阅读说明：本技术 一种水貂IFN-ε成熟肽的制备方法 (Preparation method of mink IFN-epsilon mature peptide ) 是由 张海玲 卢士英 白雪 廉士珍 胡博 章沙沙 张蕾 张东亮 李双双 李虹晔 于 2021-01-15 设计创作，主要内容包括：本发明公开了一种水貂IFN-ε成熟肽的制备方法。本发明还公开了一种优化后的水貂IFN-ε成熟肽的编码核苷酸序列,其核苷酸序列如SEQ ID NO.1所示。本发明通过构建含有优化后的编码水貂IFN-ε成熟肽基因的重组表达载体,并在大肠杆菌中成功表达。对其抗病毒活性进行分析,结果显示,得到的重组水貂IFN-ε成熟肽具有明显的抗病毒活性。本发明的提出研制开发具有抗病毒活性的基因工程干扰素制剂,提供了一种有效的技术手段。(The invention discloses a preparation method of mink IFN-epsilon mature peptide. The invention also discloses an optimized encoding nucleotide sequence of the mink IFN-epsilon mature peptide, wherein the nucleotide sequence is shown as SEQ ID NO. 1. The invention constructs a recombinant expression vector containing the optimized gene for coding the mature peptide of the mink IFN-epsilon and successfully expresses the gene in escherichia coli. The result of analyzing the antiviral activity of the recombinant mink IFN-epsilon mature peptide shows that the obtained recombinant mink IFN-epsilon mature peptide has obvious antiviral activity. The present invention provides an effective technical means for developing gene engineering interferon preparations with antiviral activity.)

1. An optimized encoding nucleotide sequence of mink IFN-epsilon mature peptide, which is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.

2. Use of the nucleotide sequence of claim 1 for the preparation of a mink IFN-epsilon mature peptide.

3. A preparation method of a mink IFN-epsilon mature peptide is characterized by comprising the following steps:

(1) synthesizing an optimized encoding nucleotide sequence of the mink IFN-epsilon mature peptide, and connecting the optimized encoding nucleotide sequence with a TA cloning vector to construct a vector containing the optimized encoding nucleotide sequence of the mink IFN-epsilon mature peptide as shown in SEQ ID No. 1;

(2) the primers for amplifying the IFN-epsilon mature peptide gene of the mink are designed, and the sequences of the primers are as follows:

upstream: 5'-CGGGGTACCGACGACGACGACAAGCTGGAACTGAAACTGG-3', respectively;

downstream: 5' -CCGCTCGAGTTATTTAGACAGTTTA;

taking a diluted vector solution containing the optimized encoding nucleotide sequence of the mink IFN-epsilon mature peptide as a template, carrying out PCR amplification by adopting the primers, recovering and purifying a PCR product, connecting the PCR product with an expression vector, converting the PCR product into E.coli competent cells, and carrying out PCR and enzyme digestion identification to obtain an expression vector containing the optimized encoding nucleotide sequence of the mink IFN-epsilon mature peptide;

(3) transforming BL21(DE3) competent cells into the expression vector containing the optimized encoding nucleotide sequence of the mink IFN-epsilon mature peptide obtained in the step (2), and obtaining the bacterial liquid OD₆₀₀When the numerical value reaches 0.4-0.6, adding IPTG (isopropyl-beta-D-thiogalactoside) for induction expression, and performing shaking culture at the constant temperature of 37 ℃;

(4) after the culture is finished, taking out the bacterial liquid, centrifuging, removing the supernatant liquid, and keeping the precipitate; and (3) resuspending the precipitate with PBS, centrifuging to remove supernatant, reserving the precipitate, resuspending with PBS of 1/10 original bacterial liquid volume, purifying protein by using Ni-NTA, wherein the purified protein is the mink IFN-epsilon mature peptide, and freezing and storing at-80 ℃.

4. The method of claim 3, wherein the TA cloning vector in step (1) is the pMD18-T vector.

5. The method of claim 3, wherein the expression vector in step (2) is a pET-32a expression vector.

6. The method of claim 3, wherein the amplification reaction system of the mink IFN-epsilon mature peptide gene in step (2) is as follows: 10 XE х Taq Buffer2.5 μ L, 2.5mmol/μ L dNTP4 μ L, 25pmol/μ L upstream and downstream primers 1 μ L each, 25pmol Taq DNA polymerase 1 μ L, adding 3 μ L pMD 18-T/MiIFN-epsilon plasmid solution diluted 100 times as template, and supplementing 25 μ L with sterilized deionized water; the reaction conditions are as follows: after 25 mul of reaction liquid is mixed evenly, amplification is carried out on a PCR instrument, the cycle parameters are pre-denaturation at 95 ℃ for 5min, pre-denaturation at 94 ℃ for 45s, amplification at 53 ℃ for 45s, amplification at 72 ℃ for 50s, and extension at 72 ℃ for 10min after 34 cycles.

7. A mink IFN-epsilon mature peptide produced by the method of any one of claims 3-6.

8. Use of the mink IFN-epsilon mature peptide of claim 7 in the preparation of an antiviral formulation.

Technical Field

The invention relates to a preparation method of interferon, in particular to a preparation method of mink IFN-epsilon mature peptide. The invention belongs to the field of biotechnology.

Background

Interferons (IFNs) are proteins with wide biological activities, have the functions of regulating the immune function of an organism, resisting viruses and the like, and are important components of a defense system of the organism. Since Isaacs and Lindenmann have found interferons for over 50 years, research, development and use of IFNs has never been stopped. At present, IFN has been proved to have significant adjuvant therapy effect on various virus diseases in human clinical practice. 2003Kotenko et al are classified into three types, i.e., type I, type II and type III interferons, based on their gene sequences, chromosomal localization and receptor specificity, type I including IFN-. alpha.beta.omega.epsilon.kappa.delta.tau.zeta.etc., and type II consisting of a single gene family of IFN-. gamma.also known as immunointerferons. Human interferon-epsilon (IFN-epsilon) was first reported in 1999 and was mainly expressed in lung, brain, small intestine and reproductive system tissues, after which mouse, pig and dog IFN-epsilon genes were gradually reported. IFN-epsilon is thought to play an important role in reproductive function, in the protection of mammalian placenta from viral infection and in early placental development. Up to now, only the biological functions of IFN-epsilon in human, rhesus monkey, cow and dog have been reported, and no related research report on the cloning and expression of IFN-epsilon gene in mink (Mustella vison) has been found.

In recent years, the fur animal industry in China develops rapidly, is one of the countries with developed fur animal breeding industry in China, and causes serious economic loss due to infectious diseases of the mink breeding industry caused by the hazards of mink canine distemper, parvovirus disease, Aleutian disease and the like. The viral infectious diseases have acute morbidity and high mortality, and the common medicaments have unsatisfactory treatment effect, so that the development of the gene engineering interferon preparation with antiviral activity is an ideal choice.

Disclosure of Invention

One of the purposes of the invention is to provide an optimized encoding nucleotide sequence of the mink IFN-epsilon mature peptide.

The second purpose of the invention is to provide a preparation method of the mink IFN-epsilon mature peptide.

In order to achieve the purpose, the invention adopts the following technical means:

the optimized encoding nucleotide sequence of the mink IFN-epsilon mature peptide is shown as SEQ ID No. 1.

Furthermore, the invention also provides application of the nucleotide sequence in preparation of the mink IFN-epsilon mature peptide.

Furthermore, the invention also provides a preparation method of the mink IFN-epsilon mature peptide, which comprises the following steps:

(1) synthesizing an optimized encoding nucleotide sequence of the mink IFN-epsilon mature peptide, and connecting the optimized encoding nucleotide sequence with a TA cloning vector to construct a vector containing the optimized encoding nucleotide sequence of the mink IFN-epsilon mature peptide as shown in SEQ ID No. 1;

(2) the primers for amplifying the IFN-epsilon mature peptide gene of the mink are designed, and the sequences of the primers are as follows:

upstream: 5'-CGGGGTACCGACGACGACGACAAG TTAGAACTGAAACTG-3', respectively;

downstream: 5' -CCGCTCGAG TCAAACAATGGTCCAG;

taking a diluted vector solution containing the optimized encoding nucleotide sequence of the mink IFN-epsilon mature peptide as a template, carrying out PCR amplification by adopting the primers, recovering and purifying a PCR product, connecting the PCR product with an expression vector, converting the PCR product into E.coli competent cells, and carrying out PCR and enzyme digestion identification to obtain an expression vector containing the optimized encoding nucleotide sequence of the mink IFN-epsilon mature peptide;

(3) transforming BL21(DE3) competent cells into the expression vector containing the optimized encoding nucleotide sequence of the mink IFN-epsilon mature peptide obtained in the step (2), and obtaining the bacterial liquid OD₆₀₀When the numerical value reaches 0.4-0.6, adding IPTG (isopropyl-beta-D-thiogalactoside) for induction expression, and performing shaking culture at the constant temperature of 37 ℃;

(4) after the culture is finished, taking out the bacterial liquid, centrifuging, removing the supernatant liquid, and keeping the precipitate; resuspending the precipitate with PBS, centrifuging to remove supernatant, keeping the precipitate, resuspending with PBS (1/10 original bacterial liquid volume), purifying protein with Ni-NTA, and freezing at-80 deg.C.

Among them, the TA cloning vector in step (1) is preferably the pMD18-T vector.

Among them, the expression vector in step (2) is preferably a pET-32a expression vector.

Preferably, the amplification reaction system of the mink IFN-epsilon mature peptide gene in the step (2) is as follows: 10 XE х Taq buffer2.5 μ L, 2.5mmol/μ L dNTP4 μ L, 25pmol/μ L upstream and downstream primers 1 μ L each, 25pmol Taq DNA polymerase 1 μ L, adding 3 μ L pMD 18-T/MiIFN-epsilon plasmid solution diluted 100 times as template, and supplementing 25 μ L with sterilized deionized water; the reaction conditions are as follows: after 25 mul of reaction liquid is mixed evenly, amplification is carried out on a PCR instrument, the cycle parameters are pre-denaturation at 95 ℃ for 5min, pre-denaturation at 94 ℃ for 45s, amplification at 53 ℃ for 45s, amplification at 72 ℃ for 50s, and extension at 72 ℃ for 10min after 34 cycles.

The mink IFN-epsilon mature peptide prepared by the method is also within the protection scope of the invention.

Finally, the invention also provides the application of the mink IFN-epsilon mature peptide in preparing an antiviral preparation.

Compared with the prior art, the invention has the beneficial effects that:

the invention constructs a recombinant expression vector containing the optimized gene for coding the mature peptide of the IFN-epsilon of the mink, and successfully expresses the gene in escherichia coli. The result of analyzing the antiviral activity of the recombinant mink IFN-epsilon mature peptide shows that the obtained recombinant mink IFN-epsilon mature peptide has obvious antiviral activity.

Drawings

FIG. 1 shows the result of IFN-. epsilon.PCR amplification;

wherein: 1. amplification of mink IFN-epsilon gene; IFN-epsilon mature peptide gene amplification; 3. negative control; 4.100bp DNAmarker;

FIG. 2 is the nucleic acid sequence and amino acid sequence of the MinkIFN-epsilon gene;

note: the signal peptide coding sequence is underlined;

FIG. 3 is a comparison of gene sequences encoding IFN-epsilon mature peptide of mink and the gene sequences after codon optimization of Escherichia coli;

FIG. 4 shows the expression and purification of mink IFN-epsilon gene in E.coli;

wherein: 1. protein molecular weight Marker; 2. no induction control; 3. expressing the recombinant mMiIFN-epsilon in escherichia coli; 4. centrifuging and precipitating after cracking; 5. splitting heart supernatant; 6. purification of expression products

FIG. 5 is an IFN-epsilon Western blotting identification;

1. a protein Marker; 2. empty vector control; 3. no induction control; IFN-epsilon inducing samples.

Detailed Description

The present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Example 1 cloning and expression of mink epsilon-interferon mature peptide Gene (MiIFN-epsilon)

1 materials and methods

1.1 Experimental materials

1.1.1 strain E.coli.DH5 alpha sensitive strain is preserved in the laboratory;

1.1.2 reagents pMD18-T vector, ExTaq polymerase, AMV reverse transcriptase and Phytohemagglutinin (PHA) were purchased from Takara bioengineering (Dalian) Co., Ltd; RPMI1640 was obtained from GIBCO, and lymphocyte isolate was obtained from Chinese academy of medicine; TRIzol cell lysate purchased from Invitrogen corporation; the glue recovery kit is purchased from Hangzhou Bori science and technology ltd; the primers were synthesized by Biotechnology engineering (Shanghai).

1.1.3 Experimental animals 6 months old minks were purchased from fur animal breeding base of specialty institute of Chinese academy of agricultural sciences;

1.2 methods

1.2.1 primer design and Synthesis

The amplification primers are designed according to the complete sequence of the IFN-epsilon cDNA of the carnivorous animal published on GenBank, and the primer sequences are as follows:

IFN-εF：5’-ATGATTAACAAGCATTT-3’；

IFN-εR：5’-TTATTTGCTTAGCTTTCTT-3’。

1.2.2 mink IFN-epsilon Induction

Sterile collecting peripheral anticoagulation isolated lymphocyte of healthy mink, using vaccine strain (TCID) containing 25 μ g/ml HA (phytohemagglutinin) and 300 μ L CDV3 (canine distemper virus)₅₀＝10^5.6) Suspending with 10% calf serum in RPM-1640 culture solution, counting cell suspension, and diluting to 1 × 10 according to counting result⁷At a concentration of one/mL, CO at 37 ℃₂Culturing in an incubator for 12-24 h.

1.2.3 amplification of mink IFN-epsilon Gene and sequence analysis

Induced lymphocytes were collected and resuspended with TRIzol Reagent. Adding 1/5TRIzol volume of chloroform, mixing, standing for 3min, centrifuging at 12000r/min for 10min, sucking supernatant, adding 1/2TRIzol volume of isopropanol, mixing, standing at-20 deg.C for 1h, centrifuging at 12000r/min for 15min, washing the precipitate with 75% ethanol for 1 time, inverting, draining, and dissolving the precipitate with 9.5 μ L of 0.1% DEPC treated sterilized water to obtain lymphocyte total RNA.

The epsilon-interferon gene is amplified by adopting an RT-PCR method. The specific operation is as follows: to 9.5. mu.L of the RNA solution was added 1. mu. LOoligo (dT)₁₅Primers (50 pmol/. mu.L) were mixed wellAnd (3) after the reaction, the mixture is placed in a 70 ℃ water bath for 5min and then immediately cooled, 10mmol/L dNTP 5 mu L, AMV Buffer 4 mu L, RNase inhibitor0.5 mu L and AMV 1 mu L are added in sequence, the mixture is fully and uniformly mixed, then the mixture is placed on a PCR instrument for 1h at 42 ℃ and the reverse transcriptase is inactivated for 3min at 95 ℃, and the mixture is directly used for PCR reaction after being cooled.

And (3) PCR reaction system: 10 × E х Taq Buffer2.5 μ L, 2.5 mmol/. mu.L dNTP 2 μ L, upstream and downstream primers (25 pmol/. mu.L) each 1 μ L, 25pmol, Taq DNA polymerase (2U/. mu.L) 0.125 μ L, 17.375 μ L sterile deionized water was added. After 25 mul of reaction liquid is mixed evenly, amplification is carried out on a PCR instrument, the cycle parameters are pre-denaturation at 95 ℃ for 5min, pre-denaturation at 94 ℃ for 45s, pre-denaturation at 52.5 ℃ for 50s and pre-denaturation at 72 ℃ for 1min, extension at 72 ℃ for 6min after 32 cycles, and after the amplification is finished, 6 mul of reaction liquid is taken out to be subjected to electrophoresis observation on 1.5 percent agarose gel. Recovering target gene with glue, constructing TA clone, and PCR, enzyme digestion and sequencing to identify the amplified sequence as the target gene. The results of PCR amplification are shown in FIG. 1, and the results of sequencing are shown in FIG. 2.

1.2.1 mature peptide Gene amplification and expression vector construction

According to software prediction (SignalP4.0 Server), the first 21 amino acids coded by the mink IFN-epsilon gene are predicted to be a signal peptide sequence, the fragment of the mink IFN-epsilon mature peptide gene (MiIFN-epsilon gene) is 501p long, and 167 amino acids are coded. Carrying out Escherichia coli expression codon optimization and gene synthesis on the original sequence, wherein the nucleotide sequence is shown as SEQ ID NO.1, and connecting the original sequence with a pMD18-T vector to construct a vector containing the optimized mature peptide gene, which is named as pMD 18-T/MiIFN-epsilon. The alignment of the optimized codon gene sequence to the original sequence is shown in FIG. 3.

The primers for amplifying the IFN-epsilon mature peptide gene of the mink are designed, and the sequences of the primers are as follows: an upstream primer: 5' -CGGGG TACCGACGACGACGACAAGCTGGAACTGAAACTG-3' (underlined is the KpnI cleavage site); a downstream primer: 5' -CCGCTCGAGTTATTTAGACAGTTTA (underlined part is XhoI cleavage site).

Mature peptide gene PCR amplification reaction system: 10 xE х Taq buffer 2.5. mu.L, 2.5 mmol/. mu.L dNTP 4. mu.L, 1. mu.L each of the upstream and downstream primers (25 pmol/. mu.L), 1. mu.L of 25pmol TaqDNA polymerase (2.5U/. mu.L), 3. mu.L of 100-fold diluted pMD 18-T/MiIFN-. epsilon.plasmid solution as a template, and make up to 25. mu.L with sterile deionized water. After 25 mul of reaction liquid is mixed evenly, amplification is carried out on a PCR instrument, the cycle parameters are pre-denaturation at 95 ℃ for 5min, pre-denaturation at 94 ℃ for 45s, amplification at 53 ℃ for 45s, amplification at 72 ℃ for 50s, and extension at 72 ℃ for 10min after 34 cycles. The results of the PCR products were visualized by electrophoresis on a 1.5% agarose gel, and are shown in FIG. 1.

The PCR product was recovered and purified by 1.5% agarose gel electrophoresis, and then digested with KpnI and XhoI. Recovering small fragments; by T₄The DNA ligase connects a target gene fragment with an expression vector pET-32a (KpnI and XhoI double-restriction enzyme purified product), transforms the target gene fragment into an E.coli./BL21 competent cell, and after PCR and restriction enzyme identification (KpnI and XhoI double-restriction enzyme), clones which are preliminarily identified to be positive are sent to the company of engineering biology (Shanghai) for sequencing, DNAStar software is used for analyzing sequencing results, and a correct recombinant plasmid is named as pET-32 a/MiIFN-epsilon.

1.2.2 expression and purification of MiIFN-epsilon Gene in E.coli

Converting the positive pET-32 a/MiIFN-epsilon plasmid identified as 1.2.1 into BL21(DE3) competent cells, identifying the positive clone bacteria conservation through PCR and double enzyme digestion, identifying the positive bacteria through sequencing, and obtaining the bacteria with OD₆₀₀And when the numerical value reaches 0.4-0.6, taking out 1mL of the bacterial liquid as an uninduced control, adding IPTG22.5 mu L of the residual 9mL of the bacterial liquid into the bacterial liquid for induction expression, wherein the final concentration is 1mmol/L, and simultaneously placing the uninduced bacterial liquid and the induced bacterial liquid on a constant temperature shaking table (shaking amplitude of 185 turns) at 37 ℃ for 5-6 hours and then taking out.

After the bacterial liquid is taken out, centrifuging for 2min at 10000r/min, removing supernatant liquid and retaining precipitate. After the sediment is resuspended by PBS, 10000r/min is centrifuged for 2min to remove the supernatant and retain the sediment. The induced bacterial liquid and the non-induced bacterial liquid are precipitated and resuspended by 1/10 original bacterial liquid volume, and 100 mul of induced bacterial liquid is taken out to be used as a whole bacterial control. And (4) performing freeze thawing on the induced heavy suspension bacteria liquid for 2-3 times. And carrying out ultrasonic crushing on the induced bacteria liquid, wherein the crushing power is 400W. And stopping ultrasonication for 3 seconds for 6 seconds until the bacterial solution is not hung on the wall. And centrifuging the bacterial liquid subjected to ultrasonic crushing at 12000r/min for 10 min. The supernatant was aspirated and the pellet resuspended in 100. mu.L. Mixing control sample, whole bacteria sample, supernatant sample, and precipitation sample with SDS-PAGE protein sample buffer (1X) at a ratio of 1: 1, boiling for 5min, and directly detecting by SDS-PAGE electrophoresis.

SDS-PAGE electrophoresis was performed using 15% separation gel and 4.5% concentration gel, and the SDS-PAGE result is shown in FIG. 4. IPTG-induced strain expressed a specific protein band at about 19kDa, which was consistent with the predicted molecular weight, and the protein yield was about 30% of the total protein of the strain. The expressed protein is in a soluble form by protein solubility analysis. And further carrying out Western-blot analysis after the SD S-PAGE is correctly identified. The electrotransfer condition is that constant current transfer printing is carried out for 1H40min under 200mA, sealing is carried out for 2H after the transfer printing is finished, then a monoclonal antibody of mouse anti-6 XHis is taken as a primary antibody, goat anti-mouse IgG-HRP is taken as a secondary antibody, the reaction is carried out for 1H at 37 ℃, and finally, a color development kit is used for developing the color, and the result is shown in figure 4. As a result, a specific reaction band was observed only at 19kDa, which is consistent with the result of SDS-PAGE, and the results are shown in FIG. 5. E.coli BL21 expression strain single colony containing positive recombinant plasmid pET32 a/mIFN-epsilon is picked, thalli are collected by large-volume induction, Ni-NTA is used for purifying protein, the product is concentrated and is dyed after SDS-PAGE electrophoresis, and the result shows that the impurity protein is little, the purity reaches more than 90 and the purification effect is good. The purified protein is stored at-80 ℃.

Example 2 determination of the antiviral Activity of the mink IFN-Epsilon mature peptide (MiIFN-Epsilon)

Example 1 the purified recombinant mink IFN-epsilon mature peptide prepared in example 1 was tested for antiviral activity in the Wish/VSV system using cytopathic inhibition to inhibit 50% of cytopathic effects (CPEI)₅₀) The highest interferon dilution of (a) is 1 activity unit (U).

The highest dilution of interferon capable of inhibiting 50% of cytopathic effect is used as a unit by adopting a cytopathic effect inhibition method, and an interferon reference standard substance control is set for each measurement. Wish cells were seeded in 96-well plates and placed at 37 ℃ in 5% CO₂Culturing in an incubator for 12-18 h, removing the nutrient solution after the culture grows into a single layer, sequentially adding diluted IFN (interferon) samples, and adding two holes for each dilution. Simultaneously, different dilutions of IFN standard controls, cell controls and virus controls were set. 37 ℃ and 5% CO₂Incubate overnight in an incubator. Removing IFN solution with 100TCID₅₀The VSV (Vesicular Stomatitis Virus) of (1) was challenged (cell control wells with a Virus-free nutrient solution), and cultured for 1-2 d. Virus to be detectedWhen the cells in the wells have obvious lesions in all or over 75 percent, the results are observed under an inverted microscope. Observations show that 100TCID was added₅₀When the VSV virus is subjected to challenge, after 24h, the control hole is observed to completely generate severe lesions, typical lesions of vesicular stomatitis virus on Wish cells are found in the control hole, and any control without the VSV virus shows normal.

Measuring the concentrated MiIFN-epsilon expression product by using a Wish/VSV system micro cytopathy inhibition method, and adding more than 4 percent of the MiIFN-epsilon expression product^-4The pathological changes are completely inhibited by the concentration of the MiIFN-epsilon recombinant protein; the antiviral activity of the recombinant MiIFN-epsilon sample is 0.23 multiplied by 10 after repeated 3 times of experiments⁴IU/mg。

Sequence listing

<110> institute of specialty products of Chinese academy of agricultural sciences

Jilin University

<120> preparation method of mink IFN-epsilon mature peptide

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