TW201734040A - Protein crud extract isolated from non-human connective tissue, manufacturing method and use thereof - Google Patents

Abstract

The invention relates to a protein crud extract isolated from non-human connective tissue, a manufacturing method and a use thereof. The manufacturing method comprises introducing a DNA contrstruct containing SEQ ID NO: 4 or SEQ ID NO: 5 into a rat embryo; transplanting the rat embryo into a female rat for developing into a rat; isolating the protein crud extract comprising a target amino acid sequence from the rat. The target amino sequence is selected from an amino acid sequence having (i) at least 91% sequence identity to SEQ ID NO: 1 and SEQ ID NO: 3, (ii) at least 93% sequence identity to SEQ ID NO: 2 and SEQ ID NO: 3, or (iii) at least 91% sequence identity to SEQ ID NO: 1, at least 93% sequence identity to SEQ ID NO: 2 and SEQ ID NO: 3.

Description

Protein crude extract isolated from non-human connective tissue and method and use thereof

The present invention relates to a crude protein extract isolated from non-human connective tissue and methods and uses thereof, in particular to a protein crude extract comprising (i) having at least 91% similarity to SEQ ID NO: 1 and SEQ ID NO: 3, (ii) has at least 93% similarity to SEQ ID NO: 2 and SEQ ID NO: 3 or (iii) has at least 91% similarity to SEQ ID NO: 1, with SEQ ID NO: 2 has an amino acid sequence of at least 93% similarity and SEQ ID NO: 3.

According to collagen, collagen is a very important protein in animals. It is an important structural protein in extracellular space. Collagen is also the main component of connective tissue. It is also found in tendons, ligaments, skin, cornea and other tissues. The total protein amount is 20% to 30%.

Collagen is a structure in which three independent collagen peptide chains form a hydrogen bond through glycine acid on a peptide chain to form and maintain a entanglement of three helixes, which is called procollagen. After several procollagens are laterally stacked, an aldol condensation reaction occurs between them to form a covalent bond, which becomes a collagen microfiber; several collagen microfibers undergo a similar reaction to produce a covalent bond to form collagen fibers, and collagen. Fiber is the basic form of collagen for physiological action.

Collagen is divided into five types. The first type of collagen is mainly found in skin, tendons, organs and bones; the second type of collagen is mainly found in cartilage; the third type of collagen is mainly composed of reticulin. The fourth type of collagen mainly constitutes basal laminin, which is present in the basement membrane of the epithelial tissue; the fifth type of collagen is present on the cell surface, hair or placenta.

Collagen has extremely high economic value and can be widely used in medical or cosmetic applications, such as support materials for stem cell culture systems to help stem cell growth, drug-coated materials on drug-containing cardiovascular stents, Burning patients' wound dressings, collagen hemostatic cotton tablets, collagen membrane bone fillers, moisturizing anti-aging cosmetics, oral nutritional supplements, etc.; currently seen in the market for collagen products, most of the raw materials are using type I collagen protein.

At present, the source of collagen is mainly obtained by automatic tissue extraction. The common collagen on the market can be extracted from pig skin, cowhide, fish skin, fish scales, etc. For example, the Taiwan patent case TW I487711 is based on carp skin. Extraction of collagen is performed. However, collagen derived from animals may cause allergic reactions. For example, people who are allergic to seafood may not be suitable for collagen products extracted from fish skin/fish scales. In order to reduce the allergic phenomenon caused by collagen, U.S. Patent No. US20120284817A1 facilitates the synthesis of human collagen by genetic engineering by genetic engineering, but since the enzyme system of plants and mammals is not identical, the resulting collagen is transcribed. The state of the post translational modification is still different from that of collagen, which is generally derived from mammals.

Nowadays, the inventor is still in the spirit of tirelessness in view of the above-mentioned existing genetically modified organisms to prepare collagen for practical use, and with its rich professional knowledge and years of practical experience. The invention was assisted and improved, and the present invention was developed based on this.

The present invention provides a crude protein extract isolated from non-human connective tissue, comprising (i) having at least 91% similarity to SEQ ID NO: 1 and SEQ ID NO: 3, (ii) and SEQ ID NO: 2 having at least 93% similarity and SEQ ID NO: 3 or (iii) having at least 91% similarity to SEQ ID NO: 1, at least 93% similarity to SEQ ID NO: 2, and SEQ ID NO: The amino acid sequence of 3.

The invention also provides a method for preparing a crude protein extract containing a labeled amino acid sequence, comprising (a) a composition comprising a DNA sequence as SEQ ID NO: 4 or SEQ ID NO: 5 (DNA construct) (b) introducing a DNA sequence construct into a rat embryo, respectively, and then transplanting the rat embryo into a homologous mother to develop into a mouse; (c) is isolated from the connective tissue of the adult mouse. a crude protein extract containing the indicated amino acid sequence, wherein the target amino acid sequence is selected from the group consisting of (i) having at least 91% similarity to SEQ ID NO: 1 and SEQ ID NO: 3, (ii) and SEQ ID NO: 2 has at least 93% similarity and SEQ ID NO: 3 or (iii) has at least 91% similarity to SEQ ID NO: 1, has at least 93% similarity to SEQ ID NO: 2, and SEQ ID NO: amino acid sequence of 3.

The invention also provides the use of a crude protein extract containing a labeled amino acid sequence for the preparation of human first type collagen, wherein the target amino acid sequence is selected from the group consisting of (i) having at least 91% with SEQ ID NO: 1. The above similarity and SEQ ID NO: 3, (ii) has at least 93% similarity to SEQ ID NO: 2, and SEQ ID NO: 3 or (iii) has at least 91% similarity with SEQ ID NO: 1, An amino acid sequence having at least 93% similarity to SEQ ID NO: 2 and SEQ ID NO: 3.

In one embodiment of the present invention, the target amino acid contained in the crude protein extract may be, for example, (i) SEQ ID NO: 1 and SEQ ID NO: 3, (ii) SEQ ID NO: 2, and SEQ ID NO: 3 or (iii) the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

In one embodiment of the invention, the crude protein extract can be specifically identified by an anti-human collagen antibody.

In one embodiment of the present invention, the crude protein extract is obtained by isolating tissue of a gene-transferred rat, and the genomic DNA of the gene-transferred rat has a human type I collagen. An oligoribonucleic acid sequence having a deoxyribonucleic acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

The object of the present invention and its structural and functional advantages will be explained in conjunction with the specific embodiments according to the structure shown in the following drawings, so that the reviewing committee can have a more in-depth and specific understanding of the present invention.

The present invention provides a crude protein extract isolated from non-human connective tissue comprising (i) at least 91% similarity to SEQ ID NO: 1 and SEQ ID NO: 3, (ii) and SEQ ID NO: 2 having at least 93% similarity and SEQ ID NO: 3 or (iii) having at least 91% similarity to SEQ ID NO: 1, at least 93% similarity to SEQ ID NO: 2, and SEQ ID NO: An amino acid sequence of 3; wherein the crude extract of the protein is specifically recognized by an anti-human collagen antibody, and is isolated from the connective tissue of a gene-transferred rat, and the genomic DNA of the gene is transferred to the rat Having a deoxyribonucleic acid sequence encoding human first type collagen, the deoxyribonucleic acid sequence encoding human type I collagen is SEQ ID NO: 4 or SEQ ID NO: 5.

The invention also provides a method for preparing a crude protein extract containing a labeled amino acid sequence, comprising (a) a composition comprising a DNA sequence as SEQ ID NO: 4 or SEQ ID NO: 5 (DNA construct) (b) introducing a DNA sequence construct into a rat embryo, respectively, and then transplanting the rat embryo into a homologous mother to develop into a mouse; (c) is isolated from the connective tissue of the adult mouse. a crude protein extract containing the indicated amino acid sequence, wherein the target amino acid sequence is selected from the group consisting of (i) having at least 91% similarity to SEQ ID NO: 1 and SEQ ID NO: 3, (ii) and SEQ ID NO: 2 has at least 93% similarity and SEQ ID NO: 3 or (iii) has at least 91% similarity to SEQ ID NO: 1, has at least 93% similarity to SEQ ID NO: 2, and SEQ ID NO: The amino acid sequence of 3, and the prepared crude protein extract can be specifically identified by the anti-human collagen antibody.

For example, the target amino acid contained in the crude protein extract comprises (i) SEQ ID NO: 1 and SEQ ID NO: 3, (ii) SEQ ID NO: 2 and SEQ ID NO: 3 or ( Iii) the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:

The invention also provides the use of a crude protein extract having a labeled amino acid sequence for the preparation of human first type collagen, wherein the target amino acid sequence is selected from the group consisting of (i) having at least 91% with SEQ ID NO: 1. The above similarity and SEQ ID NO: 3, (ii) have at least 93% similarity with SEQ ID NO: 2 and SEQ ID NO: 3 or (iii) has at least 91% similarity with SEQ ID NO: 1, SEQ ID NO: 2 has at least 93% similarity and the amino acid sequence of SEQ ID NO: 3, and the crude protein extract is specifically recognized by the anti-human collagen antibody; the crude protein extract is transferred from a gene The connective tissue of the rat is isolated, and the genomic DNA of the gene-transferred rat has a deoxyribonucleic acid sequence encoding human type I collagen and SEQ ID NO: 4 or SEQ ID NO: 5.

In addition, the scope of the invention may be further exemplified by the following specific examples, which are not intended to limit the scope of the invention.

Briefly, the present invention discloses a mixture derived from an exogenous collagen gene-transferred rat, a crude extract containing exogenous collagen and a crude extract of rat type B actin, and discloses a preparation method thereof. Use; the exogenous collagen gene can be a collagen gene of human, rabbit, cow or pig; and the collagen COL1A1 amino acid sequence of rabbit, bovine or pig has at least 91% with the human collagen COL1A1 amino acid sequence. The above similarity; the collagen COL1A2 amino acid sequence of rabbit, bovine or pig has a similarity to the human collagen COL1A2 amino acid sequence of at least 93%; the following are examples of human collagen gene transgenic mice:

Experiment 1. Preparation of human collagen gene transfer mouse

In this experiment, the human type I collagen gene hCOL1A1 or hCOL1A2 was transfected into SD rats (Sprague-Dawley Rat) using the specific CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) gene editing method to make rats The connective tissue expresses human type I collagen. The process is as follows:

( One ) Preparation of sgRNA (single guide RNA), Cas9 expressing RNA, COL1A1 or COL1A2 calibration vector

An oligonucleotide (oligo DNA) having a rat collagen gene COL1A1 or CAL1A2 is synthesized to prepare a sgRNA (single guide RNA) having the sequence of SEQ ID NO: 8 to SEQ ID NO: 15 respectively; The DNA was subjected to a slowing pairing reaction under the following conditions: 95 ° C for 3 minutes, 95 ° C for 30 seconds, 95 ° C to 85 ° C for cooling (cooling rate: 2 ° C per minute), 85 ° C to 25 ° C for cooling (cooling) Speed: 0.1 ° C per second), cooling to 4 ° C.

The plastid pUC57-T7-sgRNA is cleaved with a restriction enzyme (enzyme) to obtain a linear double-stranded nucleic acid, and the linear double-stranded nucleic acid is annealed with the above-mentioned slow-cold product as a nucleic acid ligase. Engagement to obtain a recombinant plasmid; transforming this plastid into Escherichia coli , which can be selected with recombinant plastids due to the presence of a special antibiotic gene on the recombinant plastid The strain is amplified and cultured to amplify the recombinant plastid described above. The recombinant plasmid is cleaved with a restriction enzyme to obtain a linear recombinant plastid. Another Cas9 expression plasmid was used to limit enzyme cleavage to obtain a linear Cas9 protein expressing plastid. The linear recombinant plastids and the linear Cas9 protein were expressed as plastids, subjected to in vitro transcription and concentrated and purified to obtain sgRNA and Cas9 RNA for microinjection.

Using genomic DNA, human cDNA (derived from human oral mucosal cells), rat cDNA, plastids containing Flag Tag/BGHpA (self-prepared), and ColE1_Amp+Minimal vector as templates, using primer SEQ ID NO: 16~SEQ ID NO: 27 Polymerase chain reaction (PCR) and Gibson Assembly were used to obtain a calibration recombinant vector ColE1_Amp+ Minimal vector_r/hCOL1A1 target construct carrying the chimeric gene SEQ ID NO: 6 .

Using primers SEQ ID NO: 28 to SEQ ID NO: 39, rat genomic DNA, human cDNA (derived from human oral mucosal cells), rat cDNA, plastid containing HA Tag/BGHpA (self And the ColE1_Amp+Minimal vector was used as a template for polymerase chain reaction (PCR) and Gibson Assembly to obtain a calibration recombinant vector ColE1_Amp+ Minimal vector_r/hCOL1A2 target construct carrying the chimeric gene SEQ ID NO: 7.

( b) Pronuclear microinjection or embryonic nuclear electroporation gene electroporation

Prepare a zygote on the 0.5th day of fertilization, using pronuclear microinjection or prokaryotic electroporation gene transfer, and prepare the product as a sgRNA 10ng, Cas9 50 ng of mRNA was mixed with 4 ng of the COL1A1 (or COL1A2) calibration vector, and then implanted into the zygote to obtain a 0.5-day gene transfer zygote for fertilization.

Sexually mature (full seven weeks) females who are not pregnant and estrus, mate with ligation males, and enter the pseudopregnant state. After mating, the mother's vagina is examined every other day, and the female with a plug is inserted. Defined as a 0.5 day pregnant female. 20-30 gene transfer zygotes were injected into the oviduct of a 0.5-day pseudopregnant female, allowing the gene transfer zygote to grow and differentiate in female mice. The gene-transferred mice will be born after 17-21 days, and can be weaned about three weeks after birth. The gene-transferred mice will take their tail tissue after weaning, extract DNA, and perform genotyping to select genes. The original generation of rats (F0). Genotyping analysis of hCOL1A1 transgenic mice was performed with primers SEQ ID NO: 40 to SEQ ID NO: 43, and genotype analysis of hCOL1A2 transgenic mice was performed with primers SEQ ID NO: 44 to SEQ ID NO: 47; The analysis is a well-known skill and will not be described here.

Experiment 2, genetic breeding mouse breeding method (Strategy 1 and 2)

In order to obtain a homologous strain with homozygous human collagen transfer gene, two breeding strategies were used to obtain the desired transgenic animal. Please refer to the first figure for breeding strategy I, as follows: Transfer to hCOL1A1 In the case of the rat, the F0 mouse obtained in the experiment 1 has only one gene on the gene (heterologous strain), and the genotype is represented by H1R1/R2R2 (H: human, R: rat, 1: COL1A1 gene, 2: COL1A2) In order to obtain a homologous strain in which both genes are replaced, the following breeding procedure is carried out: F0 (H1R1/R2R2) is bred with a general rat (wild type, R1R1/R2R2); F0 (H1R1/R2R2 X) Wt(R1R1/R2R2), obtained in the first generation, the genotype may be F1 (H1R1/R2R2) or F1 (R1R1/R2R2); self-breeding F1 (H1R1/R2R2) [F1 (H1R1/R2R2) X F1 (H1R1) /R2R2)], progeny of F2 (H1H1/R2R2), F2 (H1R1/R2R2) and F2 (R1R1/R2R2) can be obtained, and progeny of F2 (H1H1/R2R2) can be picked for subsequent breeding; collagen gene COL1A2 The breeding of the rats was the same as above, and the progeny of F2 (R1R1/H2H2) was finally picked for subsequent breeding.

F2 (H1H1/R1R1) of COL1A1 transgenic mice was bred with F2 (R1R1/H2H2) of COL1A2 transgenic mice to obtain F3 (H1R1/H2R2); F3 (H1R1/H2R2) was self-bred and the progeny were selected. F4 (H1H1/H2H2), a transgenic mouse obtained by self-breeding with F4 (H1H1/H2H2) [F4 (H1H1/H2H2)X F4 (H1H1/H2H2)], will be replaced by COL1A1 and COL1A2. Rat. The genotypes of the above transgenic mice were obtained by genotyping using primers as SEQ ID NO: 40 to SEQ ID NO: 43 (hCOL1A1 transgenic) and SEQ ID NO: 44 to SEQ ID NO: 47 (hCOL1A2 transfer).

The breeding strategy for obtaining homologous strains of homologous human collagen transfer gene is shown in the second figure, which is described as follows: taking the hCOL1A1 transgenic mouse as an example, the F0 (H1R1/R2R2) mouse obtained in Experiment 1 and General rat (wild type, R1R1/R2R2) breeding: F0 (H1R1/R2R2) X wt (R1R1/R2R2), to obtain primary mouse F1 (H1R1/R2R2) or F1 (R1R1/R2R2); collagen gene COL1A2 transgenic The mouse is bred in the same manner as above, and the progeny of F1 (R1R1/H2R2) or F1 (R1R1/R2R2) are obtained.

The hCOL1A1 transgenic primary mouse F1 (H1R1/R2R2) was mated with hCOL1A2 transgenic primary mouse F1 (R1R1/H2R2), and the genotypes were F2 (H1R1/H2R2), F2 (H1R1/R2R2), and F2 (R1R1/). H2R2) and F2 (R1R1/R2R2) progeny; cross-following F2 (H1R1/H2R2) [F2 (H1R1/H2R2)X F2 (H1R1/H2R2)], and picking off progeny F3 (H1H1/H2H2); Then F3 (H1H1/H2H2) was alternately bred, and [F3 (H1H1/H2H2)X F3 (H1H1/H2H2)] was obtained, and the transgenic mice were replaced by COL1A1 and COL1A2. mouse. The genotypes of the above transgenic mice were obtained by genotyping using primers as SEQ ID NO: 40 to SEQ ID NO: 43 (hCOL1A1 transgenic) and SEQ ID NO: 44 to SEQ ID NO: 47 (hCOL1A2 transfer).

Experiment 3: Preparation of crude extracts from rat tail

The crude extract of rat collagen can be extracted from the tail or skin of the rat. The following is the result of extraction and analysis using the tail material: after the rat is sacrificed, the tail is cut from the root and stored in - At 80 °C, there was no significant difference in appearance between the tail of the gene-transferred mouse and the tail of the normal rat (results not shown); the tail was thawed on ice for at least 30 minutes, the epithelial tissue was removed, and the tail was roughly cut into six. Divide and remove bones and muscles; cut the tendon and soak it in water, wash it in 1X Phosphate-buffered saline buffer, soak it in 70% ethanol for 10 minutes; remove non-tendon tissue (such as blood vessels) Or muscle), the tendon tissue is sedimented by centrifugation, 70% ethanol is removed, and a tendon initial product is obtained.

Weigh the initial product of the tendon and add 1N acetic acid solution (the weight ratio of the initial product of the tendon to the 1N acetic acid solution is 1:99~1:9, preferably 1:9), continue stirring, and act at 4 °C. 12~48 hours; after that, the tendon initial product solution was treated at -80 °C for 12 hours, and finally a freeze-drying process was performed at -10 °C for 48 hours to remove acetic acid and water, and finally obtained as shown in the three figures. Sponge lyophilized crude extract (ie the crude extract requested in this case), this crude extract will be further analyzed.

Experiment 4, Polyacrylamide gel electrophoresis (PAGE) and Western blotting (Western’s Blotting)

The crude extract was dissolved in a 0.1 M lactic acid solution containing 10% sucrose to a concentration of 1 mg/ml, and 5 μg of the crude extract was taken and analyzed by conventional non-denaturing polypropylene guanamine gel electrophoresis and western dot toner. , briefly described as follows:

Non-denaturing polyacrylamide gel electrophoresis (Native PAGE), the protein was separated in a colloid, and then stained with a solution containing carbamazee blue dye for 2 hours, and then washed with a destaining solution. 24 hours; as shown in the fourth figure (A), the samples in columns 1 to 3 are the crude extracts of the tails of the gene-transferred mice, and the columns 4 to 6 are the crude extracts of the tails of the general rats. The crude extract of the rat tail exhibits different patterns, but it can be preliminarily inferred that the crude extract of the gene contains a large amount of certain protein, and the protein is not present in the crude rat tail extract.

To further confirm the protein detected by non-denaturing gel electrophoresis, the crude extract was analyzed by Western blotting: after non-denaturing polyacrylamide gel electrophoresis (Native PAGE), the protein was transferred to the NC membrane. Antibody identification of human type I collagen (Millipore antibody, number MAB3391) was specifically identified on (nitrocellulose membrane). As shown in the fourth panel (B), columns 1 to 3 are the crude extracts of the gene-transferred rat tail. The human type I collagen antibody will display a signal, and the signal pattern and the third image (A) are presented. Corresponding to each other, it is confirmed that the crude extract of the gene-transferred rat tail contains a large amount of human type I collagen; however, in column 4 to column 6 of the third figure (B), there is no signal at all after the action of human type I collagen antibody. It was confirmed that the crude extract of the tail of the general rat did not contain human type 1 collagen.

In addition, the crude extract of the rat tail was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), and the rat type B actin (β-actin) was detected by Western blotting. The performance situation, as shown in the fifth figure, whether it is a genetically transplanted mouse or a normal rat, the crude extract contains rat beta-actin (β-actin).

The crude extract obtained in this case can be further treated with pepsin or dispase to purify human collagen for further application in products using collagen as a raw material.

In one embodiment of the present invention, the human collagen purified from the crude extract can be applied to medical materials, such as collagen hemostatic cotton, absorbable surgical sutures, wound dressings, bone fillings, artificial blood vessels, soft tissue fillers. (soft tissue filler) and so on.

In one embodiment of the present invention, the human collagen purified from the crude extract may be used in a beauty care product, and the form may include, but is not limited to, a liquid, an emulsion, a cream, a powder, a whitening agent, a spotting agent, and a cockroach. A patch or any combination of the above, such as a collagen moisturizer, a collagen moisturizer, a collagen mask, and the like.

In one embodiment of the present invention, when the human collagen purified from the crude extract is applied to a food additive, it may be added to, for example, a nutraceutical such as bone joint health care or anti-aging health care.

It can be seen from the above description that the present invention has the following advantages compared with the prior art:

1. The crude extracts requested in this case can be further purified to obtain human type 1 collagen, which can be used in medical materials or cosmetic applications to reduce the incidence of allergies to non-human collagen.

2. The crude extracts requested in this case were derived from gene-transferred rats, and the post-translational modification of the protein is more similar to the natural state of human type 1 collagen.

3. The crude extracts requested in this case are derived from experimental animals. The culture environment is closely monitored, which can reduce the concern of infectious substances in raw materials and provide a good source of collagen.

In summary, a crude protein extract of the present invention isolated from non-human connective tissue, and methods and uses thereof, can achieve the intended efficacy by the above disclosed embodiments, and the present invention has not been disclosed. Prior to the application, Cheng has fully complied with the requirements and requirements of the Patent Law.爰Issuing an application for a patent for invention in accordance with the law, and asking for a review, and granting a patent, is truly sensible.

The illustrations and descriptions of the present invention are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention; those skilled in the art, which are characterized by the scope of the present invention, Equivalent variations or modifications are considered to be within the scope of the design of the invention.

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First: Genetically-transferred mouse breeding strategy

Figure 2: Genetically-transferred mouse breeding strategy II

Figure 3: Crude extracts from the transgenic mice and the tail of the average rat

Figure 4: Results of protein analysis of crude extracts from the tail (A) non-denaturing polyacrylamide gel electrophoresis, (B) Western blotting

Figure 5: Western blotting method to detect the expression of type B actin in rats with crude extracts

SEQUENCE LISTING <110> Taiwan Medical Biotechnology Co., Ltd. <120> A crude protein extract isolated from non-human connective tissue and its method and use <160> 45 <170> PatentIn version 3. 5   <210>   1   <211>   1053   <212>   PRT   <213>   Homo   Sapiens   <223>   Human COL1A1 Amino Acid (166-1218)   <400>   1   Gly   Tyr   Asp   Glu   Lys   Ser   Thr   Gly   Gly   Ile   Ser   Val   Pro   Gly   Pro   Met   1   5   10   15   Gly   Pro   Ser   Gly   Pro   Arg   Gly   Leu   Pro   Gly   Pro   Pro   Gly   Ala   Pro   Gly     20   25   30   Pro   Gln   Gly   Phe   Gln   Gly   Pro   Pro   Gly   Glu   Pro   Gly   Glu   Pro   Gly   Ala     35   40   45   Ser   Gly   Pro   Met   Gly   Pro   Arg   Gly   Pro   Pro   Gly   Pro   Pro   Gly   Lys   Asn     50   55   60     Gly   Asp   Asp   Gly   Glu   Ala   Gly   Lys   Pro   Gly   Arg   Pro   Gly   Glu   Arg   Gly   65   70   75   80   Pro   Pro   Gly   Pro   Gln   Gly   Ala   Arg   Gly   Leu   Pro   Gly   Thr   Ala   Gly   Leu     85   90   95   Pro   Gly   Met   Lys   Gly   His   Arg   Gly   Phe   Ser   Gly   Leu   Asp   Gly   Ala   Lys     100   105   110   Gly   Asp   Ala   Gly   Pro   Ala   Gly   Pro   Lys   Gly   Glu   Pro   Gly   Ser   Pro   Gly     115   120   125   Glu   Asn   Gly   Ala   Pro   Gly   Gln   Met   Gly   Pro   Arg   Gly   Leu   Pro   Gly   Glu     130   135   140   Arg   Gly   Arg   Pro   Gly   Ala   Pro   Gly   Pro   Ala   Gly   Ala   Arg   Gly   Asn   Asp   145   150   155   160   Gly   Ala   Thr   Gly   Ala   Ala   Gly   Pro   Pro   Gly   Pro   Thr   Gly   Pro   Ala   Gly     165   170   175   Pro   Pro   Gly   Phe   Pro   Gly   Ala   Val   Gly   Ala   Lys   Gly   Glu   Ala   Gly   Pro     180   185   190   Gln   Gly   Pro   Arg   Gly   Ser   Glu   Gly   Pro   Gln   Gly   Val   Arg   Gly   Glu   Pro     195   200   205   Gly   Pro   Pro   Gly   Pro   Ala   Gly   Ala   Ala   Gly   Pro   Ala   Gly   Asn   Pro   Gly     210   215   220   Ala   Asp   Gly   Gln   Pro   Gly   Ala   Lys   Gly   Ala   Asn   Gly   Ala   Pro   Gly   Ile   225   230   235   240   Ala   Gly   Ala   Pro   Gly   Phe   Pro   Gly   Ala   Arg   Gly   Pro   Ser   Gly   Pro   Gln     245   250   255   Gly   Pro   Gly   Gly   Pro   Pro   Gly   Pro   Lys   Gly   Asn   Ser   Gly   Glu   Pro   Gly     260   265   270     Ala   Pro   Gly   Ser   Lys   Gly   Asp   Thr   Gly   Ala   Lys   Gly   Glu   Pro   Gly   Pro     275   280   285   Val   Gly   Val   Gln   Gly   Pro   Pro   Gly   Pro   Ala   Gly   Glu   Glu   Gly   Lys   Arg     290   295   300     Gly   Ala   Arg   Gly   Glu   Pro   Gly   Pro   Thr   Gly   Leu   Pro   Gly   Pro   Pro   Gly   305   310   315   320   Glu   Arg   Gly   Gly   Pro   Gly   Ser   Arg   Gly   Phe   Pro   Gly   Ala   Asp   Gly   Val     325   330   335   Ala   Gly   Pro   Lys   Gly   Pro   Ala   Gly   Glu   Arg   Gly   Ser   Pro   Gly   Pro   Ala     340   345   350   Gly   Pro   Lys   Gly   Ser   Pro   Gly   Glu   Ala   Gly   Arg   Pro   Gly   Glu   Ala   Gly     355   360   365   Leu   Pro   Gly   Ala   Lys   Gly   Leu   Thr   Gly   Ser   Pro   Gly   Ser   Pro   Gly   Pro     370   375   380   Asp   Gly   Lys   Thr   Gly   Pro   Pro   Gly   Pro   Ala   Gly   Gln   Asp   Gly   Arg   Pro   385   390   395   400   Gly   Pro   Pro   Gly   Pro   Pro   Gly   Ala   Arg   Gly   Gln   Ala   Gly   Val   Met   Gly     405   410   415   Phe   Pro   Gly   Pro   Lys   Gly   Ala   Ala   Gly   Glu   Pro   Gly   Lys   Ala   Gly   Glu     420   425   430   Arg   Gly   Val   Pro   Gly   Pro   Pro   Gly   Ala   Val   Gly   Pro   Ala   Gly   Lys   Asp     435   440   445   Gly   Glu   Ala   Gly   Ala   Gln   Gly   Pro   Pro   Gly   Pro   Ala   Gly   Pro   Ala   Gly     450   455   460   Glu   Arg   Gly   Glu   Gln   Gly   Pro   Ala   Gly   Ser   Pro   Gly   Phe   Gln   Gly   Leu   465   470   475   480   Pro   Gly   Pro   Ala   Gly   Pro   Pro   Gly   Glu   Ala   Gly   Lys   Pro   Gly   Glu   Gln     485   490   495   Gly   Val   Pro   Gly   Asp   Leu   Gly   Ala   Pro   Gly   Pro   Ser   Gly   Ala   Arg   Gly     500   505   510     Glu   Arg   Gly   Phe   Pro   Gly   Glu   Arg   Gly   Val   Gln   Gly   Pro   Pro   Gly   Pro     515   520   525   Ala   Gly   Pro   Arg   Gly   Ala   Asn   Gly   Ala   Pro   Gly   Asn   Asp   Gly   Ala   Lys     530   535   540     Gly   Asp   Ala   Gly   Ala   Pro   Gly   Ala   Pro   Gly   Ser   Gln   Gly   Ala   Pro   Gly   545   550   555   560   Leu   Gln   Gly   Met   Pro   Gly   Glu   Arg   Gly   Ala   Ala   Gly   Leu   Pro   Gly   Pro     565   570   575   Lys   Gly   Asp   Arg   Gly   Asp   Ala   Gly   Pro   Lys   Gly   Ala   Asp   Gly   Ser   Pro     580   585   590     Gly   Lys   Asp   Gly   Val   Arg   Gly   Leu   Thr   Gly   Pro   Ile   Gly   Pro   Pro   Gly     595   600   605   Pro   Ala   Gly   Ala   Pro   Gly   Asp   Lys   Gly   Glu   Ser   Gly   Pro   Ser   Gly   Pro     610   615   620   Ala   Gly   Pro   Thr   Gly   Ala   Arg   Gly   Ala   Pro   Gly   Asp   Arg   Gly   Glu   Pro   625   630   635   640   Gly   Pro   Pro   Gly   Pro   Ala   Gly   Phe   Ala   Gly   Pro   Pro   Gly   Ala   Asp   Gly     645   650   655     Gln   Pro   Gly   Ala   Lys   Gly   Glu   Pro   Gly   Asp   Ala   Gly   Ala   Lys   Gly   Asp     660   665   670   Ala   Gly   Pro   Pro   Gly   Pro   Ala   Gly   Pro   Ala   Gly   Pro   Pro   Gly   Pro   Ile     675   680   685   Gly   Asn   Val   Gly   Ala   Pro   Gly   Ala   Lys   Gly   Ala   Arg   Gly   Ser   Ala   Gly     690   695   700   Pro   Pro   Gly   Ala   Thr   Gly   Phe   Pro   Gly   Ala   Ala   Gly   Arg   Val   Gly   Pro   705   710   715   720   Pro   Gly   Pro   Ser   Gly   Asn   Ala   Gly   Pro   Pro   Gly   Pro   Pro   Gly   Pro   Ala     725   730   735   Gly   Lys   Glu   Gly   Gly   Lys   Gly   Pro   Arg   Gly   Glu   Thr   Gly   Pro   Ala   Gly     740   745   750     Arg   Pro   Gly   Glu   Val   Gly   Pro   Pro   Gly   Pro   Pro   Gly   Pro   Ala   Gly   Glu     755   760   765   Lys   Gly   Ser   Pro   Gly   Ala   Asp   Gly   Pro   Ala   Gly   Ala   Pro   Gly   Thr   Pro     770   775   780     Gly   Pro   Gln   Gly   Ile   Ala   Gly   Gln   Arg   Gly   Val   Val   Gly   Leu   Pro   Gly   785   790   795   800   Gln   Arg   Gly   Glu   Arg   Gly   Phe   Pro   Gly   Leu   Pro   Gly   Pro   Ser   Gly   Glu     805   810   815   Pro   Gly   Lys   Gln   Gly   Pro   Ser   Gly   Ala   Ser   Gly   Glu   Arg   Gly   Pro   Pro     820   825   830   Gly   Pro   Met   Gly   Pro   Pro   Gly   Leu   Ala   Gly   Pro   Pro   Gly   Glu   Ser   Gly     835   840   845   Arg   Glu   Gly   Ala   Pro   Gly   Ala   Glu   Gly   Ser   Pro   Gly   Arg   Asp   Gly   Ser     850   855   860   Pro   Gly   Ala   Lys   Gly   Asp   Arg   Gly   Glu   Thr   Gly   Pro   Ala   Gly   Pro   Pro   865   870   875   880   Gly   Ala   Pro   Gly   Ala   Pro   Gly   Ala   Pro   Gly   Pro   Val   Gly   Pro   Ala   Gly     885   890   895   Lys   Ser   Gly   Asp   Arg   Gly   Glu   Thr   Gly   Pro   Ala   Gly   Pro   Ala   Gly   Pro     900   905   910   Val   Gly   Pro   Val   Gly   Ala   Arg   Gly   Pro   Ala   Gly   Pro   Gln   Gly   Pro   Arg     915   920   925   Gly   Asp   Lys   Gly   Glu   Thr   Gly   Glu   Gln   Gly   Asp   Arg   Gly   Ile   Lys   Gly     930   935   940   His   Arg   Gly   Phe   Ser   Gly   Leu   Gln   Gly   Pro   Pro   Gly   Pro   Pro   Gly   Ser   945   950   955   960   Pro   Gly   Glu   Gln   Gly   Pro   Ser   Gly   Ala   Ser   Gly   Pro   Ala   Gly   Pro   Arg     965   970   975   Gly   Pro   Pro   Gly   Ser   Ala   Gly   Ala   Pro   Gly   Lys   Asp   Gly   Leu   Asn   Gly     980   985   990   Leu   Pro   Gly   Pro   Ile   Gly   Pro   Pro   Gly   Pro   Arg   Gly   Arg   Thr   Gly   Asp     995   1000   1005   Ala   Gly   Pro   Val   Gly   Pro   Pro   Gly   Pro   Pro   Gly   Pro   Pro   Gly   Pro     1010   1015   1020     Pro   Gly   Pro   Pro   Ser   Ala   Gly   Phe   Asp   Phe   Ser   Phe   Leu   Pro   Gln     1025   1030   1035   Pro   Pro   Gln   Glu   Lys   Ala   His   Asp   Gly   Gly   Arg   Tyr   Tyr   Arg   Ala     1040   1045   1050     <210>   2   <211>   1038   <212>   PRT   <213>   Homo   Sapiens   <213> Human COL1A2 Amino Acid (82-1119)   <400>   2   Asp   Gly   Lys   Gly   Val   Gly   Leu   Gly   Pro   Gly   Pro   Met   Gly   Leu   Met   Gly   1   5   10   15   Pro   Arg   Gly   Pro   Pro   Gly   Ala   Ala   Gly   Ala   Pro   Gly   Pro   Gln   Gly   Phe     20   25   30   Gln   Gly   Pro   Ala   Gly   Glu   Pro   Gly   Glu   Pro   Gly   Gln   Thr   Gly   Pro   Ala     35   40   45     Gly   Ala   Arg   Gly   Pro   Ala   Gly   Pro   Pro   Gly   Lys   Ala   Gly   Glu   Asp   Gly     50   55   60   His   Pro   Gly   Lys   Pro   Gly   Arg   Pro   Gly   Glu   Arg   Gly   Val   Val   Gly   Pro   65   70   75   80   Gln   Gly   Ala   Arg   Gly   Phe   Pro   Gly   Thr   Pro   Gly   Leu   Pro   Gly   Phe   Lys     85   90   95   Gly   Ile   Arg   Gly   His   Asn   Gly   Leu   Asp   Gly   Leu   Lys   Gly   Gln   Pro   Gly     100   105   110   Ala   Pro   Gly   Val   Lys   Gly   Glu   Pro   Gly   Ala   Pro   Gly   Glu   Asn   Gly   Thr     115   120   125   Pro   Gly   Gln   Thr   Gly   Ala   Arg   Gly   Leu   Pro   Gly   Glu   Arg   Gly   Arg   Val     130   135   140   Gly   Ala   Pro   Gly   Pro   Ala   Gly   Ala   Arg   Gly   Ser   Asp   Gly   Ser   Val   Gly   145   150   155   160   Pro   Val   Gly   Pro   Ala   Gly   Pro   Ile   Gly   Ser   Ala   Gly   Pro   Pro   Gly   Phe     165   170   175   Pro   Gly   Ala   Pro   Gly   Pro   Lys   Gly   Glu   Ile   Gly   Ala   Val   Gly   Asn   Ala     180   185   190   Gly   Pro   Ala   Gly   Pro   Ala   Gly   Pro   Arg   Gly   Glu   Val   Gly   Leu   Pro   Gly     195   200   205   Leu   Ser   Gly   Pro   Val   Gly   Pro   Pro   Gly   Asn   Pro   Gly   Ala   Asn   Gly   Leu     210   215   220   Thr   Gly   Ala   Lys   Gly   Ala   Ala   Gly   Leu   Pro   Gly   Val   Ala   Gly   Ala   Pro   225   230   235   240   Gly   Leu   Pro   Gly   Pro   Arg   Gly   Ile   Pro   Gly   Pro   Val   Gly   Ala   Ala   Gly     245   250   255   Ala   Thr   Gly   Ala   Arg   Gly   Leu   Val   Gly   Glu   Pro   Gly   Pro   Ala   Gly   Ser     260   265   270     Lys   Gly   Glu   Ser   Gly   Asn   Lys   Gly   Glu   Pro   Gly   Ser   Ala   Gly   Pro   Gln     275   280   285   Gly   Pro   Pro   Gly   Pro   Ser   Gly   Glu   Glu   Gly   Lys   Arg   Gly   Pro   Asn   Gly     290   295   300     Glu   Ala   Gly   Ser   Ala   Gly   Pro   Pro   Gly   Pro   Pro   Gly   Leu   Arg   Gly   Ser   305   310   315   320   Pro   Gly   Ser   Arg   Gly   Leu   Pro   Gly   Ala   Asp   Gly   Arg   Ala   Gly   Val   Met     325   330   335   Gly   Pro   Pro   Gly   Ser   Arg   Gly   Ala   Ser   Gly   Pro   Ala   Gly   Val   Arg   Gly     340   345   350   Pro   Asn   Gly   Asp   Ala   Gly   Arg   Pro   Gly   Glu   Pro   Gly   Leu   Met   Gly   Pro     355   360   365   Arg   Gly   Leu   Pro   Gly   Ser   Pro   Gly   Asn   Ile   Gly   Pro   Ala   Gly   Lys   Glu     370   375   380   Gly   Pro   Val   Gly   Leu   Pro   Gly   Ile   Asp   Gly   Arg   Pro   Gly   Pro   Ile   Gly   385   390   395   400   Pro   Ala   Gly   Ala   Arg   Gly   Glu   Pro   Gly   Asn   Ile   Gly   Phe   Pro   Gly   Pro     405   410   415   Lys   Gly   Pro   Thr   Gly   Asp   Pro   Gly   Lys   Asn   Gly   Asp   Lys   Gly   His   Ala     420   425   430     Gly   Leu   Ala   Gly   Ala   Arg   Gly   Ala   Pro   Gly   Pro   Asp   Gly   Asn   Asn   Gly     435   440   445   Ala   Gln   Gly   Pro   Pro   Gly   Pro   Gln   Gly   Val   Gln   Gly   Gly   Lys   Gly   Glu     450   455   460   Gln   Gly   Pro   Pro   Gly   Pro   Pro   Gly   Phe   Gln   Gly   Leu   Pro   Gly   Pro   Ser   465   470   475   480   Gly   Pro   Ala   Gly   Glu   Val   Gly   Lys   Pro   Gly   Glu   Arg   Gly   Leu   His   Gly     485   490   495   Glu   Phe   Gly   Leu   Pro   Gly   Pro   Ala   Gly   Pro   Arg   Gly   Glu   Arg   Gly   Pro     500   505   510   Pro   Gly   Glu   Ser   Gly   Ala   Ala   Gly   Pro   Thr   Gly   Pro   Ile   Gly   Ser   Arg     515   520   525   Gly   Pro   Ser   Gly   Pro   Pro   Gly   Pro   Asp   Gly   Asn   Lys   Gly   Glu   Pro   Gly     530   535   540     Val   Val   Gly   Ala   Val   Gly   Thr   Ala   Gly   Pro   Ser   Gly   Pro   Ser   Gly   Leu   545   550   555   560   Pro   Gly   Glu   Arg   Gly   Ala   Ala   Gly   Ile   Pro   Gly   Gly   Lys   Gly   Glu   Lys     565   570   575   Gly   Glu   Pro   Gly   Leu   Arg   Gly   Glu   Ile   Gly   Asn   Pro   Gly   Arg   Asp   Gly     580   585   590   Ala   Arg   Gly   Ala   Pro   Gly   Ala   Val   Gly   Ala   Pro   Gly   Pro   Ala   Gly   Ala     595   600   605   Thr   Gly   Asp   Arg   Gly   Glu   Ala   Gly   Ala   Ala   Gly   Pro   Ala   Gly   Pro   Ala     610   615   620   Gly   Pro   Arg   Gly   Ser   Pro   Gly   Glu   Arg   Gly   Glu   Val   Gly   Pro   Ala   Gly   625   630   635   640   Pro   Asn   Gly   Phe   Ala   Gly   Pro   Ala   Gly   Ala   Ala   Gly   Gln   Pro   Gly   Ala     645   650   655   Lys   Gly   Glu   Arg   Gly   Ala   Lys   Gly   Pro   Lys   Gly   Glu   Asn   Gly   Val   Val     660   665   670   Gly   Pro   Thr   Gly   Pro   Val   Gly   Ala   Ala   Gly   Pro   Ala   Gly   Pro   Asn   Gly     675   680   685   Pro   Pro   Gly   Pro   Ala   Gly   Ser   Arg   Gly   Asp   Gly   Gly   Pro   Pro   Gly   Met     690   695   700   Thr   Gly   Phe   Pro   Gly   Ala   Ala   Gly   Arg   Thr   Gly   Pro   Pro   Gly   Pro   Ser   705   710   715   720   Gly   Ile   Ser   Gly   Pro   Pro   Gly   Pro   Pro   Gly   Pro   Ala   Gly   Lys   Glu   Gly     725   730   735   Leu   Arg   Gly   Pro   Arg   Gly   Asp   Gln   Gly   Pro   Val   Gly   Arg   Thr   Gly   Glu     740   745   750   Val   Gly   Ala   Val   Gly   Pro   Pro   Gly   Phe   Ala   Gly   Glu   Lys   Gly   Pro   Ser     755   760   765   Gly   Glu   Ala   Gly   Thr   Ala   Gly   Pro   Pro   Gly   Thr   Pro   Gly   Pro   Gln   Gly     770   775   780   Leu   Leu   Gly   Ala   Pro   Gly   Ile   Leu   Gly   Leu   Pro   Gly   Ser   Arg   Gly   Glu   785   790   795   800   Arg   Gly   Leu   Pro   Gly   Val   Ala   Gly   Ala   Val   Gly   Glu   Pro   Gly   Pro   Leu     805   810   815   Gly   Ile   Ala   Gly   Pro   Pro   Gly   Ala   Arg   Gly   Pro   Pro   Gly   Ala   Val   Gly     820   825   830   Ser   Pro   Gly   Val   Asn   Gly   Ala   Pro   Gly   Glu   Ala   Gly   Arg   Asp   Gly   Asn     835   840   845   Pro   Gly   Asn   Asp   Gly   Pro   Pro   Gly   Arg   Asp   Gly   Gln   Pro   Gly   His   Lys     850   855   860   Gly   Glu   Arg   Gly   Tyr   Pro   Gly   Asn   Ile   Gly   Pro   Val   Gly   Ala   Ala   Gly   865   870   875   880       Ala   Pro   Gly   Pro   His   Gly   Pro   Val   Gly   Pro   Ala   Gly   Lys   His   Gly   Asn     885   890   895   Arg   Gly   Glu   Thr   Gly   Pro   Ser   Gly   Pro   Val   Gly   Pro   Ala   Gly   Ala   Val     900   905   910   Gly   Pro   Arg   Gly   Pro   Ser   Gly   Pro   Gln   Gly   Ile   Arg   Gly   Asp   Lys   Gly     915   920   925   Glu   Pro   Gly   Glu   Lys   Gly   Pro   Arg   Gly   Leu   Pro   Gly   Leu   Lys   Gly   His     930   935   940     Asn   Gly   Leu   Gln   Gly   Leu   Pro   Gly   Ile   Ala   Gly   His   His   Gly   Asp   Gln   945   950   955   960   Gly   Ala   Pro   Gly   Ser   Val   Gly   Pro   Ala   Gly   Pro   Arg   Gly   Pro   Ala   Gly     965   970   975   Pro   Ser   Gly   Pro   Ala   Gly   Lys   Asp   Gly   Arg   Thr   Gly   His   Pro   Gly   Thr     980   985   990   Val   Gly   Pro   Ala   Gly   Ile   Arg   Gly   Pro   Gln   Gly   His   Gln   Gly   Pro   Ala     995   1000   1005     Gly   Pro   Pro   Gly   Pro   Pro   Gly   Pro   Pro   Gly   Pro   Pro   Gly   Val   Ser   Gly     1010   1015   1020   Gly   Gly   Tyr   Asp   Phe   Gly   Tyr   Asp   Gly   Asp   Phe   Tyr   Arg   Ala   1025   1030   1035     <210>   3   <211>   378   <212>   PRT   <213>   Sprague   Dawley   <223>   SD rat type B actin amino acid sequence   <400>   3   Met   Phe   Ala   Met   Asp   Asp   Asp   Ile   Ala   Ala   Leu   Val   Val   Asp   Asn   Gly   1   5   10   15   Ser   Gly   Met   Cys   Lys   Ala   Gly   Phe   Ala   Gly   Asp   Asp   Ala   Pro   Arg   Ala     20   25   30   Val   Phe   Pro   Ser   Ile   Val   Gly   Arg   Pro   Arg   His   Gln   Gly   Val   Met   Val     35   40   45     Gly   Met   Gly   Gln   Lys   Asp   Ser   Tyr   Val   Gly   Asp   Glu   Ala   Gln   Ser   Lys     50   55   60     Arg   Gly   Ile   Leu   Thr   Leu   Lys   Tyr   Pro   Ile   Glu   His   Gly   Ile   Val   Thr   65   70   75   80   Asn   Trp   Asp   Asp   Met   Glu   Lys   Ile   Trp   His   His   Thr   Phe   Tyr   Asn   Glu     85   90   95   Leu   Arg   Val   Ala   Pro   Glu   Glu   His   Pro   Val   Leu   Leu   Thr   Glu   Ala   Pro     100   105   110   Leu   Asn   Pro   Lys   Ala   Asn   Arg   Glu   Lys   Met   Thr   Gln   Ile   Met   Phe   Glu     115   120   125   Thr   Phe   Asn   Thr   Pro   Ala   Met   Tyr   Val   Ala   Ile   Gln   Ala   Val   Leu   Ser     130   135   140   Leu   Tyr   Ala   Ser   Gly   Arg   Thr   Thr   Gly   Ile   Val   Met   Asp   Ser   Gly   Asp   145   150   155   160   Gly   Val   Thr   His   Thr   Val   Pro   Ile   Tyr   Glu   Gly   Tyr   Ala   Leu   Pro   His     165   170   175   Ala   Ile   Leu   Arg   Leu   Asp   Leu   Ala   Gly   Arg   Asp   Leu   Thr   Asp   Tyr   Leu     180   185   190   Met   Lys   Ile   Leu   Thr   Glu   Arg   Gly   Tyr   Ser   Phe   Thr   Thr   Thr   Ala   Glu     195   200   205   Arg   Glu   Ile   Val   Arg   Asp   Ile   Lys   Glu   Lys   Leu   Cys   Tyr   Val   Ala   Leu     210   215   220   Asp   Phe   Glu   Gln   Glu   Met   Ala   Thr   Ala   Ala   Ser   Ser   Ser   Ser   Leu   Glu   225   230   235   240   Lys   Ser   Tyr   Glu   Leu   Pro   Asp   Gly   Gln   Val   Ile   Thr   Ile   Gly   Asn   Glu     245   250   255   Arg   Phe   Arg   Cys   Pro   Glu   Ala   Leu   Phe   Gln   Pro   Ser   Phe   Leu   Gly   Met     260   265   270   Glu   Ser   Cys   Gly   Ile   His   Glu   Thr   Thr   Phe   Asn   Ser   Ile   Met   Lys   Cys     275   280   285   Asp   Val   Asp   Ile   Arg   Lys   Asp   Leu   Tyr   Ala   Asn   Thr   Val   Leu   Ser   Gly     290   295   300     Gly   Thr   Thr   Met   Tyr   Pro   Gly   Ile   Ala   Asp   Arg   Met   Gln   Lys   Glu   Ile   305   310   315   320   Thr   Ala   Leu   Ala   Pro   Ser   Thr   Met   Lys   Ile   Lys   Ile   Ile   Ala   Pro   Pro     325   330   335   Glu   Arg   Lys   Tyr   Ser   Val   Trp   Ile   Gly   Gly   Ser   Ile   Leu   Ala   Ser   Leu     340   345   350   Ser   Thr   Phe   Gln   Gln   Met   Trp   Ile   Ser   Lys   Gln   Glu   Tyr   Asp   Glu   Ser     355   360   365   Gly   Pro   Ser   Ile   Val   His   Arg   Lys   Cys   Phe     370   375   <210>   4   <211>   3359   <212>   DNA   <213>   Homo   Sapiens   <223>Human   COL1A1 gene (cDNA,  partial) <400> 4 aagtcgaccg gaggaatttc cgtgcctggc cccatgggtc cctctggtcc tcgtggtctc 60 cctggccccc ctggtgcacc tggtccccaa ggcttccaag gtccccctgg tgagcctggc 120 gagcctggag cttcaggtcc catgggtccc cgaggtcccc caggtccccc tggaaagaat 180 ggagatgatg gggaagctgg aaaacctggt cgtcctggtg agcgtgggcc tcctgggcct 240 cagggtgctc gaggattgcc cggaacagct ggcctccctg gaatgaaggg acacagaggt 300 ttcagtggtt tggatggtgc caagggagat gctggtcctg ctggtcctaa gggtgagcct 360 ggcagccctg gtgaaaatgg agctcctggt cagatgggcc cccgtggcct gcctggtgag 420 agaggtcgcc ctggagcccc tggccctgct ggtgctcgtg gaaatgatgg tgctactggt 480 gctgccgggc cccctggtcc caccggcccc gctggtcctc ctggcttccc tggtgctgtt 540 ggtgctaagg gtgaagctgg tccccaaggg ccccgaggct ctgaaggtcc ccagggtgtg 600 cgtggtgagc ctggcccccc tggccctgct ggtgctgctg gccctgctgg aaaccctggt 660 gctgatggac agcctggtgc taaaggtgcc aatggtgctc ctggtattgc tggtgctcct 720 ggcttccctg gtgcc cgagg cccctctgga ccccagggcc ccggcggccc tcctggtccc 780 aagggtaaca gcggtgaacc tggtgctcct ggcagcaaag gagacactgg tgctaaggga 840 gagcctggcc ctgttggtgt tcaaggaccc cctggccctg ctggagagga aggaaagcga 900 ggagctcgag gtgaacccgg acccactggc ctgcccggac cccctggcga gcgtggtgga 960 cctggtagcc gtggtttccc tggcgcagat ggtgttgctg gtcccaaggg tcccgctggt 1020 gaacgtggtt ctcctggccc tgctggcccc aaaggatctc ctggtgaagc tggtcgtccc 1080 ggtgaagctg gtctgcctgg tgccaagggt ctgactggaa gccctggcag ccctggtcct 1140 gatggcaaaa ctggcccccc tggtcccgcc ggtcaagatg gtcgccccgg acccccaggc 1200 ccacctggtg cccgtggtca ggctggtgtg atgggattcc ctggacctaa aggtgctgct 1260 ggagagcccg gcaaggctgg agagcgaggt gttcccggac cccctggcgc tgtcggtcct 1320 gctggcaaag atggagaggc tggagctcag ggaccccctg gccctgctgg tcccgctggc 1380 gagagaggtg aacaaggccc tgctggctcc cccggattcc agggtctccc tggtcctgct 1440 ggtcctccag gtgaagcagg caaacctggt gaacagggtg ttcctggaga ccttggcgc c 1500 cctggcccct ctggagcaag aggcgagaga ggtttccctg gcgagcgtgg tgtgcaaggt 1560 ccccctggtc ctgctggtcc ccgaggggcc aacggtgctc ccggcaacga tggtgctaag 1620 ggtgatgctg gtgcccctgg agctcccggt agccagggcg cccctggcct tcagggaatg 1680 cctggtgaac gtggtgcagc tggtcttcca gggcctaagg gtgacagagg tgatgctggt 1740 cccaaaggtg ctgatggctc tcctggcaaa gatggcgtcc gtggtctgac cggccccatt 1800 ggtcctcctg gccctgctgg tgcccctggt gacaagggtg aaagtggtcc cagcggccct 1860 gctggtccca ctggagctcg tggtgccccc ggagaccgtg gtgagcctgg tccccccggc 1920 cctgctggct ttgctggccc ccctggtgct gacggccaac ctggtgctaa aggcgaacct 1980 ggtgatgctg gtgctaaagg cgatgctggt ccccctggcc ctgccggacc cgctggaccc 2040 cctggcccca ttggtaatgt tggtgctcct ggagccaaag gtgctcgcgg cagcgctggt 2100 ccccctggtg ctactggttt ccctggtgct gctggccgag tcggtcctcc tggcccctct 2160 ggaaatgctg gaccccctgg ccctcctggt cctgctggca aagaaggcgg caaaggtccc 2220 cgtggtgaga ctggccctgc tggacgtcct ggtg aagttg gtccccctgg tccccctggc 2280 cctgctggcg agaaaggatc ccctggtgct gatggtcctg ctggtgctcc tggtactccc 2340 gggcctcaag gtattgctgg acagcgtggt gtggtcggcc tgcctggtca gagaggagag 2400 agaggcttcc ctggtcttcc tggcccctct ggtgaacctg gcaaacaagg tccctctgga 2460 gcaagtggtg aacgtggtcc ccctggtccc atgggccccc ctggattggc tggaccccct 2520 ggtgaatctg gacgtgaggg ggctcctggt gccgaaggtt cccctggacg agacggttct 2580 cctggcgcca agggtgaccg tggtgagacc ggccccgctg gaccccctgg tgctcctggt 2640 gctcctggtg cccctggccc cgttggccct gctggcaaga gtggtgatcg tggtgagact 2700 ggtcctgctg gtcccgccgg tcctgtcggc cctgttggcg cccgtggccc cgccggaccc 2760 caaggcccac gtggtgacaa gggtgagaca ggcgaacagg gcgacagagg cataaagggt 2820 caccgtggct tctctggcct ccagggtccc cctggccctc ctggctctcc tggtgaacaa 2880 ggtccctctg gagcctctgg tcctgctggt ccccgaggtc cccctggctc tgctggtgct 2940 cctggcaaag atggactcaa cggtctccct ggccccattg ggccccctgg tcctcgcggt 3000 cgcactggtg atgctggtcc tgttggtccc cccggccctc ctggacctcc tggtccccct 3060 ggtcctccca gcgctggttt cgacttcagc ttcctgcccc agccacctca agagaaggct 3120 cacgatggtg gccgctacta ccgggctgat gatgccaatg tggttcgtga ccgtgacctc 3180 gaggtggaca ccaccctcaa gagcctgagc cagcagatcg agaacatccg gagcccagag 3240 ggcagccgca agaaccccgc ccgcacctgc cgtgacctca agatgtgcca ctctgactgg 3300 aagagtggag agtactggat tgaccccaac caaggctgca acctggatgc catcaaagt 3359 <210> 5 <211> 3096 < 212> DNA <213> Homo sapiens <223> Human COL1A2 gene (cDNA,  partial) <400> 5 gatggaaaag gagttggact tggccctgga ccaatgggct taatgggacc tagaggccca 60 cctggtgcag ctggagcccc aggccctcaa ggtttccaag gacctgctgg tgagcctggt 120 gaacctggtc aaactggtcc tgcaggtgct cgtggtccag ctggccctcc tggcaaggct 180 ggtgaagatg gtcaccctgg aaaacccgga cgacctggtg agagaggagt tgttggacca 240 cagggtgctc gtggtttccc tggaactcct ggacttcctg gcttcaaagg cattagggga 300 cacaatggtc tggatggatt gaagggacag cccggtgctc ctggtgtgaa gggtgaacct 360 ggtgcccctg gtgaaaatgg aactccaggt caaacaggag cccgtgggct tcctggtgag 420 agaggacgtg ttggtgcccc tggcccagct ggtgcccgtg gcagtgatgg aagtgtgggt 480 cccgtgggtc ctgctggtcc cattgggtct gctggccctc caggcttccc aggtgcccct 540 ggccccaagg gtgaaattgg agctgttggt aacgctggtc ctgctggtcc cgccggtccc 600 cgtggtgaag tgggtcttcc aggcctctcc ggccccgttg gacctcctgg taatcctgga 660 gcaaacggcc ttactggtgc caagggtgct gctggccttc ccggcgttgc tggggctccc 720 ggcctccctg gaccc cgcgg tattcctggc cctgttggtg ctgccggtgc tactggtgcc 780 agaggacttg ttggtgagcc tggtccagct ggctccaaag gagagagcgg taacaagggt 840 gagcccggct ctgctgggcc ccaaggtcct cctggtccca gtggtgaaga aggaaagaga 900 ggccctaatg gggaagctgg atctgccggc cctccaggac ctcctgggct gagaggtagt 960 cctggttctc gtggtcttcc tggagctgat ggcagagctg gcgtcatggg ccctcctggt 1020 agtcgtggtg caagtggccc tgctggagtc cgaggaccta atggagatgc tggtcgccct 1080 ggggagcctg gtctcatggg acccagaggt cttcctggtt cccctggaaa tatcggcccc 1140 gctggaaaag aaggtcctgt cggcctccct ggcatcgacg gcaggcctgg cccaattggc 1200 ccagctggag caagaggaga gcctggcaac attggattcc ctggacccaa aggccccact 1260 ggtgatcctg gcaaaaacgg tgataaaggt catgctggtc ttgctggtgc tcggggtgct 1320 ccaggtcctg atggaaacaa tggtgctcag ggacctcctg gaccacaggg tgttcaaggt 1380 ggaaaaggtg aacagggtcc ccctggtcct ccaggcttcc agggtctgcc tggcccctca 1440 ggtcccgctg gtgaagttgg caaaccagga gaaaggggtc tccatggtga gtttggtct c 1500 cctggtcctg ctggtccaag aggggaacgc ggtcccccag gtgagagtgg tgctgccggt 1560 cctactggtc ctattggaag ccgaggtcct tctggacccc cagggcctga tggaaacaag 1620 ggtgaacctg gtgtggttgg tgctgtgggc actgctggtc catctggtcc tagtggactc 1680 ccaggagaga ggggtgctgc tggcatacct ggaggcaagg gagaaaaggg tgaacctggt 1740 ctcagaggtg aaattggtaa ccctggcaga gatggtgctc gtggtgctcc tggtgctgta 1800 ggtgcccctg gtcctgctgg agccacaggt gaccggggcg aagctggggc tgctggtcct 1860 gctggtcctg ctggtcctcg gggaagccct ggtgaacgtg gtgaggtcgg tcctgctggc 1920 cccaatggat ttgctggtcc tgctggtgct gctggtcaac ctggtgctaa aggagaaaga 1980 ggagccaaag ggcctaaggg tgaaaacggt gttgttggtc ccacaggccc cgttggagct 2040 gctggcccag ctggtccaaa tggtcccccc ggtcctgctg gaagtcgtgg tgatggaggc 2100 ccccctggta tgactggttt ccctggtgct gctggacgga ctggtccccc aggaccctct 2160 ggtatttctg gccctcctgg tccccctggt cctgctggga aagaagggct tcgtggtcct 2220 cgtggtgacc aaggtccagt tggccgaact ggag aagtag gtgcagttgg tccccctggc 2280 ttcgctggtg agaagggtcc ctctggagag gctggtactg ctggacctcc tggcactcca 2340 ggtcctcagg gtcttcttgg tgctcctggt attctgggtc tccctggctc gagaggtgaa 2400 cgtggtctac caggtgttgc tggtgctgtg ggtgaacctg gtcctcttgg cattgccggc 2460 cctcctgggg cccgtggtcc tcctggtgct gtgggtagtc ctggagtcaa cggtgctcct 2520 ggtgaagctg gtcgtgatgg caaccctggg aacgatggtc ccccaggtcg cgatggtcaa 2580 cccggacaca agggagagcg cggttaccct ggcaatattg gtcccgttgg tgctgcaggt 2640 gcacctggtc ctcatggccc cgtgggtcct gctggcaaac atggaaaccg tggtgaaact 2700 ggtccttctg gtcctgttgg tcctgctggt gctgttggcc caagaggtcc tagtggccca 2760 caaggcattc gtggcgataa gggagagccc ggtgaaaagg ggcccagagg tcttcctggc 2820 ttaaagggac acaatggatt gcaaggtctg cctggtatcg ctggtcacca tggtgatcaa 2880 ggtgctcctg gctccgtggg tcctgctggt cctaggggcc ctgctggtcc ttctggccct 2940 gctggaaaag atggtcgcac tggacatcct ggtacagttg gacctgctgg cattcgaggc 3000 cctcagggtc Accaaggccc tgctggcccc cctggtcccc ctggccctcc tggacctcca 3060 ggtgtaagcg gtggtggtta tgactttggt tacgat 3096 <210> 6 <211> 4364 <212> DNA <213> Recombinant sequence <223> Rat-human COL1A1 chimeric gene <220> <221> gene <222> ( 1). . (425) <223> Rat COL1A1(partial) <220> <221> gene <222> (426). . (3783) <223> Human COL1A1(partial) <220> <221> gene <222> (3784). . (4309) <223> Rat COL1A1(partial) <220> <221> gene <222> (4310). . (4315) <223> linker <220> <221> gene <222> (4316). . (4363) <223> Flag tag <400> 6 atgttcagct ttgtggacct ccggctcctg ctcctcttag gggccactgc cctcctgacg 60 catggccaag aagacatccc tgaagtcagc tgcatacaca atggcctaag ggtccctaat 120 ggtgagacgt ggaaacctga tgtatgcttg atctgtatct gccacaatgg cacggctgtg 180 tgcgatggcg tgctatgcaa agaagacttg gactgtccca acccccaaaa acgggagggc 240 gagtgctgtc ctttctgccc agaagaatat gtatcaccag acgcagaagt cataggagtc 300 gagggaccca agggagaccc tggcccccaa ggcccacggg gacccaaggg agacccgatg 360 ccatcaaagt tggcccccaa ggcccacgga actttgcttc ccagctgtcc tatggctatg 420 acgaaaagtc gaccggagga atttccgtgc ctggccccat gggtccctct ggtcctcgtg 480 gtctccctgg cccccctggt gcacctggtc cccaaggctt ccaaggtccc cctggtgagc 540 ctggcgagcc tggagcttca ggtcccatgg gtccccgagg tcccccaggt ccccctggaa 600 agaatggaga tgatggggaa gctggaaaac ctggtcgtcc tggtgagcgt gggcctcctg 660 ggcctcaggg tgctcgagga ttgcccggaa cagctggcct ccctggaatg aagggacaca 720 gaggtttcag tggtttggat ggtgccaagg gagatgctgg tcctgctggt cctaagggtg 780 agcctggcag ccctggtgaa aatggagctc ctggtcagat gggcccccgt ggcctgcctg 840 gtgagagagg tcgccctgga gcccctggcc ctgctggtgc tcgtggaaat gatggtgcta 900 ctggtgctgc cgggccccct ggtcccaccg gccccgctgg tcctcctggc ttccctggtg 960 ctgttggtgc taagggtgaa gctggtcccc aagggccccg aggctctgaa ggtccccagg 1020 gtgtgcgtgg tgagcctggc ccccctggcc ctgctggtgc tgctggccct gctggaaacc 1080 ctggtgctga tggacagcct ggtgctaaag gtgccaatgg tgctcctggt attgctggtg 1140 ctcctggctt ccctggtgcc cgaggcccct ctggacccca gggccccggc ggccctcctg 1200 gtcccaaggg taacagcggt gaacctggtg ctcctggcag caaaggagac actggtgcta 1260 agggagagcc tggccctgtt ggtgttcaag gaccccctgg ccctgctgga gaggaaggaa 1320 agcgaggagc tcgaggtgaa cccggaccca ctggcctgcc cggaccccct ggcgagcgtg 1380 gtggacctgg tagccgtggt ttccctggcg cagatggtgt tgctggtccc aagggtcccg 1440 ctggtgaacg tggttctcct ggccctgctg gccccaaag g atctcctggt gaagctggtc 1500 gtcccggtga agctggtctg cctggtgcca agggtctgac tggaagccct ggcagccctg 1560 gtcctgatgg caaaactggc ccccctggtc ccgccggtca agatggtcgc cccggacccc 1620 caggcccacc tggtgcccgt ggtcaggctg gtgtgatggg attccctgga cctaaaggtg 1680 ctgctggaga gcccggcaag gctggagagc gaggtgttcc cggaccccct ggcgctgtcg 1740 gtcctgctgg caaagatgga gaggctggag ctcagggacc ccctggccct gctggtcccg 1800 ctggcgagag aggtgaacaa ggccctgctg gctcccccgg attccagggt ctccctggtc 1860 ctgctggtcc tccaggtgaa gcaggcaaac ctggtgaaca gggtgttcct ggagaccttg 1920 gcgcccctgg cccctctgga gcaagaggcg agagaggttt ccctggcgag cgtggtgtgc 1980 aaggtccccc tggtcctgct ggtccccgag gggccaacgg tgctcccggc aacgatggtg 2040 ctaagggtga tgctggtgcc cctggagctc ccggtagcca gggcgcccct ggccttcagg 2100 gaatgcctgg tgaacgtggt gcagctggtc ttccagggcc taagggtgac agaggtgatg 2160 ctggtcccaa aggtgctgat ggctctcctg gcaaagatgg cgtccgtggt ctgaccggcc 2220 ccattggtcc tcct ggccct gctggtgccc ctggtgacaa gggtgaaagt ggtcccagcg 2280 gccctgctgg tcccactgga gctcgtggtg cccccggaga ccgtggtgag cctggtcccc 2340 ccggccctgc tggctttgct ggcccccctg gtgctgacgg ccaacctggt gctaaaggcg 2400 aacctggtga tgctggtgct aaaggcgatg ctggtccccc tggccctgcc ggacccgctg 2460 gaccccctgg ccccattggt aatgttggtg ctcctggagc caaaggtgct cgcggcagcg 2520 ctggtccccc tggtgctact ggtttccctg gtgctgctgg ccgagtcggt cctcctggcc 2580 cctctggaaa tgctggaccc cctggccctc ctggtcctgc tggcaaagaa ggcggcaaag 2640 gtccccgtgg tgagactggc cctgctggac gtcctggtga agttggtccc cctggtcccc 2700 ctggccctgc tggcgagaaa ggatcccctg gtgctgatgg tcctgctggt gctcctggta 2760 ctcccgggcc tcaaggtatt atctggacgt gagggggctc ctggtgccga gctggacagc gtggtgtggt cggcctgcct ggtcagagag 2820 gagagagagg cttccctggt cttcctggcc cctctggtga acctggcaaa caaggtccct 2880 ctggagcaag tggtgaacgt ggtccccctg gtcccatggg cccccctgga ttggctggac 2940 cccctggtga aggttcccct ggacgagacg 3000 gttctcctgg cgccaagggt gaccgtggtg agaccggccc cgctggaccc cctggtgctc 3060 ctggtgctcc tggtgcccct ggccccgttg gccctgctgg caagagtggt gatcgtggtg 3120 agactggtcc tgctggtccc gccggtcctg tcggccctgt tggcgcccgt ggccccgccg 3180 gaccccaagg cccacgtggt gacaagggtg agacaggcga acagggcgac agaggcataa 3240 agggtcaccg tggcttctct ggcctccagg gtccccctgg ccctcctggc tctcctggtg 3300 aacaaggtcc ctctggagcc tctggtcctg ctggtccccg aggtccccct ggctctgctg 3360 gtgctcctgg caaagatgga ctcaacggtc tccctggccc cattgggccc cctggtcctc 3420 gcggtcgcac tggtgatgct ggtcctgttg gtccccccgg ccctcctgga cctcctggtc 3480 cccctggtcc tcccagcgct ggtttcgact tcagcttcct gccccagcca cctcaagaga 3540 aggctcacga tggtggccgc tactaccggg ctgatgatgc caatgtggtt cgtgaccgtg 3600 acctcgaggt ggacaccacc ctcaagagcc tgagccagca gatcgagaac atccggagcc 3660 cagagggcag ccgcaagaac cccgcccgca cctgccgtga cctcaagatg tgccactctg 3720 actggaagag tggagagtac tggattgacc ccaacca agg ctgcaacctg gatgccatca 3780 aagtctactg caacatggag acaggtcaga cctgtgtgtt ccccactcag ccctctgtgc 3840 ctcagaagaa ctggtacatc agcccaaacc ccaaggagaa gaagcatgtc tggtttggag 3900 agagcatgac cgatggattc cagttcgagt atggaagcga aggttccgat cctgccgatg 3960 tcgctatcca gctgaccttc ctgcgcctga tgtccaccga ggcctcccag aacatcacct 4020 atcactgcaa gaacagcgta gcctacatgg accaacagac tggcaacctc aagaagtccc 4080 tgctcctcca gggctccaac gagatcgagc tcaggggcga aggcaacagt cgattcacct 4140 acagcacgct tgtggatggc tgcacgagtc acaccggaac Ttggggcaag acagtcatcg 4200 aatacaaaac caccaagacc tcccgcctgc ccatcatcga tgtggctccc ttggacattg 4260 gtgccccaga ccaggaattc ggaatggaca ttggccctgc ctgcttcgtg cctagggact 4320 ataaggacga tgatgacaag gactacaaag atgatgacga taaa 4364 <210> 7 <211> 4142 <212> DNA <213> Recombinant sequence <223> Rat-human COL1A1 chimera Gene (partial) <220> <221> Gene <222> (1). . (261) <223> Rat COL1A2(partial) <220> <221> gene <222> (262). . (3357) <223> Human COL1A2(partial) <220> <221> gene <222> (262). . (4115) <223> Rat COL1A2(partial) <220> <221> gene <222> (4116). . (4142) <223> HA tag <400> 4 atgctcagct ttgtggatac gcgaactctg ttgctgcttg cagtaacgtc gtgcctagca 60 acatgccaat ctttacaaat gggatctgta cggaagggcc ccactggaga cagaggaccg 120 cgtggacaaa ggggcccagc aggtccccga ggcagagatg gtgttgatgg tcccgttggc 180 cctcctggtc cccctggtgc ccctggcccc cctggtcccc ctggcccccc tggtcttact 240 gggaatttcg cagcccagta tgatggaaaa ggagttggac ttggccctgg accaatgggc 300 ttaatgggac ctagaggccc acctggtgca gctggagccc caggccctca aggtttccaa 360 ggacctgctg gtgagcctgg tgaacctggt caaactggtc ctgcaggtgc tcgtggtcca 420 gctggccctc ctggcaaggc tggtgaagat ggtcaccctg gaaaacccgg acgacctggt 480 gagagaggag ttgttggacc acagggtgct cgtggtttcc ctggaactcc tggacttcct 540 ggcttcaaag gcattagggg acacaatggt ctggatggat tgaagggaca gcccggtgct 600 cctggtgtga agggtgaacc tggtgcccct ggtgaaaatg gaactccagg tcaaacagga 660 gcccgtgggc ttcctggtga gagaggacgt gttggtgccc ctggcccagc tggtgcccgt 720 ggcagtgatg gaagtgtggg tcccgtgggt cctgctggtc ccattgggtc tgctggccct 780 ccaggcttcc caggtgcccc tggccccaag ggtgaaattg gagctgttgg taacgctggt 840 cctgctggtc ccgccggtcc ccgtggtgaa gtgggtcttc caggcctctc cggccccgtt 900 ggacctcctg gtaatcctgg agcaaacggc cttactggtg ccaagggtgc tgctggcctt 960 cccggcgttg ctggggctcc cggcctccct ggaccccgcg gtattcctgg ccctgttggt 1020 gctgccggtg ctactggtgc cagaggactt gttggtgagc ctggtccagc tggctccaaa 1080 ggagagagcg gtaacaaggg tgagcccggc tctgctgggc cccaaggtcc tcctggtccc 1140 agtggtgaag aaggaaagag aggccctaat ggggaagctg gatctgccgg ccctccagga 1200 cctcctgggc tgagaggtag tcctggttct cgtggtcttc ctggagctga tggcagagct 1260 ggcgtcatgg gccctcctgg tagtcgtggt gcaagtggcc ctgctggagt ccgaggacct 1320 aatggagatg ctggtcgccc tggggagcct ggtctcatgg gacccagagg tcttcctggt 1380 tcccctggaa atatcggccc cgctggaaaa gaaggtcctg tcggcctccc tggcatcgac 1440 ggcaggcctg gcccaattgg cccagctgga gcaagaggag agc ctggcaa cattggattc 1500 cctggaccca aaggccccac tggtgatcct ggcaaaaacg gtgataaagg tcatgctggt 1560 cttgctggtg ctcggggtgc tccaggtcct atggtgctca gatggaaaca ccagggcctg atggaaacaa gggtgaacct gggacctcct 1620 ggaccacagg gtgttcaagg tggaaaaggt gaacagggtc cccctggtcc tccaggcttc 1680 cagggtctgc ctggcccctc aggtcccgct ggtgaagttg gcaaaccagg agaaaggggt 1740 ctccatggtg agtttggtct ccctggtcct gctggtccaa gaggggaacg cggtccccca 1800 ggtgagagtg gtgctgccgg tcctactggt cctattggaa gccgaggtcc ttctggaccc 1860 ggtgtggttg gtgctgtggg cactgctggt 1920 ccatctggtc ctagtggact cccaggagag aggggtgctg ctggcatacc tggaggcaag 1980 ggagaaaagg gtgaacctgg tctcagaggt gaaattggta accctggcag agatggtgct 2040 cgtggtgctc ctggtgctgt aggtgcccct ggtcctgctg gagccacagg tgaccggggc 2100 gaagctgggg ctgctggtcc tgctggtcct gctggtcctc ggggaagccc tggtgaacgt 2160 ggtgaggtcg gtcctgctgg ccccaatgga tttgctggtc ctgctggtgc tgctggtcaa 2220 cctggtgcta aaggagaaa g aggagccaaa gggcctaagg gtgaaaacgg tgttgttggt 2280 cccacaggcc ccgttggagc tgctggccca gctggtccaa atggtccccc cggtcctgct 2340 ggaagtcgtg gtgatggagg cccccctggt atgactggtt tccctggtgc tgctggacgg 2400 actggtcccc caggaccctc tggtatttct ggccctcctg gtccccctgg tcctgctggg 2460 aaagaagggc ttcgtggtcc tcgtggtgac caaggtccag ttggccgaac tggagaagta 2520 ggtgcagttg gtccccctgg cttcgctggt gagaagggtc cctctggaga ggctggtact 2580 gctggacctc ctggcactcc aggtcctcag ggtcttcttg gtgctcctgg tattctgggt 2640 ctccctggct cgagaggtga acgtggtcta ccaggtgttg ctggtgctgt gggtgaacct 2700 ggtcctcttg gcattgccgg ccctcctggg gcccgtggtc ctcctggtgc tgtgggtagt 2760 cctggagtca acggtgctcc tggtgaagct ggtcgtgatg gcaaccctgg gaacgatggt 2820 cccccaggtc gcgatggtca acccggacac aagggagagc gcggttaccc tggcaatatt 2880 ggtcccgttg gtgctgcagg tgcacctggt cctcatggcc ccgtgggtcc tgctggcaaa 2940 catggaaacc gtggtgaaac tggtccttct ggtcctgttg gtcctgctgg tgctgttggc 3000 ccaagaggtc ctagtggccc acaaggcatt cgtggcgata agggagagcc cggtgaaaag 3060 gggcccagag gtcttcctgg cttaaaggga cacaatggat tgcaaggtct gcctggtatc 3120 gctggtcacc atggtgatca aggtgctcct ggctccgtgg gtcctgctgg tcctaggggc 3180 cctgctggtc cttctggccc tgctggaaaa gatggtcgca ctggacatcc tggtacagtt 3240 ggacctgctg gcattcgagg ccctcagggt caccaaggcc ctgctggccc ccctggtccc 3300 cctggccctc ctggacctcc aggtgtaagc ggtggtggtt atgactttgg ttacgatgga 3360 gacttctaca gggctgacag cctcgctcac agccttcact cagacccaag gactatgaag 3420 ttgatgcaac tctgaaatct ctcaataacc aaatcgagac ccttctcact cctgaaggct 3480 ctagaaagaa ccctgcccgc acatgccgtg acttaagact cagccaccca gagtggaaga 3540 gcgattacta ctggattgac cctaaccaag gatgcactat ggatgccatc aaagtgtact 3600 gcgatttctc tactggtgaa acctgcatcc aggcccaacc tgtcaacacc ccagccaaga 3660 atgcatacag ccgtgcccag gccaacaagc atgtctggtt aggagagacc atcaatggtg 3720 gcagccagtt tgaatacaac gcagaagggg tgtcctccaa g gaaatggca actcagctcg 3780 ccttcatgcg cctgctagcc aaccgtgctt ctcagaacat cacctaccac tgcaagaaca 3840 gcattgcgta cctggacgag gagacaggcc gcctgaataa ggctgtcatt ctgcagggct 3900 ccaacgacgt cgaacttgtt gctgagggca acagcagatt cacctacact gtccttgtcg 3960 atggctgctc caaaaagaca aatgaatggg acaagacaat cattgaatac aaaacgaata 4020 agccatctcg cctgccattc cttgacattg cacctctgga catcggtggt actaaccaag 4080 aattccgtgt ggaggttggc cctgtctgtt tcaaataccc atacgatgtt ccagattacg 4140 ct 4142 <210> 8 <211> 27 <212> DNA <213> Artificial sequence <223> Human COL1A1 sgRNA1-1 <400> 8 taggtgattt ctcatca tagccat 24 <210> 9 <211> 24 <212> DNA <213> Artificial sequence <223> Human COL1A1 sgRNA1 -2 <400> 9 aaacatggct atgatgagaa atca 24 <210> 10 <211> 24 <212> DNA <213> Artificial sequence <223> Human COL1A1 sgRNA2-1 <400> 10 taggagtttc cgtgcctggc ccca 24 <210> 11 <211> 24 <212> DNA <213> Artificial sequence <223> Human COL1A1 sgRNA2-2 <400> 11 aaactggggc caggcacgga aact 24 <210> 12 <211> 21 <212> DNA <213> Artificial sequence <223> Human COL1A2 sgRNA1- 1 <400> 12 tagggctcag tattctgaca a 21 <210> 13 <211> 21 <212> DNA <213> Artificial sequence <223> Human COL1A2 sgRNA1-2 <400> 13 aaacttgtca gaatactgag c 21 <210> 14 <211> 21 <212> DNA <213> Artificial sequence <223> Human COL1A2 sgRNA21 <400> 14 taggtttaac ttagtattgt g 21 <210> 15 <211> 21 <212> DNA <213> Artificial sequence < 223>Human COL1A2 sgRNA2-2 <400> 15 aaaccacaat actaagttaa a 21 <210> 16 <211> 27 <212> DNA <213> Artificial sequence <223> PCR primer <400> 16 cgaatgcatc tagatgatgg catccct 27 <210> 17 < 211> 27 <212> DNA <213> Artificial sequence <223> PCR primer <400> 17 tcctccggtc gacttttcgt catagcc 27 <210> 18 <211> 27 <212> DNA <213> Artificial sequence <223> PCR primer <400> 18 aagtcgaccg gaggaatttc cgtgcct 27 <210> 19 <211> 27 <212> DNA <213> Artificial sequence <223 > PCR primer <400> 19 actttgatgg catccaggtt gcagcct 27 <210> 20 <211> 27 <212> DNA <213> Artificial sequence <223> PCR primer <400> 20 ggatgccatc aaagtctact gcaacat 27 <210> 21 <211> 27 < 212> DNA <213> Artificial sequence <223> PCR primer <400> 21 cacgaagcag gcagggccaa tgtccat 27 <210> 22 <211> 27 <212> DNA <213> Artificial sequence <223> PCR primer <400> 22 cctgcctgct tcgtgcctag ggactat 27 <210> 23 <211> 27 <212> DNA <213> Artificial sequence <223> PCR primer <400> 23 cgactctaga ggatctgctc gaatcga 27 <210> 24 < 211> 27 <212> DNA <213> Artificial sequence <223> PCR primer <400> 24 gatcctctag agtcgtgcct ggcccca 27 <210> 25 <211> 27 <212> DNA <213> Artificial sequence <223> PCR primer <400> 25 taccttccag ggaattccca ccagtgg 27 <210> 26 <211> 30 <212> DNA <213> Artificial sequence <223> PCR primer <400> 26 aattccctgg aaggtagctc tcctgagtag 30 <210> 27 <211> 35 <212> DNA <213> Artificial sequence <223> PCR primer <400> 27 ctgtccaggg atgccattca cagcttgtct gtaag 35 <210> 28 <211> 25 <212> DNA <213> Artificial sequence < 223> PCR primer <400> 28 cactgagggt agccgcagcc ttcag 25 <210> 29 <211> 25 <212> DNA <213> Artificial sequence <223> PCR primer <400> 29 tactgggctg cgaaattctg gaggg 25 <210> 30 <211> 25 <212> DNA <213> Artificial sequence <223> PCR primer <400> 30 tttcgcagcc cagtatgatg gaaaa 25 <210> 31 <211> 2 5 <212> DNA <213> Artificial sequence <223> PCR primer <400> 31 ctgtgagcga ggctggtcag ccctg 25 <210> 32 <211> 25 <212> DNA <213> Artificial sequence <223> PCR primer <400> 32 ccagcctcgc Tcacagcctt cactc 25 <210> 33 <211> 25 <212> DNA <213> Artificial sequence <223> PCR primer <400> 33 tttgaaacag acagggccaa cctcc 25 <210> 34 <211> 25 <212> DNA <213> Artificial sequence <223> PCR primer <400> 34 cctgtctgtt tcaaataccc atacg 25 <210> 35 <211> 25 <212> DNA <213> Artificial sequence <223> PCR primer <400> 35 ccatggagtt Ttaaccatag agccc 25 <210> 36 <211> 25 <212> DNA <213> Artificial sequence <223> PCR primer <400> 36 gttaaaactc catggttaga tcaaa 25 <210> 37 <211> 32 <212> DNA <213> Artificial sequence <223> PCR primer <400> 37 gtcctactca ggagagcctt atagtatatt ac 32 <210> 38 <211> 32 <212> DNA <213> Artificial sequence <223> PCR primer <400> 38 gtaatatact ataaggctct cctgagtagg ac 32 <210> 39 < 211> 36 <212> DNA <213> Artificial sequence <223> PCR primer <400> 37 gaaggctgcg gctaccctca cagcagcttg tctgta 36 <210 39 <211> 40 <212> DNA <213> Artificial sequence <223> PCR primer <400> 40 cataacagcc ccctccattt c 21 <210> 41 <211> 20 <212> DNA <213> Artificial sequence <223> PCR primer< 400> 41 accagtagca ccatcatttc 20 <210> 42 <211> 18 <212> DNA <213> Artificial sequence <223> PCR primer <400> 42 agcagatcct ctagagtc 18 <210> 43 <211> 21 <212> DNA <213> Artificial sequence <223> PCR primer <400> 43 tggctttggt ttaggggaac c 21 <210> 44 <211> 18 <212> DNA <213> Artificial sequence <223> PCR primer <400> 44 cagagatggt gttgatgg 18 <210> 45 <211> 17 <212> DNA <213> Artificial sequence <223> PCR primer <400> 45 aggtccttgg aaccttg 17 <210> 46 <211> 20 < 212> DNA <213> Artificial sequence <223> PCR primer <400> 46 ctgctagcca accgtgcttc 20 <210> 47 <211> 19 <212> DNA <213> Artificial sequence <223> PCR primer <400> 47 gagtaatgcc cggcattag 19

Claims (13)

A crude protein extract isolated from non-human connective tissue, wherein the crude protein extract comprises (i) at least 91% similarity to SEQ ID NO: 1 and SEQ ID NO: 3, (ii) and SEQ ID NO: 2 has at least 93% similarity and SEQ ID NO: 3 or (iii) has at least 91% similarity to SEQ ID NO: 1, has at least 91% similarity to SEQ ID NO: 2, and SEQ ID NO: amino acid sequence of 3. The protein crude extract according to claim 1, wherein the protein crude extract comprises (i) SEQ ID NO: 1 and SEQ ID NO: 3, (ii) SEQ ID NO: 2 and SEQ ID NO: 3 or (iii) the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. The crude protein extract of claim 1, wherein the crude protein extract is specifically identified by an anti-human collagen antibody. The crude protein extract of the first aspect of the patent application is obtained by isolating the connective tissue of a gene-transferred rat, wherein the genomic DNA of the gene-transferred rat has a human type I collagen. DNA sequence. The crude protein extract of claim 4, wherein the DNA sequence encoding human type I collagen is SEQ ID NO: 4 or SEQ ID NO: 5. A method for preparing a crude protein extract containing a labeled amino acid sequence, comprising: (a) a DNA construct comprising a composition comprising SEQ ID NO: 4 or SEQ ID NO: 5; b) introducing the DNA sequence construct into a rat embryo, and then transplanting the rat embryo into a homologous mother to develop into a mouse; (c) is isolated from the connective tissue of the adult mouse a crude protein extract containing a target amino acid sequence, wherein the target amino acid sequence is selected from the group consisting of (i) having at least 91% similarity to SEQ ID NO: 1 and SEQ ID NO: 3, (ii) and SEQ ID NO: 2 has at least 93% similarity and SEQ ID NO: 3 or (iii) has at least 91% similarity to SEQ ID NO: 1, has at least 93% similarity to SEQ ID NO: 2, and The amino acid sequence of SEQ ID NO: 3. The preparation method of claim 6, wherein the target amino acid is selected from the group consisting of (i) SEQ ID NO: 1 and SEQ ID NO: 3, (ii) SEQ ID NO: 2 and SEQ ID NO: 3. Or (iii) SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. The preparation method according to claim 6, wherein the crude protein extract is specifically identified by an anti-human collagen antibody. Use of a crude protein extract having a labeled amino acid sequence for the preparation of human first type collagen, wherein the target amino acid sequence is selected from (i) at least 91% similarity to SEQ ID NO: 1. And SEQ ID NO: 3, (ii) has at least 93% similarity to SEQ ID NO: 2 and SEQ ID NO: 3 or (iii) has at least 91% similarity to SEQ ID NO: 1, with SEQ ID NO: 2 has an amino acid sequence of at least 93% similarity and SEQ ID NO: 3. The use according to claim 9, wherein the target amino acid is selected from the group consisting of (i) SEQ ID NO: 1 and SEQ ID NO: 3, (ii) SEQ ID NO: 2 and SEQ ID NO: 3. Or (iii) SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. The use according to claim 9, wherein the crude protein extract is specifically identified by an anti-human collagen antibody. The use according to claim 9, wherein the crude protein extract is obtained by isolating the connective tissue of a gene-transferred rat, and the genomic DNA of the gene-transferred rat has a coding human first Deoxyribonucleic acid sequence of collagen. The use of claim 12, wherein the deoxyriboribonucleotide sequence encoding human type I collagen is SEQ ID NO: 4 or SEQ ID NO: 5.