the recombinant antifreeze protein analog 1A; Path 2: Recombinant antifreeze protein analog 2A from the medium portion of E. coli BL21 (DE3) (pSP2Atag); and Run 3: recombinant antifreeze protein similar to the medium portion of E. coli BL2l (DE3) (pSP3Atag) 3A; 5 Figure 15 shows the cell growth of Escherichia coli BL21 (DE3) transformed with the antifreeze protein (AFP) analogs ία, 2A and 3A expression vectors pSP1Atag, pSP2Atag and pSP3Atag, and recombinant antifreeze protein analogues 1A, 2A and 3 A secretion yield; Figure 16 shows the results of the ability of recombinant antifreeze protein analogues 1 a, 2A and 3A to inhibit the recrystallization of ice crystal 1 ,, wherein the concentrations of recombinant antifreeze protein analogues ία, 2A and 3A are 400 pg/pL; C is a control group (PBS buffer, 50 mM NaH2P04, 300 mM NaCl, pH 8.0); and CAFPI is a commercially available type I antifreeze protein (A/F protein Canada Inc.), The protein is isolated from the blood of cold ocean fish with a purity between 15 and 99%; Figure 17 shows the observation of a single ice crystal configuration of the recombinant anti-beam protein analogues ia, 2A and 3A, Recombinant antifreeze protein analogs were at a concentration of 400 pg/pL for octa-8, arsenic and 3A; C was a control group (PBS buffer, 50 mM NaH2P04, 300 mM NaCl, pH 8_0); and cAFPI was a commercial sale 20 Type 1 anti-beam protein (A/F protein Canada Inc.), the protein is from The purity of the fish with cold fish fish is between 90 and 99%; Figure 18 shows the pH stability of the recombinant antifreeze protein analogues 1A, 2A and 3A, wherein the recombinant antifreeze protein is similar. The concentrations of the materials 1A, 2A and 3A are all 80 1321567. The amendment of the specification of the patent application No. 095118228 is amended on the date of October: 1 October, 400 μβ / μί; and C is the pair of Zhao έ rpF^ and (PBS ^ flushing 5 mM NaH2P04, 300 mM NaCl, pH 8.0); Figure 19, - indicates the thermal stability of the heavy-type anti-beam protein analog with 3-8, wherein the recombinant anti-freeze protein analogues 1-8, 2-8 The concentration of each of the three and eight is 4〇〇5 (four) called 'and C is the control group (PBS buffer '50 mM NaH2P04, 300 mM NaCl, pH 8.0); and Figure 20 shows the recombinant antifreeze protein analogue ία, 2A and 3A minimum inhibition concentration (MIC). [Description of main component symbols] (None) 81 1321567 Patent Application No. 095118228 Revision Date: October 1st, 1998 Sequence Listing &lt;110&gt; National Chung Hsing University &lt;120&gt; Antifreeze Protein Analogs and Nucleic Acids thereof Coding sequences and their production and application &lt;130&gt; AFP &lt;160> 44 &lt;170&gt; Patentln version 3.3

&lt;210〉 1 &lt;211&gt; 37 &lt;212&gt; PRT &lt;213> Winter Flounder &lt;400〉 1

Asp Thr Ala Ser Asp Ala Ala Ala Ala Ala Ala Leu Thr Ala Ala Asn 15 10 15

Ala Lys Ala Ala Ala Glu Leu Thr Ala Ala Asn Ala Ala Ala Ala Ala 20 25 30

Ala Ala Thr Ala Arg 35 &lt;210〉 2 &lt;211&gt; 37 &lt;212&gt; PRT &lt;213> Winter Flounder &lt;400〉 2

Asp Thr Ala Ser Asp Ala Ala Ala Ala Ala Ala Leu Thr Ala Ala Asn 15 10 15

Ala Lys Ala Ala Ala Lys Leu Thr Ala Asp Asn Ala Ala Ala Ala Ala 20 25 30

Ala Ala Thr Ala Arg 35 1321567 Revision No. 095118228 Patent Application Revision Date: October 1st, 1998 &lt;210> 3 &lt;211&gt; 56

&lt;212&gt; PRT &lt;213> Winter Flounder &lt;400&gt; 3

Asp Thr Ala Ser Asp Ala Ala Ala Ala Ala Ala Ala Thr Ala Ala Thr 15 10 15

Ala Ala Ala Ala Ala Ala Ala Thr Ala Val Thr Ala Ala Lys Ala Ala 20 25 30

Ala Leu Thr Ala Ala Asn Ala Ala Ala Ala Ala Ala Ala Thr Ala Ala 35 40 45

Ala Ala Arg Gly Ala Ala Arg Gly 50 55 &lt;210&gt; 4 &lt;211&gt; 12 &lt;212> PRT &lt;213&gt; Winter Flounder &lt;220&gt;&lt;221>MISC_Features&lt;222&gt; (1). (12) &lt;223>5--like protein N-terminal partial amino acid sequence 歹ϋ &lt;220&gt;&lt;221>MISCjt sign &lt;222>(1) _. (1) &lt; 223> Xaa can be Asn, Thr or Ala &lt;300> &lt;301&gt; CB Marshall et al. &lt;303&gt; J. Biol. Chem. &lt;304&gt; 280 2 1321567 Patent Publication No. 095118228 Revision date: October 1st, 1998&lt;305&gt; 18 &lt;306> 17920-17929 &lt;307&gt; 2005-05-06 &lt;400&gt; 4

Xaa He Asp Pro Ala Ala Arg Ala Ala Ala Ala Ala 1 5 10

&lt;210> 5 &lt;211&gt; 48 &lt;212&gt; PRT &lt;213>Yellowtail flounder &lt;400&gt;

Asp Thr Ala Ser Asp Ala Ala Ala Ala Ala Ala Ala Thr Ala Ala Ala 15 10 15

Ala Ala Lys Ala Ala Ala Asp Thr Ala Ala Ala Ala Ala Lys Ala Ala 20 25 30

Ala Asp Thr Ala Ala Ala Ala Ala Glu Ala Ala Ala Ala Thr Ala Arg 35 40 45

&lt;210> 6 &lt;211&gt; 40 &lt;212&gt; PRT &lt;213> Alaskan plaice &lt;400> 6

Asp Thr Ala Ser Asp Ala Ala Ala Ala Ala Ala Ala Thr Ala Ala Ala 15 10 15

Ala Lys Ala Ala Ala Glu Lys Thr Ala Arg Asp Ala Ala Ala Ala Ala 20 25 30 3 1321567 Revision No. 095118228 Patent Application Revision Date: October 1st, 1998

Ala Ala Thr Ala Ala Ala Ala Arg 35 40

&lt;210&gt; 7 &lt;211&gt; 32 &lt;212&gt; PRT &lt; 213 &gt; shorthorn sculpin &lt;400&gt;

Met Asn Ala Pro Ala Arg Ala Ala Ala Lys Thr Ala Ala Asp Ala Leu 15 10 15

Ala Ala Ala Lys Lys Thr Ala Ala Asp Ala Ala Ala Ala Ala Ala Ala 20 25 30

&lt;210〉 8 &lt;211&gt; 45 &lt;212&gt; PRT &lt; 213 &gt; Shorthorn sculpin &lt;400&gt; 8

Met Asn Gly Glu Thr Pro Ala Gin Lys Ala Ala Arg Leu Ala Ala Ala 15 10 15

Ala Ala Leu Ala Ala Lys Thr Ala Ala Asp Ala Ala Ala Lys Ala Ala 20 25 30

Ala Lys Ala Ala Ala lie Ala Ala Ala Ala Ala Ser Ala 35 40 45

&lt;210&gt; 9 &lt;211&gt; 33 &lt;212&gt; PRT &lt; 213 &gt; 213>Grubby sculpin &lt;400> 9 4 1321567 Patent Application No. 095118228 Revision page Revision period: 98 October 1st

Met Asp Ala Pro Ala lie Ala Ala Ala Lys Thr Ala Ala Asp Ala Leu 15 10 15

Ala Ala Ala Lys Lys Thr Ala Ala Asp Ala Ala Ala Ala Ala Ala Lys 20 25 30

Pro &lt;210&gt; 10 &lt;211&gt; 40 &lt;212> PRT &lt;213&gt;Bronze bed Dufy &lt;400&gt;

Met Asp Gly Glu Thr Pro Ala Gin Lys Ala Ala Arg Leu Ala Ala Ala 15 10 15

Ala Ala Ala Leu Ala Ala Lys Thr Ala Ala Asp Ala Ala Ala Lys Ala 20 25 30

Ala Ala He Ala Ala Ala Ala Ala 35 40

&lt;210> 11 &lt;211&gt; 39 &lt;212&gt; PRT &lt;213>Arctic sculpin &lt;400> 11

Met Asp Gly Glu Thr Pro Ala Gly Lys Ala Ala Arg Leu Ala Ala Ala 15 10 15

Ala Ala Leu Ala Ala Lys Thr Ala Ala Asp Ala Ala Ala Lys Ala Ala 20 25 30

Ala He Ala Ala Ala Ala Ala 35 5 1321567 Patent Application No. 095118228 Revision Date Amendment Date: October 1st, 1998 &lt;210> 12 &lt;211> 33 &lt;212&gt; PRT &lt;213&gt; Arctic Du Father Fish &lt;400〉 12

Met Asp Ala Pro Ala Arg Ala Ala Ala Lys Thr Ala Ala Asp Ala Leu 1 5 10 15

Ala Ala Ala Asn Lys Thr Ala Ala Asp Ala Ala Ala Ala Ala Ala Ala 20 25 30

Ala

&lt;210> 13 &lt;211&gt; 113 &lt;212> PRT &lt; 213 &gt; Atlantic Lionfish (Atlantic snailfish) &lt;300 &lt;308&gt; NCBI/AAR22528 &lt;309&gt; 2005-10-19 &lt;313&gt; (1)..(113) &lt;400〉 13

Met Ala Ala Ala Thr Pro Ala Gin Arg Ala Ala Ala Thr Ala Thr Ala 15 10 15

Ala Ala Ala Ala Ala Ala Ser Ala Ala Ala Ala Ala Ala Ala Thr Thr 20 25 30

Ala Ser Thr Ala Ala Lys Val Ser Ala Gly Ala Ala Ala Thr Ala Ala 35 40 45

Ala Ala Val Val Ala Ala Lys Asn Ala Ala Thr Ala Val Ala Pro Asn 50 55 60 6 1321567 Revision No. 095118228 Patent Application Revision Date: October 1st, 1998

Thr Gly Ala lie Thr Ala Ala Thr Ala Ala Ser Ala Thr Ala Ala Ala 65 70 75 80

Ala Ala Lys Ala Ala Gin Ala Thr Ala Asp Ala Ala Ala Thr Lys Ala 85 90 95

Ala Ala Ala Ala Val Thr Ser Lys Ala Ala Ala Ala Ala Leu Ala Ala 100 105 110

Leu

&lt;210> 14 &lt;211&gt; 113 &lt;212&gt; PRT &lt; 213 &gt; 213> lionfish (Dusky snailfish) &lt;300&gt;&lt;308&gt; NCBI/AAR22529 &lt;309&gt; 2005-10-19 &lt;313 〉 (1)..(113) &lt;400〉 14

Met Ala Ala Ala Thr Pro Ala Gin Arg Ala Ala Ala Thr Ala Thr Ala 15 10 15

Ala Ala Ala Ala Ala Ala Ser Ala Ala Ala Ala Ala Ala Ala Thr Thr 20 25 30

Ala Ser Thr Ala Ala Lys Val Ser Ala Gly Ala Ala Ala Thr Ala Ala 35 40 45 i

Ala Ala Val Val Ala Ala Lys Asn Ala Ala Thr Ala Val Ala Pro Asn 50 55 60

Thr Gly Ala lie Thr Ala Ala Thr Ala Ala Ser Ala Thr Ala Ala Ala 65 70 75 80

Ala Ala Lys Ala Ala Gin Ala Thr Ala Asp Ala Ala Ala Thr Lys Ala 85 90 95 7 1321567 Revision No. 095118228 Patent Application Revision Date: October 1st, 1998

Ala Ala Ala Ala Val Thr Ser Lys Ala Ala Ala Ala Leu Pro Arg Arg 100 105 110

Leu &lt;210&gt; 15 &lt;211&gt; 38 &lt;212&gt; PRT &lt;213&gt;&gt; chilly halibut &lt;400&gt;

Met Asp Ala Pro Ala Arg Ala Ala Ala Ala Thr Ala Ala Ala Ala Lys 15 10 15

Ala Ala Ala Glu Ala Thr Lys Ala Ala Ala Ala Lys Ala Ala Ala Ala 20 25 30

Thr Lys Ala Ala Ala His 35 &lt;210> 16 &lt;211&gt; 39 &lt;212&gt; PRT &lt;213> Winter Flounder &lt;400> 16

Met Asp Ala Pro Ala Lys Ala Ala Ala Ala Thr Ala Ala Ala Ala Lys 15 10 15

Ala Ala Ala Glu Ala Thr Ala Ala Ala Ala Ala Lys Ala Ala Ala Ala 20 25 30

Thr Lys Ala Gly Ala Ala Arg 8 35 1321567 Revision No. 095118228 Patent Application Revision Date: October 1st, 1998 &lt;210> 17 &lt;211&gt; 39

&lt;212&gt; PRT &lt;213&gt; Winter Flounder &lt;300&gt;&lt;301&gt; WK Low et al. &lt;303&gt; J. Biol. Chem. &lt;304> 276 &lt;305&gt; 15 &lt;306> 11582- 11589 &lt;307〉 2001-04-13 &lt;400&gt; 17

Met Asp Ala Pro Ala Lys Ala Ala Ala Ala Thr Ala Ala Ala Ala Lys 15 10 15

Ala Ala Ala Glu Ala Thr Ala Ala Ala Ala Ala Lys Ala Ala Ala Asp 20 25 30

Thr Lys Ala Lys Ala Ala Arg 35 &lt;210&gt; 18 &lt;211&gt; 92 &lt;212&gt; PRT &lt;213> Short-horned Dugong &lt;400&gt; 18

Met Ala Ala Ala Ala Lys Ala Ala Glu Ala Ala Ala Met Ala Ala Ala 1 5 10 15

Asn Ala Ala Glu Ala Ala Ala Thr Lys Ala Ala Asp Ala Ala Ala Ser 20 25 30

Ala Ala Ala Ala Ala lie Ala Ala lie Ala Glu Ala Ala Glu Ala Ala 35 40 45 9 1321567 Revision No. 095118228 Patent Application Manual Revision Date: October 1st, 1998

Glu Ala Ala Ala Thr Lys Ser Ala Asn Val Ala Ala Ala Ala Ala Ala 50 55 60

Thr Ser Ala Ala Ala Ala Ala Lys Ala Thr Ala Asn Ala Ala Ala Ala 65 70 75 80

Ala Ser Ala Ala Ala Ala Ala Ala Ala Ala Val Ala 85 90

&lt;210&gt; 19 &lt;211&gt; 42 &lt;212&gt; PRT &lt;213&gt; Longhorn sculpin &lt;300&gt;&lt;301&gt; WK Low et al. &lt;303&gt; J. Biol. Chera &lt;304&gt; 276 &lt;305&gt; 15 &lt;306> 11582-11589 &lt;307> 2001-04-13 &lt;400&gt; 19

Met Asp Ala Pro Ala Lys Ala Ala Ala Lys Thr Ala Ala Asp Ala Lys 15 10 15

Ala Ala Ala Ala Lys Thr Ala Ala Asp Ala Leu Ala Ala Ala Asn Lys 20 25 30

Thr Ala Ala Ala Ala Lys Ala Ala Ala Lys 35 40 &lt;210&gt; 20 &lt;211&gt; 56 &lt;212&gt; PRT &lt;213>ΛΧ 10 1321567 No. 095118228 Patent Application Manual Amendment Page Amendment Date: 98 years 10 1曰&lt;220> &lt;223>Antifreeze Protein Analog 1 &lt;400> 20

Gly Ser Asp Thr Ala Ser Asp Ala Ala Ala Ala Ala Ala Leu Thr Ala 15 10 15

Ala Asn Ala Lys Ala Ala Ala Glu Leu Thr Ala Ala Asn Ala Lys Ala 20 25 30

Ala Ala Glu Leu Thr Ala Ala Asn Ala Ala Ala Ala Ala Ala Ala Thr 35 40 45

Ala Arg His His His His His His 50 50 &lt;210> 21 &lt;211&gt; 56 &lt;212&gt; PRT &lt; 213 &gt; 213 &gt;&lt;220&gt;&lt;223&gt; 223 &quot;Antifreeze Protein Analog Γ &lt;400&gt;

Asp Pro Asp Thr Ala Ser Asp Ala Ala Ala Ala Ala Ala Leu Thr Ala 15 10 15

Ala Asn Ala Lys Ala Ala Ala Glu Leu Thr Ala Ala Asn Ala Lys Ala 20 25 30

Ala Ala Glu Leu Thr Ala Ala Asn Ala Ala Ala Ala Ala Ala Ala Thr 35 40 45

Ala Arg His His His His His His His 50 50 11 1321567 Patent Application No. 095118228 Revision Date Revision Date: October 1st, 1998&lt;210> 22 &lt;211&gt; 36 &lt;212&gt; DNA &lt;213&gt;&lt;220&gt;&lt;223&gt; Synthetic primer for PCR amplification of DNA sequence encoding AFP analog 1 &lt;220&gt;&lt;221&gt;2 restriction site &lt;222&gt; (7)..(12) &lt;;223>Recognized by BamHI&lt;400> 22 caatcaggat ccgatacagc gtcagatgcg gcagcg 36 &lt;210> 23 &lt;211> 41 &lt;212&gt; DNA &lt;213>manual &lt;220&gt;&lt;223> for encoding AFP The synthetic primer for PCR amplification of the DNA sequence of the analog 11 &lt;220> &lt;221&gt; restriction address &lt;222> (7)..(12) &lt;223&gt; is recognized by 3卩111&lt;400〉 23 aatttagcat gcaagatcct gatacagcgt cagatgcggc a 41 &lt;210> 24 &lt;211&gt; 46 &lt;212&gt; DNA &lt;213&gt; ΛΧ12 1321567 Revision Date: October 01, pp. 095118228 Patent Application Specification Revision Page &lt;220&gt;&lt;223&gt;&gt;221>Restriction of synthetic primers for the amplification of the sequence of AFP analog 1 or 1. &lt;220&gt;&lt;221&gt; Address &lt;222> (7)..(12) &lt;223> is recognized by Sail&lt;220&gt;&lt;221>RestrictedAddress&lt;222> (11)..(16) &lt;223&gt; The port 61 recognizes &lt;400&gt; 24 atttaggtcg actagttatt agtggtggtg gtggtggtgt cttgcg 46 &lt;210&gt; 25 &lt;211> 96 &lt;212&gt; DNA &lt;213&gt;manual &lt;220&gt;&lt;223&gt; Synthetic primer for PCR amplification of 1 or Γ DNA sequence &lt;400&gt; 25 gatcctgata cagcgtcaga tgcggcagcg gctgcggctc ttactgcagc taacgcaaaa 60 gccgcagctg aactgacggc tgccaatgcg aaggcg 96 &lt;210&gt; 26 &lt;211&gt; 96 &lt;212&gt; DNA &lt;213&gt;&lt;220&gt;&lt;223&gt; Synthetic primer for PCR amplification of DNA sequence of &lt;400&gt; 26, AFP analog 1 or Γ13 13 567 567 Revision No. 095118228 Patent Application Revision Date: 98 years October 1曰ttagtggtgg tggtggtggt gtcttgcggt ggcagcggct gcagcagcag cgttagctgc 60 tgtgagctcg gccgccgcct tcgcattggc agccgt 96 &lt;210> 27 &lt;211&gt; 195 &lt;212&gt; DNA &lt;213>ΛΧ&lt;220&gt;&lt;223&gt; substantially corresponds to coding AFP class The PCR product of the DNA sequence of the like 1 &lt;220&gt;&lt;221&gt; restriction address &lt;222> (7)..(12) &lt;223> is recognized by BamHI &lt;220> &lt;221&gt; Address &lt;222> (180)..(185) &lt;223&gt; is recognized by 3061&lt;220> &lt;221>Restricted Address&lt;222> (184)..(189) &lt;223> is Sail Recognized &lt;400&gt; 27 caatcaggat ccgatacagc gtcagatgcg gcagcggctg cggctcttac tgcagctaac 60 gcaaaagccg cagctgaact gacggctgcc aatgcgaagg cggcggccga gctcacagca 120 gctaacgctg ctgctgcagc cgctgccacc gcaagacacc accaccacca ccactaataa 180 ctagtcgacc taaat 195

&lt;210&gt; 28 &lt;211&gt; 203 &lt;212&gt; DNA 14 1321567 Patent Application No. 095118228 Revision page revision period: October 1st, 1998&lt;213&gt;&&lt;220&lt;223&gt; The PCR product substantially corresponding to the DNA sequence encoding 1' of the AFP analog &lt;220> &lt;221&gt; restriction site &lt;222&gt; (7)..(12) &lt;223&gt; is recognized by SphI &lt;220 〉 &lt;221&gt;Restricted Address&lt;222&gt; (188)..(193) &lt;223&gt; Recognized by Spel&lt;220&gt;&lt;221>RestrictedAddress&lt;222> (192)..(197 ) &lt; 223> are & lt the recognized Sail; 400> 28 60 120 180 203 aatttagcat gcaagatcct gatacagcgt cagatgcggc agcggctgcg gctcttactg cagctaacgc aaaagccgca gctgaactga cggctgccaa tgcgaaggcg gcggccgagc tcacagcagc taacgctgct gctgcagccg ctgccaccgc aagacaccac caccaccacc actaataact agtcgaccta aat &lt; 210> 29 &lt; 211 &gt; 38 &lt;212&gt; DNA &lt; 213 &gt; artificial &lt;220&gt;&lt;223&gt; Synthetic Bow Tweezers for PCR amplification of DNA sequences encoding AFP analogs 1A, 2A or 3A &lt;220&gt; Address &lt;222> (7)..(12) 15 1321567第09511822 Revision No. 8 Patent Application Revision Amendment: October 1st, 1998 &lt;223> Recognized by Ndel &lt;400> 29 gatatacata tgaataagaa aatggggaaa attgttgc 38 &lt;210&gt; 30 &lt;211&gt; 32 &lt;212&gt; DNA &lt;213>Artifical &lt;220&gt;&lt;223&gt;&gt;220>Recombinant primer for PCR amplification of DNA sequence encoding AFP analog 2A or 3A &lt;220> &lt;221>restricted address&lt;222&gt; 7)..(12) &lt;223> is recognized by ΚρηΙ&lt;400> 30 caatatggta ccgatacagc gtcagatgcg gc 32 &lt;210&gt; 31 &lt;211&gt; 32 &lt;212> DNA &lt;213&gt; Artificial &lt;220&gt;&lt;223&gt; Synthetic primer for PCR amplification of DNA sequence encoding AFP analog 3A &lt;220&gt;&lt;221>restrictedaddress&lt;222> (7)..(12) &lt;223> by BamHI Recognized &lt;400&gt; 31 catattggat cctgatacag cgtcagatgc gg 32 16 1321567 Patent Application No. 095118228 Revision Date: October 1st, 曰 &lt;210&gt; 32 &lt;211&gt; 31 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;&lt;223&gt; Synthetic primer for PCR amplification of DNA sequence encoding AFP analog 2A or 3A Lt220> &lt;221>Restricted address&lt;222> (7)..(12) &lt;223&gt; is recognized by ΚρηΙ&lt;400> 32 tagataggta cctcttgcgg tggcagcggc t 31 &lt;210&gt; 33 &lt;211&gt; 33 &lt;212&gt; DNA &lt;213> AX&lt;220&gt;&lt;223&gt; Synthetic primer for PCR amplification of DNA sequence encoding AFP analog 3A &lt;220&gt;&lt;221&gt; restriction site&lt; 222> (7)..(12) &lt;223&gt; is recognized by 8&1&lt;400&gt; 33 ttacttggat cccctcttgc ggtggcagcg get 33 &lt;210&gt; 34 &lt;211&gt; 42 &lt;212&gt; DNA &lt;213&gt; 1717 1321567 No. 095118228 Patent §# Case Correction Page Revision Date: October 1, 1998 &lt;220&gt;&lt;223&gt; For the compilation of the sequence of fiiAFP analogues 1A, 2 or 38 Increased Synthetic Primer &lt;220&gt;&lt;221>RestrictedAddress&lt;222&gt; (7)..(12) &lt;223>Recognized by Sail&lt;220&gt;&lt;221&gt;RestrictedAddress&lt;222&gt; (11)..(16) &lt;223&gt; is recognized by 3?61&lt;400> 34 tagatagtcg actagttatt agtggtggtg gtggtggtgt ct 42 &lt;210&gt; 35 &lt;211&gt; 174 &lt;212> DNA &lt;213&gt;&Lt; 220> &lt; 223 &gt; encoding AFP analogs 1A is the DNA sequence &lt; 400> 35 gccgagggat ccgatacagc gtcagatgcg gcagcggctg cggctcttac tgcagctaac 60 gcaaaagccg cagctgaact gacggctgcc aatgcgaagg cggcggccga gctcacagca 120 gctaacgctg ctgctgcagc cgctgccacc gcaagacacc accaccacca ccac 174 &lt; 210 &gt; 36 &lt; 211 &gt; 324 &lt;212&gt; DNA &lt;213>AX&lt;220&gt;&lt;223&gt; DNA sequence encoding AFP analog 2A 18 1321567 Patent No. 095118228 Patent application revision page Revision date: October 1st, 1998 ; 400> 36 gccgagggat ccgatacagc gtcagatgcg gcagcggctg cggctcttac tgcagctaac 60 gcaaaagccg cagctgaact gacggctgcc aatgcgaagg cggcggccga gctcacagca 120 gctaacgctg ctgctgcagc cgctgccacc gcaagaggta ccgatacagc gtcagatgcg 180 gcagcggctg cggctcttac tgcagctaac gcaaaagccg cagctgaact gacggctgcc 240 aatgcgaagg cggcggccga gctcacagca gctaacgctg ctgctgcagc cgctgccacc 300 gcaagacacc accaccacca ccac 324 &lt; 210 &gt; 37 &lt; 211 &gt; 477 &lt;212&gt; DNA &lt;213&gt;&lt;220&gt;&lt;223&gt; DNA sequence encoding AFP analog 3A &Lt; 400 &gt; 37 gccgagggat ccgatacagc gtcagatgcg gcagcggctg cggctcttac tgcagctaac 60 gcaaaagccg cagctgaact gacggctgcc aatgcgaagg cggcggccga gctcacagca 120 gctaacgctg ctgctgcagc cgctgccacc gcaagaggta ccgatacagc gtcagatgcg 180 gcagcggctg cggctcttac tgcagctaac gcaaaagccg cagctgaact gacggctgcc 240 aatgcgaagg cggcggccga gctcacagca gctaacgctg ctgctgcagc cgctgccacc 300 gcaagagggg atcctgatac agcgtcagat gcggcagcgg ctgcggctct tactgcagct 360 aacgcaaaag ccgcagctga actgacggct Gccaatgcga aggcggcggc cgagctcaca 420 gcagctaacg ctgctgctgc agccgctgcc accgcaagac accaccacca ccaccac 477 &lt;210&gt; 38 &lt;211&gt; 58 &lt;212&gt; PRT &lt;213&gt;ΛΧ&lt;220&gt;&lt;223&gt; Recombinant antifreeze protein analog 1A amine Base acid sequence 19 1321567 Revision No. 095118228 Patent Application Revision Date: October 1st, 1998 &lt;400> 38

Ala Glu Gly Ser Asp Thr Ala Ser Asp Ala Ala Ala Ala Ala Ala Leu 1 5 10 15

Thr Ala Ala Asn Ala Lys Ala Ala Ala Glu Leu Thr Ala Ala Asn Ala 20 25 30

Lys Ala Ala Ala Glu Leu Thr Ala Ala Asn Ala Ala Ala Ala Ala Ala 35 40 45

Ala Thr Ala Arg His His His His His His 5050 &lt;210&gt; 39 &lt;211&gt; 108 &lt;212&gt; PRT &lt; 213 &gt; 213 &gt;&lt;220&lt; 223 &gt; 223 &gt; Recombinant Antifreeze Protein Analog 2A Amino acid sequence &lt;400> 39

Ala Glu Gly Ser Asp Thr Ala Ser Asp Ala Ala Ala Ala Ala Ala Leu 15 10 15

Thr Ala Ala Asn Ala Lys Ala Ala Ala Glu Leu Thr Ala Ala Asn Ala 20 . 25 30

Lys Ala Ala Ala Glu Leu Thr Ala Ala Asn Ala Ala Ala Ala Ala Ala 35 40 45

Ala Thr Ala Arg Gly Thr Asp Thr Ala Ser Asp Ala Ala Ala Ala Ala 50 55 60

Ala Leu Thr Ala Ala Asn Ala Lys Ala Ala Ala Glu Leu Thr Ala Ala 65 70 75 80

Asn Ala Lys Ala Ala Ala Glu Leu Thr Ala Ala Asn Ala Ala Ala Ala 85 90 95 20 1321567 Patent Revision No. 095118228 Revision Date: October 1st, 1998

Ala Ala Ala Thr Ala Arg His His His His His His 100 105 &lt;210&gt; 40 &lt;211&gt; 159 &lt;212>PRT &lt;213&gt; Artificial &lt;220&gt;&lt;223&gt; Recombinant antifreeze protein analog Amino acid sequence of 3A &lt;400&gt; 40

Ala Glu Gly Ser Asp Thr Ala Ser Asp Ala Ala Ala Ala Ala Ala Leu 15 10 15

Thr Ala Ala Asn Ala Lys Ala Ala Ala Glu Leu Thr Ala Ala Asn Ala 20 25 30

Lys Ala Ala Ala Glu Leu Thr Ala Ala Asn Ala Ala Ala Ala Ala Ala 35 40 45

Ala Thr Ala Arg Gly Thr Asp Thr Ala Ser Asp Ala Ala Ala Ala Ala 50 55 60

Ala Leu Thr Ala Ala Asn Ala Lys Ala Ala Ala Glu Leu Thr Ala Ala 65 70 75 80

Asn Ala Lys Ala Ala Ala Glu Leu Thr Ala Ala Asn Ala Ala Ala Ala 85 90 95

Ala Ala Ala Thr Ala Arg Gly Asp Pro Asp Thr Ala Ser Asp Ala Ala 100 105 110

Ala Ala Ala Ala Leu Thr Ala Ala Asn Ala Lys Ala Ala Ala Glu Leu 115 120 125

Thr Ala Ala Asn Ala Lys Ala Ala Ala Glu Leu Thr Ala Ala Asn Ala 130 135 140

Ala Ala Ala Ala Ala Ala Thr Ala Arg His His His His His His 145 150 155 21 1321567 Patent No. 095118228 Patent Revision Revision Date: October 1st, 1998 &lt;210&gt; 41 &lt;211&gt; 42 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;&lt;223&gt;&gt;223>Synthesis primer for PCR amplification of DNA sequence containing an a1eE message peptide coding sequence&lt;220&gt;&lt;221&gt;&lt;222&gt; (7)..(12) &lt;223> is recognized by EcoRI&lt;400> 41 tagactgaat tcgtatattt cccaatagct aaaaaggttt cc 42 &lt;210> 42 &lt;211&gt; 54 &lt;212&gt; DNA &lt;213&gt;&lt;220&gt;&lt;223&gt; of AX for PCR amplification of a DNA sequence comprising an aleE message peptide coding sequence &lt;220&gt;&lt;221> restriction address &lt;222&gt; (7) ..(12) &lt;223&gt; is recognized by 3?61&lt;220&gt;&lt;221>P mandatory address &lt;222> (13)..(18) &lt;223&gt; is recognized by Xhol&lt;220&gt;&lt;221&gt;RestrictedAddress&lt;222&gt; (16)..(21) 22 1321567 Patent Application No. 095118228 Revision Date Revision Date: 98 years &lt;223> Distinguished by SacI &lt;220&gt;&lt;221&gt; Restricted Address &lt;222> (23)..(28) &lt;223&gt; Recognized by SphI &lt;220&gt;&lt;221&gt; Restricted Address &lt;222&gt; (30). (35) &lt;223&gt; Recognized by BaraHI &lt;400&gt; 42 taaatcacta gtctcgagct cagcatgctg gatccctcgg ctgcttgtgc gate &lt;210> 43 &lt;211&gt; 244 &lt;212> DNA &lt;213&gt; Artificial &lt;220&gt;223&gt; PCR product carrying an aleE message peptide coding sequence and SD sequence &lt;220> &lt;221&gt; restriction address &lt;222&gt; (7)..(12) &lt;223&gt; recognized by EcoRI&lt;220&gt;&lt;221&gt; SD sequence (Shine-Dalgarno sequence) &lt;222&gt; (108)..(113) &lt;220&gt;&lt;221&gt; Message peptide &lt;222&gt; (123)..(203) 54 23 &lt;220&gt; 1321567 Patent Application No. 095118228 Revision Date Revision Date: October 1st, 1998&lt;221&gt; Restricted Address&lt;222> (210)..(215) &lt;223&gt; by BamHI Identification &lt;220> &lt;221&gt; Restricted Address &lt;222&gt; (217)..(222) &lt;223&gt; Recognized by SphI &lt;220 &lt;221&gt; Restricted Address &lt;222&gt; (224)..(229) &lt;223&gt; Recognized by SacI &lt;220> &lt;221&gt; Restricted Address &lt;222&gt; (227).. (232) &lt;223&gt; Recognized by Xhol&lt;220&gt;&lt;221> Restricted Address&lt;222> (233)..(238) &lt;223> Recognized by Spel&lt;400&gt; 43 tagactgaat tcgtatattt cccaatagct aaaaaggttt cccaatacca aaagagttaa 60 aattttgtta attttagatt accagctgcg caggttggaa cattttgagg aggtataacc 120 atatgaataa gaaaatgggg aaaattgttg ccggaacagc actaattata tcagtagcat 180 ttagttcatc gatcgcacaa gcagccgagg gatccagcat gctgagctcg agactagtga 240 ttta 244 &lt; 210 &gt; 44 &lt; 211 &gt; 10 &lt; 212 &gt; PRT &lt; 213> AX of 241,321,567 No. 095118228 Revision date of the patent application specification revision date: October 1st, 1998&lt;220&gt;&lt;223&gt; 6X-His tag &lt;400> 44

Met Arg Gly Ser His His His His His His 1 5 10 25

Claims (1)

1321567 No. 95丨18228 Patent Application Supplement ^Repair ^分^牧线_ The description of the replacement of the revised period · Correction period: November 10, 1998, the scope of the patent application: 1. An antifreeze protein analog, which contains The amino acid sequence represented by the formula (1): YP-[AR]aWZ (I) 5 wherein: a is an integer from 1 to 20; R in each -[AR] - independently represents a direct bond (direct bond) or a spacer sequence containing 3 to 15 amino acid residues; ° A in each -[AR]- independently represents a _Qm- which forms an α_helical structure Wherein: m is an integer from 3 to 20; Q is an amine a-acid repeating unit comprising 11 amino acid residues and represented by formula (II): -Xl-X2-X3-X4-X5_X6_X7_X8 -X9-Xl〇-Xll- (II) where: Χι in each Q is a threonine (Thre), or Χι in at least one Q can be replaced with valine (Val) X4 in each Q is an amino acid residue selected from asparagine (Q Asn) and aspartic acid (asPartic ac 〖d, Asp), or at least one Q X4 may be replaced by an amino acid selected from the group consisting of threonine, leucine, and alanine; X6 and Χίο in at least one Q are a group of amino acid sequences. The formation of a bridge to stabilize the amino acid residue of the α-helical structure, the supplement of the patent application No. 095118228, the correction of the part of the line without the line of the replacement page. Date of revision: 98 years 1 month, X6 and X丨One of 0 is an amino acid residue selected from the group consisting of lysine (lysine, Lys) and arginine (Argin), and the other is glutamic acid (Glu); Χι丨 in Q is leucine (Leu), or 5 Χ π in at least one Q can be replaced with alanine; and the remaining amino acid residues in each Q are alanine, such Alanine provides the van der Waals force required for the binding of the anti-protein analog to ice crystals; Y is absent or represents a pr〇tein tag; 1〇P represents one or several amino acid residues, wherein the amino acid residue directly adjacent to -[AR]a- is one From the acid amino acid of aspartic acid and glutamic acid; W represents one or several amino acid residues, wherein at least one amino acid residue 疋 is selected from arginine , histidine (His) and arginine 15 basic amino acid; and Z is absent or represents a protein marker; but conditionally the antifreeze protein analogue is not Is a protein having an amino acid sequence as shown in Sequence Identification Number: 1. 2. An antifreeze protein analog as claimed in claim 1 wherein a is an integer from 2 to 1 to 12. 3. An antifreeze protein analog comprising an amino acid sequence represented by the formula (III): (III) YPA-[RA]nWZ wherein: 2 1321567 Patent Application No. 095,118,228 Line of instructions Replacement page Revision date: November 1997 η is 0 or an integer from 1 to 15; R/ is independently a spacer sequence containing 3 to 15 amino acid residues at each occurrence Each time it appears, it is independently a 5-Qm_ ' which forms an α-helical structure. Where: m is an integer from 3 to 20; Q is a residue containing 11 amino acid residues and The amino acid repeating unit represented by: 10 wherein: X in each Q is a threonine, or there is at least one X in Q, which can be replaced with valine; X4 in each Q is An amino acid residue selected from aspartic acid and aspartic acid, or X4 having at least one Q may be substituted 15 as an amino acid selected from the group consisting of: sulphonic acid , valine and alanine; χ6 and χ10 in at least one Q are a group of salt bridges formed within the amino acid sequence An amino acid residue of a stable helical structure, wherein one of Χ6 and Χ10 is an amino acid residue selected from the group consisting of lysine and arginine 20, and the other is a face acid; The Χη is a leucine, or at least one of the Q in the Q can be replaced with alanine; and the remaining amino acid residues in each Q are alanine, the alanine providing the antifreeze protein analog The need to combine with ice crystals 3 1321567 Patent Application No. 095118228 Supplement, Amendment Partial Lineless Manual Replacement Page Revision Date: Van der Waals Force in November 1998; Y is non-existent, or represents a protein marker P represents one or several amino acid residues, wherein the amino acid residue directly adjacent to -A-[RA]n- is an acidic amine 5-based acid selected from aspartic acid and glutamic acid W represents one or several amino acid residues, wherein at least one amino acid residue is a basic amino acid selected from the group consisting of arginine, histidine and lysine; and Z is non-existent Or a protein marker; 10 but conditionally the antifreeze protein analogue is not The sequence identification number having: a protein the amino acid sequence shown in FIG. 1. 4. The antifreeze protein analog of claim 3, wherein η is an integer from 0 to 11. 5. An antifreeze protein analog according to claim 1 or 3 wherein m is 15 an integer from 4 to 15. 6. The antifreeze protein analog of claim 1 or 3, wherein the antifreeze protein analog has a monoalanine content ranging from 50% to 85%. 7. The antifreeze protein analog according to claim 1 or 3, wherein X6 in at least one Q in each of 20-Qm- is an isoleic acid, and X1() is glutamic acid. 8. The antifreeze protein analog of claim 1 or 3 wherein there are two or more salt bridges within each -Qm-. 9. For example, the antifreeze protein analogue of the scope of patent application No. 8 is included in the patent No. 095118228, the supplementary part of the amendment, the replacement part of the no-line specification replacement page 〜8~ Revision date: November 1998- The salt bridge within Qm- is present adjacent to the amino acid repeat unit. 10. An anti-whitening analog according to item 8 of the patent application, wherein the salt bridges present in each Qm- are alternately present in the amino acid repeating unit. U_ is a protein analog of claim (4), wherein the protein marker at the position of ¥ or 2 serving as an anti-/east protein analog is selected from the group consisting of: ms tag, (7) Ding Na, Arg-tag, FLAG-tag, e_myc tag, s_tag, HAT tag, Camodulm-tag, cellulose binding field tons, SBp_tag, chitin binding field tagm maltose combination field mail. 12. The anti-conne analog according to claim 1 or 3, wherein 3 to 15 amino groups are present at the position of the anti-protein analog of the formula (1) or the R of the formula (1). Acid residue. 13_ The anti-eastern protein analog of claim 1 or 3, wherein the antifreeze protein analog has i-15 amine groups at the P position of formula (I) or formula (III) Acid residue. 14. If an anti-silk-white analog of the third or third term is applied for, the anti-freeze protein analog has ^15 amino acid residues at the 1-position of the formula (1) or the formula (111). 15. An anti-canon analog according to claim 4, which has an amino acid sequence selected from the group consisting of: sequence identification number 20., sequence identification number: 21, sequence identification number : 38, sequence identification number: 39 and sequence identification number: 4〇. ° Patent Application No. 095118228 Supplementary, Amendment Section Replacement Manual for No Line Drawing, . Amendment Date: 98 years/January 6. 6. Monthly patent gas circumference! Or 3 anti-Kang protein analogs which are prepared by chemical synthesis. 17. The anti-final analog of the first item of claim 4 (4), which is produced by recombinant DNA technology. A nucleic acid sequence having a nucleotide sequence encoding an antifreeze protein analog as set forth in claim 1 of the patent application. 19. A nucleic acid sequence having a nucleotide sequence encoding an antifreeze protein analog as in claim 3 of the scope of the patent application. 20. The nucleic acid sequence of claim 18 or 19, which has a nucleotide sequence selected from the group consisting of 10: sequence identification number: 27, sequence identification number: 28, sequence identification number : 35, sequence identification number: 36 and sequence identification number: 37. 21. A nucleic acid construct comprising a nucleic acid sequence as claimed in claim 18 or 19, and a promoter sequence operably linked upstream of 15 of the nucleic acid sequence. 22. The nucleic acid construct of claim 21, wherein the nucleic acid sequence has a nuclear acid sequence selected from the group consisting of: sequence identification number: 27, sequence identification number: 28, sequence identification Number: 35, serial identification number: 36 and serial identification number: 37. The nucleic acid construct of claim 21, wherein the promoter sequence is a natural promoter derived from any of the following: a virus, a bacterial cell, a yeast cell, a fungal cell, an algal cell, a plant cell. , insect cells, animal cells, and human cells, or a synthetic promoter engineered from the native promoter. 6 1321567 Patent Application No. 095,118,228, the entire disclosure of which is incorporated herein by reference. The following groups are composed: T5, T7, P, c, P/ac, p~, P, Pw, Pw, Pwir, pare, and, and promoters derived therefrom. 5 25. A performance carrier comprising a nucleic acid construct as in claim 21 of the patent application. 26. The performance vector of claim 25, further comprising at least one of: a marker gene, a reporter gene, an antibiotic-resistance gene, a enhancer sequence, an SD sequence, an encoding A gene of 10 selected gene products, a polyadenylation position, and a regulatory sequence. 27. The expression vector of claim 25, which is selected from the group consisting of pRP-AFP, pQE30-AFP, pET-12a-As AFP, pET-12a-0s AFP, pSP 1 Atag, pSP2 Atag 15 and pSP3Atag. 28. A recombinant host cell comprising a performance vector as claimed in claim 25. 29. The recombinant host cell of claim 28, which is selected from the group consisting of a bacterial cell, a yeast cell, a fungal cell, a 20 plant cell, an insect cell, an animal cell, or a human cell. 30. A recombinant host cell according to claim 29, which is an E. coli cell. 7