JP2016082934A - Expression promoters in pneumococci - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide promoters for high level expression of a target gene in pneumococci on the background that although high level expression systems have been developed in common bacteria such as E. coli, such high level expression systems do not work in pneumococci having a basically different expression system.SOLUTION: A promoter comprises a polynucleotide (a) or (b), where (a) is a polynucleotide comprising a specified base sequence of 131 bp (SP); and (b) is a polynucleotide that hybridizes with the polynucleotide (a) under stringent conditions, which is capable of expressing a target gene linked to downstream in pneumococci.SELECTED DRAWING: Figure 4

Description

  The present invention relates to a promoter comprising a polynucleotide capable of expressing a target gene linked downstream in Streptococcus pneumoniae.

  Pneumonia accounts for 9.9% of causes of death in Japan and is the third largest disease after cancer and heart disease. And pneumococcus is the most frequent pneumonia causing bacteria in the city.

  This pneumococcus is a facultative anaerobic Gram-positive bacilli. Streptococcus pneumoniae has various virulence factors (hemolysin, IgA-degrading enzyme, etc.), and in particular, it is protected from the host immune cells (phagocytic cells) by the capsule and protected from the membrane attack complex formed by complement. Important for colonization (infection). Since the capsule has a simple structure, its antigenicity is weak and T cell-dependent antibody production by follicular B cells is not performed, and high affinity IgG antibody-producing B cells are not expressed. Instead, T1-independent antibody production is performed by B1 cells localized in the serosal cavity and splenic marginal zone B cells, and an immediate immune defense mechanism is induced by an IgM antibody having low affinity. When IgM antibody binds to the pneumococcal capsule, it can activate the complement system and cellular immunity to eliminate pneumococci. However, in this antibody production mechanism, since memory B cells are not produced, long-term "immunity" There is no “memory” and there is always a risk of reinfection with pneumococci. Furthermore, since the capsules of pneumococcus are known, there are over 90 types, so even if pneumoniae infection occurs once, there is a possibility of infection with different serotypes.

  Thus, pneumococci establish infection by a skillful immune evasion mechanism, and are therefore particularly dangerous bacteria for elderly people with weakened immune functions and infants with undeveloped immune functions. Furthermore, in recent years, pneumococci resistant to β-lactam antibiotics used as first-line drugs have been reported, making it difficult to treat. Currently, vaccines based on capsular polysaccharides of pneumococcus and vaccines with adjuvants added to capsular polysaccharides have been developed for effective infection prevention in susceptible persons. These vaccines have a proven track record of effective prevention of pneumococcal infection overseas, and were approved in Japan several years ago, and vaccination is spreading.

  In order to understand the pathogenicity of Streptococcus pneumoniae, it is necessary to regulate and analyze the gene expression of virulence factors in Streptococcus pneumoniae. So far, several promoters for expressing foreign genes in cells have been put into practical use in humans and E. coli. For example, the CMV-IE promoter is a promoter derived from cytomegalovirus that invisibly infects humans, and is a general promoter used for high protein expression in mammalian cells (see Non-Patent Document 1). Regarding bacteria, the tac promoter (see Non-Patent Document 2) is widely used as a promoter capable of dealing with various E. coli strains. The tac promoter is a promoter in which a trp promoter derived from E. coli and a lac promoter are combined, and is useful when a target protein is expressed in E. coli. However, in Streptococcus pneumoniae, general high expression promoters such as CMV-IE promoter and tac promoter have not been put into practical use. Therefore, the present situation is that progress has not been made in the analysis of the properties and functions of pneumococci such as the analysis of the mechanism of colonization of pneumococci in lung tissue and the mechanism of avoiding the host immune system. Therefore, there has been a demand for the development of an efficient expression system that can highly express the target molecule in pneumococci.

Schmidt EV, Christoph G, Zeller R, Leder P. Mol Cell Biol. (1990) 10 (8): 4406-4411 de Boer HA, Comstock LJ, Vasser M. Proc Natl Acad Sci U S A (1983) 80 (1): 21-25

  Until now, the development of high expression systems for common bacteria such as Escherichia coli has progressed, but the basic expression system for pneumococci is different, so even if the high expression system for general bacteria is used with pneumococci, It cannot be highly expressed in cocci. Therefore, an object of the present invention is to provide a promoter capable of highly expressing a target gene in pneumococci.

  The inventors searched for a promoter highly active in Streptococcus pneumoniae. If the promoter of the PspA protein known as one of the virulence factors of Streptococcus pneumoniae is used, expression of the target protein can be induced in Streptococcus pneumoniae. Thought. This PspA protein is one of the virulence factors of pneumococci and is a cell surface molecule. It is known that when this PspA protein is deficient, it is easily phagocytosed in an infected host and its pathogenicity is reduced, and is a protein that is surely expressed in pneumococci.

  Therefore, the PspA protein promoter site (SEQ ID NO: 1) was amplified by PCR using the chromosome of Streptococcus pneumoniae GTC261 strain as a template, and the gene encoding mCherry was amplified by PCR using the pmCherry plasmid (Clontech) as a template. Using these PCR products, a recombinant E. coli / pneumococcal shuttle vector pLS5-HSG398-PpspA-mCherry (PpspA-mCherry) shown in FIG. 1 was prepared. Furthermore, Escherichia coli JM109 strain was transformed using the recombinant Escherichia coli / pneumococcal shuttle vector. As a result, a plurality of E. coli JM109 strains emitting red fluorescence were obtained. Next, in order to confirm the expression of red fluorescent protein mCherry in Streptococcus pneumoniae, an expression plasmid was extracted from Escherichia coli JM109 strain that emitted red fluorescence and transformed into Streptococcus pneumoniae. As shown in FIG. As in the case of introducing an expression plasmid (lac_promoter-mCherry: preparation method described later) ligated with mCherry downstream into pneumococci, pneumococci that strongly emit red fluorescence were not obtained. From this result, it was revealed that when trying to induce expression of a target molecule in Streptococcus pneumoniae, it is difficult to induce efficient expression even using a promoter that is considered to be theoretically possible. Therefore, in order to achieve high expression of the target gene in Streptococcus pneumoniae, we thought that it was necessary to discover a new promoter, and screened by partially cleaving the pneumococcal chromosome with restriction enzymes. From these fragments, a sequence capable of highly expressing a target gene in pneumococci was found, and the present invention was completed.

That is, the present invention is as follows.
(1) (a) a polynucleotide containing the base sequence shown in SEQ ID NO: 2; (b) hybridizing under stringent conditions with a polynucleotide containing the base sequence shown in SEQ ID NO: 2 and downstream in pneumococci A promoter comprising the polynucleotide shown in (a) or (b) of a polynucleotide capable of expressing a linked target gene.
(2) The promoter according to (1) above, wherein the polynucleotide containing the base sequence shown in SEQ ID NO: 2 is a polynucleotide containing the base sequence shown in SEQ ID NO: 3.
(3) A recombinant vector comprising the promoter according to (1) or (2) above.
(4) A transformant of Streptococcus pneumoniae into which the recombinant vector according to (3) is introduced.

  The promoter of the present invention has an effect that the target gene linked downstream in pneumococci can be highly expressed. By using such a promoter, the properties and functions of pneumococci can be analyzed, such as analysis of the protein encoded by the target gene, analysis of the mechanism of pneumococcal colonization in the lung tissue and the mechanism to avoid the host immune system, etc. It becomes possible.

The map of the Escherichia coli pneumococcal shuttle vector pLS5-HSG398-PpspA-mCherry (PpspA-mCherry) containing the promoter region of the PspA protein and the sequence encoding the red fluorescent protein mCherry is shown. It is a figure which shows the result of having introduce | transduced into pneumococcus and culture | cultivating the expression plasmid which connected mCherry downstream to the lac promoter or the PspA promoter, and observed with the fluorescence microscope. (A) is a case where lac_promoter-mCherry is introduced, and (b) is a case where PpspA-mCherry is introduced, the left is a differential interference image and the right is a fluorescence image. The maps of (a) pLS21, (b) pHSG398, and (c) pLS5-HSG398 rev-mCherry rev are shown. It is a figure which shows the arrangement | sequence of the chromosome fragment inserted in fragment | piece S-mCherry. The map of pLS5-HSG398 rev is shown. A map of pLS5-HSG398 rev-Plac-mCherry (lac_promoter-mCherry) in which the lac promoter and mCherry gene are introduced into the vector pLS5-HSG398 rev is shown. It is a figure which shows the result of having introduce | transduced the fragment | piece S-mCherry obtained in Example 1, and lac_promoter-mCherry as a control into a pneumococcus, culture | cultivating with a LBG flat plate culture medium and a TSBY flat plate culture medium, and observing with the fluorescence microscope. The map of pLS5-HSG398-rev-Sp-mCherry (Sp-mCherry) is shown. Of the fragment S, a plasmid (Sp-mCherry) containing a non-coding region sequence (Sp) upstream of the B region shown in FIG. 4 was introduced into pneumococci, cultured in a TSBY plate medium, and observed with a fluorescence microscope. FIG.

  As the promoter of the present invention, (a) a polynucleotide containing the base sequence shown in SEQ ID NO: 2; (b) a polynucleotide containing the base sequence shown in SEQ ID NO: 2 is hybridized under stringent conditions, and pneumonia A polynucleotide that can express a target gene downstream linked to a cocci is not particularly limited as long as it is a promoter comprising the polynucleotide shown in (a) or (b), and such pneumococci are both pneumococcus and pneumococcus pneumoniae. It is called a Gram-positive bacilli that causes pneumonia.

  In the present invention, a promoter means a base sequence of a region that is arranged upstream of a target gene sequence and is associated with the control of expression of a gene arranged downstream by binding of RNA polymerase, and is an enhancer region or ribosome binding. The base sequence of the region may be included.

  The base sequence shown in SEQ ID NO: 2 in the present invention is a sequence contained in a fragment obtained by treating a pneumococcal chromosome with the restriction enzyme Sau3AI. As a polynucleotide containing the base sequence shown in SEQ ID NO: 2, As long as it contains the base sequence shown in SEQ ID NO: 2, it is not particularly limited, and examples thereof include a polynucleotide containing the base sequence shown in SEQ ID NO: 3, and as long as it can express the target gene linked downstream, Alternatively, it may contain all or part of the structural gene downstream, a marker gene, other base sequences such as microRNA, a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2, It may be a polynucleotide having the base sequence shown.

  In the present invention, “stringent conditions” in a polynucleotide that can hybridize with a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 2 under stringent conditions and express a gene linked downstream in pneumococci Means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. More specifically, a homology of 70% or more, more preferably 85% or more, and still more preferably 95% or more. This is a condition in which polynucleotides having the same hybridize and polynucleotides having lower homology do not hybridize to each other, for example, 65 ° C., 1 × SSC, 0.1%, which is a washing condition for normal Southern hybridization SDS, or 0.1 × SSC, high at a salt concentration equivalent to 0.1% SDS The conditions for bridging can be mentioned.

  A polynucleotide that hybridizes under stringent conditions uses a nucleic acid such as DNA or RNA as a probe, and a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. This refers to the resulting polynucleotide. Specifically, hybridization is performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which colony or plaque-derived polynucleotide fragments are immobilized. After that, using a 0.1 to 2-fold SSC solution (the composition of a 1-fold concentration SSC solution is 150 mM sodium chloride, 15 mM sodium citrate) and washing the filter under the condition of 65 ° C. List nucleotides Door can be. Hybridization can be performed, for example, by the method described in Molecular Cloning, A laboratory manual, 3rd edition, Chapter 6.

  In the above pneumococci, the polynucleotide capable of expressing the target gene linked downstream is not particularly limited as long as it can express the target gene operably linked downstream in pneumococci, but, for example, when using the lac promoter In comparison, a polynucleotide that expresses the target gene 2 times or more, preferably 3 times or more, more preferably 4 times or more, and further preferably 5 times or more can be mentioned.

The expression of the target gene can be achieved, for example, by introducing a recombinant polynucleotide operably linked to a fluorescent gene such as mCherry or GFP downstream of the promoter into pneumococci, and transforming the resulting transformant to a final concentration of 1.5. It can be determined by measuring and analyzing the fluorescence intensity when applied to a TSBY plate medium supplemented with 5% agar powder and 5% defibrinated sheep blood and cultured overnight at 37 ° C. and 5% CO ₂ . The fluorescence intensity can be measured and analyzed by using commercially available software such as image integration software NIS-elements (manufactured by Nikon).

  The target gene may be a full-length gene or a part thereof depending on the use. The origin may be a gene isolated from any organism or an artificial gene produced by genetic engineering.

  The vector used for the recombinant vector of the present invention is not particularly limited as long as it contains the promoter of the present invention and can express the target gene operably incorporated downstream of the promoter, and may be linear or cyclic. Those that are capable of autonomous replication and those that can be integrated into a chromosome are preferred, and those that contain a regulatory sequence such as a terminator or a selection marker may be used.

  The pneumococci introduced with the recombinant vector of the present invention means a transformant of pneumococci introduced with the recombinant vector of the present invention. The method for introducing the recombinant vector of the present invention into pneumococci is not particularly limited, and is a chemical method such as lithium acetate method, lipofection method, calcium phosphate coprecipitation method, liposome method, DEAE dextran method; And known methods such as biological methods such as cell fusion methods; physical methods such as electroporation methods, microinjection methods, gene gun methods, and ultrasonic gene transfer methods.

By culturing a pneumococcal transformant containing the recombinant vector of the present invention, the protein encoded by the target gene can be efficiently produced. As a culture method, it can be carried out according to a usual method used for culture of pneumococci. For example, pneumococci introduced with the recombinant vector of the present invention can be cultured in an environment of around 37 ° C., pH around 7, and 5% CO ₂ . The protein encoded by the gene of interest can be recovered from the culture solution or disrupted pneumococci. Examples of such recovery methods include known protein recovery methods such as centrifugation, then gel filtration, ion exchange, affinity, etc. The method of collect | recovering by chromatography of these can be mentioned.

[Production of Streptococcus pneumoniae capable of expressing mCherry]
In order to search for a promoter with high expression ability in pneumococci, the pneumococcal chromosome is partially cleaved with a restriction enzyme and inserted into an E. coli / pneumococcal shuttle vector containing a gene encoding mCherry to produce an expression plasmid. The expression plasmid was introduced into Escherichia coli and pneumococci to examine the expression of mCherry.

(E. coli / pneumococcal shuttle vector)
E. coli strain JM109 was used, and S. pneumoniae strain GTC261 was used. PLS5-HSG398 rev-mCherry rev was used as the E. coli / pneumococcal shuttle vector. The shuttle vector was prepared by binding the EcoRI fragment of pLS21 (ATCC67492) and the EcoRI fragment of pHSG398 and inserting the mCherry gene (Z2522N: pmCherry origin, manufactured by Takara Bio Inc.) into the HindIII cleavage site. Such a shuttle vector has an origin of replication of E. coli and a replication start region of pneumococci (repA / B / D). Further, it contains a chloramphenicol resistance gene, a tetracycline resistance gene, and a coral-derived red fluorescent protein gene mCherry. A map of pLS21, pHSG398 and pLS5-HSG398 rev-mCherry rev is shown in FIG.

(Preparation of expression plasmid)
Restriction enzyme BamHI (15 U / μl) 0.5 μl and CIP (10-30 U / μl) 0.125 μl were added to the shuttle vector pLS5-HSG398 rev-mCherry rev 50 ng and reacted at 30 ° C. for 60 minutes (reaction system). 50 μl). Further, 1 μl of a 128-fold diluted restriction enzyme Sau3AI (10 U / μl) was added to 500 ng of chromosome of Streptococcus pneumoniae GTC261 strain, and reacted at 37 ° C. for 60 minutes (reaction system 50 μl). After the reaction, the enzyme was inactivated, and the shuttle vector reaction solution and the pneumococcal chromosome reaction solution were mixed and subjected to ethanol precipitation. 1 μl of T4 DNA ligase (350 U / μl) was added to the concentrated and dried reaction mixture and reacted overnight at 16 ° C. (reaction system 10 μl) to prepare an expression plasmid (Pneumococcal chromosome fragment library). .

(Introduction of expression plasmid into E. coli)
Expression plasmid after ligation treatment of 100 μl of E. coli JM109 strain competent cell prepared by Inoue H, Nojima H, Okayama H. Gene (1990) 96 (1): 23-28) In addition to the solution, it was left in ice for 30 minutes. Then, it left still for 45 seconds in 42 degreeC environment. After placing on ice for 5 minutes, 1 ml of LBG liquid medium (final concentration 2% LB medium, final concentration 0.1% glucose) was added and allowed to stand at 37 ° C. for 1 hour, and tetracycline (final concentration 20 μg / ml) was added. The whole amount was applied to the containing LBG plate medium (final concentration of 1.5% agar powder was added to the LBG liquid medium) and cultured at 30 ° C. overnight.

(Extraction of expression plasmid)
The colonies having an appropriate size were observed with a fluorescence microscope, and 40 colonies that emitted red fluorescence were each inoculated into 5 ml of LBG liquid medium containing tetracycline (final concentration 20 μg / ml). Cultured overnight. After confirming that the turbidity exceeds 1 using an absorptiometer, the culture broth was collected overnight by centrifugation and then collected using the QIAprep (registered trademark) Spin Miniprep Kit (manufactured by Qiagen). Escherichia coli JM109-derived expression plasmid (hereinafter also referred to as “JM109-derived mCherry expression plasmid”) was extracted to obtain 40 types of expression plasmids.

(Introduction of JM109-derived mCherry expression plasmid into pneumococci)
In order to confirm the expression of mCherry in Streptococcus pneumoniae, 40 types of JM109-derived mCherry expression plasmids were each introduced into Streptococcus pneumoniae. First, TSBY liquid medium (final concentration 3% Soybean-Casein Digest Broth, final concentration 0. 1) added with HCl (final concentration 10 mM) and glycine (final concentration 20 mM) in 1 ml of pneumococci in the logarithmic growth phase (GTC261 strain). 5% Yeast extract) was transferred to 100 ml and cultured at 37 ° C. for 3 hours. The culture broth was confirmed to have grown pneumococci to a turbidity of about 0.1 using an absorptiometer. The culture solution was neutralized with NaOH (final concentration 10 mM), and then centrifuged at 4 ° C., 7000 rpm for 5 minutes. The cells were washed 3 times with TB (Transfer Buffer, final concentration 4.88% glucose, final concentration 10 mM MgCl ₂ , pH 6.5) and suspended in 800 μl of TB. A mixture of 40 μl of the extracted JM109-derived mCherry expression plasmid 75-225 ng was added to 200 μl of this TB suspension bacterial solution, and poured into a cuvette for electroporation (0.2 cm gap). MicroPulser (manufactured by Bio-Rad) ), A current of 2.9 kV was passed for 3.70 to 3.90 ms. Next, 1 ml of TSBY liquid medium was added to the bacterial solution in the cuvette and cultured at 37 ° C. for 1 hour. 250 μl of this culture solution was collected, added to 750 μl of fresh TSBY liquid medium, and cultured at 37 ° C. for 2 hours. Thereafter, the culture solution is diluted to 1/10 and 1/1000 concentration so that the total volume is 1 ml each, and TSBY plate medium (final concentration 1.5% agar powder, 5% defibrinated sheep blood, final concentration in TSBY medium) The total amount was applied to 0.5 μg / ml tetracycline added). The applied pneumococci were cultured overnight in an environment of 37 ° C. and 5% CO ₂ . Each colony having an appropriate size was observed with a fluorescence microscope, and a colony emitting red fluorescence, that is, a strain (GTC261-S strain) that strongly expressed mCherry in Streptococcus pneumoniae was obtained. The JM109-derived mCherry expression plasmid introduced into the GTC261-S strain is hereinafter also referred to as “fragment S-mCherry”.

[Sequence analysis of chromosome fragments]
A region containing the chromosomal fragment inserted into the fragment S-mCherry was amplified by PCR, and sequence analysis was performed.

(PCR method)
For 200 ng of the fragment S-mCherry obtained in the above “extraction of expression plasmid”, PrimeSTAR HS DNA Polymerase (2.5 U / μl) 0.5 μl, 5 × PrimeSTAR Buffer 10 μl, dNTP Mixture (2.5 mMeach) 4 μl, SEQ ID NO: 4 The primer seq F shown and the primer seq R shown in SEQ ID NO: 5 (10 μmol / μl) were added in an amount of 1 μl, and the reaction was carried out in a 50 μl system. Appropriate annealing temperature and elongation temperature and reaction time were selected. The DNA after the PCR reaction was purified, measured for concentration, and used for sequence analysis.

(Sequence analysis)
Sequence analysis was performed on the DNA obtained after the PCR reaction, that is, the sequence in the range including the chromosomal fragment inserted into the fragment S-mCherry and a part of the mCherry gene. In order to search a highly homologous sequence from a genome whose base sequence has already been analyzed, the sequence obtained by the analysis is expressed by BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE= BlastSearch & LINK_LOC = blasthome).

(Sequence analysis result)
As a result of sequence analysis, it was revealed that the sequence of the chromosomal fragment inserted into the fragment S-mCherry is the base sequence shown in SEQ ID NO: 3 (FIG. 4). From these results, it was revealed that the base sequence shown in SEQ ID NO: 3 is a polynucleotide capable of expressing the target gene in pneumococci. Hereinafter, the base sequence shown in SEQ ID NO: 3 is also referred to as “fragment S” hereinafter. As a result of homologous search by BLAST, the A region of the 1st to 453rd base sequence in the base sequence shown in SEQ ID NO: 3 is UvrABC system protein A, and B of the 585th to 818th base sequence. The region is a putative transporter, the C region of the 958th to 1530th base sequences is a CorA-like Mg ²⁺ transporter protein, and the D region of the 1542th to 1995th bases is a putative membrane. It was revealed that it encodes a protein (putative membrane protein: S protein).

[Measurement of fluorescence intensity in Streptococcus pneumoniae transformed with a plasmid containing fragment S]
From the results of Example 1, it was revealed that the chromosomal fragment inserted into the fragment S-mCherry is the fragment S shown in SEQ ID NO: 3. Therefore, in order to examine the promoter activity of the fragment S in pneumococci, the fragment S-mCherry obtained in Example 1 was introduced into pneumococci by the same method as in Example 1, cultured in a TSBY plate medium, and fluorescent microscope Observed at. As a control, a plasmid (lac_promoter-mCherry) in which the lac promoter and mCherry gene were introduced into the vector pLS5-HSG398 rev was prepared by the following method, introduced into pneumococci in the same manner as above, cultured in a TSBY plate medium and fluorescent microscope Observed at. In addition, pneumococci introduced with lac_promoter-mCherry were cultured in two groups: a group without IPTG and a group cultured with a medium supplemented with IPTG to 100 μM. In the culture of pneumococci introduced with the fragment S-mCherry, there is no IPTG.

(Production of lac_promoter-mCherry)
PCR was performed using the pmCherry plasmid (manufactured by Clontech) as a template and the primer Plac-mCherry F shown in SEQ ID NO: 6 and the primer Plac-mCherry R shown in SEQ ID NO: 7 to obtain a Plac-mCherry fragment. The obtained PCR product was cleaved with restriction enzymes BglII and SacI. In addition, pLS5-HSG398 rev prepared based on pLS21 plasmid (ATCC 6749) and pHSG398 plasmid (Clontech) was cleaved with restriction enzymes BamHI and SacI. A map of pLS5-HSG398 rev is shown in FIG.

  The restriction enzyme-treated Plac-mCherry fragment and the vector pLS5-HSG398 rev were mixed, 1 μl of T4 DNA ligase (350 U / μl) was added, and the mixture was reacted at 16 ° C. overnight (reaction system 10 μl). The ligation product was mixed with the E. coli JM109 competent cell prepared by the above Inoue / Nojima method, and the gene was introduced into E. coli JM109 to transform E. coli JM109. A plasmid was extracted from the obtained colony in the same manner as the above-described extraction of the plasmid to obtain a pLS5-HSG398 rev-Plac-mCherry (lac_promoter-mCherry) plasmid (FIG. 6).

(Fluorescence observation)
The cultured medium was observed with a fluorescence microscope, and three colonies with red fluorescence were selected to prepare a preparation. Using a fluorescent microscope, photographs of the bacteria on the slide were taken with a plurality of fields of view at an exposure time of 2 seconds, and the luminance of 100 bacteria on the photograph was measured and analyzed. The value obtained by subtracting the luminance of the background portion from the average value of the luminance of 100 bacteria was defined as the fluorescence intensity of the bacteria having the plasmid. Image integration software NIS-elements (manufactured by Nikon) was used for luminance measurement and analysis.

(result)
The fluorescence intensity in pneumococci introduced with each plasmid is shown in Table 1, and a fluorescence micrograph is shown in FIG. In FIG. 7, (a) and (b) are pneumococci introduced with lac_promoter-mCherry, and (c) are pneumococci introduced with the fragment S-mCherry. The numerical value of the fluorescence intensity is an actual measurement value measured using NIS-elements and represents “luminance” per pixel.

  As shown in Table 1 and FIG. 7, in pneumococci introduced with the fragment S-mCherry, compared to pneumococci transformed with lac_promoter-mCherry, 7 times or more without IPTG and 5 times with IPTG 100 μM. As described above, the fluorescence intensity was high, and it was revealed that the fragment S highly expressed the mCherry gene in pneumococci.

[Measurement of fluorescence intensity in Streptococcus pneumoniae transformed with a plasmid containing a part of fragment S]
Since the fragment S contains the A to D regions encoding the protein as shown in FIG. 4, the sequence of the non-coding region upstream of the B region (Sp: SEQ ID NO: 2) is downstream in pneumococci. It was thought to be greatly involved in the expression of the linked target gene. Therefore, among the fragments S, a plasmid (Sp-mCherry) containing Sp shown in SEQ ID NO: 2 was prepared by the following method to transform pneumococci, and the fluorescence intensity was measured.

  Using the chromosome of GTC261 strain as a template, PCR was performed using the primer Sp F shown in SEQ ID NO: 8 and the primer Sp R shown in SEQ ID NO: 9 to obtain an Sp fragment. The obtained PCR product was cleaved with restriction enzymes SphI and NdeI. Further, PCR was performed using the pmCherry plasmid (manufactured by Clontech) as a template and the primer mCherry F shown in SEQ ID NO: 10 and the primer mCherry R shown in SEQ ID NO: 11 to obtain an mCherry fragment to be inserted downstream of the Sp fragment. The obtained PCR product was cleaved with restriction enzymes NdeI and BamHI. Furthermore, the vector pLS5-HSG398 rev was cleaved with restriction enzymes SphI and BamHI.

  The obtained restriction enzyme-treated Sp fragment, mCherry fragment and vector pLS5-HSG398 rev were mixed, 1 μl of T4 DNA ligase (350 U / μl) was added, and the mixture was reacted at 16 ° C. overnight (10 μl of reaction system). The ligation product was mixed with the E. coli JM109 competent cell prepared by the above Inoue / Nojima method, and the gene was introduced into E. coli JM109 to transform E. coli JM109. A plasmid was extracted from the obtained colony by the same method as the above-described extraction of the plasmid to obtain a pLS5-HSG398-rev-Sp-mCherry (Sp-mCherry) plasmid (FIG. 8).

  The Sp-mCherry produced above was used to transform the Streptococcus pneumoniae GTC261 strain in the same manner as in Example 1, cultured in the same manner as in Example 2, and the fluorescence intensity was measured. The results are shown in Table 2 and FIG.

(result)
As shown in Table 2 and FIG. 9, strong fluorescence intensity was also observed in pneumococci introduced with Sp-mCherry, and it was revealed that Sp highly expressed the mCherry gene in pneumococci.

  Since the promoter of the present invention can highly express the target gene in Streptococcus pneumoniae, analysis of the mechanism of colonization of Streptococcus pneumoniae in the lung tissue and the mechanism that avoids the host immune system, or a vaccine targeting the pathogenic factor of Streptococcus pneumoniae It can be used in the field of development.

Claims (4)

A promoter comprising the polynucleotide shown in the following (a) or (b).
(A) a polynucleotide comprising the base sequence represented by SEQ ID NO: 2;
(B) a polynucleotide that hybridizes with a polynucleotide containing the base sequence shown in SEQ ID NO: 2 under stringent conditions and can express a target gene linked downstream in pneumococci; The promoter according to claim 1, wherein the polynucleotide containing the base sequence shown in SEQ ID NO: 2 is a polynucleotide containing the base sequence shown in SEQ ID NO: 3. A recombinant vector comprising the promoter according to claim 1 or 2. A transformant of Streptococcus pneumoniae into which the recombinant vector according to claim 3 has been introduced.