基于绿色荧光蛋白的真菌免疫调节蛋白细胞定位方法

Fungal immunomodulatory protein cell positioning method based on green fluorescent protein

文档序号：6333 发布日期：2021-09-17 浏览：124次中文

1. A fungal immunomodulatory protein cell positioning method based on green fluorescent protein is characterized in that after a vector pUC57-GFP containing green fluorescent protein gene shown as Seq ID No.1 is cloned, the vector pUC57-GFP is recombined with pET-30a vector sequence to obtain recombinant plasmid pET-GFP, the recombinant plasmid pET-glu1-GFP is further recombined with glu1 gene sequence shown as Seq ID No.5 to obtain recombinant plasmid pET-glu1-GFP prokaryotic expression strain, and glu1-GFP recombinant protein for positioning fungal immunomodulatory protein cell is obtained by culturing;

the nucleotide sequence of the recombinant plasmid pET-GFP is shown as Seq ID No. 2.

2. The method of claim 1, wherein the recombinant plasmid pET-GFP is transformed into E.coli DH5 a competent cells and subjected to colony PCR to determine the accuracy of the recombinant plasmid;

the nucleotide sequence of the recombinant plasmid pET-glu1-GFP is shown as Seq ID No. 8.

3. The method of claim 1, wherein the prokaryotic expression strain is E.coli BL21(DE3) competent cell.

4. The method of claim 1, wherein the glu1-GFP recombinant protein is purified.

5. The method of claim 1, wherein the step of localizing the fungal immunomodulatory protein cell comprises: fungal immunomodulatory protein cells are cultured in a culture medium comprising a glu1-GFP recombinant protein, such that the glu1-GFP recombinant protein comprising fluorescence is subjected to the fungal immunomodulatory protein cells.

6. The method of claim 1, wherein the fungal immunomodulatory protein cell is a macrophage.

7. A method according to any one of claims 1 to 6, wherein the method of localising a fungal immunomodulatory protein cell comprises:

step 1) designing and synthesizing a green fluorescent protein gene according to a gene sequence of the green fluorescent protein, and cloning the gene into a pUC-57 vector to obtain pUC 57-GFP;

step 2) according to the gene sequences of the green fluorescent protein and the pET-30a vector, using BamH I and Not I to carry out enzyme digestion on pUC57-GFP and pET-30a vector plasmids, connecting enzyme digestion products to obtain a recombinant plasmid pET-GFP, and transferring the recombinant plasmid pET-GFP into a escherichia coli DH5 alpha competent cell;

step 3) carrying out colony PCR on the recombinant plasmid pET-GFP by using primers T7 and T7T to identify the accuracy of the recombinant plasmid;

step 4) digesting the pET-GFP recombinant vector and the glu1 gene by Nde I and BamH I according to the pET-GFP recombinant vector sequence and the glu1 gene sequence, and connecting digestion products to obtain a recombinant plasmid pET-glu 1-GFP;

step 5) transferring the recombinant plasmid into a competent cell of escherichia coli BL21(DE3) to construct a pET-glu1-GFP escherichia coli prokaryotic expression strain;

step 6) placing the Escherichia coli positive monoclonal bacteria on LB^KanCulturing in liquid culture medium to obtain rFIP-glu1-GFP recombinant protein and purifying;

step 7) carrying out SDS-PAGE and Western blotting detection on rFIP-glu1-GFP recombinant protein;

and 8) treating the macrophage by using rFIP-glu1-GFP recombinant protein, observing the recombinant protein under a fluorescence microscope, and carrying out cell localization.

8. The method of claim 7, wherein the LB molecules are as defined in^KanThe content of the liquid culture medium comprises the following components: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride and 25 mu g/mL of kanamycin.

Background

Green Fluorescent Protein (GFP) was originally a protein found in jellyfish when aequorin bound Ca²⁺This then fluoresces blue which further excites GFP to produce green fluorescence. The fluorescence emitted after GFP excitation has long duration, and does not need the action of any other accessory factors, and after the cDNA of GFP is successfully cloned and successfully expressed in colibacillus and mammalian cells, the application prospect is realized. The probe is applied to the processes of protein expression, pathogen infection monitoring, cell positioning and the like at present, and is an ideal probe for monitoring gene expression and protein positioning in living cells and tissues. Has been widely used as a reporter gene for cytological experimental study of living bodies.

Fungal Immunomodulatory Protein (FIP) is a small molecule protein with immunomodulatory activity isolated from higher basidiomycetes. The protein of FIP family has the functions of Anti-Tumor, Anti-allergy, and stimulating immune cells to generate various cytokines and other immune regulation effects, and has good medical health care value and application prospect (Li QZ et al, Fungal immunomodulation Proteins: Characteriostic, Potential Anti-Tumor Activities and therapeutic Molecular mechanisms. drug discovery today,2019,24: 307) and the like. Macrophages, which are mononuclear phagocytes, produce large amounts of proinflammatory or anti-inflammatory cytokines that elicit an immune response, and are often used as cellular models for evaluating natural product immunoregulatory activity. The existing research shows that the recombinant FIP-glu is a potential stimulating factor for proliferation and activation of mouse peritoneal macrophages, can regulate proinflammatory and anti-inflammatory mediators of the macrophages at the mRNA level, and can mediate the immunoregulation function of the macrophages through PI3K and MAPKs pathways. (Li QZ, et al, immunomodulating activity of biochemical protein via PI3K/Akt and MAPK signaling pathway in RAW264.7 cells. J Cell physiology.2019, 234: 23337-.

When the biological activity of the fungal immunomodulatory protein on the macrophage is researched, the identification of whether the protein enters the macrophage to play a role is the primary step. The existing protein cell positioning method mainly comprises an immunofluorescence technology, an immune colloidal gold marker, a fusion reporter gene and the like. The immunofluorescence technique is based on the principle of reaction between antigen and antibody, the known antigen or antibody is marked with fluorescein to prepare a fluorescent marker, and then the fluorescent antibody is used as a molecular probe to detect the corresponding antigen in cells or tissues. Although the approximate distribution of the target protein in the cell can be obtained by the method, the obtained image is the superposition of fluorescence signals in the whole cell, and higher resolution cannot be obtained, so that the method is not suitable for the research of intracellular protein localization. The immune colloidal gold labeling technology is to make colloidal gold attract an antibody by utilizing the property that the colloidal gold is negatively charged in an alkaline environment, so as to label the antibody. This method can very accurately reflect the precise location of different proteins in the cell structure, but it is rather complicated to operate, and requires a lot of time and cost.

The method for fusing the reporter gene is to fuse the target gene with the reporter gene such as gfp which is easy to monitor, regulate the expression of the fusion gene by using the expression regulation mechanism of the target gene, and monitor the existence state of the fusion protein in the cell under a fluorescent microscope. Many scholars construct fusion vectors by means of GFP for subcellular localization, for example, Staphylyu and the like use genome of the orf virus AH-F10 strain as a template to construct pEGFP (N1) ORFV127 fusion expression vectors, and use GFP to perform subcellular localization on ORFV127 protein (Staphylyu and the like, clone expression and subcellular localization analysis of the orf virus 127 gene, Jiangsu agricultural science, 2019,35(03): 646-. The Yang theory and others use GFP to construct Adv, CerS2, GFP recombinant adenovirus, and laser confocal microscope to locate the expression and cell location of CerS2-GFP fusion protein in liver cancer cell HepG2 (Yang theory and others, human CerS2 recombinant adenovirus construction and its effect on the cell cycle of liver cancer cell HepG 2. Chongqing medicine, 2018,47(31): 39397 8-.

From the existing research, gfp can be used as the most widely used cell marker gene at present, and can effectively perform cell localization on the fusion protein. Through the search of the prior art, no literature report on the cell localization of Fungal Immunomodulatory Protein (FIP) by using GFP has been found so far.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a fungal immunomodulatory protein cell positioning method based on green fluorescent protein, which constructs a fusion expression vector of GFP and FIP-glu protein, expresses an FIP mutant with the green fluorescent protein in a prokaryotic expression system and transfers the expression vector into macrophage RAW264.7, and uses the green fluorescent protein to perform cell positioning on the fungal immunomodulatory protein, so that whether the protein enters cells can be accurately and quickly identified, and a wider prospect is provided for the exploration of the biological activity of the fungal immunomodulatory protein.

The invention is realized by the following technical scheme:

the invention clones a vector pUC57-GFP containing a green fluorescent protein gene shown as Seq ID No.1, recombines the vector pUC57-GFP with a pET-30a vector sequence to obtain a recombinant plasmid pET-GFP, further recombines with a glu1 gene sequence shown as Seq ID No.5 to obtain a recombinant plasmid pET-glu1-GFP, constructs a pET-glu1-GFP prokaryotic expression strain based on the recombinant plasmid, and obtains the glu1-GFP recombinant protein for positioning fungal immunomodulatory protein cells by culturing.

The nucleotide sequence of the recombinant plasmid pET-GFP is shown as Seq ID No. 2.

The recombinant plasmid pET-GFP is preferably transferred into a commercially available Escherichia coli DH5 alpha competent cell and subjected to colony PCR to identify the accuracy of the recombinant plasmid.

The nucleotide sequence of the recombinant plasmid pET-glu1-GFP is shown as Seq ID No. 8.

The prokaryotic expression strain is a commercially available escherichia coli BL21(DE3) competent cell.

The glu1-GFP recombinant protein is preferably subjected to purification treatment.

The positioning of the fungal immunomodulatory protein cells refers to: fungal immunomodulatory protein cells are cultured in a culture medium comprising a glu1-GFP recombinant protein, such that the glu1-GFP recombinant protein comprising fluorescence is subjected to the fungal immunomodulatory protein cells.

The fungal immunomodulatory protein cell is not limited to a macrophage.

The positioning method specifically comprises the following steps:

step 1) designing and synthesizing a green fluorescent protein gene according to a gene sequence of the green fluorescent protein, and cloning the gene into a pUC-57 vector to obtain pUC 57-GFP;

step 3) carrying out colony PCR on the recombinant plasmid pET-GFP by using primers T7 and T7T to identify the accuracy of the recombinant plasmid;

step 5) transferring the recombinant plasmid into a competent cell of escherichia coli BL21(DE3) to construct a pET-glu1-GFP escherichia coli prokaryotic expression strain;

step 6) placing the Escherichia coli positive monoclonal bacteria on LB^KanCulturing in liquid culture medium to obtain rFIP-glu1-GFP recombinant protein and purifying;

the LB^KanThe content of the liquid culture medium comprises the following components: 10g/L Tryptone (Tryptone), 5g/L Yeast extract (Yeast extract), 10g/L sodium chloride (NaCl), 25 μ g/mL kanamycin (Kan).

And step 7) carrying out SDS-PAGE and Western blotting detection on the rFIP-glu1-GFP recombinant protein.

And 8) treating the macrophage by using rFIP-glu1-GFP recombinant protein, observing the recombinant protein under a fluorescence microscope, and carrying out cell localization.

Technical effects

The invention integrally solves the technical problem of realizing the cell localization of the protein when various immune cells or tumor cells are used for experiments through the fusion expression of the green fluorescent protein and the fungal immunomodulatory protein.

Compared with the prior art, the invention obtains the prokaryotic expression strain of the fungal immunomodulatory protein FIP-glu1-gfp (LZ-8) with a green fluorescent protein marker, and directly meets the requirement of the protein on the positioning in cells in subsequent FIP activity experiments.

Drawings

FIG. 1 shows the electrophoresis of plasmid pET-30a and GFP;

in the figure: (A) the plasmid pET-30a is digested, lane M: DNA Marker DL 15,000; lane 1: after the plasmid pET-30a is subjected to enzyme digestion; lane 2: before the enzyme digestion of the plasmid pET-30 a; (B) the restriction electrophoresis of the recombinant plasmid pUC57-GFP, lane 1: before the enzyme digestion of a recombinant plasmid pUC 57-GFP; lane 2: after the recombinant plasmid pUC57-GFP is cut by enzyme; GFP gene indicated by arrow;

FIG. 2 is a PCR electrophoretogram of recombinant plasmid pET-GFP colony;

in the figure: lane M: DNA Marker DL 2,000; lanes 1-9: detecting the result of the escherichia coli transformant containing the recombinant plasmid pET-GFP; lane-the most: a negative control is carried out, and the negative control,

FIG. 3 is a flow chart of the construction of the recombinant plasmid pET-glu 1-GFP;

FIG. 4 is a clone electrophoretogram of FIP-glu1(LZ-8) gene;

in the figure: lane M: DNA Marker DL 2,000; lane 1: FIP-glu1(LZ-8) gene; lane-the most: a negative control is carried out, and the negative control,

FIG. 5 is an electrophoretogram of recombinant plasmid pET-GFP;

in the figure: lane M: DNA Marker DL 15,000; lane 1: before the recombinant plasmid pET-GFP is digested; lane 2: after the recombinant plasmid pET-GFP is cut by enzyme;

FIG. 6 shows PCR electrophoresis of recombinant plasmid pET-glu1-GFP colonies;

in the figure: colony PCR assay, lane M: DNA Marker DL 2,000; lanes 1-12: the detection result of the E.coli transformant containing the recombinant plasmid pET-glu 1-GFP; lane-the most: negative control

FIG. 7 is a SDS-PAGE and Western blotting assay of glu1-GFP protein;

in the figure: (A) coli transformants SDS-PAGE detection, lane M: protein standard molecular weight; lane 1: e.coli transformant total protein without IPTG induction; lane 2-5: IPTG-induced total protein of e.coli transformants, (B) detection of purified recombinant glu1-GFP protein, lane M: protein standard molecular weight; lane 1: SDS-PAGE detection of the purified recombinant glu1-GFP protein; lane 2: western blotting detection is carried out on the purified recombinant glu1-GFP protein,

FIG. 8 is a map of the localization of recombinant glu1-GFP cells;

in the figure: (A) photographs of glu1-GFP treated cells under visible light; (B) photograph of glu1-GFP treated cells under UV light.

Detailed Description

The embodiment relates to a fungal immunomodulatory protein cell localization method based on green fluorescent protein, which comprises the following steps:

step 1) construction of pET-GFP recombinant plasmid

Amino acid sequences of a Linker and GFP are used as blueprints to synthesize a Linker + GFP gene sequence (synthesized by Shanghai biological engineering Co., Ltd.), a synthetic sequence is shown as Seq ID No.1, and the gene is cloned in a pUC-57 vector to obtain pUC 57-GFP. Prokaryotic expression vectors pET-30a and pUC57-GFP were extracted, and double digestion was carried out using restriction enzymes BamH I and Not I. As shown in FIG. 1, plasmid pET-30a was cleaved to give a single band, approximately 5,390bp in size, consistent with the expectation. The pUC57-GFP gene was double digested to give two bands, the larger band was the linearized vector pUC-57 and the smaller band was GFP, 779bp in size, consistent with the expectations.

pET-30a was ligated with GFP using T4 DNA ligase to construct a recombinant plasmid pET-GFP, which was transformed into E.coli DH 5. alpha. competent cells. Through LB^KanAfter the solid medium is screened, a plurality of colonies are respectively picked for colony PCR detection, and the PCR reaction procedure is as follows: 5min at 94 ℃ (30 s at 94 ℃, 1min at 55 ℃, 40s at 72 ℃)35cycles, 10min at 72 ℃; the primer sequences used for PCR were T7 and T7T as shown in Seq ID No.3 and Seq ID No.4, respectively.

The amplified gene fragment should be 1,107 bp. As a result, as shown in FIG. 2, lanes 1, 2, 3, 5, 7, 8 and 9 each had a specific band at the corresponding position. The sequencing of Shanghai biological engineering Co., Ltd shows that the sequence is correct. The success of the construction of the recombinant plasmid pET-GFP is shown.

Step 2) construction of rFIP-glu1-GFP recombinant plasmid

As shown in FIG. 3, a prokaryotic expression vector pET-glu1-GFP was constructed. As shown in FIG. 4, a single band appeared at about 400bp after PCR amplification of glu1, and the size was consistent with that expected. The PCR amplification reaction program is as follows: 5min at 94 ℃ (30 s at 94 ℃, 1min at 55 ℃, 40s at 72 ℃)35cycles, 10min at 72 ℃; the primer sequences used for PCR are shown in Seq ID No.6 and Seq ID No.7, respectively.

And extracting a recombinant plasmid pET-GFP. The recombinant plasmid and the PCR purified product were digested with restriction enzymes Nde I and BamH I. Since the PCR product was double-digested and only several bases were reduced, no change before and after digestion could be observed on agarose gel electrophoresis, and no electrophoretic pattern was shown. As shown in FIG. 5, the recombinant plasmid pET-GFP was digested to a single band, which was 6016bp in size, and was identical to the expected band, as shown in Seq ID No. 2. After the corresponding bands are recovered, the recombinant plasmid pET-glu1-GFP is constructed by connecting the bands through T4 DNA ligase, and the recombinant plasmid is transferred into Escherichia coli DH5 alpha competent cells. Through LB^KanAfter the solid medium is screened, a plurality of colonies are respectively picked to carry out colony PCR detection, and the amplified gene fragment is 1,323 bp. The PCR reaction program is: 5min at 94 ℃ (30 s at 94 ℃, 1min at 55 ℃, 40s at 72 ℃)35cycles, 10min at 72 ℃; the primer sequences used for PCR were T7 and T7T as shown in Seq ID No.3 and Seq ID No.4, respectively. The results are shown in FIG. 6, where 12 samples each had a specific band at the corresponding position. The sequencing result shows that the sequence is correct, namely as shown in Seq ID No. 8. The success of the construction of the recombinant plasmid pET-glu1-GFP is shown.

Step 3) expression and purification of rFIP-glu1-GFP protein

The recombinant plasmid pET-glu1-GFP is transformed into escherichia coli BL21(DE3) competent cells (purchased from Shanghai Biotechnology engineering Co., Ltd.), and after IPTG induction, the produced protein is subjected to 10% SDS-PADE and Western blotting detection. SDS-PAGE detection is carried out by preparing SDS-PAGE gel according to a method provided by a website of TaKaRa biotech (Beijing) Co Ltd, carrying out electrophoresis at 80V, and carrying out electrophoresis at 120V until the sample is migrated to the separation gel; western blotting detection is carried out by a semidry method, wherein the primary antibody is a mouse anti-6 XHis monoclonal antibody, and the secondary antibody is goat anti-mouse IgG marked by HRP. As a result of the detection, as shown in FIG. 7, a specific band, i.e., a fusion protein of recombinant FIP-glu1(LZ-8) and GFP, abbreviated as glu1-GFP, appeared at about 40kD in the sample. Dialyzing the supernatant with 3.5kD dialysis bag, freeze-drying the solution in the dialysis bag, dissolving with lysine Buffer, purifying with His60 Ni Superflow Resin column (purchased from Shanghai Biotech engineering Co., Ltd.), dialyzing the purified eluate with 3.5kD dialysis bag, freeze-drying the solution in the dialysis bag, and dissolving in PBS.

Step 4) cellular localization of rFIP-glu1-GFP protein

After washing macrophage RAW264.7 with PBS, cells were suspended in DMEM cell culture medium (GE Healthcare Life Sciences, HyClone) by trypsinization^TM) Count cells at 2X 10⁵At a concentration of one/mL, cells were inoculated into a new cell culture flask containing DMEM medium and placed in CO₂The incubator was incubated at 37 ℃. 2X 10⁵Cell/well concentration cells were plated in 12-, 24-or 96-well cell culture plates, cultured for 24h, medium was discarded, medium containing recombinant glu1-GFP was added, and culture was continued for 24 h. The cells were observed by fluorescence microscopy, and the results are shown in FIG. 8, indicating that recombinant glu1-GFP entered the interior of macrophages.

The method utilizes protein fusion expression to construct protein prokaryotic expression strain with markers, and directly produces the fungal immunomodulatory protein capable of cell positioning. Compared with the prior art, the method directly produces the fungal immunomodulatory protein with the fluorescent label through the fusion expression construction of GFP and FIP-glu1, thereby more conveniently obtaining the cell localization result of the fungal immunomodulatory protein.

The foregoing embodiments may be modified in many different ways by those skilled in the art without departing from the spirit and scope of the invention, which is defined by the appended claims and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Sequence listing

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gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 4080

ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 4140

aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 4200

tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 4260

ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 4320

gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 4380

tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 4440

caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 4500

ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 4560

cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 4620

ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 4680

cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 4740

gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc 4800

aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg 4860

ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg 4920

ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg 4980

ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca 5040

agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa 5100

cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg 5160

cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc 5220

gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg 5280

attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac 5340

ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta 5400

tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc 5460

gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga 5520

aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt 5580

tgatgctcga tgagtttttc taagaattaa ttcatgagcg gatacatatt tgaatgtatt 5640

tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgaaatt 5700

gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt 5760

aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg 5820

ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc 5880

aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca 5940

agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga 6000

tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa 6060

ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc 6120

gccgcgctta atgcgccgct acagggcgcg tcccattcgc ca 6162

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

taatacgact cactataggg 20

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gctagttatt gctcagcgg 19

<210> 5

<211> 648

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gatccaaacg atgagatttc cttcaatttt tactgcagtt ttattcgcag catcctccgc 60

attagctgct ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc 120

tgtcatcggt tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa 180

cagcacaaat aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga 240

agaaggggta tctctcgaga aaagagaggc tgaagcttac gtagaattca tgtctgacac 300

cgctttgatc ttcagactgg cttgggacgt caagaagttg tccttcgact acaccccaaa 360

ctggggaaga ggtaacccaa acaacttcat cgacaccgtt accttcccaa aggtcttgac 420

tgacaaggcc tacacctaca gagttgccgt ttctggtcgt aacctgggtg tcaagccatc 480

ctacgctgtt gagtccgacg gttcccagaa ggtcaacttc ttggagtaca actctggtta 540

cggtatcgct gacactaaca ccatccaagt tttcgttgtc gacccagaca ccaacaacga 600

cttcatcatt gctcaatgga acgggcccca tcatcatcat catcatca 648

<210> 6

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atatacatat gcaccatcat catcatcatg agctcatgtc tgacaccgct ttgatcttc 59

<210> 7

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

actcgagcgg atccgttcca ttgagcaatg atgaagtcg 39

<210> 8

<211> 6662

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8