CN113549642B - Mycobacterium tuberculosis integrated expression plasmid and application thereof - Google Patents

Abstract

The invention relates to an integrated expression plasmid of mycobacterium tuberculosis and application thereof, wherein the plasmid comprises the following structural elements: (1) The screening marker of the resistance gene is used for constructing and screening the recombinant plasmid; (2) Gamma delta sequences respectively positioned at two sides of the resistance gene are used for knocking out the resistance; (3) elements for insertion and expression of a gene of interest; (4) Essential tag elements, replicon elements and Mycobacterium tuberculosis genome integration elements.

Description

Mycobacterium tuberculosis integrated expression plasmid and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an integrated expression plasmid of mycobacterium tuberculosis and application thereof.

Background

Tuberculosis is a chronic infectious disease caused by infection of tubercle bacillus, and remains one of infectious diseases seriously threatening human health. BCG is the only vaccine approved for tuberculosis prevention, but BCG has unstable protective effect on adults, and research on the development of protective vaccines such as recombinant BCG vaccine around recombinant mycobacteria has been a hot spot for tuberculosis vaccine research.

Conventionally, the efficiency of finally obtaining the nonreactive recombinant mycobacterium tuberculosis is low by integrating specific over-expressed protein in the mycobacterium tuberculosis genome, and the construction of the recombinant bacteria needs 1 year or more. The method for constructing the integrated over-expression recombinant mycobacterium can greatly improve the efficiency, provides a new genetic tool for accelerating the research of functional genomes of the mycobacterium, and provides great convenience for the research and development of non-antibody and anti-tuberculosis vaccines or tuberculosis prevention vaccines with high specific antigen content.

The current construction of overexpression strains without resistant mycobacteria integrated into the genome requires roughly the following steps:

(1) Constructing an integrated overexpression plasmid containing resistance;

(2) Screening and constructing successful plasmids, and transferring the plasmids into mycobacteria;

(3) Obtaining an overexpression strain containing resistance genes through resistance and expression screening,

(4) And (3) constructing a knockout resistant plasmid, constructing a knockout resistant bacteriophage, and obtaining an overexpression strain without resistant mycobacteria.

Therefore, in the traditional method, a complete recombinant mycobacterium strain with gene overexpression and resistance knockout is constructed, plasmid construction needs to be carried out for at least 4 times, complicated steps such as enzyme digestion, connection transformation, screening and the like need to be repeated for each construction, in addition, the process often needs 1 year or more due to the characteristic of slow growth of mycobacteria, so that the traditional method is extremely low in efficiency, and the research process is greatly limited.

Disclosure of Invention

The invention firstly relates to an integrated expression plasmid of mycobacterium tuberculosis, which comprises the following structural elements:

(1) The screening marker of the resistance gene is used for constructing and screening the recombinant plasmid;

(2) Gamma delta sequences respectively positioned at two sides of the resistance gene are used for knocking out the resistance;

(3) Elements for insertion and expression of a gene of interest;

(4) Necessary tag elements, replicon elements and integration elements of the mycobacterium tuberculosis genome.

The gamma delta sequence is shown in SEQ ID NO. 33.

Preferably, the first and second liquid crystal materials are,

the resistance genes are SacB and hyg resistance genes;

the element for inserting and expressing the target gene is hsp60 promoter;

the described label element is his label, the described replicon element is OIE replicon for passage in colibacillus, and the described mycobacterium tuberculosis genome integration element is attp and int gene;

most preferably, the first and second liquid crystal display panels are,

the plasmid is pTB01 plasmid, and the nucleotide sequence of the plasmid is shown in SEQ ID NO. 1.

The invention also relates to the following application of the integrated expression plasmid of mycobacterium tuberculosis:

(1) Preparing a mycobacterium tuberculosis strain with a specific over-expression protein integrated in a genome;

(2) Preparing the anti-tuberculosis vaccine or the tuberculosis prevention vaccine with higher specific antigen content.

A method for preparing a mycobacterium tuberculosis strain having an overexpressed target protein integrated into its genome, the method comprising the steps of:

(1) Inserting an expression gene of a target protein into the integrated expression plasmid of the mycobacterium tuberculosis;

(2) Amplifying and extracting the target protein-containing mycobacterium tuberculosis integrated expression plasmid;

(3) And transferring the integrated expression plasmid containing the target protein of the mycobacterium tuberculosis into the mycobacterium tuberculosis.

A method for preparing a tuberculosis vaccine with high content of specific target protein, which comprises the following steps:

(1) Inserting the expression gene of the target protein into the integrated expression plasmid of the mycobacterium tuberculosis;

(2) Amplifying and extracting the target protein-containing mycobacterium tuberculosis integrated expression plasmid;

(3) Transferring the integrated expression plasmid containing the target protein of the mycobacterium tuberculosis into the mycobacterium tuberculosis;

(4) Preparing the vaccine by using the mycobacterium tuberculosis with high expression of the target protein.

The invention also relates to an integrated expression plasmid of the mycobacterium tuberculosis with high expression of specific target proteins, wherein the specific target proteins are as follows: rv3899c (Va 07) protein, rv0287 (Va 16) protein, rv3803c (Va 25) protein.

The plasmids are respectively as follows:

pBT01s1-Va07 containing Rv3899c (Va 07) protein, the sequence of which is shown in SEQ ID NO. 2;

pBT01s1-Va16 containing Rv0287 (Va 16) protein, and the sequence is shown as SEQ ID NO. 5;

pBT01s1-Va25 containing Rv3803c (Va 25) protein, and the sequence is shown in SEQ ID NO. 8.

The beneficial effect of the invention is that,

in the existing construction of recombinant mycobacterium tuberculosis, the insertion of an antibiotic marker gene may cause a polar effect, so that the expression or silencing of genes downstream of the insertion site is caused, and the antibiotic marker is retained in a genome, thereby limiting the further use of the antibiotic marker in the strain.

The sucrose lethal gene SacB can be applied to a gene deletion reverse screening marker and a traceless deletion suicide vector [1].

The pTB01 vector constructed by the invention contains sacB gene coding sucrose levan, the sucrose can be catalyzed by the SacB gene coding sucrose levan, the sucrose can be hydrolyzed into glucose and fructose by the sacB gene coding sucrose levan, the fructose is polymerized into high molecular weight levan, and the high molecular weight levan is accumulated to have toxic effect on cells to cause cell death. Therefore, the sucrose lethal gene SacB is used for constructing a suicide vector, a resistance gene is not needed to replace a target gene, any possible polar effect of a resistance marker on a downstream gene is avoided, and the method has important significance for the function research of the tuberculosis gene.

The invention constructs a recombinant pTB01 plasmid vector, can obtain a mycobacterium over-expression strain which is integrated on a genome and does not contain resistance only by one-time plasmid construction, and shortens the construction process which usually needs 1 year or even longer to 2-3 months.

[ REFERENCE ] to:

[1]Jackson M,Reinaldo Camacho L,Gicquel B,Guilhot C.Gene Replacement and Transposon Delivery Using the Negative Selection Marker sacB.Methods Mol Med.2001；54:59-75.doi:10.1385/1-59259-147-7:059. PMID:21341069.

drawings

FIG. 1 is a schematic diagram of a vector construction process.

FIG. 2 shows an electrophoresis diagram of a recovered fragment containing fragment 1 having a γ δ (sacB-hyg) γ δ sequence, fragment 2 having an hsp60 promoter sequence, and fragment 3 having an oriE, attP, int, etc. (fragment 1 corresponding to primer A, fragment 2 corresponding to primer B, and fragment 3 corresponding to primer C).

FIG. 3, the results of verifying the recovery of pTB01 plasmid.

FIG. 4 shows the results of electrophoretic detection of the linearized pTB01 plasmid and Va07, va16 and Va25 fragments.

FIG. 5 shows the detection results of the amplified target plasmid.

FIG. 6, verification of the PCR of the structure of plasmids pTB01s1-Va07, pTB01s1-Va16 and pTB01s1-Va 25: 6A, PCR results for plasmids pTB01s1-Va07, pTB01s1-Va16 and pTB01s1-Va25 using pMV361-sFP-1 and pTB01-sRP1 as primers; 6B, PCR results for plasmids pTB01s1-Va07, pTB01s1-Va16 and pTB01s1-Va25 using Hyg-F and Hyg-R as primers; 6C, PCR results for plasmids pTB01s1-Va07, pTB01s1-Va16 and pTB01s1-Va25 using BCG-sFP-2 and pMV361-sRP-2 as primers; 6D, PCR was performed on plasmids pTB01s1-Va07, pTB01s1-Va16 and pTB01s1-Va25 using pMV361-sFP-2 and BCG-sRP-2 as primers.

FIG. 7 is a schematic view of: carrying out protein electrophoresis verification on the BCG vaccine: 7A, rBCG-pTB01s1-Va07 protein electrophoresis picture; 7B, rBCG-pTB01s1-Va16 protein electrophoresis picture; 7C and rBCG-pTB01s1-Va16 protein electrophoresis picture.

FIG. 8 shows the results of plate screening of the anti-resorptive BCG vaccine.

FIG. 9 shows the results of amplifying sucrose-screened positive clones using primers Hyg-F and Hyg-R,

m1 represents 100bp Plus DNA ladder;

1 represents rBCG-pTB01s5-Va02-del. HygB, without target band;

2 represents rBCG-pTB01s1-Va04-del.HygB without target band;

3, rBCG-pTB01s1-Va07-del.HygB without a target band;

4 represents rBCG-pTB01s1-Va16-del.HygB without target band;

5 shows rBCG-pTB01s1-Va25-del. HygB, without target band;

6 represents rBCG-pTB01-del. HygB-1, which is a negative control and has no target band;

7' represents Trans5 alpha-pTB 01-1 as a positive control with a target band;

7 represents rBCG-pTB01-1, which is a positive control and has a target band;

ultrapure water, negative control, no strip, is indicated at 8.

Detailed Description

Example 1 construction and verification of pTB01 plasmid

1. Respectively carrying out PCR amplification on 3 fragments, wherein the fragment 1 contains a gamma delta (sacB-hyg) gamma delta sequence, the fragment 2 contains an hsp60 promoter sequence, and the fragment 3 contains oriE, attP, int and other sequences;

2. connecting three vector fragments by adopting a seamless cloning method;

3. transforming the competence of the Escherichia coli Trans5 alpha;

4. selecting single clone, extracting plasmid and enzyme digestion verification;

5. and finally, carrying out sequencing verification on the vector. The vector construction process is shown in FIG. 1.

The transformation process of the plasmid comprises the following steps:

the construction of the recombinant plasmid adopts a seamless cloning technology (In-

HD Cloning Kit User Manual，

Laboratories, Inc.Cat.Nos.Many(011614))。

The method is characterized in that the tail end of a vector and the tail end of a primer are provided with about 20 homologous bases, about 20 bases which are homologous with a vector sequence are respectively carried at two ends of an obtained PCR product, complementary pairing and cyclization are carried out by virtue of acting force between the bases, the PCR product can be directly used for transforming host bacteria without ligase, and gaps of linear plasmids (circular plasmids) entering the host bacteria are repaired by virtue of self enzyme systems. The construction method of the recombinant plasmid comprises the following steps:

1. respectively designing and synthesizing seamless cloning primers by taking a target sequence as a template;

the 3 fragments were PCR amplified separately using the designed primers using Polymerase Chain Reaction (PCR): fragment 1 contains the gamma delta (sacB-hyg) gamma delta sequence, fragment 2 contains the hsp60 promoter sequence, and fragment 3 contains the oriE, attP, int, etc. sequences:

(1) Fragment 1 contains the γ δ (sacB-hyg) γ δ signature sequence:

the gamma delta sequence is shown in SEQ ID NO. 33.

SEQ ID NO.33：

CCTGTATCCTAAATCAAATATCGGACAAGCAGTGTCTGTTATAACAAAAAATCGATTTAATAGACA CACCAACAGCATGGTTTTTATGTGTGCGATAATTTATAATATTTCGGACAGG。

Primers C1 and C2 were used with the plasmid p0004s (which was offered by the laboratory of the researchers in Wuhan Virus institute of China).

Primer C1:5 'CCAAGAATTGGCAGGTCCTG-3' (SEQ ID NO. 31)

And (3) primer C2:5 'CCAATCTTTGGCTAGAGTCTCTCTCTG-3' (SEQ ID NO. 32)

The PCR parameters were: 94 ℃/30sec,52 ℃/30sec,68 ℃/4min,30 cycles.

The PCR product is 3683bp long.

(2) Fragment 2 contains the characteristic sequences of oriE, attP, int:

primers A1 and A2 were used, and the pMV361 plasmid (which is a premium from the laboratory of the researchers in Deng, wuhan Virus, china academy of sciences) was used as a template.

Primer A1:5 'GGACTCTAGCCAAAGATTGGACCTCCACTAGTTCCATGACG 3' (SEQ ID NO. 11)

And (3) primer A2:5 'GCGCGCGTCGTTGTGGTCACCGTCTAGATTTCTGGCGGGACG 3' (SEQ ID NO. 12)

The PCR parameters were: 94 ℃/30sec,52 ℃/30sec,68 ℃/4min,30 cycles.

The PCR product was 2716bp in length.

(3) Fragment 3 was obtained containing the characteristic sequence of the hsp60 promoter:

using the primers B1 and B2, the synthetic plasmid PUC57-phsp60 (synthesized by Sommermay Biotech Co., ltd., nanjing) was used as a template.

Primer B1: CGGTGACCACAACGACGCGC (SEQ ID NO. 13)

And (3) primer B2: TACAGGACCTGCCAATTCTTGGGCTTAGCTGATCACCGCGG (SEQ ID NO. 14)

The PCR parameters were: 94 ℃/30sec,59 ℃/30sec,68 ℃/1min,30 cycles.

The PCR product is 559bp long.

(4) Detecting the amplification product by electrophoresis and cutting gel for recovery

The electrophoresis result of the amplified product is shown in figure 2, and three fragment genes are obtained by adopting a gel cutting recovery mode from three fragments respectively obtained in the three PCR steps, wherein the fragment 1 is a 3683bp gel strip, the fragment 2 is 2716bp, and the fragment 3 is 559bp. Using AxyPrep ^TM The DNA Gel Extraction Kit was recovered and stored at-20 ℃ for further use.

(5) The recovered target fragment was added to a test tube to carry out a recombination reaction to obtain pTB01 plasmid.

Using the seamless cloning technique, fragment 1 (5. Mu.l, 50 ng/. Mu.l), fragment 2 (4. Mu.l, 50 ng/. Mu.l), fragment 3 (1. Mu.l, 100 ng/. Mu.l), 5 XIn-fusion HD Enzyme Premix (4. Mu.l) were ligated to the ligation substrates and ligase, if indicated by volume only, with the addition of the instruction concentration, purified water (6. Mu.l), and the total of 20. Mu.l of the reaction system was incubated at 50 ℃ for 25min.

(6) Recombinant pTB01 plasmid transformed Escherichia coli

Transforming the reactant obtained in the step (5) into the competence of Trans5 alpha of Escherichia coli, coating the reactant in an LB plate containing kanamycin, and culturing overnight at 37 ℃, wherein the concrete steps are as follows:

adding 20 ul of seamless clone product into 100 ul of Trans5 alpha competence, carrying out ice bath for 30min, carrying out heat shock at 42 ℃ for 90sec, carrying out ice bath for 5min, adding 1mL of liquid LB, uniformly mixing, culturing at 37 ℃ and 200rpm for 1h, then centrifuging at 5000rpm for 5min, removing 1mL of supernatant, remaining 100 ul of heavy suspension thallus, completely coating an LB plate containing 150 ug/mL Hyg, and carrying out overnight culture at 37 ℃.

(7) Screening for Single clones

The positive strain is verified by enzyme digestion of the extracted plasmid, and the full-length DNA sequence is verified by sequencing reaction.

Selecting a single clone, shaking bacteria, extracting plasmids, using Van 91I to cut pTB01 plasmids by enzyme, wherein 2 fragments can be seen after the enzyme is cut by running gel, and the sizes of the 2 fragments are 3672bp containing a gamma delta (sacB-hyg) gamma delta sequence and the sizes of the other sequences are 3260bp. The electrophoresis results of the plasmid and the plasmid digestion products are shown in FIG. 3. And (3) selecting the plasmid with the correct restriction enzyme, performing whole plasmid sequencing to obtain the whole sequence information of the plasmid, wherein the sequencing result shows that the plasmid pTB01 is correctly constructed, and the full-length sequence of the plasmid is shown as SEQ ID No. 1.

Example 2 application of Mycobacterium Integrated expression plasmid pTB01

3 examples of applications using pTB01 as a vector are shown here to verify that the desired protein can be correctly expressed after electrotransformation of Mycobacterium vaccae BCG (preservation of the unit) after the recombinant vector is constructed using pTB01, and that a recombinant BCG with resistance to further inactivation can be obtained.

In example 1, rv3899c (CN 201510811331.8) is used as the target gene and is referred to as Va07 in the present invention.

Example 2, the gene with Rv0287 (CN 201310607242.2) as the target gene is named as Va16 in the invention.

Example 3, rv3803c (CN 201310608293.7) is the target gene, referred to as Va25 in the present invention.

1. Construction and amplification of pTB01s1-Va07, pTB01s1-Va16, pTB01s1-Va25

Using a vector ATG as an initiation codon, wherein the target gene does not contain the initiation codon, and the N end and the C end of the target gene simultaneously contain 8 × His tags;

in example 1, rBCG-pTB01s1-Va07 encodes Va07 protein CDS initiated by ATG vector, and the encoded protein has 434 amino acids, has a relative molecular weight of 44.0kDa and can be detected by His antibody (the nucleotide sequence is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4).

In example 2, the Va16 protein encoded by rBCG-pTB01s1-Va16 is CDS initiated by a vector ATG, and the encoded protein contains 121 amino acids, has a relative molecular weight of 13.0kDa and can be detected by a His antibody (the nucleotide sequence is shown as SEQ ID NO.6, and the amino acid sequence is shown as SEQ ID NO. 7).

In example 3, rBCG-pTB01s1-Va25 encodes Va25 protein CDS initiated by ATG vector, and the encoded protein contains 323 amino acids, has a relative molecular weight of 34.3kDa and can be detected by His antibody (the nucleotide sequence is shown in SEQ ID NO.9, and the amino acid sequence is shown in SEQ ID NO. 10).

The method comprises the following specific steps:

(1) Seamless cloning primer designed and synthesized respectively by taking pTB01 vector sequence as template

Linearizing the pTB01 vector by using a Polymerase Chain Reaction (PCR) amplification method by using a designed primer;

pTB01 linearized primer:

primers pTB01s1-LFP and pMV361-L1-RP were used with pTB01 plasmid as template.

Primer pTB01s1-LFP:5 'GAAGCTTATCGATGTCGACGTAGTC-3' (SEQ ID NO. 15)

Primer pMV361-L1-RP:5 'TGCGAAGTGATTCCTCCGGGATC-3' (SEQ ID NO. 16)

The PCR parameters were: 94 ℃/30sec,55 ℃/30sec,68 ℃/7min10sec,30 cycles.

The PCR product is 6897bp long.

(2) A seamless cloning primer was designed and synthesized using Va07, va16, and Va25 sequences as templates, respectively, and the target DNA fragment was amplified.

Va07：

Primer pTB01s1-Va07-FP:5 'GGAGGAATCACTTCGCAATGCATCATCATCATCACCACCACGTGACGGGGCAACCGG 3' (SEQ ID NO. 17)

Primer pTB01s1-Va07-RP:

5’-CGTCGACATCGATAAGCTTCGGCAGCGAACCCTCGTGAG-3’(SEQ ID NO.18)

the PCR product is 1291bp in length.

Va16：

Primer pTB01s1-Va16-FP:5' GGAGGAATCACTTCGCAATGCATCATCATCATCACCACCCAGACTTTTGGATGCTCATACC-

Primer pTB01s1-Va16-RP:

5’-CGTCGACATCGATAAGCTTCGAACCCGGTATAGGTCGAC-3’(SEQ ID NO.20)

the PCR product was 352bp in length.

Va25：

Primer pTB01s1-Va25-FP:

5’-GGAGGAATCACTTCGCAATGCATCATCATCATCATCACCACCACAAGGGTCGGTCGGCGC-3’ (SEQ ID NO.21)

primer pTB01s1-Va25-RP:

5’-CGTCGACATCGATAAGCTTCGCGGATCGCACCGACGATATCG-3’(SEQ ID NO.22)

the PCR product was 958bp in length.

(3) The collected target DNA fragment and the linearized vector are recombined to construct expression plasmids pTB01s1-Va07, pTB01s1-Va16 and pTB01s1-Va25

And (3) respectively carrying out nucleic acid electrophoresis on the amplification products obtained in the steps (1) and (2), cutting gel and recovering the amplification products, wherein the electrophoresis result is shown in figure 4.

And adding the recovered target DNA fragment and the linearized vector into a test tube to perform recombination reaction. The reaction system is as follows: 2.5. Mu.l of pTB01 vector linearized PCR product, 1.5. Mu.l of target gene PCR product, and 1. Mu.l of seamless clonase 5 XIn-fusion were added, and 5. Mu.l of the reaction system was mixed well. The reaction conditions are as follows: water bath at 50 deg.c for 25min.

(4) Transformation and amplification of a target plasmid

Transforming the recombinant plasmid vector obtained in the step (3) into a Trans5 alpha competent strain by referring to the method of example 1, selecting a single clone, amplifying a target strain and harvesting a target plasmid, and respectively selecting an upstream primer pMV361-sFP-1 and a downstream primer pTB01-sRP1 which are positioned at two ends of an inserted target gene as PCR screening primers of a screening colony to amplify a target gene fragment.

The upstream primer pMV361-sFP-1:5 'GGTCATGGGCCGAACATACTCAC-3' (SEQ ID NO. 23)

The downstream primer pTB01-sRP1:5 'TGGCTAGCTGATCACCGCG-3' (SEQ ID NO. 24)

The results show that:

va07 target gene PCR product is long: 1707bp

Va16 target gene PCR product length: 768bp

Va25 target gene PCR product length: 1374bp

The electrophoretogram of the target fragment is shown in FIG. 5, and the sequencing verification result shows that the target fragment is completely consistent with the theoretical sequence. Clones that were sequence verified to be correct were selected and retained. Plasmids were extracted with the kit. Recombinant plasmids pTB01s1-Va07, pTB01s1-Va16 and pTB01s1-Va25 were obtained.

2. Preparation of recombinant BCG vaccine

Obtaining the recombinant BCG: rBCG-pTB01s1-Va07, rBCG-pTB01s1-Va16 and rBCG-pTB01s1-Va25.

(1) And (3) transformation: the recombinant plasmids pTB01s1-Va07, pTB01s1-Va16 and pTB01s1-Va25 were electrotransformed into BCG competent cells (prepared and stored in this unit), plated on a 7H10 plate containing hygromycin B, and incubated at 37 ℃ for 3 to 4 weeks.

Taking out BCG from a refrigerator at minus 80 ℃ to be competent, firstly putting the BCG on ice for dissolving, and then putting the BCG at room temperature for standby;

adding 10ul of plasmid into 200 mul of electrotransformation competent cells, standing at room temperature for 10min, and transferring into a 2mm electrotransfer cup;

electric shock: after the voltage is 2.5kV, the resistance is 1000 omega, and the electric shock is finished under the condition of 25 muF capacitance, 1ml of 7H9 (BD-271310, BD Difco/BBL) + OADC (BD 211886, BD Difco/BBL) liquid culture medium which is placed at room temperature in advance is added;

recovering and coating plates: after being transferred to a 1.5ml centrifuge tube for recovery overnight at 37 ℃, the tube is centrifuged at 5000r/min for 10min to remove most of the supernatant, and about 250. Mu.l of the remaining solution is plated with solid medium plates containing HygB (hygromycin) antibiotic 7H10 (BD-262710, BD Difco/BBL) + OADC. Standing and culturing at 37 ℃ for 3-4 weeks.

(2) Subculturing

For liquid culture, a T25 cell culture flask was used and 10ml of the medium was contained. After the plate colonies grow, picking the monoclonal colonies in the plate to liquid medium containing HygB resistance 7H9+ OADC. Standing and culturing at 37 ℃ for 3-4 weeks.

(3) And (5) PCR screening and verifying positive clones to obtain the recombinant BCG containing the recombinant plasmid.

And (3) molecular identification: the single clone is picked, and the positive strain is verified through colony PCR.

To determine that the recombinant BCG genome has inserted a recombinant plasmid containing the gene of interest, the PCR strategy includes the following 3 aspects:

the first detection scheme is as follows: PCR amplification of target Gene with primers pMV361-sFP-1 and pTB01-sRP1

The upstream primer pMV361-sFP-1:5 'GGTCATGGGCCGAACATACTCAC-3' (SEQ ID NO. 23)

The downstream primer pTB01-sRP1:5 'TGGCTAGCTGATCACCGCG-3' (SEQ ID NO. 24)

The PCR parameters were: 94 ℃/15sec,56 ℃/30sec,72 ℃/2min,30 cycles.

Va07 target gene PCR product length: 1707bp of

Va16 target gene PCR product length: 768bp

Va25 target gene PCR product is long: 1374bp

The results show that the target gene primers pMV361-sFP-1 and pTB01-sRP1 PCR both have bands, and the sizes of the bands are consistent. The results are shown in FIG. 6A;

and (2) detection scheme II: PCR amplification of HygB Gene with primers Hyg-F and Hyg-R

An upstream primer Hyg-F:5 'AACATCTTCTGTGGACCTGGC-3' (SEQ ID NO. 25)

Downstream primer Hyg-R:5 'GTGAAGCCGGAGATCCAG-doped 3' (SEQ ID NO. 26)

The PCR parameters were: 94 ℃/15sec,52 ℃/30sec,72 ℃/30sec,30 cycles.

The PCR product is long: 257bp

Gel image results show that primers Hyg-F and Hyg-R PCR both have bands, and the sizes of the bands accord with each other. The results are shown in FIG. 6B;

and a third detection scheme: PCR is carried out by using primers BCG-sFP-2 and pMV361-sRP-2 to amplify the upstream integration site sequence of the recombinant plasmid and the BCG host genome.

The upstream primer BCG-sFP-2:5 'GCAGGTCAAGCTCACCGCGATCGAC 3' (SEQ ID NO. 27)

The downstream primer pMV361-sRP-2:5 'TCCAGGTCTCTCCATCGATGATGAGC-3' (SEQ ID NO. 28)

The PCR parameters were: 94 ℃/15sec,57 ℃/30sec,72 ℃/1min,30 cycles.

The PCR product is long: 701bp

The gel diagram result shows that the PCR of the upstream primers BCG-sFP-2 and pMV361-sRP-2 have bands, and the sizes of the bands are consistent. The results are shown in FIG. 6C;

and (4) detecting a scheme IV: the downstream integration site sequence of the recombinant plasmid and BCG host genome is amplified by PCR with primers pMV361-sFP-2 and BCG-sRP-2.

The upstream primer pMV361-sFP-2:5 'TgcAATCCGTGCAACCTTG-3' (SEQ ID NO. 29)

The downstream primer BCG-sRP-2:5 'TCAACTGCTCGACGGTGCTCTTC-3' (SEQ ID NO. 30)

The PCR parameters were: 94 ℃/15sec,57 ℃/30sec,72 ℃/1min,30 cycles.

The PCR product is long: 585bp

The gel diagram result shows that the downstream primers BCG-sRP-2 and pMV361-sFP-2PCR have bands, and the sizes of the bands are consistent. The results are shown in FIG. 6D;

(4) Protein expression assay of positive clones:

for positive clones verified by PCR, the expression of the target protein needs to be further verified. The target protein is provided with a His label, so that the expression condition of the target protein can be detected by using a His antibody through a Western blot method. The detailed method comprises the following steps:

1) Extracting thallus total protein:

a proper amount of bacterial liquid cultured until the OD600 is about 1OD is taken, and the thalli are collected centrifugally. Washing with PBS for 2 times, adding lysis buffer solution and appropriate amount of zirconium beads, shaking to break bacteria, centrifuging, and collecting supernatant to obtain thallus holoprotein.

2) Preparation of samples and electrophoresis

The protein content was measured with a microspectrophotometer A280, and the prepared electrophoretic sample was diluted to a final concentration of 4mg/ml. And (3) placing the diluted sample in a boiling water bath at 100 ℃ for 10min, placing the sample to room temperature, taking 10 mu l of sample, performing Tricine SDS-PAGE electrophoresis under the condition that 60V runs for 30min, and adjusting to 120V to run for 60min.

3) Electrophoresis gel block examination and transfer membrane

Examination and dyeing: after the electrophoresis is finished, the gel is removed, the gel is placed into Coomassie brilliant blue dye liquor for dyeing for 4 hours, and then the gel is eluted, and the result is observed.

Film transferring: soaking the membrane-transferring consumables (NC membrane and filter paper) in membrane-transferring buffer solution for at least 10min. And after the electrophoresis is finished, removing the rubber block, and rotating the membrane by using a membrane rotating instrument.

4) Western blot detection

And (3) sealing: and (3) carrying out temperature sealing on the NC membrane subjected to the membrane transfer for 1h by using a 5% skim milk sealing solution.

Incubating the primary antibody: after blocking, primary antibody was added and incubated overnight (16-18 h) at 2-8 ℃ and washed 6 times with TBST for 5min each.

The primary antibody against the target protein was Anti-6 XHis-McAb Mouse, diluted with blocking solution 1 at a ratio of 2000 for use.

The primary antibody of the internal reference protein is Anti-Ecoli RNA polymerase beta, is derived from mice, and is used after being diluted by a ratio of 1.

5) Incubation of secondary antibody: after incubation for 2h at 37 ℃ with the addition of secondary antibody, wash 6 times with TBST for 5min each time.

6) Developing and fixing: adding ECL chemiluminescence liquid into a darkroom, covering with an X-ray film, exposing for a proper time, taking out, developing in a developing solution, and immediately transferring to a fixing solution for fixing after the target strip is developed. The film is dried to observe the result.

The rBCG-pTB01s1-Va07 strain has normal total protein check bands, and the expression of target proteins (44 kDa) expressing Va07 can be seen by Western blot, while BCG and rBCG-pTB01 controls have no expression and are in line with the expectation. The results are shown in FIG. 7A:

the rBCG-pTB01s1-Va16 strain has normal total protein check bands, and the expression of target protein (13 kDa) of expressed Va16 can be seen by Western blot, while BCG and rBCG-pTB01 controls have no expression and are in line with the expectation. The results are shown in FIG. 7B:

the rBCG-pTB01s1-Va25 strain has normal total protein check bands, and the expression of target protein (34.3 kDa) of Va25 can be seen by Western blot, while BCG and rBCG-pTB01 controls have no expression and are in line with the expectation. The results are shown in FIG. 7C:

3. obtaining the recombinant BCG vaccine rBCG-pTB01s1-Va07-del

The recombinant BCG vaccine rBCG-pTB01s1-Va07, rBCG-pTB01s1-Va16 and rBCG-pTB01s1-Va25 obtained in the last step contain hygromycin B resistance genes. The pJH532 plasmid is electrically transformed into the competence of the recombinant BCG, and the competence is coated on a 7H10 flat plate containing a sucrose screening pressure, so that the recombinant BCG without hygromycin B resistance can be obtained, and the detailed method comprises the following steps:

(1) rBCG-pTB01s1-Va07, rBCG-pTB01s1-Va16 and rBCG-pTB01s1-Va25 competence were prepared, respectively.

3 recombinant BCG strains were inoculated into liquid medium containing HygB resistance 7H9+ OADC at a ratio of 1. For liquid culture, a T75 cell culture flask was used and 50ml of the medium was filled. The culture was allowed to stand at 37 ℃ until the OD600 value became about 0.8, which was about 3 to 4 weeks.

And (3) collecting thalli: 50ml of the bacterial solution is poured into a 50ml centrifuge tube, centrifuged for 20min at 4000rpm, and the supernatant is discarded.

Washing for 3 times: adding 50ml sterilized 10% glycerol, mixing, centrifuging at 4000rpm for 25min, and removing supernatant;

resuspending the cells: 5-10 ml of 10% glycerol is added to dissolve and precipitate, and 210ul of the solution is subpackaged into 6 tubes for storage.

(2) Electrotransformation pJH532 plasmid

The pJH532 plasmid containing a dissociation enzyme gene is transformed into the recombinant BCG integrated with the recombinant plasmid (rBCG-pTB 01s1-Va07, rBCG-pTB01s1-Va16 and rBCG-pTB01s1-Va 25), the sacB-HygB gene clamped between two dissociation enzyme sites is dissociated from the genome of the recombinant BCG under the action of a dissociation enzyme and is integrated into the pJH532 plasmid, and the original recombinant BCG genome is deleted for sacB-Hyg, so that two strains are generated, wherein one strain is rBCG-pJH 532-sacB-HygB containing an episomal plasmid pJH532-sacB, and the strain cannot grow under the sucrose screening pressure; one is recombinant BCG vaccine with lost sacB-HygB gene, and the strain can grow normally under the screening pressure of cane sugar.

Sample adding: adding the 5ul pJH532 plasmid into the recombinant BCG competent cells respectively, and incubating for 10min at 37 ℃;

electric conversion: transferring the mixture into an electric rotating cup, and according to the parameters: the voltage is 2.5kV, the resistance is 1000 omega, and the capacitance is 25 muF for electric conversion.

And (3) resuscitation: immediately after electric shock, 1ml of 7H9+ OADC medium was added, transferred to a 1.5ml centrifuge tube and incubated overnight at 37 ℃ and 100 rpm.

Culturing: centrifuging at 5000rpm for 5min, removing the culture medium, uniformly mixing the residual about 250ul of bacterial liquid, respectively coating 50ul and 200ul of the mixture in a 7H9+ OADC plate containing 2% sucrose, and standing and culturing at 37 ℃ for 3-4 weeks.

Through 2% sucrose 7H9+ OADC plate culture, the recombinant BCG vaccine without sacB-HygB gene is screened. Due to the loss of the sacB-HygB gene, the strain grew normally in a medium containing 2% sucrose and failed to grow in a medium containing hygromycin B. Thereby obtaining the following recombinant BCG vaccine:

antibiotic rBCG-pTB01s1-Va07-del

Anti-disease rBCG-pTB01s1-Va16-del

Anti-disease rBCG-pTB01s1-Va25-del

(3) Culture characteristic verification of recombinant BCG vaccine for eliminating antibiotics

The results were different when the disarmed rBCG was cultured on solid plates containing different resistance 7H10+ OADC.

Growing in a plate containing 2% sucrose 7H10 +OADC; does not grow in the plate containing HygB 7H10+OADC; no growth in the OADC plate containing Kana 7H10 +; growing in an OADC plate without resistance of 7H10 +; the results are shown in fig. 8 and table 1:

TABLE 1 Flat-plate screening table for antibiotic recombinant BCG vaccine

(4) Molecular characterization

The primers Hyg-F and Hyg-R are used, the sucrose-screened positive clone strain is used as a template, the HygB gene is amplified by PCR, the result is shown in figure 9, the result is in line with expectation, and the result shows that the sucrose-screened positive clone strain is in line with the expectation.

An upstream primer Hyg-F:5' AACATCTTTCGTGGACCTGGC-3

Downstream primer Hyg-R:5' GTGAAGCCGGAGATCCAG-doped 3

The PCR parameters were: 94 ℃/15sec,52 ℃/30sec,72 ℃/30sec,30 cycles.

The PCR product is long: 257bp.

Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not used to limit the protection scope of the present invention.

SEQUENCE LISTING

<110> Guangdong body Bi kang Biotech Co., ltd

<120> integration type expression plasmid combined with mycobacterium and application thereof

<160> 33

<170> PatentIn version 3.3

<210> 1

<211> 6934

<212> DNA

<213> Artificial sequence

<400> 1

ccaagaattg gcaggtcctg tatcctaaat caaatatcgg acaagcagtg tctgttataa 60

caaaaaatcg atttaataga cacaccaaca gcatggtttt tatgtgtgcg ataatttata 120

atatttcgga caggactcta gggtcgtcga ggtccacagg ccctcggcga cgttccgccg 180

ggcctcggcg accgccgcgt cgaggcgccg gtcgaagggg cagtcctcca cgggcagctc 240

gtggagggcg cgggccagct ccgccatcgc ctcgaccacg gcgaaccgct ggtgctcggg 300

ccactcctcg gccgccgcga cgccggggac ggcctccgtg acgagccacg cggcggtgtc 360

gtcggcaccg cgctcgacga cgcgggggac ggggatcggc ggggcctggg acggcgcctc 420

gccgtcgcag aaccaggcgg tgctttttaa cccatcacat atacctgccg ttcactatta 480

tttagtgaaa tgagatatta tgatattttc tgaattgtga ttaaaaaggc aactttatgc 540

ccatgcaaca gaaactataa aaaatacaga gaatgaaaag aaacagatag attttttagt 600

tctttaggcc cgtagtctgc aaatcctttt atgattttct atcaaacaaa agaggaaaat 660

agaccagttg caatccaaac gagagtctaa tagaatgagg tcgaaaagta aatcgcgcgg 720

gtttgttact gataaagcag gcaagaccta aaatgtgtaa agggcaaagt gtatactttg 780

gcgtcacccc ttacatattt taggtctttt tttattgtgc gtaactaact tgccatcttc 840

aaacaggagg gctggaagaa gcagaccgct aacacagtac ataaaaaagg agacatgaac 900

gatgaacatc aaaaagtttg caaaacaagc aacagtatta acctttacta ccgcactgct 960

ggcaggaggc gcaactcaag cgtttgcgaa agaaacgaac caaaagccat ataaggaaac 1020

atacggcatt tcccatatta cacgccatga tatgctgcaa atccctgaac agcaaaaaaa 1080

tgaaaaatat caagttcctg aattcgattc gtccacaatt aaaaatatct cttctgcaaa 1140

aggcctggac gtttgggaca gctggccatt acaaaacgct gacggcactg tcgcaaacta 1200

tcacggctac cacatcgtct ttgcattagc cggagatcct aaaaatgcgg atgacacatc 1260

gatttacatg ttctatcaaa aagtcggcga aacttctatt gacagctgga aaaacgctgg 1320

ccgcgtcttt aaagacagcg acaaattcga tgcaaatgat tctatcctaa aagaccaaac 1380

acaagaatgg tcaggttcag ccacatttac atctgacgga aaaatccgtt tattctacac 1440

tgatttctcc ggtaaacatt acggcaaaca aacactgaca actgcacaag ttaacgtatc 1500

agcatcagac agctctttga acatcaacgg tgtagaggat tataaatcaa tctttgacgg 1560

tgacggaaaa acgtatcaaa atgtacagca gttcatcgat gaaggcaact acagctcagg 1620

cgacaaccat acgctgagag atcctcacta cgtagaagat aaaggccaca aatacttagt 1680

atttgaagca aacactggaa ctgaagatgg ctaccaaggc gaagaatctt tatttaacaa 1740

agcatactat ggcaaaagca catcattctt ccgtcaagaa agtcaaaaac ttctgcaaag 1800

cgataaaaaa cgcacggctg agttagcaaa cggcgctctc ggtatgattg agctaaacga 1860

tgattacaca ctgaaaaaag tgatgaaacc gctgattgca tctaacacag taacagatga 1920

aattgaacgc gcgaacgtct ttaaaatgaa cggcaaatgg tacctgttca ctgactcccg 1980

cggatcaaaa atgacgattg acggcattac gtctaacgat atttacatgc ttggttatgt 2040

ttctaattct ttaactggcc catacaagcc gctgaacaaa actggccttg tgttaaaaat 2100

ggatcttgat cctaacgatg taacctttac ttactcacac ttcgctgtac ctcaagcgaa 2160

aggaaacaat gtcgtgatta caagctatat gacaaacaga ggattctacg cagacaaaca 2220

atcaacgttt gcgccaagct tcctgctgaa catcaaaggc aagaaaacat ctgttgtcaa 2280

agacagcatc cttgaacaag gacaattaac agttaacaaa taaaaacgca aaagaaaatg 2340

ccgatctcag gcggtggcgt acaccgtcgc ctcggtcggc ccgtagagat tggcgatccc 2400

gaccgcagca ccaccgagaa cgtccccgac gtggccgacc agcccgtcat cgtcaacgca 2460

tgatccgcgg tgcggacagg ccgtgtcgcg accggccgtg cggaattaag ccggcccgta 2520

ccctgtgaat agaggtccgc tgtgacacaa gaatccctgt tacttctcga ccgtattgat 2580

tcggatgatt cctacgcgag cctgcggaac gaccaggaat tctgggagcc gctggcccgc 2640

cgagccctgg aggagctcgg gctgccggtg ccgccggtgc tgcgggtgcc cggcgagagc 2700

accaaccccg tactggtcgg cgagcccggc ccggtcatca agctgttcgg cgagcactgg 2760

tgcggtccgg agagcctcgc gtcggagtcg gaggcgtacg cggtcctggc ggacgccccg 2820

gtgccggtgc cccgcctcct cggccgcggc gagctgcggc ccggcaccgg agcctggccg 2880

tggccctacc tggtgatgag ccggatgacc ggcaccacct ggcggtccgc gatggacggc 2940

acgaccgacc ggaacgcgct gctcgccctg gcccgcgaac tcggccgggt gctcggccgg 3000

ctgcacaggg tgccgctgac cgggaacacc gtgctcaccc cccattccga ggtcttcccg 3060

gaactgctgc gggaacgccg cgcggcgacc gtcgaggacc accgcgggtg gggctacctc 3120

tcgccccggc tgctggaccg cctggaggac tggctgccgg acgtggacac gctgctggcc 3180

ggccgcgaac cccggttcgt ccacggcgac ctgcacggga ccaacatctt cgtggacctg 3240

gccgcgaccg aggtcaccgg gatcgtcgac ttcaccgacg tctatgcggg agactcccgc 3300

tacagcctgg tgcaactgca tctcaacgcc ttccggggcg accgcgagat cctggccgcg 3360

ctgctcgacg gggcgcagtg gaagcggacc gaggacttcg cccgcgaact gctcgccttc 3420

accttcctgc acgacttcga ggtgttcgag gagaccccgc tggatctctc cggcttcacc 3480

gatccggagg aactggcgca gttcctctgg gggccgccgg acaccgcccc cggcgcctga 3540

cgccccggtc ctgtatccta aatcaaatat cggacaagca gtgtctgtta taacaaaaaa 3600

tcgatttaat agacacacca acagcatggt ttttatgtgt gcgataattt ataatatttc 3660

ggacaggact ctagccaaag attggacctc actagttcca tgagcgtcag accccgtaga 3720

aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 3780

aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 3840

tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc 3900

gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 3960

cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 4020

acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 4080

cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 4140

cgccacgctt cccgagggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 4200

aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 4260

gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 4320

atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 4380

tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga 4440

gtgagctgat accgctcgcc gcagccgaac gaccgagcgc aacgcgtgcg gccgcggtac 4500

ccggggatcc tctagagtcg accaccaagg gcaccatctc tgcttgggcc accccgttgg 4560

ccgcagccag ctcgctgaga gccgtgaacg acagggcgaa cgccagcccg ccgacggcga 4620

gggttccgac cgctgcaact cccggtgcaa ccttgtcccg gtctattctc ttcactgcac 4680

cagctccaat ctggtgtgaa tgcccctcgt ctgttcgcgc aggcgggggg ctctattcgt 4740

ttgtcagcat cgaaagtagc cagatcaggg atgcgttgca accgcgtatg cccaggtcag 4800

aagagtcgca caagagttgc agacccctgg aaagaaaaat ggccagaggg cgaaaacacc 4860

ctctgaccag cggagcgggc gacgggaatc gaacccgcgt agctagtttg gaagaatggg 4920

tgtctgccga ccacatatgg gccggtcaag ataggttttt accccctctc ggctgcatcc 4980

tctaagtgga aagaaattgc aggtcgtaga agcgcgttga agcctgagag ttgcacagga 5040

gttgcaaccc ggtagccttg ttcacgacga gaggagacct agttggcacg tcgcggatgg 5100

ggatcgctga agactcagcg cagcgggagg atccaagcct catacgtcaa cccgcaggac 5160

ggtgtgaggt actacgcgct gcagacctac gacaacaaga tggacgccga agcctggctc 5220

gcgggcgaga agcggctcat cgagatggag acctggaccc ctccacagga ccgggcgaag 5280

aaggcagccg ccagcgccat cacgctggag gagtacaccc ggaagtggct cgtggagcgc 5340

gacctcgcag acggcaccag ggatctgtac agcgggcacg cggagcgccg catctacccg 5400

gtgctaggtg aagtggcggt cacagagatg acgccagctc tggtgcgtgc gtggtgggcc 5460

gggatgggta ggaagcaccc gactgcccgc cggcatgcct acaacgtcct ccgggcggtg 5520

atgaacacag cggtcgagga caagctgatc gcagagaacc cgtgccggat cgagcagaag 5580

gcagccgatg agcgcgacgt agaggcgctg acgcctgagg agctggacat cgtcgccgct 5640

gagatcttcg agcactaccg gatcgcggca tacatcctgg cgtggacgag cctccggttc 5700

ggagagctga tcgagcttcg ccgcaaggac atcgtggacg acggcatgac gatgaagctc 5760

cgggtgcgcc gtggcgcttc ccgcgtgggg aacaagatcg tcgttggcaa cgccaagacc 5820

gtccggtcga agcgtcctgt gacggttccg cctcacgtcg cggagatgat ccgagcgcac 5880

atgaaggacc gtacgaagat gaacaagggc cccgaggcat tcctggtgac cacgacgcag 5940

ggcaaccggc tgtcgaagtc cgcgttcacc aagtcgctga agcgtggcta cgccaagatc 6000

ggtcggccgg aactccgcat ccacgacctc cgcgctgtcg gcgctacgtt cgccgctcag 6060

gcaggtgcga cgaccaagga gctgatggcc cgtctcggtc acacgactcc taggatggcg 6120

atgaagtacc agatggcgtc tgaggcccgc gacgaggcta tcgctgaggc gatgtccaag 6180

ctggccaaga cctcctgaaa cgcaaaaagc ccccctccca aggacactga gtcctaaaga 6240

ggggggtttc ttgtcagtac gcgaagaacc acgcctggcc gcgagcgcca gcaccgccgc 6300

tctgtgcgga gacctgggca ccagccccgc cgccgccagg agcattgccg ttcccgccag 6360

aaatctagac ggtgaccaca acgacgcgcc cgctttgatc ggggacgtct gcggccgacc 6420

atttacgggt cttgttgtcg ttggcggtca tgggccgaac atactcaccc ggatcggagg 6480

gccgaggaca aggtcgaacg aggggcatga cccggtgcgg ggcttcttgc actcggcata 6540

ggcgagtgct aagaataacg ttggcactcg cgaccggtga gtgctaggtc gggacggtga 6600

ggccaggccc gtcgtcgcag cgagtggcag cgaggacaac ttgagccgtc cgtcgcgggc 6660

actgcgcccg gccagcgtaa gtagcggggt tgccgtcacc cggtgacccc cgtttcatcc 6720

ccgatccgga ggaatcactt cgcaatggcc aagacaattg cggatccagc tgcagaattc 6780

gaagcttatc gatgtcgacg tagtcatcat catcatcatc accaccacta actagcgtac 6840

gatcgactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt 6900

ttatgccatc atggccgcgg tgatcagcta gcca 6934

<210> 2

<211> 8150

<212> DNA

<213> Artificial sequence

<400> 2

ccaagaattg gcaggtcctg tatcctaaat caaatatcgg acaagcagtg tctgttataa 60

caaaaaatcg atttaataga cacaccaaca gcatggtttt tatgtgtgcg ataatttata 120

atatttcgga caggactcta gggtcgtcga ggtccaccaa gccctcggcg acgttccgcc 180

gggcctcggc gaccgccgcg tcgaggcgcc ggtcggaggg gcagtcctcc acgggcagct 240

cgtggagggc gcgggccagc tccgccatcg cctcgaccac ggcgaaccgc tggtgctcgg 300

gccactcctc ggccgccgcg acgccgggga cggcctccgt gacgagccac gcggcggtgt 360

cgtcggcacc gcgctcgacg acgcggggga cggggatcgg cggggcctgg cggcgcctcg 420

ccgtcgcaga accaggcggt gctttttaac ccatcacata tacctgccgt tcactattat 480

ttagtgaaat gagatattat gatattttct gaattgtgat taaaaaggca actttatgcc 540

catgcaacag aaactataaa aaatacagag aatgaaaaga aacagataga ttttttagtt 600

ctttaggccc gtagtctgca aatcctttta tgattttcta tcaaacaaaa gaggaaaata 660

gaccagttgc aatccaaacg agagtctaat agaatgaggt cgaaaagtaa atcgcgcggg 720

tttgttactg ataaagcagg caagacctaa aatgtgtaaa gggcaaagtg tatactttgg 780

cgtcacccct tacatatttt aggtcttttt ttattgtgcg taactaactt gccatcttca 840

aacaggaggg ctggaagaag cagaccgcta acacagtaca taaaaaagga gacatgaacg 900

atgaacatca aaaagtttgc aaaacaagca acagtattaa cctttactac cgcactgctg 960

gcaggaggcg caactcaagc gtttgcgaaa gaaacgaacc aaaagccata taaggaaaca 1020

tacggcattt cccatattac acgccatgat atgctgcaaa tccctgaaca gcaaaaaaat 1080

gaaaaatatc aagttcctga attcgattcg tccacaatta aaaatatctc ttctgcaaaa 1140

ggcctggacg tttgggacag ctggccatta caaaacgctg acggcactgt cgcaaactat 1200

cacggctacc acatcgtctt tgcattagcc ggagatccta aaaatgcgga tgacacatcg 1260

atttacatgt tctatcaaaa agtcggcgaa acttctattg acagctggaa aaacgctggc 1320

cgcgtcttta aagacagcga caaattcgat gcaaatgatt ctatcctaaa agaccaaaca 1380

caagaatggt caggttcagc cacatttaca tctgacggaa aaatccgttt attctacact 1440

gatttctccg gtaaacatta cggcaaacaa acactgacaa ctgcacaagt taacgtatca 1500

gcatcagaca gctctttgaa catcaacggt gtagaggatt ataaatcaat ctttgacggt 1560

gacggaaaaa cgtatcaaaa tgtacagcag ttcatcgatg aaggcaacta cagctcaggc 1620

gacaaccata cgctgagaga tcctcactac gtagaagata aaggccacaa atacttagta 1680

tttgaagcaa acactggaac tgaagatggc taccaaggcg aagaatcttt atttaacaaa 1740

gcatactatg gcaaaagcac atcattcttc cgtcaagaaa gtcaaaaact tctgcaaagc 1800

gataaaaaac gcacggctga gttagcaaac ggcgctctcg gtatgattga gctaaacgat 1860

gattacacac tgaaaaaagt gatgaaaccg ctgattgcat ctaacacagt aacagatgaa 1920

attgaacgcg cgaacgtctt taaaatgaac ggcaaatggt acctgttcac tgactcccgc 1980

ggatcaaaaa tgacgattga cggcattacg tctaacgata tttacatgct tggttatgtt 2040

tctaattctt taactggccc atacaagccg ctgaacaaaa ctggccttgt gttaaaaatg 2100

gatcttgatc ctaacgatgt aacctttact tactcacact tcgctgtacc tcaagcgaaa 2160

ggaaacaatg tcgtgattac aagctatatg acaaacagag gattctacgc agacaaacaa 2220

tcaacgtttg cgccaagctt cctgctgaac atcaaaggca agaaaacatc tgttgtcaaa 2280

gacagcatcc ttgaacaagg acaattaaca gttaacaaat aaaaacgcaa aagaaaatgc 2340

cgatctcagg cggtggcgta caccgtcgcc tcggtcggcc cgtagagatt ggcgatcccg 2400

accgcagcac caccgagaac gtccccgacg tggccgacca gcccgtcatc gtcaacgcct 2460

gaccgcggtg cggacaggcc gtgtcgcgac cggccgtgcg gaattaagcc ggcccgtacc 2520

ctgtgaatag aggtccgctg tgacacaaga atccctgtta cttctcgacc gtattgattc 2580

ggatgattcc tacgcgagcc tgcggaacga ccaggaattc tgggagccgc tggcccgccg 2640

agccctggag gagctcgggc tgccggtgcc gccggtgctg cgggtgcccg gcgagagcac 2700

caaccccgta ctggtcggcg agcccgaccc ggtcatcaag ctgttcggcg agcactggtg 2760

cggtccggag agcctcgcgt cggagtcgga ggcgtacgcg gtcctggcgg acgccccggt 2820

gccggtgccc cgcctcctcg gccgcggcga gctgcggccc ggcaccggag cctggccgtg 2880

gccctacctg gtgatgagcc ggatgaccgg caccacctgg cggtccgcga tggacggcac 2940

gaccgaccgg aacgcgctgc tcgccctggc ccgcgaactc ggccgggtgc tcggccggct 3000

gcacagggtg ccgctgaccg ggaacaccgt gctcaccccc cattccgagg tcttcccgga 3060

actgctgcgg gaacgccgcg cggcgaccgt cgaggaccac cgcgggtggg gctacctctc 3120

gccccggctg ctggaccgcc tggaggactg gctgccggac gtggacacgc tgctggccgg 3180

ccgcgaaccc cggttcgtcc acggcgacct gcacgggacc aacatcttcg tggacctggc 3240

cgcgaccgag gtcaccggga tcgtcgactt caccgacgtc tatgcgggag actcccgcta 3300

cagcctggtg caactgcatc tcaacgcctt ccggggcgac cgcgagatcc tggccgcgct 3360

gctcgacggg gcgcagtgga agcggaccga ggacttcgcc cgcgaactgc tcgccttcac 3420

cttcctgcac gacttcgagg tgttcgagga gaccccgctg gatctctccg gcttcaccga 3480

tccggaggaa ctggcgcagt tcctctgggg gccgccggac accgcccccg gcgcctgacg 3540

ccccggtcct gtatcctaaa tcaaatatcg gacaagcagt gtctgttata acaaaaaatc 3600

gatttaatag acacaccaac agcatggttt ttatgtgtgc gataatttat aatatttcgg 3660

acaggactct agccaaagat tggacctcac tagttccatg agcgtcagac cccgtagaaa 3720

agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 3780

aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 3840

cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 3900

agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 3960

tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 4020

gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 4080

gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 4140

ccacgcttcc cgaggggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 4200

gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 4260

ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 4320

ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 4380

acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 4440

gagctgatac cgctcgccgc agccgaacga ccgagcgcaa cgcgtgcggc cgcggtaccc 4500

ggggatcctc tagagtcgac caccaagggc accatctctg cttgggccac cccgttggcc 4560

gcagccagct cgctgagagc cgtgaacgac agggcgaacg ccagcccgcc gacggcgagg 4620

gttccgaccg ctgcaactcc cggtgcaacc ttgtcccggt ctattctctt cactgcacca 4680

gctccaatct ggtgtgaatg cccctcgtct gttcgcgcag gcggggggct ctattcgttt 4740

gtcagcatcg aaagtagcca gatcagggat gcgttgcaac cgcgtatgcc caggtcagaa 4800

gagtcgcaca agagttgcag acccctggaa agaaaaatgg ccagagggcg aaaacaccct 4860

ctgaccagcg gagcgggcga cgggaatcga acccgcgtag ctagtttgga agaatgggtg 4920

tctgccgacc acatatgggc cggtcaagat aggtttttac cccctctcgg ctgcatcctc 4980

taagtggaaa gaaattgcag gtcgtagaag cgcgttgaag cctgagagtt gcacaggagt 5040

tgcaacccgg tagccttgtt cacgacgaga ggagacctag ttggcacgtc gcggatgggg 5100

atcgctgaag actcagcgca gcgggaggat ccaagcctca tacgtcaacc cgcaggacgg 5160

tgtgaggtac tacgcgctgc agacctacga caacaagatg gacgccgaag cctggctcgc 5220

gggcgagaag cggctcatcg agatggagac ctggacccct ccacaggacc gggcgaagaa 5280

ggcagccgcc agcgccatca cgctggagga gtacacccgg aagtggctcg tggagcgcga 5340

cctcgcagac ggcaccaggg atctgtacag cgggcacgcg gagcgccgca tctacccggt 5400

gctaggtgaa gtggcggtca cagagatgac gccagctctg gtgcgtgcgt ggtgggccgg 5460

gatgggtagg aagcacccga ctgcccgccg gcatgcctac aacgtcctcc gggcggtgat 5520

gaacacagcg gtcgaggaca agctgatcgc agagaacccg tgccggatcg agcagaaggc 5580

agccgatgag cgcgacgtag aggcgctgac gcctgaggag ctggacatcg tcgccgctga 5640

gatcttcgag cactaccgga tcgcggcata catcctggcg tggacgagcc tccggttcgg 5700

agagctgatc gagcttcgcc gcaaggacat cgtggacgac ggcatgacga tgaagctccg 5760

ggtgcgccgt ggcgcttccc gcgtggggaa caagatcgtc gttggcaacg ccaagaccgt 5820

ccggtcgaag cgtcctgtga cggttccgcc tcacgtcgcg gagatgatcc gagcgcacat 5880

gaaggaccgt acgaagatga acaagggccc cgaggcattc ctggtgacca cgacgcaggg 5940

caaccggctg tcgaagtccg cgttcaccaa gtcgctgaag cgtggctacg ccaagatcgg 6000

tcggccggaa ctccgcatcc acgacctccg cgctgtcggc gctacgttcg ccgctcaggc 6060

aggtgcgacg accaaggagc tgatggcccg tctcggtcac acgactccta ggatggcgat 6120

gaagtaccag atggcgtctg aggcccgcga cgaggctatc gctgaggcga tgtccaagct 6180

ggccaagacc tcctgaaacg caaaaagccc ccctcccaag gacactgagt cctaaagagg 6240

ggggtttctt gtcagtacgc gaagaaccac gcctggccgc gagcgccagc accgccgctc 6300

tgtgcggaga cctgggcacc agccccgccg ccgccaggag cattgccgtt cccgccagaa 6360

atctagacgg tgaccacaac gacgcgcccg ctttgatcgg ggacgtctgc ggccgaccat 6420

ttacgggtct tgttgtcgtt ggcggtcatg ggccgaacat actcacccgg atcggagggc 6480

cgaggacaag gtcgaacgag gggcatgacc cggtgcgggg cttcttgcac tcggcatagg 6540

cgagtgctaa gaataacgtt ggcactcgcg accggtgagt gctaggtcgg gacggtgagg 6600

ccaggcccgt cgtcgcagcg agtggcagcg aggacaactt gagccgtccg tcgcgggcac 6660

tgcgcccggc cagcgtaagt agcggggttg ccgtcacccg gtgacccccg tttcatcccc 6720

gatccggagg aatcacttcg caatgcatca tcatcatcat caccaccacg tgacggggca 6780

accggccgcg gctggcgcgc attcgctgtc ggaaggggca atgacggcga tgcagtcggg 6840

gtcggttccc ccgccgcagg ccactcctcc gataacgaca ccgcccgtgg tgtctgcgcc 6900

gaccatggct gcgggcatcg aagccacaca cgggccagtt gacacgccgg cgaacacctc 6960

gggcgctcca ccggcgtcga ccggtactac cgggccggtc gcgccgaccg tggtgaccgc 7020

cgggccggtg gcggcacccg ctgcgccggt ggttggtggc tcggctgtcc ccgcgggacc 7080

gctgccggct tacggctctg atctacggcc ccccgtcgtg gcagcccccg ccgtgccctc 7140

ggttcctacg gcgcccgtat ccggcgcgcc ggtggcgccc tcggcgtcat cggccccatc 7200

ggcgggtggg gcgctggttt ctccggtgga gcgcgcagcc tcgaaagctg tggctggaca 7260

ggctggtgcg agctcgtcga caatggccgg cgcctcggca ctgtcggcca ccgccggcgc 7320

gacggcgggc gcggtatcgg ctcgggcggc tgagcagcaa cgcctacagc gaatcgtgga 7380

tgccgtggcg cgccaggagc cgcgaatctc atgggcggcc gggctgcgcg acgacggcac 7440

caccaccctg ctggtcaccg atttggccgg cgggtggatt ccgccccacg tccggctgcc 7500

cgcgaacgtg acgctgctgg agccaaccgc gcgacgccgt gatgccgacg tgatcgactt 7560

gctgggcgcc gtcgtcgccg tggcagccca cgagtccaac acctacgttg ccgagccggg 7620

gccagatgcg cctgcgctga ccggtgatcg gtcagcgcgc tcggcgatac ccaaggtgga 7680

cgagttcggg ccgaccttgg tcgaagctgt gcgccgccgc gatagcctgc cgcggatcgc 7740

gcaggcgatc gcgctgccgg cggtacgcaa aaccggcgtg ctggaaaacg aagccgagct 7800

gctgcacggc tgcatcaccg cggtcaagga gtcggtgctc aaggcctatc ccagtcacga 7860

gctcaccgct gtcggggatt ggatgctgct ggcggcgatc gaggcactga tcgacgagca 7920

ggactacctc gccaactatc acctggcctg gtatgccgta accaccaggc gtggcggctc 7980

acgagggttc gctgccgaag cttatcgatg tcgacgtagt catcatcatc atcatcacca 8040

ccactaacta gcgtacgatc gactgccagg catcaaataa aacgaaaggc tcagtcgaaa 8100

gactgggcct ttcgttttat gccatcatgg ccgcggtgat cagctagcca 8150

<210> 3

<211> 1305

<212> DNA

<213> Artificial sequence

<400> 3

atgcatcatc atcatcatca ccaccacgtg acggggcaac cggccgcggc tggcgcgcat 60

tcgctgtcgg aaggggcaat gacggcgatg cagtcggggt cggttccccc gccgcaggcc 120

actcctccga taacgacacc gcccgtggtg tctgcgccga ccatggctgc gggcatcgaa 180

gccacacacg ggccagttga cacgccggcg aacacctcgg gcgctccacc ggcgtcgacc 240

ggtactaccg ggccggtcgc gccgaccgtg gtgaccgccg ggccggtggc ggcacccgct 300

gcgccggtgg ttggtggctc ggctgtcccc gcgggaccgc tgccggctta cggctctgat 360

ctacggcccc ccgtcgtggc agcccccgcc gtgccctcgg ttcctacggc gcccgtatcc 420

ggcgcgccgg tggcgccctc ggcgtcatcg gccccatcgg cgggtggggc gctggtttct 480

ccggtggagc gcgcagcctc gaaagctgtg gctggacagg ctggtgcgag ctcgtcgaca 540

atggccggcg cctcggcact gtcggccacc gccggcgcga cggcgggcgc ggtatcggct 600

cgggcggctg agcagcaacg cctacagcga atcgtggatg ccgtggcgcg ccaggagccg 660

cgaatctcat gggcggccgg gctgcgcgac gacggcacca ccaccctgct ggtcaccgat 720

ttggccggcg ggtggattcc gccccacgtc cggctgcccg cgaacgtgac gctgctggag 780

ccaaccgcgc gacgccgtga tgccgacgtg atcgacttgc tgggcgccgt cgtcgccgtg 840

gcagcccacg agtccaacac ctacgttgcc gagccggggc cagatgcgcc tgcgctgacc 900

ggtgatcggt cagcgcgctc ggcgataccc aaggtggacg agttcgggcc gaccttggtc 960

gaagctgtgc gccgccgcga tagcctgccg cggatcgcgc aggcgatcgc gctgccggcg 1020

gtacgcaaaa ccggcgtgct ggaaaacgaa gccgagctgc tgcacggctg catcaccgcg 1080

gtcaaggagt cggtgctcaa ggcctatccc agtcacgagc tcaccgctgt cggggattgg 1140

atgctgctgg cggcgatcga ggcactgatc gacgagcagg actacctcgc caactatcac 1200

ctggcctggt atgccgtaac caccaggcgt ggcggctcac gagggttcgc tgccgaagct 1260

tatcgatgtc gacgtagtca tcatcatcat catcaccacc actaa 1305

<210> 4

<211> 434

<212> PRT

<213> Artificial sequence

<400> 4

Met His His His His His His His His Val Thr Gly Gln Pro Ala Ala

1 5 10 15

Ala Gly Ala His Ser Leu Ser Glu Gly Ala Met Thr Ala Met Gln Ser

20 25 30

Gly Ser Val Pro Pro Pro Gln Ala Thr Pro Pro Ile Thr Thr Pro Pro

35 40 45

Val Val Ser Ala Pro Thr Met Ala Ala Gly Ile Glu Ala Thr His Gly

50 55 60

Pro Val Asp Thr Pro Ala Asn Thr Ser Gly Ala Pro Pro Ala Ser Thr

65 70 75 80

Gly Thr Thr Gly Pro Val Ala Pro Thr Val Val Thr Ala Gly Pro Val

85 90 95

Ala Ala Pro Ala Ala Pro Val Val Gly Gly Ser Ala Val Pro Ala Gly

100 105 110

Pro Leu Pro Ala Tyr Gly Ser Asp Leu Arg Pro Pro Val Val Ala Ala

115 120 125

Pro Ala Val Pro Ser Val Pro Thr Ala Pro Val Ser Gly Ala Pro Val

130 135 140

Ala Pro Ser Ala Ser Ser Ala Pro Ser Ala Gly Gly Ala Leu Val Ser

145 150 155 160

Pro Val Glu Arg Ala Ala Ser Lys Ala Val Ala Gly Gln Ala Gly Ala

165 170 175

Ser Ser Ser Thr Met Ala Gly Ala Ser Ala Leu Ser Ala Thr Ala Gly

180 185 190

Ala Thr Ala Gly Ala Val Ser Ala Arg Ala Ala Glu Gln Gln Arg Leu

195 200 205

Gln Arg Ile Val Asp Ala Val Ala Arg Gln Glu Pro Arg Ile Ser Trp

210 215 220

Ala Ala Gly Leu Arg Asp Asp Gly Thr Thr Thr Leu Leu Val Thr Asp

225 230 235 240

Leu Ala Gly Gly Trp Ile Pro Pro His Val Arg Leu Pro Ala Asn Val

245 250 255

Thr Leu Leu Glu Pro Thr Ala Arg Arg Arg Asp Ala Asp Val Ile Asp

260 265 270

Leu Leu Gly Ala Val Val Ala Val Ala Ala His Glu Ser Asn Thr Tyr

275 280 285

Val Ala Glu Pro Gly Pro Asp Ala Pro Ala Leu Thr Gly Asp Arg Ser

290 295 300

Ala Arg Ser Ala Ile Pro Lys Val Asp Glu Phe Gly Pro Thr Leu Val

305 310 315 320

Glu Ala Val Arg Arg Arg Asp Ser Leu Pro Arg Ile Ala Gln Ala Ile

325 330 335

Ala Leu Pro Ala Val Arg Lys Thr Gly Val Leu Glu Asn Glu Ala Glu

340 345 350

Leu Leu His Gly Cys Ile Thr Ala Val Lys Glu Ser Val Leu Lys Ala

355 360 365

Tyr Pro Ser His Glu Leu Thr Ala Val Gly Asp Trp Met Leu Leu Ala

370 375 380

Ala Ile Glu Ala Leu Ile Asp Glu Gln Asp Tyr Leu Ala Asn Tyr His

385 390 395 400

Leu Ala Trp Tyr Ala Val Thr Thr Arg Arg Gly Gly Ser Arg Gly Phe

405 410 415

Ala Ala Glu Ala Tyr Arg Cys Arg Arg Ser His His His His His His

420 425 430

His His

<210> 5

<211> 7211

<212> DNA

<213> Artificial sequence

<400> 5

ccaagaattg gcaggtcctg tatcctaaat caaatatcgg acaagcagtg tctgttataa 60

caaaaaatcg atttaataga cacaccaaca gcatggtttt tatgtgtgcg ataatttata 120

atatttcgga caggactcta gggtcgtcga ggtccaccaa gccctcggcg acgttccgcc 180

gggcctcggc gaccgccgcg tcgaggcgcc ggtcggaggg gcagtcctcc acgggcagct 240

cgtggagggc gcgggccagc tccgccatcg cctcgaccac ggcgaaccgc tggtgctcgg 300

gccactcctc ggccgccgcg acgccgggga cggcctccgt gacgagccac gcggcggtgt 360

cgtcggcacc gcgctcgacg acgcggggga cggggatcgg cggggcctgg cggcgcctcg 420

ccgtcgcaga accaggcggt gctttttaac ccatcacata tacctgccgt tcactattat 480

ttagtgaaat gagatattat gatattttct gaattgtgat taaaaaggca actttatgcc 540

catgcaacag aaactataaa aaatacagag aatgaaaaga aacagataga ttttttagtt 600

ctttaggccc gtagtctgca aatcctttta tgattttcta tcaaacaaaa gaggaaaata 660

gaccagttgc aatccaaacg agagtctaat agaatgaggt cgaaaagtaa atcgcgcggg 720

tttgttactg ataaagcagg caagacctaa aatgtgtaaa gggcaaagtg tatactttgg 780

cgtcacccct tacatatttt aggtcttttt ttattgtgcg taactaactt gccatcttca 840

aacaggaggg ctggaagaag cagaccgcta acacagtaca taaaaaagga gacatgaacg 900

atgaacatca aaaagtttgc aaaacaagca acagtattaa cctttactac cgcactgctg 960

gcaggaggcg caactcaagc gtttgcgaaa gaaacgaacc aaaagccata taaggaaaca 1020

tacggcattt cccatattac acgccatgat atgctgcaaa tccctgaaca gcaaaaaaat 1080

gaaaaatatc aagttcctga attcgattcg tccacaatta aaaatatctc ttctgcaaaa 1140

ggcctggacg tttgggacag ctggccatta caaaacgctg acggcactgt cgcaaactat 1200

cacggctacc acatcgtctt tgcattagcc ggagatccta aaaatgcgga tgacacatcg 1260

atttacatgt tctatcaaaa agtcggcgaa acttctattg acagctggaa aaacgctggc 1320

cgcgtcttta aagacagcga caaattcgat gcaaatgatt ctatcctaaa agaccaaaca 1380

caagaatggt caggttcagc cacatttaca tctgacggaa aaatccgttt attctacact 1440

gatttctccg gtaaacatta cggcaaacaa acactgacaa ctgcacaagt taacgtatca 1500

gcatcagaca gctctttgaa catcaacggt gtagaggatt ataaatcaat ctttgacggt 1560

gacggaaaaa cgtatcaaaa tgtacagcag ttcatcgatg aaggcaacta cagctcaggc 1620

gacaaccata cgctgagaga tcctcactac gtagaagata aaggccacaa atacttagta 1680

tttgaagcaa acactggaac tgaagatggc taccaaggcg aagaatcttt atttaacaaa 1740

gcatactatg gcaaaagcac atcattcttc cgtcaagaaa gtcaaaaact tctgcaaagc 1800

gataaaaaac gcacggctga gttagcaaac ggcgctctcg gtatgattga gctaaacgat 1860

gattacacac tgaaaaaagt gatgaaaccg ctgattgcat ctaacacagt aacagatgaa 1920

attgaacgcg cgaacgtctt taaaatgaac ggcaaatggt acctgttcac tgactcccgc 1980

ggatcaaaaa tgacgattga cggcattacg tctaacgata tttacatgct tggttatgtt 2040

tctaattctt taactggccc atacaagccg ctgaacaaaa ctggccttgt gttaaaaatg 2100

gatcttgatc ctaacgatgt aacctttact tactcacact tcgctgtacc tcaagcgaaa 2160

ggaaacaatg tcgtgattac aagctatatg acaaacagag gattctacgc agacaaacaa 2220

tcaacgtttg cgccaagctt cctgctgaac atcaaaggca agaaaacatc tgttgtcaaa 2280

gacagcatcc ttgaacaagg acaattaaca gttaacaaat aaaaacgcaa aagaaaatgc 2340

cgatctcagg cggtggcgta caccgtcgcc tcggtcggcc cgtagagatt ggcgatcccg 2400

accgcagcac caccgagaac gtccccgacg tggccgacca gcccgtcatc gtcaacgcct 2460

gaccgcggtg cggacaggcc gtgtcgcgac cggccgtgcg gaattaagcc ggcccgtacc 2520

ctgtgaatag aggtccgctg tgacacaaga atccctgtta cttctcgacc gtattgattc 2580

ggatgattcc tacgcgagcc tgcggaacga ccaggaattc tgggagccgc tggcccgccg 2640

agccctggag gagctcgggc tgccggtgcc gccggtgctg cgggtgcccg gcgagagcac 2700

caaccccgta ctggtcggcg agcccgaccc ggtcatcaag ctgttcggcg agcactggtg 2760

cggtccggag agcctcgcgt cggagtcgga ggcgtacgcg gtcctggcgg acgccccggt 2820

gccggtgccc cgcctcctcg gccgcggcga gctgcggccc ggcaccggag cctggccgtg 2880

gccctacctg gtgatgagcc ggatgaccgg caccacctgg cggtccgcga tggacggcac 2940

gaccgaccgg aacgcgctgc tcgccctggc ccgcgaactc ggccgggtgc tcggccggct 3000

gcacagggtg ccgctgaccg ggaacaccgt gctcaccccc cattccgagg tcttcccgga 3060

actgctgcgg gaacgccgcg cggcgaccgt cgaggaccac cgcgggtggg gctacctctc 3120

gccccggctg ctggaccgcc tggaggactg gctgccggac gtggacacgc tgctggccgg 3180

ccgcgaaccc cggttcgtcc acggcgacct gcacgggacc aacatcttcg tggacctggc 3240

cgcgaccgag gtcaccggga tcgtcgactt caccgacgtc tatgcgggag actcccgcta 3300

cagcctggtg caactgcatc tcaacgcctt ccggggcgac cgcgagatcc tggccgcgct 3360

gctcgacggg gcgcagtgga agcggaccga ggacttcgcc cgcgaactgc tcgccttcac 3420

cttcctgcac gacttcgagg tgttcgagga gaccccgctg gatctctccg gcttcaccga 3480

tccggaggaa ctggcgcagt tcctctgggg gccgccggac accgcccccg gcgcctgacg 3540

ccccggtcct gtatcctaaa tcaaatatcg gacaagcagt gtctgttata acaaaaaatc 3600

gatttaatag acacaccaac agcatggttt ttatgtgtgc gataatttat aatatttcgg 3660

acaggactct agccaaagat tggacctcac tagttccatg agcgtcagac cccgtagaaa 3720

agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 3780

aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 3840

cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 3900

agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 3960

tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 4020

gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 4080

gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 4140

ccacgcttcc cgaggggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 4200

gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 4260

ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 4320

ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 4380

acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 4440

gagctgatac cgctcgccgc agccgaacga ccgagcgcaa cgcgtgcggc cgcggtaccc 4500

ggggatcctc tagagtcgac caccaagggc accatctctg cttgggccac cccgttggcc 4560

gcagccagct cgctgagagc cgtgaacgac agggcgaacg ccagcccgcc gacggcgagg 4620

gttccgaccg ctgcaactcc cggtgcaacc ttgtcccggt ctattctctt cactgcacca 4680

gctccaatct ggtgtgaatg cccctcgtct gttcgcgcag gcggggggct ctattcgttt 4740

gtcagcatcg aaagtagcca gatcagggat gcgttgcaac cgcgtatgcc caggtcagaa 4800

gagtcgcaca agagttgcag acccctggaa agaaaaatgg ccagagggcg aaaacaccct 4860

ctgaccagcg gagcgggcga cgggaatcga acccgcgtag ctagtttgga agaatgggtg 4920

tctgccgacc acatatgggc cggtcaagat aggtttttac cccctctcgg ctgcatcctc 4980

taagtggaaa gaaattgcag gtcgtagaag cgcgttgaag cctgagagtt gcacaggagt 5040

tgcaacccgg tagccttgtt cacgacgaga ggagacctag ttggcacgtc gcggatgggg 5100

atcgctgaag actcagcgca gcgggaggat ccaagcctca tacgtcaacc cgcaggacgg 5160

tgtgaggtac tacgcgctgc agacctacga caacaagatg gacgccgaag cctggctcgc 5220

gggcgagaag cggctcatcg agatggagac ctggacccct ccacaggacc gggcgaagaa 5280

ggcagccgcc agcgccatca cgctggagga gtacacccgg aagtggctcg tggagcgcga 5340

cctcgcagac ggcaccaggg atctgtacag cgggcacgcg gagcgccgca tctacccggt 5400

gctaggtgaa gtggcggtca cagagatgac gccagctctg gtgcgtgcgt ggtgggccgg 5460

gatgggtagg aagcacccga ctgcccgccg gcatgcctac aacgtcctcc gggcggtgat 5520

gaacacagcg gtcgaggaca agctgatcgc agagaacccg tgccggatcg agcagaaggc 5580

agccgatgag cgcgacgtag aggcgctgac gcctgaggag ctggacatcg tcgccgctga 5640

gatcttcgag cactaccgga tcgcggcata catcctggcg tggacgagcc tccggttcgg 5700

agagctgatc gagcttcgcc gcaaggacat cgtggacgac ggcatgacga tgaagctccg 5760

ggtgcgccgt ggcgcttccc gcgtggggaa caagatcgtc gttggcaacg ccaagaccgt 5820

ccggtcgaag cgtcctgtga cggttccgcc tcacgtcgcg gagatgatcc gagcgcacat 5880

gaaggaccgt acgaagatga acaagggccc cgaggcattc ctggtgacca cgacgcaggg 5940

caaccggctg tcgaagtccg cgttcaccaa gtcgctgaag cgtggctacg ccaagatcgg 6000

tcggccggaa ctccgcatcc acgacctccg cgctgtcggc gctacgttcg ccgctcaggc 6060

aggtgcgacg accaaggagc tgatggcccg tctcggtcac acgactccta ggatggcgat 6120

gaagtaccag atggcgtctg aggcccgcga cgaggctatc gctgaggcga tgtccaagct 6180

ggccaagacc tcctgaaacg caaaaagccc ccctcccaag gacactgagt cctaaagagg 6240

ggggtttctt gtcagtacgc gaagaaccac gcctggccgc gagcgccagc accgccgctc 6300

tgtgcggaga cctgggcacc agccccgccg ccgccaggag cattgccgtt cccgccagaa 6360

atctagacgg tgaccacaac gacgcgcccg ctttgatcgg ggacgtctgc ggccgaccat 6420

ttacgggtct tgttgtcgtt ggcggtcatg ggccgaacat actcacccgg atcggagggc 6480

cgaggacaag gtcgaacgag gggcatgacc cggtgcgggg cttcttgcac tcggcatagg 6540

cgagtgctaa gaataacgtt ggcactcgcg accggtgagt gctaggtcgg gacggtgagg 6600

ccaggcccgt cgtcgcagcg agtggcagcg aggacaactt gagccgtccg tcgcgggcac 6660

tgcgcccggc cagcgtaagt agcggggttg ccgtcacccg gtgacccccg tttcatcccc 6720

gatccggagg aatcacttcg caatgcatca tcatcatcat caccaccaca gccttttgga 6780

tgctcatatc ccacagttgg tggcctccca gtcggcgttt gccgccaagg cggggctgat 6840

gcggcacacg atcggtcagg ccgagcaggc ggcgatgtcg gctcaggcgt ttcaccaggg 6900

ggagtcgtcg gcggcgtttc aggccgccca tgcccggttt gtggcggcgg ccgccaaagt 6960

caacaccttg ttggatgtcg cgcaggcgaa tctgggtgag gccgccggta cctatgtggc 7020

cgccgatgct gcggccgcgt cgacctatac cgggttcgaa gcttatcgat gtcgacgtag 7080

tcatcatcat catcatcacc accactaact agcgtacgat cgactgccag gcatcaaata 7140

aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tgccatcatg gccgcggtga 7200

tcagctagcc a 7211

<210> 6

<211> 366

<212> DNA

<213> Artificial sequence

<400> 6

atgcatcatc atcatcatca ccaccacagc cttttggatg ctcatatccc acagttggtg 60

gcctcccagt cggcgtttgc cgccaaggcg gggctgatgc ggcacacgat cggtcaggcc 120

gagcaggcgg cgatgtcggc tcaggcgttt caccaggggg agtcgtcggc ggcgtttcag 180

gccgcccatg cccggtttgt ggcggcggcc gccaaagtca acaccttgtt ggatgtcgcg 240

caggcgaatc tgggtgaggc cgccggtacc tatgtggccg ccgatgctgc ggccgcgtcg 300

acctataccg ggttcgaagc ttatcgatgt cgacgtagtc atcatcatca tcatcaccac 360

cactaa 366

<210> 7

<211> 121

<212> PRT

<213> Artificial sequence

<400> 7

Met His His His His His His His His Ser Leu Leu Asp Ala His Ile

1 5 10 15

Pro Gln Leu Val Ala Ser Gln Ser Ala Phe Ala Ala Lys Ala Gly Leu

20 25 30

Met Arg His Thr Ile Gly Gln Ala Glu Gln Ala Ala Met Ser Ala Gln

35 40 45

Ala Phe His Gln Gly Glu Ser Ser Ala Ala Phe Gln Ala Ala His Ala

50 55 60

Arg Phe Val Ala Ala Ala Ala Lys Val Asn Thr Leu Leu Asp Val Ala

65 70 75 80

Gln Ala Asn Leu Gly Glu Ala Ala Gly Thr Tyr Val Ala Ala Asp Ala

85 90 95

Ala Ala Ala Ser Thr Tyr Thr Gly Phe Glu Ala Tyr Arg Cys Arg Arg

100 105 110

Ser His His His His His His His His

115 120

<210> 8

<211> 7817

<212> DNA

<213> Artificial sequence

<400> 8

ccaagaattg gcaggtcctg tatcctaaat caaatatcgg acaagcagtg tctgttataa 60

caaaaaatcg atttaataga cacaccaaca gcatggtttt tatgtgtgcg ataatttata 120

atatttcgga caggactcta gggtcgtcga ggtccaccaa gccctcggcg acgttccgcc 180

gggcctcggc gaccgccgcg tcgaggcgcc ggtcggaggg gcagtcctcc acgggcagct 240

cgtggagggc gcgggccagc tccgccatcg cctcgaccac ggcgaaccgc tggtgctcgg 300

gccactcctc ggccgccgcg acgccgggga cggcctccgt gacgagccac gcggcggtgt 360

cgtcggcacc gcgctcgacg acgcggggga cggggatcgg cggggcctgg cggcgcctcg 420

ccgtcgcaga accaggcggt gctttttaac ccatcacata tacctgccgt tcactattat 480

ttagtgaaat gagatattat gatattttct gaattgtgat taaaaaggca actttatgcc 540

catgcaacag aaactataaa aaatacagag aatgaaaaga aacagataga ttttttagtt 600

ctttaggccc gtagtctgca aatcctttta tgattttcta tcaaacaaaa gaggaaaata 660

gaccagttgc aatccaaacg agagtctaat agaatgaggt cgaaaagtaa atcgcgcggg 720

tttgttactg ataaagcagg caagacctaa aatgtgtaaa gggcaaagtg tatactttgg 780

cgtcacccct tacatatttt aggtcttttt ttattgtgcg taactaactt gccatcttca 840

aacaggaggg ctggaagaag cagaccgcta acacagtaca taaaaaagga gacatgaacg 900

atgaacatca aaaagtttgc aaaacaagca acagtattaa cctttactac cgcactgctg 960

gcaggaggcg caactcaagc gtttgcgaaa gaaacgaacc aaaagccata taaggaaaca 1020

tacggcattt cccatattac acgccatgat atgctgcaaa tccctgaaca gcaaaaaaat 1080

gaaaaatatc aagttcctga attcgattcg tccacaatta aaaatatctc ttctgcaaaa 1140

ggcctggacg tttgggacag ctggccatta caaaacgctg acggcactgt cgcaaactat 1200

cacggctacc acatcgtctt tgcattagcc ggagatccta aaaatgcgga tgacacatcg 1260

atttacatgt tctatcaaaa agtcggcgaa acttctattg acagctggaa aaacgctggc 1320

cgcgtcttta aagacagcga caaattcgat gcaaatgatt ctatcctaaa agaccaaaca 1380

caagaatggt caggttcagc cacatttaca tctgacggaa aaatccgttt attctacact 1440

gatttctccg gtaaacatta cggcaaacaa acactgacaa ctgcacaagt taacgtatca 1500

gcatcagaca gctctttgaa catcaacggt gtagaggatt ataaatcaat ctttgacggt 1560

gacggaaaaa cgtatcaaaa tgtacagcag ttcatcgatg aaggcaacta cagctcaggc 1620

gacaaccata cgctgagaga tcctcactac gtagaagata aaggccacaa atacttagta 1680

tttgaagcaa acactggaac tgaagatggc taccaaggcg aagaatcttt atttaacaaa 1740

gcatactatg gcaaaagcac atcattcttc cgtcaagaaa gtcaaaaact tctgcaaagc 1800

gataaaaaac gcacggctga gttagcaaac ggcgctctcg gtatgattga gctaaacgat 1860

gattacacac tgaaaaaagt gatgaaaccg ctgattgcat ctaacacagt aacagatgaa 1920

attgaacgcg cgaacgtctt taaaatgaac ggcaaatggt acctgttcac tgactcccgc 1980

ggatcaaaaa tgacgattga cggcattacg tctaacgata tttacatgct tggttatgtt 2040

tctaattctt taactggccc atacaagccg ctgaacaaaa ctggccttgt gttaaaaatg 2100

gatcttgatc ctaacgatgt aacctttact tactcacact tcgctgtacc tcaagcgaaa 2160

ggaaacaatg tcgtgattac aagctatatg acaaacagag gattctacgc agacaaacaa 2220

tcaacgtttg cgccaagctt cctgctgaac atcaaaggca agaaaacatc tgttgtcaaa 2280

gacagcatcc ttgaacaagg acaattaaca gttaacaaat aaaaacgcaa aagaaaatgc 2340

cgatctcagg cggtggcgta caccgtcgcc tcggtcggcc cgtagagatt ggcgatcccg 2400

accgcagcac caccgagaac gtccccgacg tggccgacca gcccgtcatc gtcaacgcct 2460

gaccgcggtg cggacaggcc gtgtcgcgac cggccgtgcg gaattaagcc ggcccgtacc 2520

ctgtgaatag aggtccgctg tgacacaaga atccctgtta cttctcgacc gtattgattc 2580

ggatgattcc tacgcgagcc tgcggaacga ccaggaattc tgggagccgc tggcccgccg 2640

agccctggag gagctcgggc tgccggtgcc gccggtgctg cgggtgcccg gcgagagcac 2700

caaccccgta ctggtcggcg agcccgaccc ggtcatcaag ctgttcggcg agcactggtg 2760

cggtccggag agcctcgcgt cggagtcgga ggcgtacgcg gtcctggcgg acgccccggt 2820

gccggtgccc cgcctcctcg gccgcggcga gctgcggccc ggcaccggag cctggccgtg 2880

gccctacctg gtgatgagcc ggatgaccgg caccacctgg cggtccgcga tggacggcac 2940

gaccgaccgg aacgcgctgc tcgccctggc ccgcgaactc ggccgggtgc tcggccggct 3000

gcacagggtg ccgctgaccg ggaacaccgt gctcaccccc cattccgagg tcttcccgga 3060

actgctgcgg gaacgccgcg cggcgaccgt cgaggaccac cgcgggtggg gctacctctc 3120

gccccggctg ctggaccgcc tggaggactg gctgccggac gtggacacgc tgctggccgg 3180

ccgcgaaccc cggttcgtcc acggcgacct gcacgggacc aacatcttcg tggacctggc 3240

cgcgaccgag gtcaccggga tcgtcgactt caccgacgtc tatgcgggag actcccgcta 3300

cagcctggtg caactgcatc tcaacgcctt ccggggcgac cgcgagatcc tggccgcgct 3360

gctcgacggg gcgcagtgga agcggaccga ggacttcgcc cgcgaactgc tcgccttcac 3420

cttcctgcac gacttcgagg tgttcgagga gaccccgctg gatctctccg gcttcaccga 3480

tccggaggaa ctggcgcagt tcctctgggg gccgccggac accgcccccg gcgcctgacg 3540

ccccggtcct gtatcctaaa tcaaatatcg gacaagcagt gtctgttata acaaaaaatc 3600

gatttaatag acacaccaac agcatggttt ttatgtgtgc gataatttat aatatttcgg 3660

acaggactct agccaaagat tggacctcac tagttccatg agcgtcagac cccgtagaaa 3720

agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 3780

aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 3840

cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 3900

agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 3960

tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 4020

gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 4080

gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 4140

ccacgcttcc cgaggggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 4200

gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 4260

ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 4320

ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 4380

acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 4440

gagctgatac cgctcgccgc agccgaacga ccgagcgcaa cgcgtgcggc cgcggtaccc 4500

ggggatcctc tagagtcgac caccaagggc accatctctg cttgggccac cccgttggcc 4560

gcagccagct cgctgagagc cgtgaacgac agggcgaacg ccagcccgcc gacggcgagg 4620

gttccgaccg ctgcaactcc cggtgcaacc ttgtcccggt ctattctctt cactgcacca 4680

gctccaatct ggtgtgaatg cccctcgtct gttcgcgcag gcggggggct ctattcgttt 4740

gtcagcatcg aaagtagcca gatcagggat gcgttgcaac cgcgtatgcc caggtcagaa 4800

gagtcgcaca agagttgcag acccctggaa agaaaaatgg ccagagggcg aaaacaccct 4860

ctgaccagcg gagcgggcga cgggaatcga acccgcgtag ctagtttgga agaatgggtg 4920

tctgccgacc acatatgggc cggtcaagat aggtttttac cccctctcgg ctgcatcctc 4980

taagtggaaa gaaattgcag gtcgtagaag cgcgttgaag cctgagagtt gcacaggagt 5040

tgcaacccgg tagccttgtt cacgacgaga ggagacctag ttggcacgtc gcggatgggg 5100

atcgctgaag actcagcgca gcgggaggat ccaagcctca tacgtcaacc cgcaggacgg 5160

tgtgaggtac tacgcgctgc agacctacga caacaagatg gacgccgaag cctggctcgc 5220

gggcgagaag cggctcatcg agatggagac ctggacccct ccacaggacc gggcgaagaa 5280

ggcagccgcc agcgccatca cgctggagga gtacacccgg aagtggctcg tggagcgcga 5340

cctcgcagac ggcaccaggg atctgtacag cgggcacgcg gagcgccgca tctacccggt 5400

gctaggtgaa gtggcggtca cagagatgac gccagctctg gtgcgtgcgt ggtgggccgg 5460

gatgggtagg aagcacccga ctgcccgccg gcatgcctac aacgtcctcc gggcggtgat 5520

gaacacagcg gtcgaggaca agctgatcgc agagaacccg tgccggatcg agcagaaggc 5580

agccgatgag cgcgacgtag aggcgctgac gcctgaggag ctggacatcg tcgccgctga 5640

gatcttcgag cactaccgga tcgcggcata catcctggcg tggacgagcc tccggttcgg 5700

agagctgatc gagcttcgcc gcaaggacat cgtggacgac ggcatgacga tgaagctccg 5760

ggtgcgccgt ggcgcttccc gcgtggggaa caagatcgtc gttggcaacg ccaagaccgt 5820

ccggtcgaag cgtcctgtga cggttccgcc tcacgtcgcg gagatgatcc gagcgcacat 5880

gaaggaccgt acgaagatga acaagggccc cgaggcattc ctggtgacca cgacgcaggg 5940

caaccggctg tcgaagtccg cgttcaccaa gtcgctgaag cgtggctacg ccaagatcgg 6000

tcggccggaa ctccgcatcc acgacctccg cgctgtcggc gctacgttcg ccgctcaggc 6060

aggtgcgacg accaaggagc tgatggcccg tctcggtcac acgactccta ggatggcgat 6120

gaagtaccag atggcgtctg aggcccgcga cgaggctatc gctgaggcga tgtccaagct 6180

ggccaagacc tcctgaaacg caaaaagccc ccctcccaag gacactgagt cctaaagagg 6240

ggggtttctt gtcagtacgc gaagaaccac gcctggccgc gagcgccagc accgccgctc 6300

tgtgcggaga cctgggcacc agccccgccg ccgccaggag cattgccgtt cccgccagaa 6360

atctagacgg tgaccacaac gacgcgcccg ctttgatcgg ggacgtctgc ggccgaccat 6420

ttacgggtct tgttgtcgtt ggcggtcatg ggccgaacat actcacccgg atcggagggc 6480

cgaggacaag gtcgaacgag gggcatgacc cggtgcgggg cttcttgcac tcggcatagg 6540

cgagtgctaa gaataacgtt ggcactcgcg accggtgagt gctaggtcgg gacggtgagg 6600

ccaggcccgt cgtcgcagcg agtggcagcg aggacaactt gagccgtccg tcgcgggcac 6660

tgcgcccggc cagcgtaagt agcggggttg ccgtcacccg gtgacccccg tttcatcccc 6720

gatccggagg aatcacttcg caatgcatca tcatcatcat caccaccaca agggtcggtc 6780

ggcgctgctg cgggcgctct ggattgccgc actgtcattc gggttgggcg gtgtcgcggt 6840

agccgcggaa cccaccgcca aggccgcccc atacgagaac ctgatggtgc cgtcgccctc 6900

gatgggccgg gacatcccgg tggccttcct agccggtggg ccgcacgcgg tgtatctgct 6960

ggacgccttc aacgccggcc cggatgtcag taactgggtc accgcgggta acgcgatgaa 7020

cacgttggcg ggcaagggga tttcggtggt ggcaccggcc ggtggtgcgt acagcatgta 7080

caccaactgg gagcaggatg gcagcaagca gtgggacacc ttcttgtccg ctgagctgcc 7140

cgactggctg gccgctaacc ggggcttggc ccccggtggc catgcggccg ttggcgccgc 7200

tcagggcggt tacggggcga tggcgctggc ggccttccac cccgaccgct tcggcttcgc 7260

tggctcgatg tcgggctttt tgtacccgtc gaacaccacc accaacggtg cgatcgcggc 7320

gggcatgcag caattcggcg gtgtggacac caacggaatg tggggagcac cacagctggg 7380

tcggtggaag tggcacgacc cgtgggtgca tgccagcctg ctggcgcaaa acaacacccg 7440

ggtgtgggtg tggagcccga ccaacccggg agccagcgat cccgccgcca tgatcggcca 7500

agccgccgag gcgatgggta acagccgcat gttctacaac cagtatcgca gcgtcggcgg 7560

gcacaacgga cacttcgact tcccagccag cggtgacaac ggctggggct cgtgggcgcc 7620

ccagctgggc gctatgtcgg gcgatatcgt cggtgcgatc cgcgaagctt atcgatgtcg 7680

acgtagtcat catcatcatc atcaccacca ctaactagcg tacgatcgac tgccaggcat 7740

caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatgcc atcatggccg 7800

cggtgatcag ctagcca 7817

<210> 9

<211> 972

<212> DNA

<213> Artificial sequence

<400> 9

atgcatcatc atcatcatca ccaccacaag ggtcggtcgg cgctgctgcg ggcgctctgg 60

attgccgcac tgtcattcgg gttgggcggt gtcgcggtag ccgcggaacc caccgccaag 120

gccgccccat acgagaacct gatggtgccg tcgccctcga tgggccggga catcccggtg 180

gccttcctag ccggtgggcc gcacgcggtg tatctgctgg acgccttcaa cgccggcccg 240

gatgtcagta actgggtcac cgcgggtaac gcgatgaaca cgttggcggg caaggggatt 300

tcggtggtgg caccggccgg tggtgcgtac agcatgtaca ccaactggga gcaggatggc 360

agcaagcagt gggacacctt cttgtccgct gagctgcccg actggctggc cgctaaccgg 420

ggcttggccc ccggtggcca tgcggccgtt ggcgccgctc agggcggtta cggggcgatg 480

gcgctggcgg ccttccaccc cgaccgcttc ggcttcgctg gctcgatgtc gggctttttg 540

tacccgtcga acaccaccac caacggtgcg atcgcggcgg gcatgcagca attcggcggt 600

gtggacacca acggaatgtg gggagcacca cagctgggtc ggtggaagtg gcacgacccg 660

tgggtgcatg ccagcctgct ggcgcaaaac aacacccggg tgtgggtgtg gagcccgacc 720

aacccgggag ccagcgatcc cgccgccatg atcggccaag ccgccgaggc gatgggtaac 780

agccgcatgt tctacaacca gtatcgcagc gtcggcgggc acaacggaca cttcgacttc 840

ccagccagcg gtgacaacgg ctggggctcg tgggcgcccc agctgggcgc tatgtcgggc 900

gatatcgtcg gtgcgatccg cgaagcttat cgatgtcgac gtagtcatca tcatcatcat 960

caccaccact aa 972

<210> 10

<211> 323

<212> PRT

<213> Artificial sequence

<400> 10

Met His His His His His His His His Lys Gly Arg Ser Ala Leu Leu

1 5 10 15

Arg Ala Leu Trp Ile Ala Ala Leu Ser Phe Gly Leu Gly Gly Val Ala

20 25 30

Val Ala Ala Glu Pro Thr Ala Lys Ala Ala Pro Tyr Glu Asn Leu Met

35 40 45

Val Pro Ser Pro Ser Met Gly Arg Asp Ile Pro Val Ala Phe Leu Ala

50 55 60

Gly Gly Pro His Ala Val Tyr Leu Leu Asp Ala Phe Asn Ala Gly Pro

65 70 75 80

Asp Val Ser Asn Trp Val Thr Ala Gly Asn Ala Met Asn Thr Leu Ala

85 90 95

Gly Lys Gly Ile Ser Val Val Ala Pro Ala Gly Gly Ala Tyr Ser Met

100 105 110

Tyr Thr Asn Trp Glu Gln Asp Gly Ser Lys Gln Trp Asp Thr Phe Leu

115 120 125

Ser Ala Glu Leu Pro Asp Trp Leu Ala Ala Asn Arg Gly Leu Ala Pro

130 135 140

Gly Gly His Ala Ala Val Gly Ala Ala Gln Gly Gly Tyr Gly Ala Met

145 150 155 160

Ala Leu Ala Ala Phe His Pro Asp Arg Phe Gly Phe Ala Gly Ser Met

165 170 175

Ser Gly Phe Leu Tyr Pro Ser Asn Thr Thr Thr Asn Gly Ala Ile Ala

180 185 190

Ala Gly Met Gln Gln Phe Gly Gly Val Asp Thr Asn Gly Met Trp Gly

195 200 205

Ala Pro Gln Leu Gly Arg Trp Lys Trp His Asp Pro Trp Val His Ala

210 215 220

Ser Leu Leu Ala Gln Asn Asn Thr Arg Val Trp Val Trp Ser Pro Thr

225 230 235 240

Asn Pro Gly Ala Ser Asp Pro Ala Ala Met Ile Gly Gln Ala Ala Glu

245 250 255

Ala Met Gly Asn Ser Arg Met Phe Tyr Asn Gln Tyr Arg Ser Val Gly

260 265 270

Gly His Asn Gly His Phe Asp Phe Pro Ala Ser Gly Asp Asn Gly Trp

275 280 285

Gly Ser Trp Ala Pro Gln Leu Gly Ala Met Ser Gly Asp Ile Val Gly

290 295 300

Ala Ile Arg Glu Ala Tyr Arg Cys Arg Arg Ser His His His His His

305 310 315 320

His His His

<210> 11

<211> 40

<212> DNA

<213> Artificial sequence

<400> 11

ggactctagc caaagattgg acctcactag ttccatgacg 40

<210> 12

<211> 40

<212> DNA

<213> Artificial sequence

<400> 12

gcgcgtcgtt gtggtcaccg tctagatttc tggcgggacg 40

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<400> 13

cggtgaccac aacgacgcgc 20

<210> 14

<211> 42

<212> DNA

<213> Artificial sequence

<400> 14

tacaggacct gccaattctt ggtggctagc tgatcaccgc gg 42

<210> 15

<211> 25

<212> DNA

<213> Artificial sequence

<400> 15

gaagcttatc gatgtcgacg tagtc 25

<210> 16

<211> 22

<212> DNA

<213> Artificial sequence

<400> 16

tgcgaagtga ttcctccgga tc 22

<210> 17

<211> 60

<212> DNA

<213> Artificial sequence

<400> 17

ggaggaatca cttcgcaatg catcatcatc atcatcacca ccacgtgacg gggcaaccgg 60

<210> 18

<211> 39

<212> DNA

<213> Artificial sequence

<400> 18

cgtcgacatc gataagcttc ggcagcgaac cctcgtgag 39

<210> 19

<211> 66

<212> DNA

<213> Artificial sequence

<400> 19

ggaggaatca cttcgcaatg catcatcatc atcatcacca ccacagcctt ttggatgctc 60

atatcc 66

<210> 20

<211> 39

<212> DNA

<213> Artificial sequence

<400> 20

cgtcgacatc gataagcttc gaacccggta taggtcgac 39

<210> 21

<211> 60

<212> DNA

<213> Artificial sequence

<400> 21

ggaggaatca cttcgcaatg catcatcatc atcatcacca ccacaagggt cggtcggcgc 60

<210> 22

<211> 42

<212> DNA

<213> Artificial sequence

<400> 22

cgtcgacatc gataagcttc gcggatcgca ccgacgatat cg 42

<210> 23

<211> 23

<212> DNA

<213> Artificial sequence

<400> 23

ggtcatgggc cgaacatact cac 23

<210> 24

<211> 19

<212> DNA

<213> Artificial sequence

<400> 24

tggctagctg atcaccgcg 19

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence

<400> 25

aacatcttcg tggacctggc 20

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence

<400> 26

gtgaagccgg agagatccag 20

<210> 27

<211> 22

<212> DNA

<213> Artificial sequence

<400> 27

gcaggtcaag ctcaccgcga tc 22

<210> 28

<211> 23

<212> DNA

<213> Artificial sequence

<400> 28

tccaggtctc catctcgatg agc 23

<210> 29

<211> 22

<212> DNA

<213> Artificial sequence

<400> 29

tgcaactccc ggtgcaacct tg 22

<210> 30

<211> 23

<212> DNA

<213> Artificial sequence

<400> 30

tcaactgctc gacggtgctc ttc 23

<210> 31

<211> 20

<212> DNA

<213> Artificial sequence

<400> 31

ccaagaattg gcaggtcctg 20

<210> 32

<211> 22

<212> DNA

<213> Artificial sequence

<400> 32

ccaatctttg gctagagtcc tg 22

<210> 33

<211> 118

<212> DNA

<213> Artificial sequence

<400> 33

cctgtatcct aaatcaaata tcggacaagc agtgtctgtt ataacaaaaa atcgatttaa 60

tagacacacc aacagcatgg tttttatgtg tgcgataatt tataatattt cggacagg 118

Claims (5)

1. An integrated expression plasmid of mycobacterium tuberculosis, which comprises the following structural elements:

(1) The screening marker of the resistance gene is used for constructing and screening the recombinant plasmid; the resistance genes are SacB and hyg resistance genes;

(2) Gamma delta sequences respectively positioned at two sides of the resistance gene are used for knocking out the resistance; the gamma delta sequence is shown as SEQ ID NO. 33;

(3) Elements for insertion and expression of a gene of interest; the element for inserting and expressing the target gene is hsp60 promoter;

(4) Essential tag elements, replicons and mycobacterium tuberculosis genome integration elements; the tag element is a his tag, the replicon is an oriE gene, and the mycobacterium tuberculosis genome integration element is attp and int genes;

the plasmid is pTB01 plasmid, and the nucleotide sequence of the plasmid is shown in SEQ ID NO. 1.

2. The use of the integrative expression plasmid of Mycobacterium tuberculosis of claim 1, which comprises:

(1) Preparing a mycobacterium tuberculosis strain with a specific over-expression protein integrated in the genome;

or (2) preparing an anti-tuberculosis vaccine or a tuberculosis prevention vaccine with higher specific antigen content;

the strain or the vaccine contains the integrated expression plasmid of the mycobacterium tuberculosis with high expression of specific target protein, and the integrated expression plasmid of the mycobacterium tuberculosis with high expression of the specific target protein is respectively as follows:

a pBT01s1-Va07 plasmid containing an Rv3899c protein, and the sequence of the plasmid is shown as SEQ ID NO. 2; a pBT01s1-Va16 plasmid containing an Rv0287 protein, and the sequence of the plasmid is shown as SEQ ID NO. 5; the plasmid pBT01s1-Va25 containing the Rv3803c protein has the sequence shown in SEQ ID NO. 8.

3. A method for preparing a mycobacterium tuberculosis strain having a genome integrated with an overexpressed target protein, the method comprising the steps of:

(1) Inserting an expression gene of a target protein into the mycobacterium tuberculosis integrative expression plasmid of claim 1;

(2) Amplifying and extracting the target protein-containing mycobacterium tuberculosis integrated expression plasmid;

(3) Transferring the integrated expression plasmid containing the target protein of the mycobacterium tuberculosis into the mycobacterium tuberculosis;

the target protein-containing mycobacterium tuberculosis integrated expression plasmids are respectively as follows:

a pBT01s1-Va07 plasmid containing an Rv3899c protein, and the sequence of the plasmid is shown as SEQ ID NO. 2; a pBT01s1-Va16 plasmid containing an Rv0287 protein, and the sequence of the plasmid is shown as SEQ ID NO. 5; the plasmid pBT01s1-Va25 containing the Rv3803c protein has the sequence shown in SEQ ID NO. 8.

4. A method for preparing a tuberculosis vaccine with high content of specific target protein, which comprises the following steps:

(1) Inserting an expression gene of a target protein into the mycobacterium tuberculosis integrative expression plasmid of claim 1;

(2) Amplifying and extracting the target protein-containing mycobacterium tuberculosis integrated expression plasmid;

(3) Transferring the integrated expression plasmid containing the target protein of the mycobacterium tuberculosis into the mycobacterium tuberculosis;

(4) Preparing the vaccine by using the mycobacterium tuberculosis obtained in the step (3);

the target protein-containing mycobacterium tuberculosis integrated expression plasmids are respectively as follows:

a pBT01s1-Va07 plasmid containing an Rv3899c protein, the sequence of which is shown in SEQ ID NO. 2; a pBT01s1-Va16 plasmid containing an Rv0287 protein, and the sequence of the plasmid is shown as SEQ ID NO. 5;

the plasmid pBT01s1-Va25 containing the Rv3803c protein has the sequence shown in SEQ ID NO. 8.

5. A set of mycobacterium tuberculosis integrated expression plasmids which are prepared by taking the mycobacterium tuberculosis integrated expression plasmid as the basic vector of claim 1 and highly express specific target proteins, wherein the mycobacterium tuberculosis integrated expression plasmids which are highly expressed by the specific target proteins are respectively as follows:

pBT01s1-Va07 containing an Rv3899c protein, and the sequence of the pBT01s1-Va07 is shown in SEQ ID NO. 2;

pBT01s1-Va16 containing the Rv0287 protein, and the sequence of the pBT01s1-Va16 is shown as SEQ ID NO. 5;

pBT01s1-Va25 containing Rv3803c protein, and the sequence is shown in SEQ ID NO. 8.

Images