CN108823193B

CN108823193B - Efficient streptococcus pneumoniae chimeric lyase, and mutant and application thereof

Info

Publication number: CN108823193B
Application number: CN201810751086.XA
Authority: CN
Inventors: 危宏平; 杨航
Original assignee: Wuhan Institute of Virology of CAS
Current assignee: Wuhan Institute of Virology of CAS
Priority date: 2018-07-10
Filing date: 2018-07-10
Publication date: 2021-06-29
Anticipated expiration: 2038-07-10
Also published as: CN108823193A

Abstract

The invention relates to the technical field of genetic engineering, in particular to a high-efficiency streptococcus pneumoniae chimeric lyase, a mutant and an application thereof. The invention adopts a gene splicing means to construct a brand-new chimeric lyase ClyJ, and constructs mutants ClyJ-1, ClyJ-2 and ClyJ-3 thereof by means of gene fragment insertion and deletion. The ClyJ and the mutant thereof have good stability and can effectively kill streptococcus pneumoniae in vitro and in vivo; ClyJ and the mutant thereof can be well expressed in Escherichia coli BL21(DE3), and the high-efficiency cleavage activity of the ClyJ on Streptococcus pneumoniae suggests that the ClyJ has the potential capability of preventing and treating Streptococcus pneumoniae infection. Therefore, the ClyJ and the mutant thereof can be used independently or used as additives to be compatible with reagents and solutions in different forms, are used for controlling streptococcus pneumoniae and treating infection caused by the streptococcus pneumoniae, and have wide application prospect.

Description

Efficient streptococcus pneumoniae chimeric lyase, and mutant and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a high-efficiency streptococcus pneumoniae chimeric lyase, a mutant and an application thereof.

Background

Streptococcus pneumoniae (Streptococcus pneumoniae) is a type of gram-positive bacteria with a capsule, belongs to conditioned pathogens, and is widely distributed in the nasopharynx of humans and animals. Infection with streptococcus pneumoniae causes a range of invasive diseases, the most common of which are acquired pneumonia, bacteremia and meningitis. In addition, streptococcus pneumoniae can cause other mucosal infectious diseases such as acute otitis media, sinusitis, bronchitis and the like due to air transmission. Due to the overuse of antibiotics, multi-drug resistant streptococcus pneumoniae isolates continue to emerge worldwide, posing serious challenges to clinical treatment. Plus as many as 92 serotypes of streptococcus pneumoniae, the vaccines currently developed are not completely effective against all serotypes of streptococcus pneumoniae. Therefore, the development of novel antibacterial drugs against streptococcus pneumoniae is of great significance.

The phage lyase is a kind of peptidoglycan hydrolase expressed late after double-stranded DNA phage infects host, and is used for hydrolyzing the cell wall of host bacteria to release progeny phage. More and more studies have shown that lytic enzymes can be used alone or in combination with antibiotics to treat infections caused by gram-positive bacteria in vitro as well as in animal infection models. Therefore, the lyase molecules with protein properties are expected to be a substitute of antibiotics for controlling drug-resistant bacterial infection, and part of lyase drugs enter clinical research.

Not only natural lyase has wide application prospect, but also chimeric lyase with better enzymology property can be obtained by a functional domain recombination mode, and more successful examples are available. However, previous practice has also shown that not any arbitrary combination of 2 lyase domains can achieve a better functioning lyase. In addition, single and multiple point mutations in the lyase may alter one enzyme's properties, such as activity and thermostability.

Some streptococcus pneumoniae lyases have been reported to have bactericidal activity and stability, but the quantity is small, and some physicochemical properties cannot meet the requirement of actual control of streptococcus pneumoniae infection. Therefore, the development and modification of lyase enzymes with better properties and application potential are of great importance.

Disclosure of Invention

The ClyJ and the mutant thereof have good stability and can kill the streptococcus pneumoniae efficiently in vitro and in vivo. The recombinant protein ClyJ and the mutant thereof can be well expressed in Escherichia coli BL21(DE3), and the high-efficiency cleavage activity of the ClyJ and the mutant thereof on Streptococcus pneumoniae suggests that the ClyJ and the mutant thereof have potential capability of preventing and treating Streptococcus pneumoniae infection.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the invention firstly provides a high-efficiency streptococcus pneumoniae chimeric lyase ClyJ, and the amino acid sequence of the high-efficiency streptococcus pneumoniae chimeric lyase ClyJ is shown as SEQ ID No. 1.

The nucleotide sequence of the encoding gene of the high-efficiency streptococcus pneumoniae chimeric lyase ClyJ is shown as SEQ ID NO. 2.

The second purpose of the invention is to provide a preparation method of the high-efficiency streptococcus pneumoniae chimeric lyase ClyJ, which comprises the following specific steps:

(1) construction of recombinant expression vector for chimeric lyase ClyJ: cloning a target gene of the chimeric lyase ClyJ to a pET28b (+) vector to construct an expression vector pET28b-ClyJ of the ClyJ;

(2) transformation of expression vector pET28 b-ClyJ: transforming a host bacterium escherichia coli BL21(DE3) by using an expression vector pET28b-ClyJ, culturing the transformed bacterium on a bacterial culture medium, and screening a high-expression strain after a transformant appears;

(3) expression purification of ClyJ: and (3) selecting a single colony in the high-expression strain, inoculating the single colony in a culture medium for induction culture, collecting the induced thallus, crushing, separating, purifying and identifying to obtain the chimeric lyase ClyJ.

Further, the target gene of the chimeric lyase ClyJ in the step (1) comprises a CHAP functional domain of a PlyC catalytic domain of the lyase and a cell wall binding domain of ACQ35-gp20, wherein the amino acid sequence of the cell wall binding domain of the ACQ35-gp20 is shown as SEQ ID No.3, and the nucleotide sequence thereof is shown as SEQ ID No. 4.

The third purpose of the invention is to provide the application of the high-efficiency streptococcus pneumoniae chimeric lyase ClyJ, and the streptococcus pneumoniae chimeric lyase ClyJ is applied to animals and humans for controlling and killing streptococcus pneumoniae in vitro and in vivo.

Furthermore, the streptococcus pneumoniae chimeric lyase ClyJ is applied to preparation of in vivo medicines for resisting streptococcus pneumoniae infection.

The fourth purpose of the invention is to provide a mutant of the high-efficiency streptococcus pneumoniae chimeric lyase ClyJ, which comprises ClyJ-1, ClyJ-2 and ClyJ-3, wherein the amino acid sequence of the ClyJ-1 is shown as SEQ ID NO.5, and the nucleotide sequence thereof is shown as SEQ ID NO. 6; the ClyJ-2 amino acid sequence is shown as SEQ ID NO.7, and the nucleotide sequence is shown as SEQ ID NO. 8; the ClyJ-3 amino acid sequence is shown as SEQ ID NO.9, and the nucleotide sequence is shown as SEQ ID NO. 10.

The fifth purpose of the invention is to provide a preparation method of a mutant of the high-efficiency streptococcus pneumoniae chimeric lyase ClyJ, which comprises the following specific steps:

(1) construction of recombinant expression vector for chimeric lyase ClyJ mutant: using the ClyJ gene as a template, and carrying out PCR amplification to obtain full-length ClyJ-1, ClyJ-2 and ClyJ-3 genes; respectively cloning target genes of ClyJ-1, ClyJ-2 and ClyJ-3 into pET28b (+) vectors to construct expression vectors pET28b-ClyJ-1, pET28b-ClyJ-2 and pET28 b-ClyJ-3;

(2) transformation of the expression vector: respectively transforming host bacteria escherichia coli BL21(DE3) by the expression vectors pET28b-ClyJ-1, pET28b-ClyJ-2 and pET28b-ClyJ-3, respectively, culturing three kinds of transformation bacteria on a bacterial culture medium, and respectively screening high-expression strains of the three kinds of transformation bacteria after transformants appear;

(3) expression purification of chimeric lyase ClyJ mutant: selecting corresponding single colonies in the three mutant high-expression strains, respectively inoculating the single colonies to a culture medium for induction culture, collecting induced thalli, ultrasonically crushing, separating, purifying and identifying to obtain chimeric lyase ClyJ mutants ClyJ-1, ClyJ-2 and ClyJ-3.

A final object of the invention is to propose the use of mutants of the highly potent Streptococcus pneumoniae chimeric lyase ClyJ, which are ClyJ-1, ClyJ-2 and ClyJ-3, for killing Streptococcus pneumoniae in vitro and in vivo in animals and humans.

Furthermore, the Streptococcus pneumoniae chimeric lyase ClyJ mutants ClyJ-1, ClyJ-2 and ClyJ-3 are applied to preparation of in vivo medicines for resisting Streptococcus pneumoniae infection.

The invention has the beneficial effects that:

compared with the prior art, the efficient streptococcus pneumoniae chimeric lyase ClyJ is constructed by adopting a gene splicing method, and the cell wall binding domain is a new cell wall binding domain verified by the inventor experiment. And further constructs mutants of ClyJ-1, ClyJ-2 and ClyJ-3 by means of gene fragment insertion and deletion. The ClyJ and the mutant thereof disclosed by the invention have good stability and can kill streptococcus pneumoniae in vitro and in vivo with high efficiency. ClyJ and the mutant thereof can be well expressed in Escherichia coli BL21(DE3), and the high-efficiency cleavage activity of the ClyJ on Streptococcus pneumoniae suggests that the ClyJ has the potential capability of preventing and treating Streptococcus pneumoniae infection. Therefore, the ClyJ and the mutant thereof can be used independently or used as additives to be compatible with reagents and solutions in different forms, are used for controlling streptococcus pneumoniae and treating infection caused by the streptococcus pneumoniae, and have wide application prospect.

Drawings

FIG. 1 shows the results of EGFP-GPB in example 2 after the induction expression and purification of E.coli strain, where M is a standard molecular weight marker.

FIG. 2 shows the results of the examination of the binding of EGFP-GPB to Streptococcus pneumoniae in example 3, wherein the left side shows the fluorescence microscopic image of the binding of the bacterial cells to EGFP, and the right side shows the fluorescence microscopic image of the binding of the bacterial cells to EGFP-GPB.

FIG. 3 shows the result of PCR verification of the ClyJ encoding gene in example 4, where M is a standard molecular weight marker.

FIG. 4 shows the result of PCR verification of the gene encoding the ClyJ mutant in example 4, wherein M is a standard molecular weight marker.

FIG. 5 shows the result of purification of ClyJ and its mutant induced in E.coli in example 4, where M is standard molecular weight marker.

FIG. 6 is a graphical representation of the killing effect of ClyJ against S.pneumoniae NS26 over time in example 5, wherein PBS indicates the trend of OD600 over time after S.pneumoniae mixing with buffer; the solid line indicated by ClyJ is the trend of OD600 over time after streptococcus pneumoniae incorporation with ClyJ.

FIG. 7 is a graph of the results of ClyJ killing Streptococcus pneumoniae NS26 in example 6 at various NaCl concentrations, with the ordinate indicating the relative activity of ClyJ in killing Streptococcus pneumoniae in the presence of various NaCl.

FIG. 8 is the results of ClyJ killing activity against Streptococcus pneumoniae NS26 at various pH's in example 7, with the ordinate indicating the relative activity of ClyJ in killing Streptococcus pneumoniae at various pH's.

FIG. 9 is the results of the activity of ClyJ in example 8 at various EDTA concentrations to kill Streptococcus pneumoniae NS26, the ordinate indicating the relative activity of ClyJ in the presence of various EDTA concentrations to kill Streptococcus pneumoniae.

FIG. 10 is a graph of the results of ClyJ killing Streptococcus pneumoniae NS26 in example 9 at various temperatures, with the ordinate indicating the relative activity of ClyJ killing Streptococcus pneumoniae at various temperatures.

FIG. 11 is a graph showing the results of the induction of resistance by ClyJ and penicillin G in example 10, and the results of the detection of the resistance of Streptococcus pneumoniae to ClyJ and penicillin G during 8 consecutive passages are shown, in which the MICs of antibiotics to the cultured strain are, from left to right, the MICs of ClyJ to NS26, of penicillin G to NS26, of ClyJ to NS63, and of penicillin G to NS63, respectively.

Fig. 12 is a graph showing the results of the protective effect of ClyJ in the mouse infection model in example 11.

FIG. 13 is a graph showing the results of comparing the activity of ClyJ against other lytic enzymes to kill Streptococcus pneumoniae NS26 in example 12.

FIG. 14 is a graph showing the results of comparison of the activity of ClyJ and its mutants against Streptococcus pneumoniae NS26 in example 13.

Detailed Description

The following examples are shown to illustrate certain embodiments of the invention in detail and should not be construed as limiting the scope of the invention. The present disclosure may be modified from materials, methods, and reaction conditions at the same time, and all such modifications are intended to be within the spirit and scope of the present invention.

Through the analysis of the catalytic domain and the cell structure domain amino acid sequences of the streptococcal phage lyase, the invention designs and artificially constructs a novel streptococcus pneumoniae chimeric lyase ClyJ and a mutant thereof. The methods used in the following examples are conventional experimental methods unless otherwise specified. The primers used in the experiment are all provided by Wuhan Tianyihui biology technology Limited company, and the sequencing is all completed by Wuhan Tianyihui biology technology Limited company.

Example 1: synthesis of the cell wall-binding domain GPB of ACQ35_ gp20

The ACQ35-gp20 gene of streptococcal phage SpSL1 was synthesized in the complete sequence of Nanjing Kingsri Biotechnology, Inc., and the synthetic sequence was packaged into pUC57 plasmid. The ACQ35-gp20 gene is used as a template, gp20-F/gp20-R is used as a primer, and the cell wall binding domain GPB of ACQ35-gp20 is obtained by amplification, wherein the amino acid sequence of the GPB is shown as SEQ ID NO.3, and the nucleotide sequence of the GPB is shown as SEQ ID NO. 4. The primers and restriction site information used in the cloning construction were as follows:

SEQ ID NO.11 gp20-F:5-tataggatccgttgatccgtatccgtatctg-3BamHI

SEQ ID NO.12 gp20-R:5-tatactcgagtttggtggtaatcagaccgtcc-3XhoI

the reaction system for PCR amplification of the gene fragment is as follows:

the PCR amplification procedure was as follows:

1)94 ℃ for 5 min; 2)94 ℃, 30sec, 62 ℃, 45sec, 72 ℃, 45sec, 30 cycles; 3)72 ℃ for 10 min.

Example 2: EGFP-GPB and construction and expression of EGFP

1. The ACQ35-gp20 gene of streptococcal phage SpSL1 was synthesized in the complete sequence of Nanjing Kingsri Biotechnology, Inc., and the synthetic sequence was packaged into pUC57 plasmid. And (2) amplifying to obtain a sequence of the enhanced green fluorescent protein EGFP by taking a pEGFP plasmid as a template and an EGFP-F/EGFP-R as a primer, wherein the primers and enzyme cutting site information used in construction of the clone are as follows:

SEQ ID NO.13 EGFP-F:5-tataccatggtgagcaagggcgaggagc-3NcoI

SEQ ID NO.14 EGFP-R:5-tataggatcccttgtacagctcgtccatgc-3BamHI

the reaction system for PCR amplification of the gene fragment is as follows:

the PCR amplification procedure was as follows:

2. The above fragment and GPB, the cell wall binding domain of ACQ35-gp20 in example 1, were digested with the corresponding enzymes and ligated into pET28b (+) vector digested with NcoI and XhoI to obtain expression vector pET28b-EGFP-GPB for EGFP-GPB. The EGFP fragment was digested with the corresponding enzymes and ligated into pET28b (+) vector digested with NcoI and BamHI to obtain the expression vector pET28b-EGFP of EGFP. Then, the ligation products were transformed into E.coli BL21(DE3), and after transformants appeared, high-expression strains of the electrotransport cells were selected.

3. Selecting single colonies from EGFP-GPB and EGFP high-expression strains, inoculating the single colonies into 5mL of liquid LB culture medium containing 50 mu g/mL Kan, and carrying out shaking culture at 37 ℃ for overnight at 200 r/min; according to the following steps of 1: 100 percent of the culture medium is transferred into 200mL LB culture medium containing 50 mu g/mL Kan, cultured at 200r/min and 37 ℃ to OD₆₀₀And (3) adding IPTG with the final concentration of 0.2mM when the temperature is 0.6-0.8, cooling for 20min at room temperature, performing 120r/min, and performing induction for 16-20 h at 16 ℃. The induced bacteria were collected and sonicated, the supernatant was applied to a Ni affinity column, and the impurity proteins were eluted with 20mM and 60mM imidazole, respectively, and the 250mM imidazole peak was collected and dialyzed overnight in PBS. The dialyzed protein was detected by 12% SDS-PAGE electrophoresis, as shown in FIG. 1. The results show that both EGFP-GPB and EGFP can be expressed in Escherichia coli in a soluble way.

Example 3: verification of EGFP-GPB binding to streptococcus pneumoniae

Overnight cultured streptococci including Streptococcus pneumoniae NS26(Spn NS26), Streptococcus pneumoniae NS63(Spn NS63), Streptococcus agalactiae (Sag), Streptococcus dysgalactiae (Sdy), Streptococcus pyogenes (Spy), and Streptococcus mutans (Smu) were each washed once with PBS and resuspended to OD600 ═ 0.5-0.8, and then 0.01ml of the treated bacterial suspension was incubated with 20. mu.M EGFP-GPB protein at 37 ℃ for 1 hour. Unbound protein was removed by 3 washes with PBS and the cells were observed under a fluorescent microscope. Meanwhile, the EGFP protein was used as a control protein for the binding assay under the same assay conditions. The results obtained are shown in FIG. 2. The results show that EGFP-GPB can specifically bind to the streptococcus pneumoniae bacterial strain.

Example 4: construction and expression of lyase ClyJ and mutant thereof

1. A chimeric lyase ClyT gene synthesized by Nanjing Kingsler Biotechnology Limited is used as a template (the ClyT gene synthesis method is shown in Hang Yang, Sara B. linden, Jung Wang, Junping Yu, Daniel C.Nelson & HongPing We, A Chimeolys with extended-specific polypeptide host transformed by an induced lysine-based polypeptide screening method, Scientific reporting volume 5, and oligonucleotide number 17257(2015)), PyCAC-F/PyCAC-R is used as a primer to amplify a sequence of a catalytic domain of the lyase PyCAC, and ACQ35-gp20 gene is used as a template, gp20-F/gp20-R is used as a primer to amplify a cell wall binding domain of ACQ 57-gp 20. The primers and restriction site information used in the cloning construction were as follows:

SEQ ID NO.15 PlyCAC-F:5-tataccatgggcatggcagcaaatctgg-3NcoI

SEQ ID NO.16 PlyCAC-R:5-tataggatcctttgaaggtaatcaggcccgtc-3BamHI

SEQ ID NO.11 gp20-F:5-tataggatccgttgatccgtatccgtatctg-3BamHI

SEQ ID NO.12 gp20-R:5-tatactcgagtttggtggtaatcagaccgtcc-3XhoI

the reaction system for PCR amplification of the gene fragment is as follows:

the PCR amplification procedure was as follows:

2. The above fragments were digested with the corresponding enzymes and ligated into pET28b (+) vector digested with NcoI and XhoI to obtain expression vector pET28b-ClyJ for ClyJ. Then, it was transformed into E.coli BL21(DE3), and after the transformant appeared, a high-expression strain of the transgenic cell was selected. PCR verification is carried out on the transformant by taking PlyCAC-F/gp20-R as a primer, and the full-length ClyJ gene can be obtained through amplification. After the reaction, the PCR product was subjected to 1% agarose gel electrophoresis, and the purification result was as shown in FIG. 3, which corresponded to 972bp, the target size.

3. Construction of ClyJ mutants

(1) Using ClyJ gene as a template and PlyCAC-F/ClyJ-1-R1 as a primer to amplify a catalytic domain segment ClyJ-1C of lyase ClyJ; the binding domain segment ClyJ-1B of the lyase ClyJ is obtained by amplification by using the ClyJ gene as a template and ClyJ-1-F2/gp20-R as primers. And (3) taking products obtained by two times of amplification as a template, and taking PlyCAC-F/gp20-R as a primer to obtain full-length ClyJ-1 by amplification.

(2) Using the ClyJ gene as a template and ClyJ-2-F1/ClyJ-2-R1 as primers to amplify a catalytic domain segment ClyJ-2C of the lyase ClyJ; and (3) amplifying a binding domain segment ClyJ-2B of the lyase ClyJ by using the ClyJ gene as a template and ClyJ-1-F2/gp20-R as primers. And (3) taking products obtained by two times of amplification as a template, and taking PlyCAC-F/gp20-R as a primer to obtain full-length ClyJ-2.

(3) Using ClyJ gene as a template and PlyCAC-F/ClyJ-3-R1 as a primer to amplify a catalytic domain segment ClyJ-3C of lyase ClyJ; the binding domain segment ClyJ-3B of the lyase ClyJ is obtained by amplification by using the ClyJ gene as a template and ClyJ-3-F2/gp20-R as primers. And (3) taking products obtained by two amplifications as a template, and taking PlyCAC-F/gp20-R as a primer to amplify to obtain full-length ClyJ-3.

(4) The above mutant fragments were digested with the corresponding enzymes and ligated into pET28b (+) vector digested with NcoI and XhoI to obtain expression vectors pET28b-ClyJ-1, pET28b-ClyJ-2 and pET28b-ClyJ-3, respectively, for ClyJ mutants. Then, the strains are respectively transformed into escherichia coli BL21(DE3) for induction expression, and after transformants appear, three high-expression strains of the electrotransfer cells are screened out. PCR verification is carried out on the transformant by taking PlyCAC-F/gp20-R as a primer, each mutant gene of ClyJ can be obtained through amplification, 1% agarose gel electrophoresis is carried out on a PCR product after the reaction is finished, and the purification result is shown in figure 4.

(5) The primers and restriction site information used in the cloning construction were as follows:

SEQ ID NO.17 ClyJ-1-R1:5-tataggatccggaggatcctcctttgaaggtaatc-3

SEQ ID NO.18 ClyJ-1-F2:5-tataggatccgttgatccgtatccgtatctg-3

SEQ ID NO.19 ClyJ-2-F1:5-tataccatgggcatggcagcaaatctggcaaacgcacaagcaca-3NcoI

SEQ ID NO.20 ClyJ-2-R1:5-tataggatccggaggatcctccggatccggaggatcctcc-3

SEQ ID NO.21 ClyJ-3-R1:5-cccagggatccggaggatcctccttt-3

SEQ ID NO.22 ClyJ-3-F2:5-aaaggaggatcctccggatccctgg-3

(6) the reaction system for PCR amplification of the gene fragment is as follows:

(7) the PCR amplification procedure was as follows:

4. Expression purification of ClyJ and its mutants

Selecting a single colony from a high-expression strain of ClyJ and a mutant thereof, inoculating the single colony in 5mL of liquid LB culture medium containing 50 mu g/mL Kan, and carrying out shake culture at 37 ℃ at 200r/min for overnight; according to the following steps of 1: 100 percent of the culture medium is transferred into 200mL LB culture medium containing 50 mu g/mL Kan, cultured at 200r/min and 37 ℃ to OD₆₀₀And (3) adding IPTG with the final concentration of 0.2mM when the temperature is 0.6-0.8, cooling for 20min at room temperature, performing 120r/min, and performing induction for 16-20 h at 16 ℃. Collection of InductionThe cells were sonicated, the supernatant was applied to a Ni affinity column, and the impurity proteins were eluted with 20mM and 60mM imidazole, respectively, and the 250mM imidazole peak was collected and dialyzed overnight in PBS. The dialyzed protein was detected by 12% SDS-PAGE electrophoresis, as shown in FIG. 5. The results show that ClyJ and the mutant thereof can be expressed in Escherichia coli in a soluble way.

Example 5: verification that ClyJ kills streptococcus pneumoniae NS26

The overnight cultures of Streptococcus pneumoniae were collected by centrifugation, washed once with PBS, and then dissolved in PBS. 0.01ml of ClyJ was mixed with 0.19ml of the above-mentioned bacterial suspension, and the change in the absorbance at 600nm of the mixture was monitored by a microplate reader. Meanwhile, a negative control was prepared by mixing 0.01ml of PBS buffer with 0.19ml of the above-mentioned Streptococcus pneumoniae. The resulting cleavage curve is shown in FIG. 6. The results show that ClyJ rapidly cleaves streptococcus pneumoniae leading to a rapid decrease in absorbance at 600 nm.

Example 6: verification that ClyJ kills streptococcus pneumoniae NS26 activity under different NaCl concentrations

The overnight Streptococcus pneumoniae cultures were collected by centrifugation, washed once with Tris-HCl and then dissolved in 20mM Tris-HCl (pH 7.4). 0.01ml of ClyJ was mixed with 0.19ml of the above-mentioned bacterial suspension, NaCl was added to the mixture to give final concentrations of 0, 50, 100, 150, 300, 500 and 750mM, and the change in absorbance at 600nm of the mixture was monitored by a microplate reader. Meanwhile, a mixture of PBS buffer and streptococcus pneumoniae was used as a negative control. The resulting cleavage curves were processed and the numbers of the decrease in OD600nm in each group over 60min were calculated to give the maximum decrease of 100% activity, to which the other groups compared the relative enzyme activity, see FIG. 7. The results showed that high concentrations of NaCl (500mM) had no significant inhibitory effect on ClyJ enzyme activity.

Example 7: verification that ClyJ kills Streptococcus pneumoniae NS26 activity at different pH values

After centrifugation and collection, the overnight culture of Streptococcus pneumoniae was washed once with PBS, then resuspended in buffers of different pH (pH3.5-pH11.5), 0.01ml of ClyJ was mixed with 0.19ml of the above-mentioned bacteria of different pH, and the change in absorbance at 600nm of the mixture was monitored by a microplate reader. Meanwhile, a mixture of PBS buffer and streptococcus pneumoniae was used as a negative control. The resulting cleavage curves were processed and the numbers of the decrease in OD600nm in each group over 60min were calculated to give the maximum decrease of 100% activity, to which the other groups compared the relative enzyme activity, see FIG. 8. The results show that ClyJ has the activity of rapidly cleaving Streptococcus pneumoniae in the pH range of 5-9.

Example 8: verification of ClyJ activity in killing streptococcus pneumoniae NS26 under different EDTA concentrations

After centrifugation and collection, the overnight culture of Streptococcus pneumoniae was washed once with PBS, and then resuspended in 2ml of PBS, and 0.1ml of ClyJ was mixed with the above-mentioned bacterial solution to prepare a mixture. And (3) taking 7 parts of mixed solution, wherein each part of mixed solution is 0.1ml, adding EDTA (ethylene diamine tetraacetic acid) to the mixed solution until the final concentration is 0, 50, 100, 200, 300, 400 and 500 mu M, and monitoring the change of the absorption value of the mixed solution at 600nm by using a microplate reader. Meanwhile, a mixture of PBS buffer and streptococcus pneumoniae was used as a negative control. The resulting cleavage curves were processed and the numbers of the decrease in OD600nm in each group over 60min were calculated to give the maximum decrease of 100% activity, to which the other groups compared the relative enzyme activity, see FIG. 9. The results show no significant effect of EDTA on the activity of ClyJ to lyse NS26 strain.

Example 9 validation of ClyJ killing of Streptococcus pneumoniae NS26 Activity at different temperatures

After the overnight culture of streptococcus pneumoniae was collected by centrifugation, it was washed once with PBS, then resuspended in 2ml PBS, 0.01ml of ClyJ was taken out and left at different temperatures for 1 hour, and then mixed with 0.19ml of the above-mentioned bacterial liquid, and the change in the absorbance at 600nm of the mixture was monitored by a microplate reader. Meanwhile, a mixed solution of buffer and streptococcus pneumoniae was used as a negative control. The resulting cleavage curves were processed and the numbers of the decrease in OD600nm in each group over 60min were calculated to give the maximum decrease of 100% activity, to which the other groups compared the relative enzyme activity, see FIG. 10. The results show that ClyJ has higher activity of cracking the NS26 strain below 37 ℃.

Example 10 verification of the results of ClyJ-induced drug resistance

Streptococcus pneumoniae NS26 and NS63 were cultured to log phase and then transferred to fresh medium containing 1/32 × MIC ClyJ, and after 12 hours of culture, the bacterial fluid was divided into two portions, one portion was used for inoculation into fresh medium containing ClyJ at the previous 2-fold concentration, and the other portion was used for broth dilution to test the MIC of ClyJ for the cultured strain. The above steps were repeated until the culture concentration of ClyJ reached 4 × MIC, and the ratio of the actual MIC to the original MIC during each passage was calculated. Meanwhile, penicillin G was used as a parallel control. The results obtained are shown in FIG. 11. The results showed that none of the S.pneumoniae developed resistance to ClyJ but significant resistance to penicillin G during the 8 consecutive days (ClyJ concentrations ranged from 1/32 XMIC to 4 XMIC) of passage.

Example 11 verification of the protective Effect of ClyJ in mouse infection model

BALB/C female mice were intraperitoneally injected with a lethal dose of Streptococcus pneumoniae NS26 for 5-6 weeks, and ClyJ was intraperitoneally injected at different concentrations after 1 hour to observe the survival rate of the mice, and the results obtained are shown in FIG. 12. As can be seen from the figure, PBS can not kill the streptococcus pneumoniae NS26 in the mouse, and the intraperitoneal injection of ClyJ with low concentration can obviously improve the survival rate of the mouse infected by the streptococcus pneumoniae.

Example 12 validation of ClyJ in comparison to other lytic enzymes for killing Streptococcus pneumoniae NS26 Activity

The overnight cultures of Streptococcus pneumoniae were collected by centrifugation, washed once with PBS, and then dissolved in PBS. 0.01ml of ClyJ and 0.01ml of other lytic enzymes with the same molar concentration are respectively mixed with 0.19ml of the bacterial liquid, the mixture is incubated at 37 ℃ for 1 hour, and the survival rate of the bacteria in a treatment group is calculated by a dilution plate. The survival rate of the resulting different treatment groups is shown in figure 13. The result shows that the bactericidal activity of ClyJ on streptococcus pneumoniae is higher than that of ClyR and Cpl-1, and the bactericidal activity of ClyJ and Cpl-1 have a synergistic effect.

Example 13 verification of comparison of ClyJ and its mutants against Streptococcus pneumoniae NS26 Activity

The overnight cultures of Streptococcus pneumoniae were collected by centrifugation, washed once with PBS, and then dissolved in PBS. 0.01ml of ClyJ and 0.01ml of ClyJ mutant with the same molar concentration are respectively mixed with 0.19ml of the bacterial liquid, the mixture is incubated for 1 hour at 37 ℃, and the survival rate of the bacteria in a treatment group is calculated by a dilution plate. The survival rates of the resulting different treatment groups are shown in figure 14. The result shows that the bactericidal activity of the mutant ClyJ-3 on the streptococcus pneumoniae is obviously higher than that of ClyJ, and the activities of the mutants ClyJ-1 and ClyJ-2 are equivalent to that of ClyJ.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Sequence listing

<110> Wuhan Virus institute of Chinese academy of sciences

<120> high-efficiency streptococcus pneumoniae chimeric lyase, and mutant and application thereof

<160> 22

<170> SIPOSequenceListing 1.0

<210> 1

<211> 324

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Ala Ala Asn Leu Ala Asn Ala Gln Ala Gln Val Gly Lys Tyr Ile

1 5 10 15

Gly Asp Gly Gln Cys Tyr Ala Trp Val Gly Trp Trp Ser Ala Arg Val

20 25 30

Cys Gly Tyr Ser Ile Ser Tyr Ser Thr Gly Asp Pro Met Leu Pro Leu

35 40 45

Ile Gly Asp Gly Met Asn Ala His Ser Ile His Leu Gly Trp Asp Trp

50 55 60

Ser Ile Ala Asn Thr Gly Ile Val Asn Tyr Pro Val Gly Thr Val Gly

65 70 75 80

Arg Lys Glu Asp Leu Arg Val Gly Ala Ile Trp Cys Ala Thr Ala Phe

85 90 95

Ser Gly Ala Pro Phe Tyr Thr Gly Gln Tyr Gly His Thr Gly Ile Ile

100 105 110

Glu Ser Trp Ser Asp Thr Thr Val Thr Val Leu Glu Gln Asn Ile Leu

115 120 125

Gly Ser Pro Val Ile Arg Ser Thr Tyr Asp Leu Asn Thr Phe Leu Ser

130 135 140

Thr Leu Thr Gly Leu Ile Thr Phe Lys Gly Ser Val Asp Pro Tyr Pro

145 150 155 160

Tyr Leu Ala Lys Trp Gly Val Ser Arg Glu Gln Phe Lys Arg Asp Ile

165 170 175

Glu Asn Gly Leu Gly Ala Glu Thr Gly Trp Gln Lys Asn Asp Lys Gly

180 185 190

Tyr Trp Tyr Val His Ser Asp Gly Ser Tyr Pro Lys Asp Lys Phe Glu

195 200 205

Lys Ile Asn Gly Thr Trp Tyr Tyr Phe Asp Gly Ser Gly Tyr Met Leu

210 215 220

Ala Asp Arg Trp Lys Lys His Ser Asp Gly Asn Trp Tyr Tyr Phe Asp

225 230 235 240

Gln Ser Gly Glu Met Ala Thr Gly Trp Lys Lys Ile Val Glu Lys Trp

245 250 255

Tyr Tyr Phe Asp Val Glu Gly Ala Met Lys Thr Gly Trp Val Lys Tyr

260 265 270

Lys Asp Thr Trp Tyr Tyr Leu Asp Ser Lys Gly Gly Asn Met Val Ser

275 280 285

Asn Glu Phe Val Arg Ala Gly Gln Gly Trp Tyr Tyr Ile Lys Pro Asp

290 295 300

Gly Ser Met Ala Asp Lys Pro Glu Phe Thr Val Glu Pro Asp Gly Leu

305 310 315 320

Ile Thr Thr Lys

<210> 2

<211> 972

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggcagcaa atctggcaaa cgcacaagca caagtgggca aatacatcgg cgacggtcaa 60

tgttacgcat gggttggttg gtggagcgcg cgtgtgtgcg gctatagcat ttcttacagt 120

accggtgatc cgatgctgcc gctgattggc gacggtatga acgcccattc gatccacctg 180

ggctgggatt ggagcattgc aaacaccggt atcgtcaatt atccggtcgg cacggtgggt 240

cgtaaagaag acctgcgcgt gggtgcaatc tggtgtgcaa ccgcttttag cggtgccccg 300

ttctataccg gccagtacgg tcatacgggc attatcgaat cctggtcaga taccacggtg 360

accgttctgg aacaaaacat tctgggctct ccggttatcc gtagtaccta tgacctgaat 420

acgtttctgt ccaccctgac gggcctgatt accttcaaag gatccgttga tccgtatccg 480

tatctggcaa aatggggcgt tagccgcgaa cagttcaaac gcgatattga aaacggtctg 540

ggcgcagaaa ccggttggca gaaaaacgac aaaggctact ggtatgtcca ttctgacggt 600

agctatccga aagacaaatt cgagaaaatc aacggcacct ggtactactt cgacggtagc 660

ggctatatgc tggcagatcg ttggaaaaaa cacagcgacg gcaactggta ttactttgat 720

cagtccggcg aaatggcgac cggctggaaa aagattgtcg agaaatggta ctacttcgac 780

gtcgaaggcg caatgaaaac cggctgggtc aaatacaaag acacctggta ctatctggat 840

agcaaaggcg gcaacatggt cagcaacgaa tttgttcgcg caggtcaggg ctggtattat 900

atcaaaccgg acggtagcat ggcggataaa ccggaattta ccgttgaacc ggacggtctg 960

attaccacca aa 972

<210> 3

<211> 169

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Val Asp Pro Tyr Pro Tyr Leu Ala Lys Trp Gly Val Ser Arg Glu Gln

1 5 10 15

Phe Lys Arg Asp Ile Glu Asn Gly Leu Gly Ala Glu Thr Gly Trp Gln

20 25 30

Lys Asn Asp Lys Gly Tyr Trp Tyr Val His Ser Asp Gly Ser Tyr Pro

35 40 45

Lys Asp Lys Phe Glu Lys Ile Asn Gly Thr Trp Tyr Tyr Phe Asp Gly

50 55 60

Ser Gly Tyr Met Leu Ala Asp Arg Trp Lys Lys His Ser Asp Gly Asn

65 70 75 80

Trp Tyr Tyr Phe Asp Gln Ser Gly Glu Met Ala Thr Gly Trp Lys Lys

85 90 95

Ile Val Glu Lys Trp Tyr Tyr Phe Asp Val Glu Gly Ala Met Lys Thr

100 105 110

Gly Trp Val Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Ser Lys Gly

115 120 125

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

130 135 140

Tyr Ile Lys Pro Asp Gly Ser Met Ala Asp Lys Pro Glu Phe Thr Val

145 150 155 160

Glu Pro Asp Gly Leu Ile Thr Thr Lys

165

<210> 4

<211> 507

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gttgatccgt atccgtatct ggcaaaatgg ggcgttagcc gcgaacagtt caaacgcgat 60

attgaaaacg gtctgggcgc agaaaccggt tggcagaaaa acgacaaagg ctactggtat 120

gtccattctg acggtagcta tccgaaagac aaattcgaga aaatcaacgg cacctggtac 180

tacttcgacg gtagcggcta tatgctggca gatcgttgga aaaaacacag cgacggcaac 240

tggtattact ttgatcagtc cggcgaaatg gcgaccggct ggaaaaagat tgtcgagaaa 300

tggtactact tcgacgtcga aggcgcaatg aaaaccggct gggtcaaata caaagacacc 360

tggtactatc tggatagcaa aggcggcaac atggtcagca acgaatttgt tcgcgcaggt 420

cagggctggt attatatcaa accggacggt agcatggcgg ataaaccgga atttaccgtt 480

gaaccggacg gtctgattac caccaaa 507

<210> 5

<211> 328

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Ala Ala Asn Leu Ala Asn Ala Gln Ala Gln Val Gly Lys Tyr Ile

1 5 10 15

Gly Asp Gly Gln Cys Tyr Ala Trp Val Gly Trp Trp Ser Ala Arg Val

20 25 30

Cys Gly Tyr Ser Ile Ser Tyr Ser Thr Gly Asp Pro Met Leu Pro Leu

35 40 45

Ile Gly Asp Gly Met Asn Ala His Ser Ile His Leu Gly Trp Asp Trp

50 55 60

Ser Ile Ala Asn Thr Gly Ile Val Asn Tyr Pro Val Gly Thr Val Gly

65 70 75 80

Arg Lys Glu Asp Leu Arg Val Gly Ala Ile Trp Cys Ala Thr Ala Phe

85 90 95

Ser Gly Ala Pro Phe Tyr Thr Gly Gln Tyr Gly His Thr Gly Ile Ile

100 105 110

Glu Ser Trp Ser Asp Thr Thr Val Thr Val Leu Glu Gln Asn Ile Leu

115 120 125

Gly Ser Pro Val Ile Arg Ser Thr Tyr Asp Leu Asn Thr Phe Leu Ser

130 135 140

Thr Leu Thr Gly Leu Ile Thr Phe Lys Gly Gly Ser Ser Gly Ser Val

145 150 155 160

Asp Pro Tyr Pro Tyr Leu Ala Lys Trp Gly Val Ser Arg Glu Gln Phe

165 170 175

Lys Arg Asp Ile Glu Asn Gly Leu Gly Ala Glu Thr Gly Trp Gln Lys

180 185 190

Asn Asp Lys Gly Tyr Trp Tyr Val His Ser Asp Gly Ser Tyr Pro Lys

195 200 205

Asp Lys Phe Glu Lys Ile Asn Gly Thr Trp Tyr Tyr Phe Asp Gly Ser

210 215 220

Gly Tyr Met Leu Ala Asp Arg Trp Lys Lys His Ser Asp Gly Asn Trp

225 230 235 240

Tyr Tyr Phe Asp Gln Ser Gly Glu Met Ala Thr Gly Trp Lys Lys Ile

245 250 255

Val Glu Lys Trp Tyr Tyr Phe Asp Val Glu Gly Ala Met Lys Thr Gly

260 265 270

Trp Val Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Ser Lys Gly Gly

275 280 285

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

290 295 300

Ile Lys Pro Asp Gly Ser Met Ala Asp Lys Pro Glu Phe Thr Val Glu

305 310 315 320

Pro Asp Gly Leu Ile Thr Thr Lys

325

<210> 6

<211> 984

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atggcagcaa atctggcaaa cgcacaagca caagtgggca aatacatcgg cgacggtcaa 60

tgttacgcat gggttggttg gtggagcgcg cgtgtgtgcg gctatagcat ttcttacagt 120

accggtgatc cgatgctgcc gctgattggc gacggtatga acgcccattc gatccacctg 180

ggctgggatt ggagcattgc aaacaccggt atcgtcaatt atccggtcgg cacggtgggt 240

cgtaaagaag acctgcgcgt gggtgcaatc tggtgtgcaa ccgcttttag cggtgccccg 300

ttctataccg gccagtacgg tcatacgggc attatcgaat cctggtcaga taccacggtg 360

accgttctgg aacaaaacat tctgggctct ccggttatcc gtagtaccta tgacctgaat 420

acgtttctgt ccaccctgac gggcctgatt accttcaaag gaggatcctc cggatccgtt 480

gatccgtatc cgtatctggc aaaatggggc gttagccgcg aacagttcaa acgcgatatt 540

gaaaacggtc tgggcgcaga aaccggttgg cagaaaaacg acaaaggcta ctggtatgtc 600

cattctgacg gtagctatcc gaaagacaaa ttcgagaaaa tcaacggcac ctggtactac 660

ttcgacggta gcggctatat gctggcagat cgttggaaaa aacacagcga cggcaactgg 720

tattactttg atcagtccgg cgaaatggcg accggctgga aaaagattgt cgagaaatgg 780

tactacttcg acgtcgaagg cgcaatgaaa accggctggg tcaaatacaa agacacctgg 840

tactatctgg atagcaaagg cggcaacatg gtcagcaacg aatttgttcg cgcaggtcag 900

ggctggtatt atatcaaacc ggacggtagc atggcggata aaccggaatt taccgttgaa 960

ccggacggtc tgattaccac caaa 984

<210> 7

<211> 334

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Met Ala Ala Asn Leu Ala Asn Ala Gln Ala Gln Val Gly Lys Tyr Ile

1 5 10 15

Gly Asp Gly Gln Cys Tyr Ala Trp Val Gly Trp Trp Ser Ala Arg Val

20 25 30

Cys Gly Tyr Ser Ile Ser Tyr Ser Thr Gly Asp Pro Met Leu Pro Leu

35 40 45

Ile Gly Asp Gly Met Asn Ala His Ser Ile His Leu Gly Trp Asp Trp

50 55 60

Ser Ile Ala Asn Thr Gly Ile Val Asn Tyr Pro Val Gly Thr Val Gly

65 70 75 80

Arg Lys Glu Asp Leu Arg Val Gly Ala Ile Trp Cys Ala Thr Ala Phe

85 90 95

Ser Gly Ala Pro Phe Tyr Thr Gly Gln Tyr Gly His Thr Gly Ile Ile

100 105 110

Glu Ser Trp Ser Asp Thr Thr Val Thr Val Leu Glu Gln Asn Ile Leu

115 120 125

Gly Ser Pro Val Ile Arg Ser Thr Tyr Asp Leu Asn Thr Phe Leu Ser

130 135 140

Thr Leu Thr Gly Leu Ile Thr Phe Lys Gly Gly Ser Ser Gly Ser Gly

145 150 155 160

Gly Ser Ser Gly Ser Val Asp Pro Tyr Pro Tyr Leu Ala Lys Trp Gly

165 170 175

Val Ser Arg Glu Gln Phe Lys Arg Asp Ile Glu Asn Gly Leu Gly Ala

180 185 190

Glu Thr Gly Trp Gln Lys Asn Asp Lys Gly Tyr Trp Tyr Val His Ser

195 200 205

Asp Gly Ser Tyr Pro Lys Asp Lys Phe Glu Lys Ile Asn Gly Thr Trp

210 215 220

Tyr Tyr Phe Asp Gly Ser Gly Tyr Met Leu Ala Asp Arg Trp Lys Lys

225 230 235 240

His Ser Asp Gly Asn Trp Tyr Tyr Phe Asp Gln Ser Gly Glu Met Ala

245 250 255

Thr Gly Trp Lys Lys Ile Val Glu Lys Trp Tyr Tyr Phe Asp Val Glu

260 265 270

Gly Ala Met Lys Thr Gly Trp Val Lys Tyr Lys Asp Thr Trp Tyr Tyr

275 280 285

Leu Asp Ser Lys Gly Gly Asn Met Val Ser Asn Glu Phe Val Arg Ala

290 295 300

Gly Gln Gly Trp Tyr Tyr Ile Lys Pro Asp Gly Ser Met Ala Asp Lys

305 310 315 320

Pro Glu Phe Thr Val Glu Pro Asp Gly Leu Ile Thr Thr Lys

325 330

<210> 8

<211> 1002

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atggcagcaa atctggcaaa cgcacaagca caagtgggca aatacatcgg cgacggtcaa 60

tgttacgcat gggttggttg gtggagcgcg cgtgtgtgcg gctatagcat ttcttacagt 120

accggtgatc cgatgctgcc gctgattggc gacggtatga acgcccattc gatccacctg 180

ggctgggatt ggagcattgc aaacaccggt atcgtcaatt atccggtcgg cacggtgggt 240

cgtaaagaag acctgcgcgt gggtgcaatc tggtgtgcaa ccgcttttag cggtgccccg 300

ttctataccg gccagtacgg tcatacgggc attatcgaat cctggtcaga taccacggtg 360

accgttctgg aacaaaacat tctgggctct ccggttatcc gtagtaccta tgacctgaat 420

acgtttctgt ccaccctgac gggcctgatt accttcaaag gaggatcctc cggatccgga 480

ggatcctccg gatccgttga tccgtatccg tatctggcaa aatggggcgt tagccgcgaa 540

cagttcaaac gcgatattga aaacggtctg ggcgcagaaa ccggttggca gaaaaacgac 600

aaaggctact ggtatgtcca ttctgacggt agctatccga aagacaaatt cgagaaaatc 660

aacggcacct ggtactactt cgacggtagc ggctatatgc tggcagatcg ttggaaaaaa 720

cacagcgacg gcaactggta ttactttgat cagtccggcg aaatggcgac cggctggaaa 780

aagattgtcg agaaatggta ctacttcgac gtcgaaggcg caatgaaaac cggctgggtc 840

aaatacaaag acacctggta ctatctggat agcaaaggcg gcaacatggt cagcaacgaa 900

tttgttcgcg caggtcaggg ctggtattat atcaaaccgg acggtagcat ggcggataaa 960

ccggaattta ccgttgaacc ggacggtctg attaccacca aa 1002

<210> 9

<211> 304

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Ala Ala Asn Leu Ala Asn Ala Gln Ala Gln Val Gly Lys Tyr Ile

1 5 10 15

Gly Asp Gly Gln Cys Tyr Ala Trp Val Gly Trp Trp Ser Ala Arg Val

20 25 30

Cys Gly Tyr Ser Ile Ser Tyr Ser Thr Gly Asp Pro Met Leu Pro Leu

35 40 45

Ile Gly Asp Gly Met Asn Ala His Ser Ile His Leu Gly Trp Asp Trp

50 55 60

Ser Ile Ala Asn Thr Gly Ile Val Asn Tyr Pro Val Gly Thr Val Gly

65 70 75 80

Arg Lys Glu Asp Leu Arg Val Gly Ala Ile Trp Cys Ala Thr Ala Phe

85 90 95

Ser Gly Ala Pro Phe Tyr Thr Gly Gln Tyr Gly His Thr Gly Ile Ile

100 105 110

Glu Ser Trp Ser Asp Thr Thr Val Thr Val Leu Glu Gln Asn Ile Leu

115 120 125

Gly Ser Pro Val Ile Arg Ser Thr Tyr Asp Leu Asn Thr Phe Leu Ser

130 135 140

Thr Leu Thr Gly Leu Ile Thr Phe Lys Gly Gly Ser Ser Gly Ser Leu

145 150 155 160

Gly Ala Glu Thr Gly Trp Gln Lys Asn Asp Lys Gly Tyr Trp Tyr Val

165 170 175

His Ser Asp Gly Ser Tyr Pro Lys Asp Lys Phe Glu Lys Ile Asn Gly

180 185 190

Thr Trp Tyr Tyr Phe Asp Gly Ser Gly Tyr Met Leu Ala Asp Arg Trp

195 200 205

Lys Lys His Ser Asp Gly Asn Trp Tyr Tyr Phe Asp Gln Ser Gly Glu

210 215 220

Met Ala Thr Gly Trp Lys Lys Ile Val Glu Lys Trp Tyr Tyr Phe Asp

225 230 235 240

Val Glu Gly Ala Met Lys Thr Gly Trp Val Lys Tyr Lys Asp Thr Trp

245 250 255

Tyr Tyr Leu Asp Ser Lys Gly Gly Asn Met Val Ser Asn Glu Phe Val

260 265 270

Arg Ala Gly Gln Gly Trp Tyr Tyr Ile Lys Pro Asp Gly Ser Met Ala

275 280 285

Asp Lys Pro Glu Phe Thr Val Glu Pro Asp Gly Leu Ile Thr Thr Lys

290 295 300

<210> 10

<211> 912

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atggcagcaa atctggcaaa cgcacaagca caagtgggca aatacatcgg cgacggtcaa 60

tgttacgcat gggttggttg gtggagcgcg cgtgtgtgcg gctatagcat ttcttacagt 120

accggtgatc cgatgctgcc gctgattggc gacggtatga acgcccattc gatccacctg 180

ggctgggatt ggagcattgc aaacaccggt atcgtcaatt atccggtcgg cacggtgggt 240

cgtaaagaag acctgcgcgt gggtgcaatc tggtgtgcaa ccgcttttag cggtgccccg 300

ttctataccg gccagtacgg tcatacgggc attatcgaat cctggtcaga taccacggtg 360

accgttctgg aacaaaacat tctgggctct ccggttatcc gtagtaccta tgacctgaat 420

acgtttctgt ccaccctgac gggcctgatt accttcaaag gaggatcctc cggatccctg 480

ggcgcagaaa ccggttggca gaaaaacgac aaaggctact ggtatgtcca ttctgacggt 540

agctatccga aagacaaatt cgagaaaatc aacggcacct ggtactactt cgacggtagc 600

ggctatatgc tggcagatcg ttggaaaaaa cacagcgacg gcaactggta ttactttgat 660

cagtccggcg aaatggcgac cggctggaaa aagattgtcg agaaatggta ctacttcgac 720

gtcgaaggcg caatgaaaac cggctgggtc aaatacaaag acacctggta ctatctggat 780

agcaaaggcg gcaacatggt cagcaacgaa tttgttcgcg caggtcaggg ctggtattat 840

atcaaaccgg acggtagcat ggcggataaa ccggaattta ccgttgaacc ggacggtctg 900

attaccacca aa 912

<210> 11

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tataggatcc gttgatccgt atccgtatct g 31

<210> 12

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tatactcgag tttggtggta atcagaccgt cc 32

<210> 13

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tataccatgg tgagcaaggg cgaggagc 28

<210> 14

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tataggatcc cttgtacagc tcgtccatgc 30

<210> 15

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tataccatgg gcatggcagc aaatctgg 28

<210> 16

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tataggatcc tttgaaggta atcaggcccg tc 32

<210> 17

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tataggatcc ggaggatcct cctttgaagg taatc 35

<210> 18

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tataggatcc gttgatccgt atccgtatct g 31

<210> 19

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

tataccatgg gcatggcagc aaatctggca aacgcacaag caca 44

<210> 20

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tataggatcc ggaggatcct ccggatccgg aggatcctcc 40

<210> 21

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

cccagggatc cggaggatcc tccttt 26

<210> 22

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

aaaggaggat cctccggatc cctgg 25

Claims

1. A high efficiency streptococcus pneumoniae chimeric lyase ClyJ characterized by: the amino acid sequence is shown in SEQ ID NO. 1.

2. The gene encoding the chimeric streptococcus pneumoniae lyase ClyJ of claim 1, wherein: the nucleotide sequence is shown in SEQ ID NO. 2.

3. The method of claim 1, wherein the method comprises the steps of: the method comprises the following specific steps:

4. The method for preparing the high efficiency Streptococcus pneumoniae chimeric lyase ClyJ according to claim 3, wherein the method comprises the following steps: the target gene of the chimeric lyase ClyJ in the step (1) comprises a CHAP functional domain of a PlyC catalytic domain of the lyase and a cell wall binding domain of ACQ35_ gp20, wherein the amino acid sequence of the cell wall binding domain of the ACQ35_ gp20 is shown as SEQ ID No.3, and the nucleotide sequence is shown as SEQ ID No. 4.

5. The use of the highly potent chimeric Streptococcus pneumoniae lyase ClyJ according to claim 1 for the manufacture of a medicament for killing Streptococcus pneumoniae in vitro and in vivo in animals and humans.

6. Use according to claim 5, characterized in that: the streptococcus pneumoniae chimeric lyase ClyJ is applied to preparation of in vivo medicines for resisting streptococcus pneumoniae infection.

7. The mutant streptococcus pneumoniae chimeric lyase ClyJ of claim 1, wherein: the mutants are three mutants of ClyJ-1, ClyJ-2 and ClyJ-3, wherein the amino acid sequence of ClyJ-1 is shown as SEQ ID NO.5, and the nucleotide sequence is shown as SEQ ID NO. 6; the ClyJ-2 amino acid sequence is shown as SEQ ID NO.7, and the nucleotide sequence is shown as SEQ ID NO. 8; the ClyJ-3 amino acid sequence is shown as SEQ ID NO.9, and the nucleotide sequence is shown as SEQ ID NO. 10.

8. The mutant according to claim 7, which is prepared by the following steps:

(3) expression purification of chimeric lyase ClyJ mutant: selecting corresponding single colonies in high-expression strains of the three mutants, respectively inoculating the single colonies in a culture medium for induction culture, collecting induced thalli, ultrasonically crushing, separating, purifying and identifying to obtain chimeric lyase ClyJ mutants ClyJ-1, ClyJ-2 and ClyJ-3.

9. Use of the mutant according to claim 7 for the manufacture of a medicament for killing streptococcus pneumoniae in vitro and in vivo in animals and humans.

10. Use according to claim 9, characterized in that: the Streptococcus pneumoniae chimeric lyase ClyJ mutants ClyJ-1, ClyJ-2 and ClyJ-3 are applied to preparation of in vivo medicines for resisting Streptococcus pneumoniae infection.