CN112608932A - Method for efficiently expressing avian adenovirus Fiber-2 protein in escherichia coli - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and discloses a method for efficiently expressing avian adenovirus Fiber-2 protein in escherichia coli. Respectively taking the PET-21a-GX-1-Fiber2 plasmid and the PXMJ19-C1786T-hexon (T7) plasmid as templates, and carrying out amplification by using designed primers to obtain a Fiber-2 target fragment and a PXMJ19-C1786T-T7 carrier fragment; then carrying out homologous recombination to obtain a recombinant plasmid PXMJ19-T7-his-fiber 2; finally, the cells are transformed into Shuffle T7-B competent cells for expressing the Fiber-2 protein. The invention adopts the plasmid capable of improving the copy number of the exogenous gene as a vector to construct the PXMJ19-T7-his-Fiber2 recombinant plasmid so as to enhance the soluble expression of the Fiber-2 protein.

Description

Method for efficiently expressing avian adenovirus Fiber-2 protein in escherichia coli

Technical Field

The invention relates to the technical field of bioengineering, in particular to a method for efficiently expressing avian adenovirus Fiber-2 protein in escherichia coli.

Background

The avian adenovirus belongs to the family of adenoviridae and the genus avian adenovirus, exists in poultry, and chickens, ducks, geese and the like are all susceptible hosts. The avian adenovirus has strong infectivity and stable transmission, is not pathogenic in a host body generally, and is an opportunistic pathogen. In recent years, the main prevalent strains in china are avian adenovirus serotype 4 and 8b, with the serotype 4 strain predominant. FADV4 was first reported at 8 months 1987 and was found in the region of Pakistan Ampalas. The serum 4 epidemic strain HN strain which is outbreaked in 2015 has strong pathogenicity and high lethality, has larger difference with a classical strain, and brings huge loss to poultry industry, so the research on the epidemic strain is urgent and necessary. The Fiber protein of the avian adenovirus comprises Fiber-1 and Fiber-2, wherein the Fiber-1 protein is longer, and the Fiber-2 protein is shorter, which are closely related to the infectivity and pathogenicity of the virus. The spike protein not only contains main species-specific antigenic determinants and minor subgeneric-specific antigenic determinants of the avian adenovirus, but also can determine the preference of the virus for host cells and initiate virus infection. Fiber-1 deletion, once it causes a decrease in the number of viruses in the host, while Fiber-2 deletion causes the virions to fail. Thus, the Fiber-2 protein of the serotype 4 circulating strain (HN strain) may be associated with enhanced viral virulence and may have protective potency as a subunit vaccine. In conclusion, the antibody preparation of Fiber-2 protein of avian adenovirus serotype 4 (HN strain) is a precondition for researching whether Fiber-2 protein is related to the enhancement of the toxicity of avian adenovirus, is a basis for further exploring the immunological function and pathogenesis of avian adenovirus serotype 4, and has important significance for the monitoring and prevention of avian adenovirus and the targeted treatment after virus infection.

Guohoran et al disclose the expression and purification of avian adenovirus type 4Fiber2 protein and the preparation of polyclonal antibody, journal of agricultural biotechnology 10 in 2019. The document discloses that the complete gene synthesis sequence of FAdV-4Fiber2 after codon optimization is subjected to in vitro amplification, and then the complete gene synthesis sequence is cloned into a p Cold expression vector to construct a prokaryotic expression vector pCold-Fiber 2. The pCold-Fiber2 recombinant plasmid is transformed into Escherichia coli (Escherichia coli) PGTf2 competent cells, isopropyl thiogalactoside (IPTG) is used for inducing expression, the Fiber2 protein is purified, and the protein is used for immunizing rabbits (Oryctolagus cuniculus) to successfully prepare the polyclonal antibody of the anti-FAdV-4 Fiber2 protein. Western blot and indirect immunofluorescence assay (IFA) results show that the polyclonal antibody against the FAdV-4Fiber2 protein can perform specific reaction with the Fiber2 protein and the FAdV-4 protein after being purified, and the Fiber2 protein is proved to have good immunogenicity. The established indirect ELISA detection method of the FAdV-4 antibody shows that the detection sensitivity of the method is 96 percent, and the specificity is 100 percent. Patent CN 110128508A discloses an avian adenovirus fiber protein subunit vaccine. The sequence of the novel antigen fiber protein of the avian adenovirus is optimized to ensure that the fiber protein obtains soluble expression in escherichia coli, and the expression product is used for preparing subunit vaccine. Patent CN 109750036A discloses a nucleotide sequence, a method for improving protein expression efficiency by using the nucleotide sequence and application thereof. The method comprises the following steps: transforming the recombinant vector containing the protein gene into escherichia coli, wherein the recombinant vector is a recombinant expression vector; and step (2) expressing the protein; the expression of the protein is tandem co-expression or fusion expression of multiple proteins; the recombinant expression vector of the protein gene is a recombinant pET28 plasmid, a pColdIII plasmid or a pCDFDuet plasmid, and the Escherichia coli is Escherichia coli BL21(DE 3); the protein gene is one or more of chicken infectious bursal disease virus VP2 protein, avian adenovirus Penton protein, avian adenovirus Fiber-2 protein, avian egg drop syndrome virus Penton protein, or avian egg drop syndrome virus Fiber protein.

Although the above-mentioned documents and patent methods achieve the expression of Fiber2 protein, there is room for further improvement in the preparation of expression vectors and in the optimization of soluble expression.

Disclosure of Invention

Aiming at the defects and shortcomings of the prior art, the invention aims to provide a method for efficiently expressing avian adenovirus Fiber-2 protein in Escherichia coli. The method adopts the modified PXMJ19 plasmid PXMJ19-C1786T-hexon (T7) as a prokaryotic expression vector (from CN 110951767A, a corynebacterium and escherichia coli double expression vector with high copy capacity and a PXMJ19-C1786T plasmid in the construction method thereof) to construct a PXMJ19-T7-his-Fiber2 recombinant plasmid to promote the efficient soluble expression of the Fiber-2 protein. And the enhanced strain Shuffle T7-B is adopted to promote the high-efficiency expression of heterologous proteins.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for efficiently expressing avian adenovirus Fiber-2 protein in Escherichia coli comprises the following steps:

(1) carrying out amplification by taking the PET-21a-GX-1-Fiber2 plasmid as a template and Fiber-2-F and Fiber-2-R as primers to obtain a Fiber-2 target fragment;

(2) PXMJ19-C17 1786T-hexon (T7) plasmid is used as a template, PXMJ19-T7-his-F and PXMJ19-T7-his-R are used as primers for amplification, and a PXMJ19-C1786T-T7 vector fragment is obtained;

(3) carrying out homologous recombination on the Fiber-2 target fragment and the PXMJ19-C17 1786T-T7 vector fragment to obtain a recombinant plasmid PXMJ19-T7-his-Fiber 2;

(4) the recombinant plasmid PXMJ19-T7-his-Fiber2 is transformed into Shuffle T7-B competent cells for expressing the Fiber-2 protein;

the primer sequences are respectively as follows:

Fiber-2-F：AAGAAGGAGATATAATGCTGCGTGCGC；

Fiber-2-R：AGCCGGATCTCATCAGTGGTGGTGG；

PXMJ19-T7-his-F：CCACCACCACTGATGAGATCCGGCT；

PXMJ19-T7-his-R：CACGCAGCATTATATCTCCTTCTTA；

the nucleotide sequence of the PET-21a-GX-1-fiber2 plasmid is SEQ ID NO. 6, and the plasmid map is shown in figure 1.

The nucleotide sequence of the PXMJ19-C17 1786T-hexon (T7) plasmid is SEQ ID NO. 7, and the plasmid map of the PXMJ 19-C173532-hexon plasmid is shown in FIG. 2.

Further, the nucleotide sequence of the Fiber-2 target fragment obtained in the step (1) is SEQ ID NO: 8.

Further, the nucleotide sequence of the PXMJ19-C1786T-T7 vector fragment obtained in the step (2) is SEQ ID NO. 9.

Further, the homologous recombination conditions in step (3) are as follows:

adding the Fiber-2 target fragment and the PXMJ19-C17 1786T-T7 vector fragment into the reaction solution according to the volume ratio of 1:2, shaking up, incubating in a water bath kettle at 37 ℃ for 30min, and then cooling at 4 ℃ or on ice to obtain the recombinant product.

Further, the step of transforming the recombinant plasmid PXMJ19-T7-his-fiber2 into Shuffle T7-B competent cells described in step (4) is: adding the recombinant plasmid PXMJ19-T7-his-fiber2 into the Shuffle T7-B competent cells, uniformly mixing, standing on ice for 30min, performing water bath heat shock at 42 ℃ for 45sec, immediately placing on ice for cooling for 2-3min, adding an SOC culture medium, and shaking the cells at 37 ℃ and the rotation speed of 200-; preheating LB solid culture medium containing chloramphenicol resistance in a 37 ℃ incubator, centrifuging to remove supernatant, re-suspending the thallus with the rest culture medium, uniformly coating on a flat culture medium containing chloramphenicol resistance with a sterile coating rod, and performing inverted culture in the 37 ℃ incubator for 12-16 h.

Further, the expression steps of the Fiber-2 protein in the step (4) are as follows:

the single colony after transformation culture was picked up, inoculated into LB solid medium with chloramphenicol resistance, the bacteria were shake-cultured at 200rpm at 30 ℃ until the logarithmic phase (OD600 ═ 0.4-0.8), IPTG (isopropylthio-. beta. -D-galactoside) was added to a final concentration of 1mM, and induced in a shaker at 16 ℃ for 15 hours.

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

(1) a plasmid PXMJ19-C1786T-hexon (T7) (derived from CN 110951767A, a corynebacterium and escherichia coli double expression vector with high copy capacity and a PXMJ19-C1786T plasmid in the construction method thereof, wherein a specific nucleotide sequence is SEQ ID NO:6, and a plasmid map is shown in figure 2) are adopted as a vector to construct a PXMJ19-T7-his-Fiber2 recombinant plasmid, so that the high-efficiency soluble expression of the Fiber-2 protein is enhanced.

(2) Respectively amplifying a target fragment and a vector fragment by adopting a specific plasmid template, and directly obtaining a recombinant plasmid PXMJ19-T7-his-fiber2 by adopting homologous recombination; enzyme digestion and ligase are not needed, and the operation steps are reduced.

(3) The method does not need to optimize or change the Fiber-2 target fragment, but adopts PXMJ19-C1786T-T7 modified by mutating one base as a vector, improves the replication efficiency of the target protein, is simpler than the optimization of the target fragment and reduces the influence caused by a heterologous sequence.

(4) The invention adopts an enhanced strain, namely, the Shuffle bacterium Shuffle T7-B, is in Lon and OmpT protease defect type, is suitable for protein expression started by a T7 promoter and enhances the expression of Fiber-2 in escherichia coli.

(5) The invention adopts different transformation conditions and expression conditions to optimize the soluble expression of the Fiber-2 protein.

Drawings

FIG. 1 is a structural diagram of the template plasmid PET-21a-GX-1-fiber2 of the present invention;

FIG. 2 is a structural diagram of the template plasmid PXMJ19-C17 1786T-hexon (T7) of the present invention;

FIG. 3 is a diagram showing the results of gel electrophoresis identification of the amplification products of Fiber-2 target fragments in the examples;

FIG. 4 is a diagram showing the results of gel electrophoresis identification of the product of the amplification of the PXMJ19-C1786T-T7 vector fragment in the example;

FIG. 5 is a diagram showing the identification result of the recombinant product in the examples;

FIG. 6 is a diagram showing the results of gel electrophoresis of the recombinant products of examples after double digestion with NdeI and SacI;

FIG. 7 is a graph showing the results of SDS-PAGE gel electrophoresis of the pellet and supernatant after sonication of the cells transformed in different competent cells and expressed at different temperatures and times in the examples.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

The following examples illustrate the major reagents and sources:

TaqDNA polymerase, 10 XPCR Buffer (with Mg)²⁺：100mM Tris-HCl pH 8.8at 25℃；500mM KCl,0.8％(v/v)Nonidet)、MgCl₂(25mM), dNTP (10mM), Marker, 6 XDNA Loading Dye (sangon); 10 XTAE (400mM Tris-acetate and 10mM EDTA, pH8.0) (sangon); primers (sangon); 4S Red Plus nucleic acid stain (BBI a 606695); a column type DNA gel recovery kit (sangon SK 8131); one-step method rapid competent cell preparation kit (SK9307 sangon); a plasmid petiole kit (Tiangen DP 103-03); DL-8000DNA Size maker, DL-15000DNA Size maker (all-type Jinbiol Ltd.); PageRuler^TM Prestained Protein Ladder(26619，Thermo)；BeyoBlue^TMCoomassie brilliant blue ultrafast staining solution (Beyotime); SDS-PAGE gel preparation kit (Taraka); 2xRapid Taq Master Mix (Vazyme); NdeI; SacI (NEB).

The following examples illustrate the major instrument models and sources:

a dual temperature control dry thermostat (Hangzhou mi-Ou instruments Co., Ltd.); HC-2518R high-speed refrigerated centrifuge (Anhui Zhongzhongjia instruments, Inc.); DYY-6C type voltage and current stabilized electrophoresis apparatus (Beijing Liuyi); h6-1 mini electrophoresis tank (Shanghai Jingyi organic glass products apparatus factory); FR980 gel imaging system (shanghai complex science ltd); SMA4000 microphotometer (merinton); PCR reaction amplifier (BIO Co.); a pipette (range 100-; PCR apparatus T100(BIO-RAD), electronic balance (Sadoris); a chemostat incubator; a constant temperature culture shaking table (constant); three-hole electric heating constant temperature water tank (constant); decoloration shaker (its Linbel).

Primer design and source description in the following examples:

according to SnapGene software, a homologous recombination technology is adopted to design a gene Fiber-2, a vector PXMJ19-T7-his and a general primer L4440-F for bacterial liquid identification, and the primers are synthesized by the company Limited in the biological engineering (Shanghai), and are designed as the following table 1:

TABLE 1 primer sets and sequences

Primer name	Primer sequence (5 '-3')
		Fiber-2-F	AAGAAGGAGATATAATGCTGCGTGCGC；(SEQ ID NO:1)
Fiber-2-R	AGCCGGATCTCATCAGTGGTGGTGG；(SEQ ID NO:2)
		PXMJ19-T7-his-F	CCACCACCACTGATGAGATCCGGCT；(SEQ ID NO:3)
PXMJ19-T7-his-R	CACGCAGCATTATATCTCCTTCTTA；(SEQ ID NO:4)
		L4440-F	AGCGAGTCAGTGAGCGAG；(SEQ ID NO:5)
Fiber-2-R	AGCCGGATCTCATCAGTGGTGGTGG；(SEQ ID NO:2)

Examples

(1) Amplification of the fragment of Fiber-2 interest:

the PCR amplification system and the PCR amplification program are respectively shown in the following tables 2 and 3 by using PET-21a-GX-1-Fiber2 (the nucleotide sequence is SEQ ID NO:6, and the plasmid map is shown in figure 1) as a template and Fiber-2-F and Fiber-2-R as primers to amplify a Fiber-2 target fragment.

TABLE 2 Fiber-2 amplification PCR System

TABLE 3 Fiber-2 amplification PCR program

After the PCR amplification, the results of the 1% nucleic acid gel electrophoresis identification are shown in FIG. 3. The gel containing the target band is cut under ultraviolet light, the size of the cut gel is just enough to contain all the target bands, the gel is not too large, the cutting is rapid, the ultraviolet is immediately turned off after the cutting, and the target fragment is prevented from being degraded due to the over-irradiation of the ultraviolet. The cut gel was transferred to a 2mL EP tube, gel recovery was performed, and the DNA concentration was measured. The nucleotide sequence of the amplification product is SEQ ID NO. 8.

(2) Amplification of vector fragment PXMJ 19-C1786T-T7:

PXMJ19-C17 1786T-hexon (T7) plasmid (nucleotide sequence is SEQ ID NO:7, and the plasmid map is shown in figure 2) is used as a template, and PXMJ19-T7-his-F and PXMJ19-T7-his-R are used as primers to amplify the PXMJ19-C1786T-T7 vector fragment. The amplification PCR system and procedure are shown in tables 4 and 5 below, respectively.

TABLE 4 PXMJ19-C1786T-T7 amplification PCR System

TABLE 5 PXMJ19-C1786T-T7 amplification PCR program

After the PCR amplification, 0.8% nucleic acid gel electrophoresis was performed to identify the DNA fragment, and the results are shown in FIG. 4. And (2) an obvious target band appears at about 6516bp, the gel containing the target band is cut under ultraviolet light, the size of the cut gel is ensured to be just equal to all the target bands to the greatest extent, the gel is not excessively large, the ultraviolet light is quickly cut when the gel is cut, and the target fragment is prevented from being degraded due to over-irradiation of the ultraviolet light. The cut gel was transferred to a 2mL EP tube, gel recovery was performed, and the DNA concentration was measured. The nucleotide sequence of the amplification product is SEQ ID NO. 9.

(3) Carrying out homologous recombination on the Fiber-2 target fragment and the PXMJ19-C17 1786T-T7 vector fragment:

after the recovery of the gel, the target fragment and the vector fragment were prepared in the appropriate volumes on ice as shown in Table 6.

TABLE 6 homologous recombination System

Gently shaking, incubating in water bath at 37 deg.C for 30min, and cooling at 4 deg.C or on ice to obtain recombinant product.

1. And (3) identifying the bacterial liquid of the recombinant product, which comprises the following specific steps:

chemically competent cells for cloning, shuffle-T7-B cells, were thawed on ice, 10. mu.l of the recombinant product was added to 100. mu.l of the competent cells, gently flicked against the vessel wall (Do not shake well), and allowed to stand on ice for 30 min. After heat shock in 42 deg.C water bath for 45sec, immediately cooling on ice for 2-3 min. 900 μ l SOC medium was added and shaken at 37 ℃ for 1h (rotation speed 200-. LB plate solid media, which are resistant to chloramphenicol, were preheated in a 37 ℃ incubator. Centrifuge at 5,000rpm for 5min and discard 900. mu.l of supernatant. The cells were resuspended in the remaining medium and spread gently on chloramphenicol resistant plates using a sterile spreading rod. Culturing at 37 deg.C in incubator by inverting overnight (12-16 h). After overnight culture, the colonies on the plate were examined and 9 of them were picked up, placed in a 2mL EP tube containing a chloramphenicol-resistant LP medium, cultured at 37 ℃ for about 5 hours in a shaker, and then identified with a bacterial suspension, and the sizes were all around 2389bp, which is consistent with the theoretical values, as shown in FIG. 5. Identification of PCR System As shown in Table 7 below, the nucleotide sequence of the amplification product is SEQ ID NO 10.

TABLE 7 bacteria liquid identification PCR System

2. Carrying out enzyme digestion identification and sequencing on the recombinant product, and specifically comprising the following steps:

a) extracting recombinant plasmids:

taking 100uL of the bacteria liquid with positive identification, and carrying out large shaking and overnight culture in a 50mL centrifuge tube added with LB culture medium with chloramphenicol resistance. 15ml of overnight-cultured broth was added to a centrifuge tube and centrifuged at 12,000rpm (13,400 Xg) for 1min using a conventional tabletop centrifuge, and the supernatant was aspirated as much as possible. To the tube containing the pellet was added 500. mu.l of solution P1 (Tiangen DP103-03 plasmid miniprep kit) and the pellet was suspended thoroughly using a pipette or vortex shaker. The cells were lysed by gently inverting the tube for 6-8 times by adding 500. mu.l of solution P2 (Tiangen DP103-03 plasmid miniprep kit) to the tube. Note that: gently mix without vigorous shaking to avoid disrupting the genomic DNA and resulting in mixing of genomic DNA fragments with the extracted plasmid. At this time, the bacterial liquid should be clear and viscous, and the time for using the bacterial liquid should not exceed 5min so as to prevent the plasmid from being damaged. If the cells are not clear, the cells may be too much and the lysis is incomplete, so that the cell mass should be reduced. Add 700. mu.l of solution P3 (Tiangen DP103-03 plasmid miniprep kit) to the centrifuge tube, gently invert up and down 6-8 times immediately, mix well, at which time white flocculent precipitate will appear. Centrifuge at 12,000rpm (. about.13,400 Xg) for 10 min. Note that: the P3 should be mixed immediately after addition to avoid local precipitation. If there is a small white precipitate in the supernatant, the supernatant can be centrifuged again. The collected supernatant was transferred to an adsorption column CP3 (column equilibration before use: 500. mu.l of equilibration solution BL was added to adsorption column CP3 (adsorption column was put into collection tube), centrifuged at 12,000rpm (. about.13,400 Xg) for 1min, the waste solution in the collection tube was discarded, the adsorption column was put back into the collection tube), centrifuged at 12,000rpm (. about.13,400 Xg) for 30-60sec, the waste solution in the collection tube was discarded, and adsorption column CP3 was put into the collection tube. 600 mul of rinsing liquid PW (anhydrous ethanol is added firstly) is added into the adsorption column CP3, centrifugation is carried out for 30-60sec at 12,000rpm (-13,400 Xg), waste liquid in the collection tube is poured out, and the adsorption column CP3 is placed into the collection tube. The PW rinsing step was repeated, and then the adsorption column CP3 was placed in the collection tube and centrifuged at 12,000rpm (. about.13,400 Xg) for 2min to remove the residual rinse from the adsorption column. The adsorption column CP3 was uncapped and left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material. Placing the adsorption column CP3 in a clean centrifuge tube, dripping 50-100 μ l elution buffer EB into the middle part of the adsorption membrane, placing for 2min at room temperature, centrifuging for 2min at 12,000rpm (13,400 Xg), and collecting the plasmid solution in the centrifuge tube to obtain the recombinant plasmid.

b) Enzyme digestion identification and sequencing:

the double digestion with NdeI and SacI is carried out, the digestion system is 300uL, and the digestion system is divided into 6 tubes with 50uL of each tube, and the system is shown in the following table 8:

TABLE 8 enzyme digestion System

After completion of the digestion, the DNA fragment was identified by 1% gel electrophoresis, and the results are shown in FIG. 6 (M: 8000, 5000, 3000, 1500, 1000, 500; 1: NdeI, SacI double digestion fragment). The sizes of the fragments are 2714bp and 5258bp respectively, which are consistent with the size of a theoretical value. After the bands are identified correctly, the bands are sent to the company Limited of Biotechnology engineering (Shanghai) for sequencing, and the sequencing results are SEQ ID NO. 11 and SEQ ID NO. 12 which are completely the same as the theoretical values.

(4) The recombinant plasmid PXMJ19-T7-his-Fiber2 was transformed into Shuffle T7-B competent cells for expression of the Fiber-2 protein (and BL21 competent cells as a control):

a) chemical conversion:

the recombinant plasmid PXMJ19-T7-his-fiber 210 ng was added to 100. mu.l of BL21 and Shuffle T7-B competent cells, respectively, gently flicked to mix the walls of the tubes (Do Shake mix well), and left on ice for 30 min. After heat shock in 42 deg.C water bath for 45sec, immediately cooling on ice for 2-3 min. 900 μ l SOC medium was added and shaken at 37 ℃ for 1h (rotation speed 200-. LB plate solid media, which are resistant to chloramphenicol, were preheated in a 37 ℃ incubator. Centrifuge at 5,000rpm for 5min and discard 900. mu.l of supernatant. The cells were resuspended in the remaining medium and spread gently on chloramphenicol resistant plates using a sterile spreading rod. Culturing in 37 deg.C incubator for 12-16 h. The plate labeled for transformation into competent cells BL21 was A and the plate transformed into competent cells Shuffle T7-B was B.

b) Protein expression:

single colonies were picked in plate A, B and inoculated into 10ml LB medium with chloramphenicol resistance; all cultured overnight, 1mL is extracted and added into 20mL of chloramphenicol resistant LB culture medium; culturing the bacteria at 30 ℃ and 200rpm with shaking until logarithmic phase (OD600 is 0.4-0.8); dividing two bottles of bacterial liquid into 2 bottles, each bottle is 10mL, adding IPTG into each bottle to the final concentration of 1mM, respectively transferring to a shaking table, and performing experiments according to 30 ℃ induction for 8 hours and 16 ℃ induction overnight (15 hours); after induction is finished, centrifugally collecting thalli; sterile PBS and one in ten thousand EDTA were added; performing ultrasonic lysis until the solution is clear, centrifuging at 12000rpm for 1min, and respectively collecting the inclusion body precipitate and lysis supernatant; the inclusion body pellet was resuspended in PBS (one ten thousandth of EDTA).

The resulting pellet and supernatant were subjected to SDS-PAGE gel electrophoresis, and the results are shown in FIG. 7 (from left to right, 180kDa maker, Fiber-2 positive sample (+), expression of recombinant plasmid PXMJ19-T7-Fiber2 in supernatant and pellet of Shuffle cells, expression of PXMJ19-T7-Fiber2 in supernatant and pellet of BL21 cells; where s denotes supernatant and p denotes pellet, respectively). From the results of FIG. 7, it can be seen that:

1) the recombinant plasmid in BL21 cells, the protein expression of Fiber-2 in the supernatant and the precipitate of the bacterial liquid is less, and is obviously lower than the protein expression of Fiber-2 in the shuffle cells.

2) Recombinant plasmid is expressed in shuffle cells, Fiber-2 protein in supernatant and sediment of bacterial liquid is induced and cultured for 15h at 16 ℃ and then for 8h at the temperature higher than 30 ℃, so that the dissolution and folding of a protein structure are facilitated due to low temperature and increased culture time; and for protein expression under two different conditions, the expression of the supernatant of the bacterial liquid is better than that of the supernatant of the bacterial liquid.

To sum up: by using a PXMJ19-C1786T-hexon (T7) plasmid for enhancing copy level of foreign proteins by point mutation as a template, a recombinant plasmid PXMJ19-T7-his-Fiber2 is constructed, and then prokaryotic expression is carried out in an enhanced strain Shuffle T7-B cell, and higher Fiber-2 protein expression can be seen in the supernatant of bacterial liquid, thus: the optimized Fiber-2 protein has better solubility, and lays an experimental foundation for the development of subsequent vaccines.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> Chongqing research institute of BioIndustrial technology, Inc

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caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620

aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680

accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740

aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800

ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860

agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920

accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980

gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040

tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100

cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160

cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220

cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280

ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340

taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400

gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460

tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520

cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580

gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640

gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700

catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760

tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820

ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880

tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940

ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000

aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060

gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120

tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180

acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240

cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300

cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360

gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420

cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480

gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540

tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600

atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660

tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720

gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3780

tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840

cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900

tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta 3960

atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020

atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080

tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140

cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200

aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260

ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320

tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4380

tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440

gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500

gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560

gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4620

ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680

taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740

ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800

atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860

tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920

gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4980

gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040

aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100

ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc 5160

cctctagaaa taattttgtt taactttaag aaggagatat acatatggct agcatgactg 5220

gtggacagca aatgggtcgc ggatccatgc tgcgtgcgcc gaaacgccgt cactctgaaa 5280

acggtaaacc ggaaaccgaa gcgggtccgt ctccggcgcc gatcaaacgt gcgaaacgta 5340

tggtgcgcgc gtctcagctg gacctggttt acccgttcga ttacgtggcg gacccggttg 5400

gcggcctgaa cccgccgttc ctgggtggct ctggcccgct ggttgaccag ggcggccagc 5460

tgaccctgaa cgttaccgat ccgatcatca ttaaaaaccg ttccgttgac ctggctcacg 5520

atccgtctct ggatgtgaac gcgcagggcc agctggcagt ggctgttgat ccggaaggcg 5580

cgctggatat caccccggat ggtctggatg ttaaagtgga tggcgttacc gtgatggtta 5640

acgacgattg ggaactggcg gttaaagttg acccgtccgg tggcctggat tctaccgcgg 5700

gcggtctggg tgtgtctgtt gatgacaccc tgctggttga tcagggtgaa ctgggtgttc 5760

acctgaacca gcagggcccg atcacggcgg attctagcgg catcgacctg gaaatcaacc 5820

cgaacatgtt caccgtgaac acctccaccg gttccggtgt gctggaactg aacctgaaag 5880

cccagggcgg tatccaggcg gcttctagcg gtgttggtgt tagcgttgat gaatctctgc 5940

agattgttaa caacaccctg gaagttaaac cagatccgtc tggtccgctg accgttagcg 6000

cgaacggtct cggcctgaaa tatgacacca acaccctggc ggtgaccgcg ggtgcactga 6060

ccgtggttgg cggtggtagc gtttccaccc cgatcgccac cttcgtgtcc ggtagcccgt 6120

ctctgaacac ctacaacgca accaccgtta actcttccgc taacgcgttc tcctgcgcgt 6180

actacctgca gcagtggaac atccagggcc tgctggttac ctctctgtac ctgaaactgg 6240

actccgcgac catgggtaac cgtccgggtg acctgaactc tgccaacgcg aaatggttca 6300

ccttctgggt tagcgcctac ctgcagcagt gcaacccgtc cggtatccag gcgggtaccg 6360

ttagcccgag caccgcaacc ctgaccgact tcgaaccgat ggcgaaccgt agcgtgacca 6420

gcccgtggac ctactctgca aacggttact acgaaccgtc catcggcgaa ttccaggttt 6480

tctctccggt tgttaccggc gcgtggaacc caggtaacat tggtatccgt gtgctgccgg 6540

tgccggtgag cgcgagcggt gaacgttaca ccctgctgtg ctacagcctg cagtgcacca 6600

acgcgagcat cttcaacccg aacaactctg gtaccatgat cgttggtccg gtgctgtact 6660

cttgcccggc ggcgagcctg ccgaagcttg cggccgcact cgagcaccac caccaccacc 6720

actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg 6780

agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga 6840

aaggaggaac tatatccgga t 6861

<210> 7

<211> 7711

<212> DNA

<213> Artificial sequence ()

<400> 7

ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 60

agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 120

cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 180

caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 240

tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 300

ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 360

ctacaccgaa ctgagatacc tacagcgtga gcattgagaa agcgccacgc ttcccgaagg 420

gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 480

gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 540

tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 600

cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 660

gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 720

ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcaaaag tgctcatcat 780

tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 840

gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 900

tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 960

atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 1020

tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaaaga gtttgtagaa 1080

acgcaaaaag gccatccgtc aggatggcct tctgcttaat ttgatgcctg gcagtttatg 1140

gcgggcgtcc tgcccgccac cctccgggcc gttgcttcgc aacgttcaaa tccgctcccg 1200

gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc 1260

agtctttcga ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcatgg 1320

ggagacccca cactaccatc ggcgctacgg cgtttcactt ctgagttcgg catggggtca 1380

ggtgggacca ccgcgctact gccgccaggc aaattctgtt ttatcagacc gcttctgcgt 1440

tctgatttaa tctgtatcag gctgaaaatc ttctctcatc cgccaaaaca gccaagctga 1500

attcgagctc ggtacccggg gatcctctag agtcgacctg caggcatgca agctttaata 1560

cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt 1620

aactttaaga aggagatata atggctgcac taactccaga tttaacaact gctacaccca 1680

ggctacaata ttttcacatt gcgggcccgg gcacccgtga gtatctgagc gaggatctgc 1740

agcaattcat cagcgcgacc ggtagctatt tcgacctgaa gaacaaattt cgtcagaccg 1800

tggttgcgcc gacccgtaac gttaccaccg aaaaggcgca gcgtctgcaa atccgttttt 1860

acccgattca aaccgacgat accagcaccg gttatcgtgt gcgttacaac attaacgttg 1920

gtgacggctg ggtgctggat atgggcagca cctatttcga catcaagggt attctggatc 1980

gtggcccgag ctttaaaccg tattgcggta ccgcgtacaa cccgctggcg ccgaaagaga 2040

gcatgttcaa caactggagc gaaaccgcgc cgggtcagaa cgttagcgcg agcggccaac 2100

tgagcaacgt gtacaccaac accagcacca gcaaggatac caccgcggcg caggttacca 2160

aaatcagcgg cgtgtttccg aacccgaacc aaggtccggg tcgtaacccg ctgcgtcgtg 2220

ttcaaaacgc gaacaccggt gtgctgggcc gttttgcgaa gagccaatac aactatgcgt 2280

acggtgcgta cgttaaaccg gtggcggcgg atggcagcca gagcctgacc caaaccccgt 2340

attggattat ggacaacacc ggtaccaact acctgggcgc ggttgcggtg gaggactata 2400

ccaacagcct gagctatccg gataccattg tggtgccgcc gccggaagac tatgacgact 2460

acaacattgg taccacccgt gcgctgcgtc cgaactatat cggcttccgt gataacttta 2520

ttaacctgct gtaccacgac agcggtgttt gcagcggcac cctgaacagc gagcgtagcg 2580

gtatgaacgt ggttgtggag ctgccggatc gtaacaccga actgagctat cagtacatgc 2640

tggcggacat gatgagccgt caccactatt ttgcgctgtg gaaccaggcg gttgaccaat 2700

acgatccgga agtgcgtgtg ttcagcaacg acggttatga ggaaggcgcg ccgagctacg 2760

cgtttaaccc ggaagcggtg ggcgcgggcg agggttacgg tccggatctg agccaaatca 2820

aactgtatac caacaacacc caccaccacc accaccactg agatccggct gctaacaaag 2880

cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg 2940

gggcctctaa acgggtcttg aggggttttt tggacaccat cgaatggtgc aaaacctttc 3000

gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag 3060

taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg 3120

tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg 3180

agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga 3240

ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta 3300

aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg 3360

tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca 3420

ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc 3480

cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg 3540

aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc 3600

tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat 3660

atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt 3720

ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg 3780

ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg 3840

ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc 3900

cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccacg tggaccgctt 3960

gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt 4020

gaaaagaaaa accaccctgg cgccggggat cagccccgga tgctttggat acggtctatg 4080

agctggcagc gtatttgacc gatccggaca cctgggataa tgtgtggatt ttgtcggatc 4140

agcttgagta ggacaaatcc gccgagcttc gacgagattt tcaggagcta aggaagctaa 4200

aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc atcgtaaaga 4260

acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg ttcagctgga 4320

tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc cggcctttat 4380

tcacattctt gcccgcctga tgaatgctca tccggagttc cgtatggcaa tgaaagacgg 4440

tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg agcaaactga 4500

aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc tacacatata 4560

ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag ggtttattga 4620

gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg atttaaacgt 4680

ggccaatatg gacaacttct tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg 4740

cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtctgtg atggcttcca 4800

tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg gcggggcgta 4860

atttttttaa ggcagttatt ggtgcccttc gaaatgaccg accaagcgac gcccaacctg 4920

ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt 4980

ttccgggacg ccaacaacaa gacccatcat agtttgcccc cgcgacattg accataaatt 5040

catcgcacaa aatatcgaac ggggtttatg ccgcttttag tgggtgcgaa gaatagtctg 5100

ctcattaccc gcgaacaccg ccgcattcag atcacgctta gtagcgtccc catgagtagg 5160

cagaaccgcg tccaagtcca catcatccat aacgatcatg cacggggtgg aatccacacc 5220

cagacttgcc agcacctcat tagcgacacg ttgcgcagcg gccacgtcct tagccttatc 5280

cacgcaatcg agaacgtact gcctaaccgc gaaatcagac tgaatcagtt tccaatcatc 5340

gggcttcacc aaagcaacag caacgcgggt tgattcgacc cgttccggtg cttccagacc 5400

ggcgagcttg tacagttctt cttccatttc acgacgtaca tcagcgtcta tgtaatcaat 5460

gcccaaagca cgcttagccc cacgtgacca ggacgaacgc aggtttttag aaccaacctc 5520

atactcacgc caccgagcca ccaaaacagc gtccatatcc tcgccggcgt cgctttgatc 5580

ggccaacata tccaacatct gaaacggcgt gtacgacccc ttagacgcgg ttttagtagc 5640

ggagccagtc agttcctgag acatgccctt agcgaggtag gttgccattt tcgcagcgtc 5700

tccaccccag gtagacacct gatcaagttt gaccccgtgc tcacgcagtg gcgcgtccat 5760

accggcctta accacaccag cagaccagcg ggaaaacatg gaatcctcaa acgccttgag 5820

ttcatcgtca gacagtggac gatccaagaa caacagcatg ttgcggtgca agtgccaacc 5880

gttcgcccaa gagtctgtga cctcatagtc actataggtg tgctccaccc cgtaccgtgc 5940

acgttctttc ttccactgag atgttttcac catcgaagag tacgcagtct taatacccgc 6000

ttcaacctgc gcaaatgact gtgagcggtt gtgtcgaaca gtgcccacaa acatcatgag 6060

cgcgccaccc gccgccaagt gattcttagt agcaatagcc agctcaatgc ggcgttcgcc 6120

catgacttcc aattcagcca gaggtgaccc ccagcgagag tgagagtttt gcagaccctc 6180

aaactgcgaa gcaccgttag acgaccagga caccgcaaca gcttcgtccc tgcgccacct 6240

atggcacccc gccagagcct tactattggt gatcttgtac atgacgtttt gcctacgcca 6300

cgccctagcg cgagtgacct tagaaccctc attgacctgc ggttccttag aggtgttcac 6360

ttctatttca gtgttactca gtgttaccta gacccgatgt tgtgcggggt tgcgcagtgc 6420

gagtttgtgc gggtgttgtg cccgttgtct tagctagtgc tatggttgtc aattgaaacc 6480

ccttcgggtt atgtggcccc cgtgcatatg agttagtagc tcgcacgggg gtttgtcttg 6540

tctagggact attaattttt agtggtgttt ggtggccgcc tagcttggct atgcgtgcca 6600

gcttacccgt actcaatgtt aaagatttgc atcgacatgg gagggttacg tgtccgatac 6660

ctaggggggg tatccgcgac taggtgcccc ggtgctcact gtctgtaccg gcggggcaag 6720

ccccacaccc cgcatggaca gggtggctcc gccccctgca cccccagcaa tctgcatgta 6780

catgttttac acattagcac gacatgactg catgtgcatg cactgcatgc agactaggta 6840

aatatgagta tgtacgacta gtaacaggag cactgcacat aatgaatgag ttgcaggaca 6900

atgtttgcta cgcatgcgca tgacatatcg caggaaagct actagagtct taaagcatgg 6960

caaccaaggc acagctagaa cagcaactac aagaagctca acaggcacta caggcgcagc 7020

aagcgcaggc acaagccacc atcgaagcac tagaagcgca ggcaaaggct aagcccgtcg 7080

tggtcaccgc acgcgttcct ttggcactac gtgaggacat gaagcgcgca ggcatgcaga 7140

acggtgaaaa cctccaagag ttcatgatcg ccgcgtttac cgagcggcta gaaaagctca 7200

ccaccaccga caacgaggaa aacaatgtct aacccactag ttctctttgc ccaccgtgac 7260

ccggtaaatg acgtgacgtt cgagtgcatt gagcacgcca cctacgacac actttcacac 7320

gctaaagacc agatcaccgc ccaaatgcaa gccctagacg aagaagccgc cctactgccc 7380

taatgggtgt ttcatgggtg tttccctagt gtttcatggt gttttcacct aagctaggga 7440

attgcgcgag aagtctcgca aaaatcagca acccccggaa ccacacagtt cacgggggtt 7500

cttctatgcc agaaatcaga aaggggaacc agtgaacgac cccgaatggc tggatgatcc 7560

tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caaaaggatc taggtgaaga 7620

tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 7680

cagaccccgt agaaaagatc aaaggatctt c 7711

<210> 8

<211> 1505

<212> DNA

<213> Artificial sequence ()

<400> 8

aagaaggaga tataatgctg cgtgcgccga aacgccgtca ctctgaaaac ggtaaaccgg 60

aaaccgaagc gggtccgtct ccggcgccga tcaaacgtgc gaaacgtatg gtgcgcgcgt 120

ctcagctgga cctggtttac ccgttcgatt acgtggcgga cccggttggc ggcctgaacc 180

cgccgttcct gggtggctct ggcccgctgg ttgaccaggg cggccagctg accctgaacg 240

ttaccgatcc gatcatcatt aaaaaccgtt ccgttgacct ggctcacgat ccgtctctgg 300

atgtgaacgc gcagggccag ctggcagtgg ctgttgatcc ggaaggcgcg ctggatatca 360

ccccggatgg tctggatgtt aaagtggatg gcgttaccgt gatggttaac gacgattggg 420

aactggcggt taaagttgac ccgtccggtg gcctggattc taccgcgggc ggtctgggtg 480

tgtctgttga tgacaccctg ctggttgatc agggtgaact gggtgttcac ctgaaccagc 540

agggcccgat cacggcggat tctagcggca tcgacctgga aatcaacccg aacatgttca 600

ccgtgaacac ctccaccggt tccggtgtgc tggaactgaa cctgaaagcc cagggcggta 660

tccaggcggc ttctagcggt gttggtgtta gcgttgatga atctctgcag attgttaaca 720

acaccctgga agttaaacca gatccgtctg gtccgctgac cgttagcgcg aacggtctcg 780

gcctgaaata tgacaccaac accctggcgg tgaccgcggg tgcactgacc gtggttggcg 840

gtggtagcgt ttccaccccg atcgccacct tcgtgtccgg tagcccgtct ctgaacacct 900

acaacgcaac caccgttaac tcttccgcta acgcgttctc ctgcgcgtac tacctgcagc 960

agtggaacat ccagggcctg ctggttacct ctctgtacct gaaactggac tccgcgacca 1020

tgggtaaccg tccgggtgac ctgaactctg ccaacgcgaa atggttcacc ttctgggtta 1080

gcgcctacct gcagcagtgc aacccgtccg gtatccaggc gggtaccgtt agcccgagca 1140

ccgcaaccct gaccgacttc gaaccgatgg cgaaccgtag cgtgaccagc ccgtggacct 1200

actctgcaaa cggttactac gaaccgtcca tcggcgaatt ccaggttttc tctccggttg 1260

ttaccggcgc gtggaaccca ggtaacattg gtatccgtgt gctgccggtg ccggtgagcg 1320

cgagcggtga acgttacacc ctgctgtgct acagcctgca gtgcaccaac gcgagcatct 1380

tcaacccgaa caactctggt accatgatcg ttggtccggt gctgtactct tgcccggcgg 1440

cgagcctgcc gaagcttgcg gccgcactcg agcaccacca ccaccaccac tgatgagatc 1500

cggct 1505

<210> 9

<211> 6516

<212> DNA

<213> Artificial sequence ()

<400> 9

ccaccaccac tgatgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc 60

tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg 120

ttttttggac accatcgaat ggtgcaaaac ctttcgcggt atggcatgat agcgcccgga 180

agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta 240

tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc 300

gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt 360

ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc 420

cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc 480

cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca 540

caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga 600

tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga 660

ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga 720

gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt 780

ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc 840

gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat 900

gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg 960

cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg 1020

atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga 1080

ttttcgcctg ctggggcaaa ccacgtggac cgcttgctgc aactctctca gggccaggcg 1140

gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccg 1200

gggatcagcc ccggatgctt tggatacggt ctatgagctg gcagcgtatt tgaccgatcc 1260

ggacacctgg gataatgtgt ggattttgtc ggatcagctt gagtaggaca aatccgccga 1320

gcttcgacga gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat 1380

accaccgttg atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt 1440

gctcaatgta cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta 1500

aagaaaaata agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat 1560

gctcatccgg agttccgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt 1620

cacccttgtt acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa 1680

taccacgacg atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt 1740

gaaaacctgg cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat 1800

ccctgggtga gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc 1860

cccgttttca ccatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg 1920

attcaggttc atcatgccgt ctgtgatggc ttccatgtcg gcagaatgct taatgaatta 1980

caacagtact gcgatgagtg gcagggcggg gcgtaatttt tttaaggcag ttattggtgc 2040

ccttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg 2100

ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccaac aacaagaccc 2160

atcatagttt gcccccgcga cattgaccat aaattcatcg cacaaaatat cgaacggggt 2220

ttatgccgct tttagtgggt gcgaagaata gtctgctcat tacccgcgaa caccgccgca 2280

ttcagatcac gcttagtagc gtccccatga gtaggcagaa ccgcgtccaa gtccacatca 2340

tccataacga tcatgcacgg ggtggaatcc acacccagac ttgccagcac ctcattagcg 2400

acacgttgcg cagcggccac gtccttagcc ttatccacgc aatcgagaac gtactgccta 2460

accgcgaaat cagactgaat cagtttccaa tcatcgggct tcaccaaagc aacagcaacg 2520

cgggttgatt cgacccgttc cggtgcttcc agaccggcga gcttgtacag ttcttcttcc 2580

atttcacgac gtacatcagc gtctatgtaa tcaatgccca aagcacgctt agccccacgt 2640

gaccaggacg aacgcaggtt tttagaacca acctcatact cacgccaccg agccaccaaa 2700

acagcgtcca tatcctcgcc ggcgtcgctt tgatcggcca acatatccaa catctgaaac 2760

ggcgtgtacg accccttaga cgcggtttta gtagcggagc cagtcagttc ctgagacatg 2820

cccttagcga ggtaggttgc cattttcgca gcgtctccac cccaggtaga cacctgatca 2880

agtttgaccc cgtgctcacg cagtggcgcg tccataccgg ccttaaccac accagcagac 2940

cagcgggaaa acatggaatc ctcaaacgcc ttgagttcat cgtcagacag tggacgatcc 3000

aagaacaaca gcatgttgcg gtgcaagtgc caaccgttcg cccaagagtc tgtgacctca 3060

tagtcactat aggtgtgctc caccccgtac cgtgcacgtt ctttcttcca ctgagatgtt 3120

ttcaccatcg aagagtacgc agtcttaata cccgcttcaa cctgcgcaaa tgactgtgag 3180

cggttgtgtc gaacagtgcc cacaaacatc atgagcgcgc cacccgccgc caagtgattc 3240

ttagtagcaa tagccagctc aatgcggcgt tcgcccatga cttccaattc agccagaggt 3300

gacccccagc gagagtgaga gttttgcaga ccctcaaact gcgaagcacc gttagacgac 3360

caggacaccg caacagcttc gtccctgcgc cacctatggc accccgccag agccttacta 3420

ttggtgatct tgtacatgac gttttgccta cgccacgccc tagcgcgagt gaccttagaa 3480

ccctcattga cctgcggttc cttagaggtg ttcacttcta tttcagtgtt actcagtgtt 3540

acctagaccc gatgttgtgc ggggttgcgc agtgcgagtt tgtgcgggtg ttgtgcccgt 3600

tgtcttagct agtgctatgg ttgtcaattg aaaccccttc gggttatgtg gcccccgtgc 3660

atatgagtta gtagctcgca cgggggtttg tcttgtctag ggactattaa tttttagtgg 3720

tgtttggtgg ccgcctagct tggctatgcg tgccagctta cccgtactca atgttaaaga 3780

tttgcatcga catgggaggg ttacgtgtcc gatacctagg gggggtatcc gcgactaggt 3840

gccccggtgc tcactgtctg taccggcggg gcaagcccca caccccgcat ggacagggtg 3900

gctccgcccc ctgcaccccc agcaatctgc atgtacatgt tttacacatt agcacgacat 3960

gactgcatgt gcatgcactg catgcagact aggtaaatat gagtatgtac gactagtaac 4020

aggagcactg cacataatga atgagttgca ggacaatgtt tgctacgcat gcgcatgaca 4080

tatcgcagga aagctactag agtcttaaag catggcaacc aaggcacagc tagaacagca 4140

actacaagaa gctcaacagg cactacaggc gcagcaagcg caggcacaag ccaccatcga 4200

agcactagaa gcgcaggcaa aggctaagcc cgtcgtggtc accgcacgcg ttcctttggc 4260

actacgtgag gacatgaagc gcgcaggcat gcagaacggt gaaaacctcc aagagttcat 4320

gatcgccgcg tttaccgagc ggctagaaaa gctcaccacc accgacaacg aggaaaacaa 4380

tgtctaaccc actagttctc tttgcccacc gtgacccggt aaatgacgtg acgttcgagt 4440

gcattgagca cgccacctac gacacacttt cacacgctaa agaccagatc accgcccaaa 4500

tgcaagccct agacgaagaa gccgccctac tgccctaatg ggtgtttcat gggtgtttcc 4560

ctagtgtttc atggtgtttt cacctaagct agggaattgc gcgagaagtc tcgcaaaaat 4620

cagcaacccc cggaaccaca cagttcacgg gggttcttct atgccagaaa tcagaaaggg 4680

gaaccagtga acgaccccga atggctggat gatcctccag cgcggggatc tcatgctgga 4740

gttcttcgcc caccccaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 4800

aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 4860

atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 4920

gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 4980

tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca 5040

ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 5100

ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 5160

ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 5220

aacgacctac accgaactga gatacctaca gcgtgagcat tgagaaagcg ccacgcttcc 5280

cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 5340

gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 5400

ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 5460

cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 5520

tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 5580

cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag caaaagtgct 5640

catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 5700

cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 5760

cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 5820

acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 5880

ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaaagagttt 5940

gtagaaacgc aaaaaggcca tccgtcagga tggccttctg cttaatttga tgcctggcag 6000

tttatggcgg gcgtcctgcc cgccaccctc cgggccgttg cttcgcaacg ttcaaatccg 6060

ctcccggcgg atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa 6120

aggcccagtc tttcgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc 6180

gcatggggag accccacact accatcggcg ctacggcgtt tcacttctga gttcggcatg 6240

gggtcaggtg ggaccaccgc gctactgccg ccaggcaaat tctgttttat cagaccgctt 6300

ctgcgttctg atttaatctg tatcaggctg aaaatcttct ctcatccgcc aaaacagcca 6360

agctgaattc gagctcggta cccggggatc ctctagagtc gacctgcagg catgcaagct 6420

ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt 6480

ttgtttaact ttaagaagga gatataatgc tgcgtg 6516

<210> 10

<211> 2389

<212> DNA

<213> Artificial sequence ()

<400> 10

agcgagtcag tgagcgagga agcaaaagtg ctcatcattg gaaaacgttc ttcggggcga 60

aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 120

aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 180

caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 240

ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 300

gaatgtattt agaaaaataa acaaaagagt ttgtagaaac gcaaaaaggc catccgtcag 360

gatggccttc tgcttaattt gatgcctggc agtttatggc gggcgtcctg cccgccaccc 420

tccgggccgt tgcttcgcaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag 480

agcgttcacc gacaaacaac agataaaacg aaaggcccag tctttcgact gagcctttcg 540

ttttatttga tgcctggcag ttccctactc tcgcatgggg agaccccaca ctaccatcgg 600

cgctacggcg tttcacttct gagttcggca tggggtcagg tgggaccacc gcgctactgc 660

cgccaggcaa attctgtttt atcagaccgc ttctgcgttc tgatttaatc tgtatcaggc 720

tgaaaatctt ctctcatccg ccaaaacagc caagctgaat tcgagctcgg tacccgggga 780

tcctctagag tcgacctgca ggcatgcaag ctttaatacg actcactata ggggaattgt 840

gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataat 900

gctgcgtgcg ccgaaacgcc gtcactctga aaacggtaaa ccggaaaccg aagcgggtcc 960

gtctccggcg ccgatcaaac gtgcgaaacg tatggtgcgc gcgtctcagc tggacctggt 1020

ttacccgttc gattacgtgg cggacccggt tggcggcctg aacccgccgt tcctgggtgg 1080

ctctggcccg ctggttgacc agggcggcca gctgaccctg aacgttaccg atccgatcat 1140

cattaaaaac cgttccgttg acctggctca cgatccgtct ctggatgtga acgcgcaggg 1200

ccagctggca gtggctgttg atccggaagg cgcgctggat atcaccccgg atggtctgga 1260

tgttaaagtg gatggcgtta ccgtgatggt taacgacgat tgggaactgg cggttaaagt 1320

tgacccgtcc ggtggcctgg attctaccgc gggcggtctg ggtgtgtctg ttgatgacac 1380

cctgctggtt gatcagggtg aactgggtgt tcacctgaac cagcagggcc cgatcacggc 1440

ggattctagc ggcatcgacc tggaaatcaa cccgaacatg ttcaccgtga acacctccac 1500

cggttccggt gtgctggaac tgaacctgaa agcccagggc ggtatccagg cggcttctag 1560

cggtgttggt gttagcgttg atgaatctct gcagattgtt aacaacaccc tggaagttaa 1620

accagatccg tctggtccgc tgaccgttag cgcgaacggt ctcggcctga aatatgacac 1680

caacaccctg gcggtgaccg cgggtgcact gaccgtggtt ggcggtggta gcgtttccac 1740

cccgatcgcc accttcgtgt ccggtagccc gtctctgaac acctacaacg caaccaccgt 1800

taactcttcc gctaacgcgt tctcctgcgc gtactacctg cagcagtgga acatccaggg 1860

cctgctggtt acctctctgt acctgaaact ggactccgcg accatgggta accgtccggg 1920

tgacctgaac tctgccaacg cgaaatggtt caccttctgg gttagcgcct acctgcagca 1980

gtgcaacccg tccggtatcc aggcgggtac cgttagcccg agcaccgcaa ccctgaccga 2040

cttcgaaccg atggcgaacc gtagcgtgac cagcccgtgg acctactctg caaacggtta 2100

ctacgaaccg tccatcggcg aattccaggt tttctctccg gttgttaccg gcgcgtggaa 2160

cccaggtaac attggtatcc gtgtgctgcc ggtgccggtg agcgcgagcg gtgaacgtta 2220

caccctgctg tgctacagcc tgcagtgcac caacgcgagc atcttcaacc cgaacaactc 2280

tggtaccatg atcgttggtc cggtgctgta ctcttgcccg gcggcgagcc tgccgaagct 2340

tgcggccgca ctcgagcacc accaccacca ccactgatga gatccggct 2389

<210> 11

<211> 2714

<212> DNA

<213> Artificial sequence ()

<400> 11

tatgagttag tagctcgcac gggggtttgt cttgtctagg gactattaat ttttagtggt 60

gtttggtggc cgcctagctt ggctatgcgt gccagcttac ccgtactcaa tgttaaagat 120

ttgcatcgac atgggagggt tacgtgtccg atacctaggg ggggtatccg cgactaggtg 180

ccccggtgct cactgtctgt accggcgggg caagccccac accccgcatg gacagggtgg 240

ctccgccccc tgcaccccca gcaatctgca tgtacatgtt ttacacatta gcacgacatg 300

actgcatgtg catgcactgc atgcagacta ggtaaatatg agtatgtacg actagtaaca 360

ggagcactgc acataatgaa tgagttgcag gacaatgttt gctacgcatg cgcatgacat 420

atcgcaggaa agctactaga gtcttaaagc atggcaacca aggcacagct agaacagcaa 480

ctacaagaag ctcaacaggc actacaggcg cagcaagcgc aggcacaagc caccatcgaa 540

gcactagaag cgcaggcaaa ggctaagccc gtcgtggtca ccgcacgcgt tcctttggca 600

ctacgtgagg acatgaagcg cgcaggcatg cagaacggtg aaaacctcca agagttcatg 660

atcgccgcgt ttaccgagcg gctagaaaag ctcaccacca ccgacaacga ggaaaacaat 720

gtctaaccca ctagttctct ttgcccaccg tgacccggta aatgacgtga cgttcgagtg 780

cattgagcac gccacctacg acacactttc acacgctaaa gaccagatca ccgcccaaat 840

gcaagcccta gacgaagaag ccgccctact gccctaatgg gtgtttcatg ggtgtttccc 900

tagtgtttca tggtgttttc acctaagcta gggaattgcg cgagaagtct cgcaaaaatc 960

agcaaccccc ggaaccacac agttcacggg ggttcttcta tgccagaaat cagaaagggg 1020

aaccagtgaa cgaccccgaa tggctggatg atcctccagc gcggggatct catgctggag 1080

ttcttcgccc accccaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 1140

atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 1200

tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 1260

ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 1320

ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 1380

cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 1440

gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 1500

gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 1560

acgacctaca ccgaactgag atacctacag cgtgagcatt gagaaagcgc cacgcttccc 1620

gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 1680

agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 1740

tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 1800

agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 1860

cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 1920

gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc aaaagtgctc 1980

atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 2040

agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 2100

gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 2160

cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 2220

tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aaagagtttg 2280

tagaaacgca aaaaggccat ccgtcaggat ggccttctgc ttaatttgat gcctggcagt 2340

ttatggcggg cgtcctgccc gccaccctcc gggccgttgc ttcgcaacgt tcaaatccgc 2400

tcccggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa 2460

ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctggcagttc cctactctcg 2520

catggggaga ccccacacta ccatcggcgc tacggcgttt cacttctgag ttcggcatgg 2580

ggtcaggtgg gaccaccgcg ctactgccgc caggcaaatt ctgttttatc agaccgcttc 2640

tgcgttctga tttaatctgt atcaggctga aaatcttctc tcatccgcca aaacagccaa 2700

gctgaattcg agct 2714

<210> 12

<211> 5258

<212> DNA

<213> Artificial sequence ()

<400> 12

cggtacccgg ggatcctcta gagtcgacct gcaggcatgc aagctttaat acgactcact 60

ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag 120

aaggagatat aatgctgcgt gcgccgaaac gccgtcactc tgaaaacggt aaaccggaaa 180

ccgaagcggg tccgtctccg gcgccgatca aacgtgcgaa acgtatggtg cgcgcgtctc 240

agctggacct ggtttacccg ttcgattacg tggcggaccc ggttggcggc ctgaacccgc 300

cgttcctggg tggctctggc ccgctggttg accagggcgg ccagctgacc ctgaacgtta 360

ccgatccgat catcattaaa aaccgttccg ttgacctggc tcacgatccg tctctggatg 420

tgaacgcgca gggccagctg gcagtggctg ttgatccgga aggcgcgctg gatatcaccc 480

cggatggtct ggatgttaaa gtggatggcg ttaccgtgat ggttaacgac gattgggaac 540

tggcggttaa agttgacccg tccggtggcc tggattctac cgcgggcggt ctgggtgtgt 600

ctgttgatga caccctgctg gttgatcagg gtgaactggg tgttcacctg aaccagcagg 660

gcccgatcac ggcggattct agcggcatcg acctggaaat caacccgaac atgttcaccg 720

tgaacacctc caccggttcc ggtgtgctgg aactgaacct gaaagcccag ggcggtatcc 780

aggcggcttc tagcggtgtt ggtgttagcg ttgatgaatc tctgcagatt gttaacaaca 840

ccctggaagt taaaccagat ccgtctggtc cgctgaccgt tagcgcgaac ggtctcggcc 900

tgaaatatga caccaacacc ctggcggtga ccgcgggtgc actgaccgtg gttggcggtg 960

gtagcgtttc caccccgatc gccaccttcg tgtccggtag cccgtctctg aacacctaca 1020

acgcaaccac cgttaactct tccgctaacg cgttctcctg cgcgtactac ctgcagcagt 1080

ggaacatcca gggcctgctg gttacctctc tgtacctgaa actggactcc gcgaccatgg 1140

gtaaccgtcc gggtgacctg aactctgcca acgcgaaatg gttcaccttc tgggttagcg 1200

cctacctgca gcagtgcaac ccgtccggta tccaggcggg taccgttagc ccgagcaccg 1260

caaccctgac cgacttcgaa ccgatggcga accgtagcgt gaccagcccg tggacctact 1320

ctgcaaacgg ttactacgaa ccgtccatcg gcgaattcca ggttttctct ccggttgtta 1380

ccggcgcgtg gaacccaggt aacattggta tccgtgtgct gccggtgccg gtgagcgcga 1440

gcggtgaacg ttacaccctg ctgtgctaca gcctgcagtg caccaacgcg agcatcttca 1500

acccgaacaa ctctggtacc atgatcgttg gtccggtgct gtactcttgc ccggcggcga 1560

gcctgccgaa gcttgcggcc gcactcgagc accaccacca ccaccactga tgagatccgg 1620

ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag 1680

cataacccct tggggcctct aaacgggtct tgaggggttt tttggacacc atcgaatggt 1740

gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga 1800

atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg 1860

tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag 1920

cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac 1980

agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg 2040

tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag 2100

aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca 2160

gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct 2220

gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta 2280

ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc 2340

agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg 2400

gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact 2460

ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca 2520

ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt 2580

ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct 2640

catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca 2700

cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc 2760

cgtctcactg gtgaaaagaa aaaccaccct ggcgccgggg atcagccccg gatgctttgg 2820

atacggtcta tgagctggca gcgtatttga ccgatccgga cacctgggat aatgtgtgga 2880

ttttgtcgga tcagcttgag taggacaaat ccgccgagct tcgacgagat tttcaggagc 2940

taaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg 3000

gcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac 3060

cgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta 3120

tccggccttt attcacattc ttgcccgcct gatgaatgct catccggagt tccgtatggc 3180

aatgaaagac ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca 3240

tgagcaaact gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt 3300

tctacacata tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa 3360

agggtttatt gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt 3420

tgatttaaac gtggccaata tggacaactt cttcgccccc gttttcacca tgggcaaata 3480

ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtctg 3540

tgatggcttc catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca 3600

gggcggggcg taattttttt aaggcagtta ttggtgccct tcgaaatgac cgaccaagcg 3660

acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc 3720

ttcggaatcg ttttccggga cgccaacaac aagacccatc atagtttgcc cccgcgacat 3780

tgaccataaa ttcatcgcac aaaatatcga acggggttta tgccgctttt agtgggtgcg 3840

aagaatagtc tgctcattac ccgcgaacac cgccgcattc agatcacgct tagtagcgtc 3900

cccatgagta ggcagaaccg cgtccaagtc cacatcatcc ataacgatca tgcacggggt 3960

ggaatccaca cccagacttg ccagcacctc attagcgaca cgttgcgcag cggccacgtc 4020

cttagcctta tccacgcaat cgagaacgta ctgcctaacc gcgaaatcag actgaatcag 4080

tttccaatca tcgggcttca ccaaagcaac agcaacgcgg gttgattcga cccgttccgg 4140

tgcttccaga ccggcgagct tgtacagttc ttcttccatt tcacgacgta catcagcgtc 4200

tatgtaatca atgcccaaag cacgcttagc cccacgtgac caggacgaac gcaggttttt 4260

agaaccaacc tcatactcac gccaccgagc caccaaaaca gcgtccatat cctcgccggc 4320

gtcgctttga tcggccaaca tatccaacat ctgaaacggc gtgtacgacc ccttagacgc 4380

ggttttagta gcggagccag tcagttcctg agacatgccc ttagcgaggt aggttgccat 4440

tttcgcagcg tctccacccc aggtagacac ctgatcaagt ttgaccccgt gctcacgcag 4500

tggcgcgtcc ataccggcct taaccacacc agcagaccag cgggaaaaca tggaatcctc 4560

aaacgccttg agttcatcgt cagacagtgg acgatccaag aacaacagca tgttgcggtg 4620

caagtgccaa ccgttcgccc aagagtctgt gacctcatag tcactatagg tgtgctccac 4680

cccgtaccgt gcacgttctt tcttccactg agatgttttc accatcgaag agtacgcagt 4740

cttaataccc gcttcaacct gcgcaaatga ctgtgagcgg ttgtgtcgaa cagtgcccac 4800

aaacatcatg agcgcgccac ccgccgccaa gtgattctta gtagcaatag ccagctcaat 4860

gcggcgttcg cccatgactt ccaattcagc cagaggtgac ccccagcgag agtgagagtt 4920

ttgcagaccc tcaaactgcg aagcaccgtt agacgaccag gacaccgcaa cagcttcgtc 4980

cctgcgccac ctatggcacc ccgccagagc cttactattg gtgatcttgt acatgacgtt 5040

ttgcctacgc cacgccctag cgcgagtgac cttagaaccc tcattgacct gcggttcctt 5100

agaggtgttc acttctattt cagtgttact cagtgttacc tagacccgat gttgtgcggg 5160

gttgcgcagt gcgagtttgt gcgggtgttg tgcccgttgt cttagctagt gctatggttg 5220

tcaattgaaa ccccttcggg ttatgtggcc cccgtgca 5258

Claims (6)

1. A method for efficiently expressing avian adenovirus Fiber-2 protein in Escherichia coli is characterized by comprising the following steps:

(1) carrying out amplification by taking the PET-21a-GX-1-Fiber2 plasmid as a template and Fiber-2-F and Fiber-2-R as primers to obtain a Fiber-2 target fragment;

(2) PXMJ19-C17 1786T-hexon (T7) plasmid is used as a template, PXMJ19-T7-his-F and PXMJ19-T7-his-R are used as primers for amplification, and a PXMJ19-C1786T-T7 vector fragment is obtained;

(3) carrying out homologous recombination on the Fiber-2 target fragment and the PXMJ19-C17 1786T-T7 vector fragment to obtain a recombinant plasmid PXMJ19-T7-his-Fiber 2;

(4) the recombinant plasmid PXMJ19-T7-his-Fiber2 is transformed into Shuffle T7-B competent cells for expressing the Fiber-2 protein;

the primer sequences are respectively as follows:

Fiber-2-F：AAGAAGGAGATATAATGCTGCGTGCGC；

Fiber-2-R：AGCCGGATCTCATCAGTGGTGGTGG；

PXMJ19-T7-his-F：CCACCACCACTGATGAGATCCGGCT；

PXMJ19-T7-his-R：CACGCAGCATTATATCTCCTTCTTA；

the nucleotide sequence of the PET-21a-GX-1-fiber2 plasmid is SEQ ID NO 6;

the nucleotide sequence of the PXMJ19-C17 1786T-hexon (T7) plasmid is SEQ ID NO. 7.

2. The method for efficiently expressing avian adenovirus Fiber-2 protein in Escherichia coli as claimed in claim 1, wherein: the nucleotide sequence of the Fiber-2 target fragment obtained in the step (1) is SEQ ID NO: 8.

3. The method for efficiently expressing avian adenovirus Fiber-2 protein in Escherichia coli as claimed in claim 1, wherein: the nucleotide sequence of the PXMJ19-C17 1786T-T7 vector fragment obtained in the step (2) is SEQ ID NO: 9.

4. The method for efficiently expressing avian adenovirus Fiber-2 protein in Escherichia coli as claimed in claim 1, wherein: the homologous recombination conditions in the step (3) are as follows:

adding the Fiber-2 target fragment and the PXMJ19-C17 1786T-T7 vector fragment into the reaction solution according to the volume ratio of 1:2, shaking up, incubating in a water bath kettle at 37 ℃ for 30min, and then cooling at 4 ℃ or on ice to obtain the recombinant product.

5. The method for efficiently expressing avian adenovirus Fiber-2 protein in Escherichia coli as claimed in claim 1, wherein: the step of transforming the recombinant plasmid PXMJ19-T7-his-fiber2 into Shuffle T7-B competent cells in the step (4) is as follows: adding the recombinant plasmid PXMJ19-T7-his-fiber2 into the Shuffle T7-B competent cells, uniformly mixing, standing on ice for 30min, performing water bath heat shock at 42 ℃ for 45sec, immediately placing on ice for cooling for 2-3min, adding an SOC culture medium, and shaking the cells at 37 ℃ and the rotation speed of 200-; preheating LB solid culture medium containing chloramphenicol resistance in a 37 ℃ incubator, centrifuging to remove supernatant, re-suspending the thallus with the rest culture medium, uniformly coating on a flat culture medium containing chloramphenicol resistance with a sterile coating rod, and performing inverted culture in the 37 ℃ incubator for 12-16 h.

6. The method for efficiently expressing avian adenovirus Fiber-2 protein in Escherichia coli according to claim 5, wherein the step of expressing the Fiber-2 protein in step (4) is as follows:

and (3) selecting a single colony after transformation culture, inoculating the single colony into an LB solid culture medium with chloramphenicol resistance, carrying out shake culture on the bacteria at 30 ℃, and 200rpm until the bacteria reach an exponential phase, adding IPTG (isopropyl thiogalactoside) with the final concentration of 1mM, and carrying out induction for 15h at 16 ℃ in a shaking table.

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