KR100493824B1 - Antibiotics screening composition comprising Streptococcus pneumoniae spr0049 gene or its protein product, and antibiotics screening method using said composition - Google Patents

Abstract

The present invention relates to an antibiotic screening composition comprising a spr0049 gene or a protein expressed therefrom of pneumococcus and an antibiotic screening method using the same.

The spr0049 gene and the protein expressed therefrom included in the composition for screening of the present invention perform an essential function for survival of pneumococci.

Therefore, the screening method using the composition of the present invention can be usefully used for the search and development of antibiotics that can effectively act against pathogenic bacteria, including pneumococci.

Description

Antibiotic screening composition comprising Streptococcus sp. 0049 gene or protein expressed therefrom, and antibiotic screening method using the same

The present invention relates to an antibiotic screening composition comprising an essential gene of Streptococcus pneumoniae or a protein expressed therefrom and an antibiotic screening method using the same.

The development of new antibiotics is an important field in that it can create enormous added value in the industrial economy, in addition to the clinical importance of the treatment of antibiotic resistant bacterial infections.

However, traditional methods of developing antibiotics, such as randomly searching for antibiotics from natural products or bacteria, or partially modifying the structure of a conventionally synthesized compound, have already faced limitations and have developed a new class of antibiotics for the past 30 years. This is almost no state.

In recent years, new technologies such as bioinformatics, microbial genomics, proteomics, and high throughput screening (HTS) have been developed to explore new antibiotic targets in pathogenic bacteria and to prevent them. Research is underway to develop materials.

At this time, a representative gene is a representative gene that can be a target gene, the essential gene is a gene essential for the survival or pathogenesis of bacteria. Thus, substances capable of inhibiting the expression mechanism of essential genes or the activity of proteins expressed from essential genes have the potential as ideal novel antibiotics. In addition, the substance inhibits the activity of genes and proteins that are required only for the growth of bacteria, and has the advantage of being safe in human toxicity.

In addition, the discovery of target genes will lead to the development of a new concept of antibiotics, which will provide the basis for genome-based drug development, creating a global new drug market and creating enormous industrial and economic added value.

However, since little information on the essential genes of a particular pathogen is not known so far, it is urgent to invent the discovery of the essential genes and the antibiotic screening method using the same.

The present invention provides a screening composition comprising a spr0049 gene, which is an essential gene of pneumococcus, or a protein expressed therefrom, and a screening method using the same, thereby establishing a system for developing a new antibiotic having both effective antibacterial activity and human safety. .

The present invention provides a composition for screening comprising the spr0049 gene of pneumococcal.

The spr0049 gene is a gene essential for the growth of pneumococci, and includes one or more disruptions, deletions, insertions, or dots in the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 1. point, substitution, nonsense, misense, polymorphism, rearrangement mutation may be selected.

The present invention provides a composition for screening comprising a protein expressed from the spr0049 gene of pneumococcal.

The protein expressed from the spr0049 gene is 5-phospho ribosylglycinamide transformylase 1.

5-phosphoribosylglycineamide transformylase 1 is an enzyme involved in purine metabolism, specifically 10-formyltetrahydrofolate and 5'-phosphoribosylglycinamide. Tetrahydrofolate and 5'-phosphoribosyl-N-formylglycinamide catalyze the reaction produced.

The protein expressed from the spr0049 gene in the present invention is one or more disruptions, deletions, insertions, points, substitutions, nonsense, missenses, polymorphisms, or rearrangement mutations in the amino acid structure of SEQ ID NO: 2, or the nucleotide sequence of SEQ ID NO: 1 It can be expressed from a selected one of the nucleotide sequence has occurred, and may be a polypeptide fragment that exhibits the physiological activity equivalent to 5-phosphoribosilglycine amide transformylase 1.

The necessity of the spr0049 gene for pneumococcus is described below.

In order to confirm whether the gene is essential for the growth of pneumococcal, first, two PCRs are performed on the spr0049 gene to prepare a cassette in which the gene is deleted (FIG. 1).

In the primary PCR, three types of primary PCR products are obtained by amplifying the gene to replace spr0049, the flanking region and the right flank of spr0049 gene.

In the present invention, the gene for substituting spr0049 uses a marker gene to obtain a trait that was not present in the original pneumococcal strain, so that the substitution gene in the subsequent process can be correctly inserted into the genome of the transgenic strain so as to check on the selection medium. do. In the present invention, the KanR gene is used that has resistance to kanamycin.

Among the primers used in the present invention, the reverse primers used for PCR of the left side region of the spr0049 gene and the forward primers used for PCR of the right side region are respectively the promoter region of the 5 'side of the kanR gene and the 3' end of the kanR gene. It contains some.

In secondary PCR, a PCR reaction is performed on the primary PCR products using the primers, thereby obtaining a single polynucleotide in which the primary PCR products are linked together.

Finally, the obtained polynucleotide is inserted into the original spr0049 gene region, the kanR gene is deleted, and the spr0049 gene is deleted, and the left and right portions of the spr0049 gene are preserved as it is, and then used as a cassette to be inserted into the genome of the strain.

To delete the spr0049 gene in the strain genome using the cassette prepared above, the cassette is used to transform pneumococci. At this time, the selective medium for culturing the transformed strain is prepared by the addition of kanamycin.

When transformation occurs, as shown in Figure 2, the spr0049 gene existing on the genome of the bacteria is replaced by the KanR gene in the cassette introduced from the outside by homologous recombination mechanism, consequently the subject on the genome The gene is knocked out.

If the deleted gene is an essential gene, the transformed strain cannot form a colony in the selection medium because it is impossible to grow (FIG. 3). On the other hand, if the deleted gene is a non-essential gene, the transformed strain is able to survive without the target gene, thereby forming a colony in the selection medium (FIG. 3).

Transgenic strains lacking the spr0049 gene do not grow in kanamycin-containing selective media. In other words, the spr0049 gene is an essential gene necessary for the growth of pneumococcal, so if the gene is deleted or the abnormal function of the protein expressed therefrom has a fatal effect on the growth of pneumococcal.

Using this principle, the present invention provides an antibiotic screening method using the composition of the present invention comprising a spr0049 gene or a protein expressed therefrom as a target.

The screening method of the present invention comprises contacting the composition with a test substance, and then confirming a reaction therebetween to determine whether to inhibit the expression of the gene or to inhibit the function of the protein.

In the screening method of the present invention, confirmation of the reaction between the composition containing the spr0049 gene and the test substance may use conventional methods used to confirm the reaction between DNA-DNA, DNA-RNA, and DNA-protein.

For example, a hybridization test for confirming the binding between the gene and the test substance in vitro, a method of measuring the expression rate of the gene through Northern analysis after reacting pneumococcal cells and the test substance, Alternatively, the reporter gene may be linked to the gene, introduced into the bacterial cell, reacted with the test substance, and a method of measuring the expression rate of the reporter protein may be used.

In this case, the composition of the present invention, in addition to the spr0049 gene, may include distilled water or a buffer to keep the structure of the nucleic acid stable.

In the screening method of the present invention, for confirming the reaction between the composition including the protein expressed from the spr0049 gene and the test substance, conventional methods used to confirm the reaction between the protein and the protein may be used.

For example, a method of measuring enzyme activity after reacting the protein with a test substance, or a yeast double hybrid method (yeast two-hybird) may be used.

In this case, the composition of the present invention may include, in addition to the protein expressed from spr0049, a buffer or a reaction solution which stably maintains the structure or physiological activity of the protein. In addition, the composition of the present invention may include pneumococcal pneumoniae expressing the protein, or pneumococci containing the plasmid expressing the protein under a promoter capable of controlling transcription rate for in vivo experiments.

In the screening method of the present invention, the test substance may be individual nucleic acids, proteins, other extracts, or natural products that are assumed to have antibiotic potential or randomly selected according to a conventional selection method. Chemical libraries are used to enable mass retrieval in a short time.

The chemical library is a combination of thousands and tens of thousands of chemicals having various structures on a solid chip. When the composition of the present invention is reacted with a chemical library, the chemical screening method is a conventional screening method of only 1: 1 confirmation. Compared with the antimicrobial candidates, it is possible to detect candidates that have an antibiotic effect much faster and more effectively.

The candidate material obtained by searching the chemical library acts as a leading compound in the future development of antibiotics, so that the inhibitory effect of the gene of the present invention or the protein expressed therefrom may be expressed on the leading material. By modifying and optimizing its structure, new antibiotics can be developed.

The substance thus obtained exhibits a partial or complete activity inhibitory effect on the spr0049 gene of pneumococcal or the protein expressed therefrom, and thus can inhibit the growth of pneumococcal. In addition, pneumococcal and other pathogenic bacteria similar in their gene expression structure may have a growth inhibitory effect.

Therefore, the composition of the present invention and the screening method using the same can be usefully used for the search and development of antibiotics against pathogenic bacteria, including pneumococci.

Hereinafter, the present invention will be described in more detail, but the scope of the present invention is not limited to the following examples.

EXAMPLES Confirmation of Essentiality of the spr0049 Gene Used in the Present Invention

In order to confirm the necessity of the spr0049 gene used in the present invention, the deletion cassette of the gene was prepared as follows, and after deletion of the spr0049 gene of pneumococcus, the effect was observed.

1) Preparation of deletion cassette of spr0049 gene

A deletion cassette of the spr0049 gene and a deletion cassette of the non-essential gene spr0476 to be used as a comparison group were prepared as follows.

For cassette production, kanamycin resistance gene (KanR), and primers capable of amplifying the left side region and the right side region of the gene of interest were prepared as shown in Table 1 below.

Part to be amplified primer Sequence SEQ ID NO: KanR  Kan F  5'-aac agt gaa ttg gag ttc gtc ttg tt-3 ' 3  Kan R  5'-gct ttt tag aca tct aaa tct agg ta-3 ' 4 spr0049 left  spr0049 L-F  5'-accgtgtcatctatctaggcgct-3 ' 5  spr0049 L-R  5'-gacgaactccaattcactgttcaggacctacacttgctccatgc-3 ' 6 Right side  spr0049 R-F  5'-agatttagatgtctaaaaagccttcctgcaggccatgacaatgc-3 ' 7 spr0049 R-R  5'-gcttgactgtaaccatggtctgc-3 ' 8 spr0476 left  spr0476 L-F  5'-catcagtggaaggaatggttgacc-3 ' 9 spr0476 L-R  5'-gacgaactccaattcactgttatctacccacaagagcttga-3 ' 10 Right side spr0476 R-F  5'-agatttagatgtctaaaaagccatgaaaagcgtcgtttgac-3 ' 11 spr0476 R-R  5'-gttgcgattgcgtccacctcctca-3 ' 12

In this case, the reverse primer of the left side region includes 21 nucleotide sequences (5'-GACGAACTCCAATTCACTGTT-3 ') which is identical to the promoter region of the 5' side of the kanR gene, and the forward primer of the right side region is 3 of the kanR gene. It was designed to include 21 nucleotide sequences ('5'-AGATTTAGATGTCTAAAAAGC-3') identical to the terminal.

PCR reactions using the primers were repeated 30 times under conditions of 1 minute at 94 ° C, 1 minute at 55 ° C, and 1 minute at 72 ° C.

In order to connect the three kinds of primary PCR products thus obtained into one polynucleotide, secondary PCR was performed.

Since the 21 nucleotide sequences of the left region PCR product of the gene of interest have the same nucleotide sequence as the promoter region of the kanR gene, complementary strands can be synthesized by PCR. In addition, since 21 nucleotide sequences of the right region PCR product of the gene of interest are identical to the 3 'terminal region of the kanR gene, complementary strands can be synthesized by PCR.

Using this principle, secondary PCR was performed using primers 3 and 6 on three PCR products obtained in the primary PCR reaction. The secondary PCR reaction was repeated 30 times under conditions of 1 minute at 94 ° C, 1 minute at 55 ° C, and 2 minutes at 72 ° C.

As a result, the kanR gene was inserted into the original target gene region, and the target gene was deleted, and the left and right portions of the target gene were finally obtained as a cassette.

2) Knockout of Target Gene Using Gene Deletion Cassette

Each of the genes of interest was knocked out by transforming pneumococci using the gene deletion cassette prepared in 1) as follows.

In this example, pneumococcal D39 strain and R6 strain were selected as strains to be used for transformation.

The strains, which were stored frozen, were inoculated in Todd-Hewitt agar medium containing 0.5% yeast extract, and the bacteria were cultured in an incubator maintained at 5-10% CO ₂ . Each strain was incubated overnight in 10 ml agar medium, and then 5 ml of this culture solution was added to 45 ml fresh agar medium and incubated at 37 ° C until the absorbance at 600 nm was 0.25. At this time, the incubation time was about 4-5 hours. Glycerol (glycerol) was added to the bacterial culture to a final concentration of 10%, and each aliquot was aliquoted and frozen in a mixture of dry ice and ethanol, and stored at -80 ° C until the transformation experiment.

Pneumococcal transformation was performed as follows.

First, 1 μg of DNA and 200 μl of transformed cells were diluted in a ratio of 1:10 in a transformation medium (Infant-Hewitt medium containing 0.5% yeast extract, 0.2% BSA, and 0.01% calcium chloride). It was. After diluting the solution for about 1 hour at 37 ° C., 100 ng / ml peptide pheromone Csp-1 (EMRLSKFFRDFILQRKK) was added. Thereafter, incubated at 37 ° C. for about 3 hours, plated on a selective medium containing 400 μg / ml kanamycin, and incubated at 37 ° C. for 48 hours.

3) Confirmation of the essentiality of the spr0049 gene

In order to confirm the essentiality of the spr0049 gene, it was observed whether the strains of 2) grow in the kanamycin-containing medium.

As a result, in the case of the bacteria transformed with the PCR product from which spr0049 was deleted, no colonies were produced (FIG. 4, left). Therefore, it can be seen that spr0049 is an essential gene essential for the survival of pneumococci.

On the other hand, in the case of bacteria transformed with the PCR product which deleted the non-essential gene spr0476 as a control group, hundreds of colonies were observed (FIG. 4, right).

In order to confirm that the genome of these colonies was actually replaced by the KanR gene instead of spr0476, several colonies were randomly selected and confirmed to be knocked out. As a result, the KanR gene was detected in both the transformed pneumococcal D39 and R6 strains. The insertion was confirmed (FIG. 5).

Therefore, using the spr0049 gene as a target may be a good way to develop new antibiotics.

The spr0049 gene and the protein expressed therefrom contained in the composition for screening of the present invention are essential for the survival of pneumococci.

Therefore, the screening method of the present invention can be usefully used for the search and development of effective antibiotics against pneumococci and, moreover, pathogenic bacteria.

Figure 1 is a diagram showing the manufacturing process of the cassette for deleting the spr0049 gene of pneumococcal.

Figure 2 is a diagram showing the process of deleting the spr0049 gene of pneumococci through transformation.

Figure 3 is a diagram showing the process of confirming the deletion of essential genes by culturing the pneumococcal transforming strains in which a specific gene is deleted in a selective medium.

4 is a diagram showing that the pneumococcal transforming strain lacking the spr0049 gene did not grow in the selection medium.

5 is a diagram showing that the deletion cassette was correctly inserted in the pneumococcal transforming strain which deleted the spr0476 gene, which is a non-essential gene.

<110> SONG, Jae Hoon <120> Antibiotics screening method using Streptococcus pneumoniae spr0049 gene or its protein product as a target <130> 03P-12 <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 543 <212> DNA <213> Streptococcus pneumoniae <220> <221> gene (222) (1) .. (543) <223> Streptococcus pneumoniae spr0049 gene <400> 1 atgaaaaaaa tagcggtttt tgcctctggt aatggctcaa attttcaggt gattgccgaa 60 gaatttccag tggagtttgt cttttcagac catcgtgatg cctatgtgct tgagcgtgca 120 aagcagctcg gcgttctgtc ctatgctttt gaactcaagg agtttgagag caagacagac 180 tacgaagcag cccttgtcga actcttggaa gaacaccaga ttgacttggt ttgcctagca 240 ggctacatga aaatcgttgg accaacctta ttgtcggctt atgaaggtcg gattgtcaac 300 attcatccag cctacttgcc agaatttcca ggagctcatg ggattgagga tgcttggaat 360 gctggcgtgg gtcagtctgg tgtgaccatt cactgggtgg attcgggtgt ggatacaggc 420 caggtcatca aacaggttcg tgtgccacga ctagctgatg ataccattga cagatttgaa 480 gctcgcatcc atgaagcaga gtacaggttg tatccggaag tagtgaaggc tctatttaca 540 gat 543 <210> 2 <211> 181 <212> PRT <213> Streptococcus pneumoniae <220> <221> CHAIN (222) (1) .. (181) <223> 5-phospho ribosylglycinamide transformylase 1 <400> 2 Met Lys Lys Ile Ala Val Phe Ala Ser Gly Asn Gly Ser Asn Phe Gln 1 5 10 15 Val Ile Ala Glu Glu Phe Pro Val Glu Phe Val Phe Ser Asp His Arg 20 25 30 Asp Ala Tyr Val Leu Glu Arg Ala Lys Gln Leu Gly Val Leu Ser Tyr 35 40 45 Ala Phe Glu Leu Lys Glu Phe Glu Ser Lys Thr Asp Tyr Glu Ala Ala 50 55 60 Leu Val Glu Leu Leu Glu Glu His Gln Ile Asp Leu Val Cys Leu Ala 65 70 75 80 Gly Tyr Met Lys Ile Val Gly Pro Thr Leu Leu Ser Ala Tyr Glu Gly 85 90 95 Arg Ile Val Asn Ile His Pro Ala Tyr Leu Pro Glu Phe Pro Gly Ala 100 105 110 His Gly Ile Glu Asp Ala Trp Asn Ala Gly Val Gly Gln Ser Gly Val 115 120 125 Thr Ile His Trp Val Asp Ser Gly Val Asp Thr Gly Gln Val Ile Lys 130 135 140 Gln Val Arg Val Pro Arg Leu Ala Asp Asp Thr Ile Asp Arg Phe Glu 145 150 155 160 Ala Arg Ile His Glu Ala Glu Tyr Arg Leu Tyr Pro Glu Val Val Lys 165 170 175 Ala Leu Phe Thr Asp 180 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplifying KanR gene <400> 3 aacagtgaat tggagttcgt cttgtt 26 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplifying KanR gene <400> 4 gctttttaga catctaaatc taggta 26 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplifying left flanking region of spr0049 gene <400> 5 accgtgtcat ctatctaggc gct 23 <210> 6 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplifying left flanking region of spr0049 gene <400> 6 gacgaactcc aattcactgt tcaggaccta cacttgctcc atgc 44 <210> 7 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplifying right flanking region of spr0049 gene <400> 7 agatttagat gtctaaaaag ccttcctgca ggccatgaca atgc 44 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplifying right flanking region of spr0049 gene <400> 8 gcttgactgt aaccatggtc tgc 23 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplifying left flanking region of spr0476 gene <400> 9 catcagtgga aggaatggtt gacc 24 <210> 10 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplifying left flanking region of spr0476 gene <400> 10 gacgaactcc aattcactgt tatctaccca caagagcttg a 41 <210> 11 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplifying right flanking region of spr0476 gene <400> 11 agatttagat gtctaaaaag ccatgaaaag cgtcgtttga c 41 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplifying right flanking region of spr0476 gene <400> 12 gttgcgattg cgtccacctc ctca 24

Claims (9)

Composition for screening antibiotics comprising spr0049 gene of Streptococcus pneumoniae having the nucleotide sequence of SEQ ID NO: 1 delete delete Composition for antibiotic screening comprising a protein having the amino acid sequence of SEQ ID NO: 2, expressed from the spr0049 gene of pneumococcal having the nucleotide sequence of SEQ ID NO: 1 The composition of claim 4, wherein the protein is 5-phospho ribosylglycinamide transformylase 1 (5-phospho ribosylglycinamide transformylase 1). delete delete Antibiotic screening method comprising the following steps; 1) a first step of using the composition of any one of claims 1, 4 and 5 as a target material, the contact of the composition with the test material; 2) a second step of confirming the reaction between them to determine whether the activity of inhibiting the expression of the gene or inhibiting the function of the protein 10. The method of claim 8, wherein the antibiotic screening method is characterized by screening a chemical library.