Recombinant human collagen and preparation method and application thereof
阅读说明：本技术 一种重组人源胶原蛋白及其制备方法和应用 (Recombinant human collagen and preparation method and application thereof ) 是由 郭伟 于 2021-08-30 设计创作，主要内容包括：本发明公开了一种重组人源胶原蛋白及其制备方法和应用,属于生物工程领域,该制备方法包括如下步骤：(1)获得重组载体：选取人类三型胶原保守区域肽段,将该序列重复8次进行人工全基因合成RHIIIC8,将其构建到pET22b的表达载体中获得重组载体；(2)构建重组基因工程菌：提取质粒、将其转化到BL21-DE3-PLySs感受态细胞中,挑取单克隆并对其扩大培养,获得重组基因工程菌；(3)制备重组人源胶原蛋白：经过诱导表达目的蛋白,然后对表达产物进行分离纯化,得到高纯度的可溶于水的重组人源胶原蛋白。本发明提供的重组人源胶原蛋白具有活性高的特点,可以广泛应用于生物医药和化妆品行业。(The invention discloses a recombinant human collagen and a preparation method and application thereof, belonging to the field of bioengineering, wherein the preparation method comprises the following steps: (1) obtaining a recombinant vector: selecting a human triple-collagen conserved region peptide segment, repeating the sequence for 8 times, carrying out artificial total gene synthesis of RHIIC 8, and constructing the sequence into an expression vector of pET22b to obtain a recombinant vector; (2) constructing recombinant gene engineering bacteria: extracting plasmids, transforming the plasmids into BL21-DE3-PLySs competent cells, selecting monoclone and carrying out expanded culture on the monoclone to obtain recombinant gene engineering bacteria; (3) preparing recombinant human collagen: the target protein is induced and expressed, and then the expression product is separated and purified, so that the high-purity water-soluble recombinant human collagen is obtained. The recombinant human collagen provided by the invention has the characteristic of high activity, and can be widely applied to the industries of biomedicine and cosmetics.)
1. The recombinant human collagen is characterized in that the nucleotide sequence of the encoding gene RHIiC 8 and RHIiC 8 of the recombinant human collagen is SEQ ID No. 1; the amino acid sequence of RHIiC 8 is SEQ ID NO. 2.
2. An expression vector comprising the coding gene of claim 1.
3. A genetically engineered bacterium containing the coding gene of claim 1.
4. The method for preparing recombinant human collagen according to claim 1, comprising the steps of:
(1) obtaining a recombinant vector: selecting a human triple-collagen conserved region peptide segment, repeating the sequence for 8 times, carrying out artificial total gene synthesis of RHIIC 8, and constructing the sequence into an expression vector of pET22b to obtain a recombinant vector;
(2) constructing recombinant gene engineering bacteria: extracting plasmids, transforming the plasmids into BL21-DE3-PLySs competent cells, selecting monoclone and carrying out expanded culture on the monoclone to obtain recombinant gene engineering bacteria;
(3) preparing recombinant human collagen: the target protein is induced and expressed, and then the expression product is separated and purified, so that the high-purity water-soluble recombinant human collagen is obtained.
5. The method for preparing recombinant human collagen according to claim 2, wherein the specific steps of separation and purification in step (3) are as follows:
(3.1) inoculating the engineering bacteria in the step (2) into an LB culture medium, culturing at 37 ℃ overnight, transferring the LB culture medium according to the volume ratio of 1:100, culturing in a shake flask at 37 ℃ until OD600 is 0.6, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.1mM, culturing at 25 ℃ for 6-8 hours, centrifuging at the centrifugal force of 5000Xg for 5 minutes, and collecting thallus precipitates;
(3.2) washing the thallus precipitate with PBS buffer solution, then resuspending the thallus precipitate, selectively adding Triton X-100 solution with the final concentration of 1-5mg/ml lysozyme to help to crack cells, ultrasonically breaking the bacteria in ice bath, centrifuging at 12000rpm for 20min, and collecting supernatant; the amount of the Triton X-100 solution of lysozyme added is 10ml/40ml of the volume of PBS buffer solution used for resuspension;
(3.3) washing the nickel ion affinity column with PBS buffer solution, then gently shaking the affinity column and the supernatant liquid for 30min at room temperature or on ice, loading the column, washing the hybrid protein with PBS solution containing 15mM imidazole, preferably eluting for 1 column volume, washing until only pure RHIiC 8 protein is left on the column, adding Prescission Protease, namely PPase, on the column, gently shaking for 2 hours at room temperature or on ice, releasing the RHIC 8 protein from the column, eluting with PBS solution, preferably 1/2 column volume, collecting the eluent, dialyzing the obtained product overnight, and freeze-drying to obtain purified recombinant human collagen;
wherein the protease is added in an amount of 1. mu.L/5 mL based on the column volume.
6. The method for preparing recombinant human collagen according to claim 5, wherein the LB medium liquid in step (3.1) consists of: 10g/L tryptone, 5g/L, NaCl 5g/L yeast extract and deionized water as solvent, wherein the pH value is 7.5, 15g/L agar is added into LB culture medium liquid and evenly mixed to obtain an LB culture medium plate.
7. The method for preparing recombinant human collagen according to claim 5, wherein the conditions for ultrasonic disruption in step (3.2) are as follows: ultrasonic treatment at 280W for 2s for 5s at a protection temperature of 25 ℃ for 15 min.
8. The method for preparing recombinant human collagen according to claim 5, wherein the step (3.3) of lyophilization comprises the following steps: adding 10%, 5%, 3%, 2%, 1% mannitol aqueous solution 0.5ml into each 5mg protein in turn, pre-freezing at-40 deg.C for 10min, and freeze-drying at-35 deg.C.
9. A biomaterial for tissue engineering, skin care and beauty, comprising the recombinant human collagen of the coding gene of claim 1 or the recombinant human collagen prepared by the preparation method of any one of claims 4 to 8.
Technical Field
The invention relates to the field of bioengineering, and in particular relates to recombinant human collagen and a preparation method and application thereof.
Background
Collagen is one of the most abundant proteins in the human body, and collagen hydrolysate has significant biological activities, such as antioxidant properties, antihypertensive activity, lipid-lowering activity, and the property of repairing damaged skin. Since collagen has a dual role in the skin: providing a building block formed by elastin and collagen, acting as a ligand or binding to a fibroblast receptor to stimulate the synthesis of secreted hyaluronic acid; therefore, the skin care product is widely applied to skin care products. Collagen is a dextrorotatory triple helix structure, consisting of a repeating amino acid triplet sequence: glycine-X-Y, wherein X is typically proline and Y is typically hydroxyproline. The presence of the small amino acid glycine at every third position of the polypeptide chain allows for close packing of the triple helix along the central axis of the collagen molecule. Non-helical, globular regions are present at either end of the collagen molecule, preventing fibril formation prior to cleavage with an appropriate protease. Properties that have led to their popularity as scaffold materials include their relatively low toxicity, natural degradation by matrix metalloproteinases, ease of cross-linking and functionalization, and homology of amino acid sequences present between species.
Animal extracted collagen is widely applied to drug delivery and tissue engineering in the field of biological materials. From skin wound healing, to bone scaffolds, to collagen hydrogels, due to their injectable nature and self-assembly in situ, have also been used for applications including cardiac muscle, intervertebral disc, dermal replacement and regenerating cartilage, but there are many problems in clinical applications. The main obstacle is the source of the collagen used, and the major disadvantages of collagen from animal or human sources are the possible transmission of diseases, allergic reactions, contamination by pathogens. Furthermore, there are also batch-to-batch variations and cultural reasons that increase the use of collagen from animal sources. Therefore, in the technical field of human collagen preparation, there is a need to develop human collagen with high purity, high safety, strong hydrophilicity and good biocompatibility.
Disclosure of Invention
The invention aims to provide a recombinant human collagen and a preparation method and application thereof.
The technical scheme of the invention is as follows: a recombinant human collagen, the encoding gene RHIiC 8 of the recombinant human collagen, the nucleotide sequence of RHIiC 8 is SEQ ID NO. 1; the amino acid sequence of RHIiC 8 is SEQ ID NO. 2.
An expression vector containing a coding gene RHIiMC 8.
A genetically engineered bacterium containing a coding gene RHIiC 8.
A preparation method of recombinant human collagen comprises the following steps:
(1) obtaining a recombinant vector: selecting a human type III collagen conserved region peptide segment:
GERGGPGGPGPQGPPGKNGETGPQGPPGPT, repeating the sequence for 8 times, carrying out artificial total gene synthesis to obtain RHIiC 8, and constructing the RHIiC 8 into an expression vector of pET22b to obtain a recombinant vector;
(2) constructing recombinant gene engineering bacteria: extracting plasmids, transforming the plasmids into BL21-DE3-PLySs competent cells, selecting monoclone and carrying out expanded culture on the monoclone to obtain recombinant gene engineering bacteria;
(3) preparing recombinant human collagen: the target protein is induced and expressed, and then the expression product is separated and purified, so that the high-purity water-soluble recombinant human collagen is obtained.
According to a further technical scheme, the specific steps of separation and purification in the step (3) are as follows:
(3.1) inoculating the engineering bacteria in the step (2) into an LB culture medium, culturing at 37 ℃ overnight, transferring the LB culture medium according to the volume ratio of 1:100, culturing in a shake flask at 37 ℃ until OD600 is 0.6, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.1mM, culturing at 25 ℃ for 6-8 hours, centrifuging at the centrifugal force of 5000Xg for 5 minutes, and collecting thallus precipitates;
(3.2) washing the thallus precipitate with PBS buffer solution, then resuspending the thallus precipitate, selectively adding Triton X-100 solution with the final concentration of 1-5mg/ml lysozyme to help to crack cells, ultrasonically breaking the bacteria in ice bath, centrifuging at 12000rpm for 20min, and collecting supernatant; the amount of the Triton X-100 solution of lysozyme added is 10ml/40ml of the volume of PBS buffer solution used for resuspension;
(3.3) washing the nickel ion affinity column with PBS buffer solution, then gently shaking the affinity column and the supernatant liquid for 30min at room temperature or on ice, loading the column, washing the hybrid protein with PBS solution containing 15mM imidazole, preferably eluting for 1 column volume, washing until only pure RHIiC 8 protein is left on the column, adding Prescission Protease, namely PPase, on the column, gently shaking for 2 hours at room temperature or on ice, releasing the RHIC 8 protein from the column, eluting with PBS solution, preferably 1/2 column volume, collecting the eluent, dialyzing the obtained product overnight, and freeze-drying to obtain purified recombinant human collagen;
wherein the protease is added in an amount of 1. mu.L/5 mL based on the column volume.
According to a further technical scheme, the LB culture medium liquid in the step (3.1) comprises the following components: 10g/L tryptone, 5g/L, NaCl 5g/L yeast extract and deionized water as solvent, wherein the pH value is 7.5, 15g/L agar is added into LB culture medium liquid and evenly mixed to obtain an LB culture medium plate.
According to a further technical scheme, the ultrasonic bacteria breaking conditions in the step (3.2) are as follows: ultrasonic treatment at 280W for 2s for 5s at a protection temperature of 25 ℃ for 15 min.
According to a further technical scheme, the freeze-drying in the step (3.3) comprises the following specific steps: adding 10%, 5%, 3%, 2%, 1% mannitol aqueous solution 0.5ml into each 5mg protein in turn, pre-freezing at-40 deg.C for 10min, and freeze-drying at-35 deg.C.
A biomaterial for tissue engineering, beauty and skin care, comprising the recombinant human collagen of the coding gene of claim 1 or the recombinant human collagen prepared by the preparation method of any one of claims 4 to 8.
The invention has the beneficial effects that:
1. the selected sequence is completely from a human type III collagen conserved region, and the sequence is subjected to codon optimization on the premise of not influencing an amino acid sequence; an escherichia coli expression system is adopted, large-scale amplification is realized, one round of fermentation can be completed within 20 hours, and the application of the escherichia coli expression system in production has the advantages of low cost and short period, BL21-DE3-PLySs expression strains can reduce endogenous protein expression of bacteria and further improve the purity and yield of target protein, the amount of expressed protein can account for about 25% of total protein of the bacteria, and 0.25 mg of target protein can be contained in each milligram of bacterial liquid precipitate;
2. through sequence analysis and experimental screening, the amino acid composition of the collagen is 100 percent same as the corresponding part of the amino acid sequence of the natural collagen, and the collagen does not generate immunological rejection and anaphylactic reaction when being applied to a human body, has very good hydrophilicity and stability, and can perform the function of natural protein in the human body;
3. through activity detection, the activity of the recombinant human collagen prepared by the method is not obviously reduced after being stored for 30 days at room temperature, and the recombinant human collagen can be widely applied to the industries of biological medicines and cosmetics.
Drawings
FIG. 1 is a plasmid map constructed by pET22 b-RHIiC 8 vector in the invention,
FIG. 2 shows the result of the triple collagen peptide fragment screening of the present invention,
FIG. 3 shows the amino acid alignment result of the RHIiC 8 of the present invention and human collagen,
FIG. 4 is an electrophoretogram of the purified RHIiC 8 protein of the invention,
FIG. 5 shows the results of the comparison of the biological activity of the RHIiC 8 protein of the present invention and human collagen.
Detailed Description
The invention will be further illustrated and understood by the following non-limiting examples.
The room temperature of the invention is 25-30 ℃.
Example 1: construction and expression of RHIIC 8 gene expression vector
Since the characteristics of collagen function and stability depend on its triple-helical structure, it was found that the amino acid sequence plays a decisive role in the triple-helical formation of the collagen peptide chain, based on previous studies on its structural stability. Hydroxylated proline in the sequence can react with glycine to form a stable hydrogen bond, and because the peptide chain of the collagen contains a large number of charged amino acids, the special arrangement of positive and negative charged amino acids promotes the formation of a stable structure between different peptide chains.
Therefore, the invention finally obtains the peptide fragment of RHIC 8 with the best activity in the candidate sequences RHIC 1-RHIC 10 which meet the two characteristics by taking the two factors as conditions for screening potential peptide chains, as shown in FIG. 2. The sequences of RHIiC 1-RHIiC 10 are shown below:
RHIIIC1.GPPGPPGAIGPSGPAGKDGESGRPGRPGER
RHIIIC2.GKDGESGRPGRPGERGLPGPPGIKGPAGIP
RHIIIC3.GERGRPGLPGAAGARGNDGARGSDGQPGPP
RHIIIC4.GESGKPGANGLSGERGPPGPQGLPGLAGTA
RHIIIC5.GSDGKPGPPGSQGESGRPGPPGPSGPRGQP
RHIIIC6.GPKGDAGAPGAPGGKGDAGAPGERGPPGLA
RHIIIC7.GERGAPGEKGEGGPPGVAGPPGGSGPAGPP
RHIIIC8.GERGGPGGPGPQGPPGKNGETGPQGPPGPT
RHIIIC9.GPPGPVGPAGKSGDRGESGPAGPAGAPGPA
RHIIIC10.GPPGKDGTSGHPGPIGPPGPRGNRGERGSE
the preparation method of the recombinant human collagen comprises the following specific preparation steps:
(1) acquisition of human-like collagen RHIiC 8 gene
Selecting a human triple-collagen conserved region peptide segment, repeating the sequence of the segment for 8 times, optimizing a codon, entrusting Shanghai Senno biotechnology limited to carry out whole-gene synthesis, adding Xba I and Sal I enzyme cutting sites at two ends of a gene, wherein the nucleotide sequence is shown in SEQ ID NO.1 and is marked as a gene RHIiC 8:
atgggagaacgaggcggccctggaggacctggccctcagggtcctcctggtaagaatggtgaaaccggacctcagggacctccaggacctactggtgagcgaggtggtccaggtggccctggcccacaaggcccacccggaaagaacggtgagactggtccacaaggtcctccaggtccaaccggcgaacgaggaggacctggaggacctggtcctcagggtccgcctgggaagaatggtgaaacaggaccgcagggacctcctggacctacgggtgagcgaggtggtccaggcggtcctggccctcaaggacctccaggcaagaacggtgagactggccctcaaggaccaccaggtccaactggcgaacgaggcggccctggaggacctggcccacagggtccgcctggaaagaatggtgaaactggacctcagggtcctccaggacctactggtgaacgaggtggacctggtggacctggcccacaaggtccaccaggcaagaacggtgagacaggccctcagggacctccaggtcctactggagagcgaggaggcccaggaggccctggccctcagggacctcctggtaagaatggtgaaaccggaccacaagggccaccaggacctactggcgagcgaggtggtcctggcggtcctggccctcagggtccaccaggaaagaacggtgagacgggtcctcagggacctccagggccaaca。
the amino acid sequence of the protein coded by the gene RHIiC 8 is shown in SEQ ID NO. 2.
MGERGGPGGPGPQGPPGKNGETGPQGPPGPTGERGGPGGPGPQGPPGKNGETGPQGPPGPTGERGGPGGPGPQGPPGKNGETGPQGPPGPTGERGGPGGPGPQGPPGKNGETGPQGPPGPTGERGGPGGPGPQGPPGKNGETGPQGPPGPTGERGGPGGPGPQGPPGKNGETGPQGPPGPTGERGGPGGPGPQGPPGKNGETGPQGPPGPTGERGGPGGPGPQGPPGKNGETGPQGPPGPT。
(2) Construction of recombinant expression vector pET22 b-RHIiC 8
Inoculating a gene single clone with a sequence of GERGGPGGPGPQGPPGKNGETGPQGPPGPT into 5ml LB culture medium, performing amplification culture at 37 ℃ overnight, extracting a plasmid with a target gene, performing double enzyme digestion by using Xba I and Sal I, recovering the target gene by agarose gel electrophoresis, co-incubating the target gene with a pET22b expression vector (containing a His label) subjected to corresponding enzyme digestion treatment at 37 ℃ for 2h, adding T4 DNA ligase as shown in figure 1, connecting for 4 h at 25 ℃, transforming Escherichia coli DH5 alpha, screening and picking out a white spot clone by using an LB plate containing 0.1mg/ml ampicillin, identifying the positive clone to be correct by using a PCR method, picking out the positive clone for sequencing, determining that a recombinant plasmid contains a correct gene sequence reading frame, and successfully constructing a recombinant expression vector pET22 b-RHIC 8; the correct clone was inoculated into 5ml LB medium, expanded cultured overnight at 37 ℃ and plasmid extracted using Kangshi plasmid extraction kit to obtain plasmid pET22 b-RHIiC 8.
(3) Induction and expression of recombinant human collagen
Transforming the plasmid pET22 b-RHIiC 8 in the step (2) into Escherichia coli BL21-DE3-PLySs (purchased from the exclusively-living organism EC1003S), screening out a single clone by using an LB plate containing 0.1mg/ml ampicillin, culturing overnight at 37 ℃ in 5ml LB medium, transferring to the LB medium according to the volume ratio of 1:100, culturing in a shake flask at 37 ℃ until the OD600 is 0.6, adding IPTG with the final concentration of 0.1mM, culturing at 25 ℃ for 6-8 hours, centrifuging at 5000Xg for 5 minutes, collecting bacterial precipitates, preserving at-20 ℃ or immediately entering the next step for purification.
(4) Purification of recombinant human collagen RHIiC 8
And (3) washing 5g of the thallus precipitate in the step (3) by using PBS buffer solution, re-suspending the thallus precipitate by using 40ml of PBS buffer solution, adding 10ml of Triton X-100 solution containing 5mg/ml of lysozyme in a final concentration to help to crack bacteria, ultrasonically breaking the bacteria (280W, 2s ultrasonic, 5s intermittent, the total length of 45min, the protection temperature of 25 ℃), centrifuging at 12000rpm for 20min, and collecting supernatant, wherein the supernatant contains a large amount of recombinant human collagen RHIiC 8. Washing a nickel ion affinity column (Ni-NTA pre-loaded gravity column, 5ml (Bio-C600793)) with PBS buffer, gently shaking the affinity column and the supernatant for 30min at room temperature or ice, loading the column, washing the hybrid protein with 5ml of PBS solution containing 15mM imidazole, leaving pure recombinant human collagen RHIiC 8 on the column, adding 1. mu.L of prescision Protease (PPase) with His label to the column, gently shaking for 2 hours at room temperature or ice, releasing the RHIC 8 protein from the column, eluting with 2.5ml of PBS solution, collecting the eluate and dialyzing overnight (dialysis bag, RC membrane, 14KD, flat width 44mM (Bio-F600112)), freeze-drying (adding different volumes of 10%, 5%, 3%, 2%, 1%) of aqueous mannitol solutions each at-40 ℃ for 10min, and finally freeze-drying at-35 ℃), obtaining the purified recombinant human collagen RHIiC 8. A part of the purified sample is subjected to PAGE gel electrophoresis to detect the size and purity of the protein, the detection result is shown in figure 4, the size of the protein is close to 23KDa, and a single clear target protein band is arranged under a marker which is close to 28 KDa.
Example 2 detection of Activity of recombinant human collagen RHIiC 8
Protein concentration was measured using BCA method protein concentration measurement kit (pelagic day P0006).
Principle of BCA method: under alkaline conditions, the peptide bond structure in the protein molecule is complexed with Cu2+ and reduces Cu2+ to Cu1 +. BCA specifically binds to Cu1+ to form a stable blue-violet complex with a maximum light absorption at 562nm and is directly proportional to protein concentration. The assay achieves simplicity, high stability, high sensitivity and high compatibility of protein concentration determination.
The specific steps for determining the protein concentration are as follows:
(1) BCA working solution: preparing a BCA working solution by using the BCA reagent A and the BCA reagent B according to the volume ratio of 50:1, and fully mixing.
(2) Sample detection
Preparing the purified recombinant human collagen RHIiC 8 prepared in the step (4) in the example 1 into a sample of 0.5mg/ml by using PBS, taking 20 mu L of the sample to sample wells of a 96-well plate, adding 200 mu L of BCA working solution into each well, and standing at 37 ℃ for 30 minutes;
the protein concentration in the sample was calculated to be 10mg/ml based on the protein concentration (a562 value-0.16)/0.712, a562 measurement was performed.
(3) Adherence rate of cells
Culture of 3T3 cells: murine fibroblasts 3T3 (purchased from Zao and Bio JH-M2003) were inoculated into DMEM medium containing 10% FBS, cultured at 37 ℃ for 5-6 h or overnight, and then cell adhesion was observed. And changing the culture solution every two days, and culturing until the cells grow to 70-80%, namely 3T3 cells.
The purified recombinant human collagen RHIIC 8 prepared in step (4) of example 1 and the human collagen standard (purchased from manufacturer A001654) were prepared into 0.5mg/ml protein solutions with PBS, respectively.
Adding 100 mu L of 0.5mg/ml recombinant human collagen RHIiC 8 solution and human collagen standard solution into a 96-well plate as a cell to be detected and a control cell respectively, taking PBS as a blank control, and standing at room temperature for 60 min. 105 of the 3T3 cells were added to each well and incubated at 37 ℃ for 60 min. Each well was washed 4 times with PBS. The absorbance of OD450nm was measured using a cell adhesion assay kit (Beboc BB-48120). According to the value of the blank control, the adherence degree of the cells can be calculated.
The anchorage rate of the cells can reflect the activity of the collagen. The higher the activity of the protein, the better the external environment can be provided for the cells in a short time, and the cells are attached to the wall. The relative cell adhesion activity of the RHIiC 8 sample can be measured by taking the adherence rate of the human collagen standard as 1, wherein the calculation formula of the cell adhesion rate is as follows:
as shown in fig. 5, the results of the calculation of the% cell adhesion rate [ ((test cell OD-blank OD)/(control cell OD-blank OD) ] x100 indicate that the cell adhesion efficiency of RHIIIC8 can reach 90% compared with the cell adhesion efficiency of 40% of human collagen;
in conclusion, the protein of the human collagen designed and prepared by the invention has high activity, and can be widely applied to the industries of biological medicine and cosmetics.
Sequence listing
<110> Jiangsu Henry biomedical science and technology Co., Ltd
<120> recombinant human collagen and preparation method and application thereof
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atgggagaac gaggcggccc tggaggacct ggccctcagg gtcctcctgg taagaatggt 60
gaaaccggac ctcagggacc tccaggacct actggtgagc gaggtggtcc aggtggccct 120
ggcccacaag gcccacccgg aaagaacggt gagactggtc cacaaggtcc tccaggtcca 180
accggcgaac gaggaggacc tggaggacct ggtcctcagg gtccgcctgg gaagaatggt 240
gaaacaggac cgcagggacc tcctggacct acgggtgagc gaggtggtcc aggcggtcct 300
ggccctcaag gacctccagg caagaacggt gagactggcc ctcaaggacc accaggtcca 360
actggcgaac gaggcggccc tggaggacct ggcccacagg gtccgcctgg aaagaatggt 420
gaaactggac ctcagggtcc tccaggacct actggtgaac gaggtggacc tggtggacct 480
ggcccacaag gtccaccagg caagaacggt gagacaggcc ctcagggacc tccaggtcct 540
actggagagc gaggaggccc aggaggccct ggccctcagg gacctcctgg taagaatggt 600
gaaaccggac cacaagggcc accaggacct actggcgagc gaggtggtcc tggcggtcct 660
ggccctcagg gtccaccagg aaagaacggt gagacgggtc ctcagggacc tccagggcca 720
aca 723
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Met Gly Glu Arg Gly Gly Pro Gly Gly Pro Gly Pro Gln Gly Pro Pro
1 5 10 15
Gly Lys Asn Gly Glu Thr Gly Pro Gln Gly Pro Pro Gly Pro Thr Gly
20 25 30
Glu Arg Gly Gly Pro Gly Gly Pro Gly Pro Gln Gly Pro Pro Gly Lys
35 40 45
Asn Gly Glu Thr Gly Pro Gln Gly Pro Pro Gly Pro Thr Gly Glu Arg
50 55 60
Gly Gly Pro Gly Gly Pro Gly Pro Gln Gly Pro Pro Gly Lys Asn Gly
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Glu Thr Gly Pro Gln Gly Pro Pro Gly Pro Thr Gly Glu Arg Gly Gly
85 90 95
Pro Gly Gly Pro Gly Pro Gln Gly Pro Pro Gly Lys Asn Gly Glu Thr
100 105 110
Gly Pro Gln Gly Pro Pro Gly Pro Thr Gly Glu Arg Gly Gly Pro Gly
115 120 125
Gly Pro Gly Pro Gln Gly Pro Pro Gly Lys Asn Gly Glu Thr Gly Pro
130 135 140
Gln Gly Pro Pro Gly Pro Thr Gly Glu Arg Gly Gly Pro Gly Gly Pro
145 150 155 160
Gly Pro Gln Gly Pro Pro Gly Lys Asn Gly Glu Thr Gly Pro Gln Gly
165 170 175
Pro Pro Gly Pro Thr Gly Glu Arg Gly Gly Pro Gly Gly Pro Gly Pro
180 185 190
Gln Gly Pro Pro Gly Lys Asn Gly Glu Thr Gly Pro Gln Gly Pro Pro
195 200 205
Gly Pro Thr Gly Glu Arg Gly Gly Pro Gly Gly Pro Gly Pro Gln Gly
210 215 220
Pro Pro Gly Lys Asn Gly Glu Thr Gly Pro Gln Gly Pro Pro Gly Pro
225 230 235 240
Thr