CN108707185B

CN108707185B - Polypeptide for prolonging blood circulation time of bacteriophage

Info

Publication number: CN108707185B
Application number: CN201810509415.XA
Authority: CN
Inventors: 温龙平; 金佩佩; 沙锐; 张云娇
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2018-05-24
Filing date: 2018-05-24
Publication date: 2022-02-11
Anticipated expiration: 2038-05-24
Also published as: CN108707185A

Abstract

The invention discloses a polypeptide for prolonging the blood circulation time of a bacteriophage, the bacteriophage carrying the polypeptide and having the function of prolonging the blood circulation time, a method for screening the polypeptide, and applications of the polypeptide and the bacteriophage. The invention has very important significance for phage therapy.

Description

Polypeptide for prolonging blood circulation time of bacteriophage

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a polypeptide for prolonging the blood circulation time of a bacteriophage.

Background

Phage therapy is a therapeutic approach to combat bacterial infections using bacteriophages, and has been applied in clinical practice since the beginning of the 20 th century. For example, in 1919, d' Herelle attempted the use of phages to treat avian influenza (salmonella), rabbit dysentery (shigella) and bovine hemorrhagic septicemia (pasteurella), which was the first attempt to use phage therapy. In 1921, Richard Bruynoghe and Joseph Maisin used phages to treat human skin diseases caused by Staphylococcus aureus. In 1932, under the requirement of the British government, d' Herelle arrives at the epidemic area of Indian cholera to try to use bacteriophage for prevention and control, quickly control the spread of epidemic and effectively inhibit the secondary outbreak of the epidemic. In the same year, some scientific researchers in eastern Europe have developed scientific doses of phage therapy through a number of animal and human tests. Successful treatment of enterococcal infections with bacteriophages was reported by scientists in 1934. In 1930-1939, many researchers and companies commercialized phage therapy, such as Parke-Davis and Lily in the United states, began to produce phage preparations for treating staphylococci and Escherichia coli, and phage research entered into a rapid development period.

However, with the advent of antibiotics such as penicillin during world war ii, scientists in the united states and western europe have essentially abandoned intensive research on phage therapy and began to invest in the flood of antibiotic research. Since 1950-1980, there are few research articles on phage therapy. The problem of antibiotic resistance has increased after 1980. In fact, the problem of resistance to antibiotics has now seriously impacted global public health. Statistically, two million people in the united states are affected by antibiotic-resistant infections each year and directly result in about 23000 deaths each year. Therefore, the development of a new antibacterial therapy has become more and more urgent. The bacteriophage has incomparable advantages of antibiotics in the aspect of treatment of bacterial infection, particularly multiple drug-resistant bacterial infection as bacterial virus, scientists aim at returning the attention to bacteriophage treatment again, an effective antibiotic substitution treatment scheme is expected to be obtained from the bacteriophage, and the increasingly intensified global drug resistance problem is controlled.

Despite the great potential in addressing the ever-worsening crisis of antibiotic resistance, phage therapy currently presents a number of limiting factors. Among the major problems hindering their clinical use are the rapid clearance of the phage in the blood circulation after administration and the short residence time of the blood, which makes it ineffective for the target treatment. Thus, engineering therapeutic bacteriophages to extend blood circulation time is a focus of research and would be a more effective phage therapy to treat existing bacterial infections or prevent bacterial reinfection. In this regard, the strategies reported in the related studies to prolong the blood circulation time of phages include: escape from complement-mediated inactivation by naturally occurring mutations, chemical modification of the phage surface with polyethylene glycol, and by altering the phage capsid proteins, and the like. Nevertheless, the above scheme still has its limitations, and applying more innovative strategies to obtain long-circulating bacteriophages with better antibacterial effects is yet to be broken through.

Phage display has been used as a powerful technique for screening polypeptide sequences for certain properties. Typically phage libraries (containing phage displaying >109 different sequence polypeptides) are panned in vivo or in vitro to rapidly obtain polypeptides with specific interactions, such as material interactions, tissue targeting, organ targeting, and barrier penetration. In particular, in vivo phage display technology has been successfully applied to the screening of tumor vascular targeting peptides, blood brain barrier penetrating peptides, transdermal peptides, and the like.

Disclosure of Invention

The inventors of the present application utilized the phage display peptide library ph.d of New England Biolabs.^TMC7C was subjected to a series of screening, and finally, a short peptide having the function of prolonging the blood circulation time of the phage was obtained.

Accordingly, in a first aspect of the invention, there is provided a polypeptide which extends the blood circulation time of a bacteriophage.

According to the invention, the polypeptide for prolonging the blood circulation time of the bacteriophage contains an amino acid sequence shown in SEQ ID NO. 1, 2 or 3 or an analogue thereof.

In a second aspect of the invention, there is provided the use of the polypeptide for increasing the blood circulation time of a bacteriophage.

According to the present invention, the polypeptide for prolonging the blood circulation time of bacteriophage can be used for preparing bacteriophage with the function of prolonging the blood circulation time.

In a third aspect of the present invention, there is provided a bacteriophage having a function of prolonging a blood circulation time.

According to the present invention, the phage having the function of prolonging blood circulation time carries the polypeptide for prolonging blood circulation time of phage.

In a fourth aspect of the present invention, there is provided a use of the bacteriophage with a function of prolonging blood circulation time.

According to the invention, the bacteriophage with the function of prolonging the blood circulation time can be used for preparing bacteriophage antibacterial drugs.

In a fifth aspect of the invention, a pharmaceutical composition is provided.

According to the present invention, the pharmaceutical composition comprises at least one of the polypeptides for prolonging the blood circulation time of bacteriophage, and at least one pharmaceutically active protein, drug or material in an amount effective for treating a disease.

In a sixth aspect of the invention, a bacteriophage antibacterial agent is provided.

According to the present invention, the bacteriophage antibiotic agent comprises the bacteriophage having the function of prolonging the blood circulation time.

In a seventh aspect of the invention, a time-lapse antibacterial bacteriophage is provided.

According to the invention, the time-delay antibacterial phage carries the polypeptide for prolonging the blood circulation time of the phage and the nucleotide sequence corresponding to the antibacterial protein BglII.

In an eighth aspect of the present invention, there is provided a method for screening a polypeptide having an ability to prolong the circulation time in a phage, comprising the steps of:

a. injecting the phage library into the animal body through tail vein;

b. recovering the phage particles from the circulatory system, organs, tissues and cells of said animal or human after a suitable time;

c. amplifying the recovered phage for the next round of in vivo screening;

d. repeating steps a to c at least twice;

e. and (3) selecting monoclonals from the recovered phage, respectively amplifying, verifying the time delay capability, and sequencing to obtain the polypeptide with the capability of prolonging the in-vivo circulation time of the phage.

The invention screens the polypeptide capable of obviously prolonging the in vivo circulation time of the phage by the phage display technology, and proves that the function has sequence specificity, and the action principle is achieved by combining with the platelet; the phage displaying the polypeptide is engineered by means of genetic engineering to express the antibacterial protein BglII at the same time, so that the phage with time delay function and antibacterial activity is obtained, and the phage has better antibacterial activity in vitro and in vivo of rats.

The polypeptide for prolonging the in vivo circulation time of the phage has the functions of prolonging the blood circulation time and delaying and resisting bacteria, and has very important significance for phage therapy.

Drawings

Figure 1 shows the screening process of delayed phage BCP1, enrichment of long circulating phage during in vivo screening, mean and SEM (standard error) of the number of phage per ml of blood 48h after three rounds of tail vein injection of the enriched product into rats (n-3,. p <0.01,. p < 0.001).

FIG. 2 shows a comparison of the in vivo delay capacity of long circulating phage monoclonals, equivalent (1X 10)¹¹) Three BCP phages and random phage SC were mixed and injected into rats, 30 plaques were randomly picked at 0h and 72h, respectively, and the number of repetitions of the four phages was determined by sequencing (n-3).

FIG. 3 shows the long-circulating nature of the delayed phages, equivalent (1X 10)¹¹) BCP1 and SC phages were injected into different rats through the tail vein, mean and SEM of the number of phages per ml of blood at different time points, respectively.

FIG. 4 shows the mean and SEM of the number of two phages per ml of blood at different time points in rats injected with BCP1 and REW phage 1:1 mixed tail vein (n.gtoreq.3,. p <0.05,. p <0.01,. p < 0.001).

FIG. 5 shows the sequence specificity of the time-delayed phages, equivalent (1X 10)¹¹) Different phages BCP1, SC and three BCP1 mutant phages were injected into different rats tail vein separately and the mean value and SEM of the number of phages per ml of blood at different time points were examined.

Fig. 6 shows the mean value and SEM of the number of phages per ml of blood at different time points (number of blue white spots, n-3, p <0.001) from BCP1 and three BCP1 mutant phages mixed with the REW phages at an equal ratio of 1:1, respectively, and injected into rats in tail vein.

FIG. 7 shows complement system mediated inactivation of phage, BCP1 and SC phage (1X 10)⁹) In vitro co-incubation with plasma treated at 37 deg.C or 56 deg.C in advance, and detection of in vivo phage in plasma at different time pointsAverage number of volumes and SEM (n ═ 3).

FIG. 8 shows a preliminary validation of the binding of BCP1 phage to blood cells, equivalent (1X 10)⁸) The number of phages in blood at different time points (mean ± SEM, n ═ 3,. times.p) was measured by tail vein injection of rats with BCP1 and SC phages previously bound to PBC and unbound BCP1 and SC phages<0.01，***p< 0.001)。

FIGS. 9A-9C are graphs showing the distribution of different phages in plasma and blood cells over time; in each of FIGS. 9A, 9B and 9C, the same numbers (1X 10) correspond to the same numbers, respectively¹¹) Distribution of SC, BCP1 and TB2 phages in plasma and PBC at different time points after injection of the phages, respectively, into rats (n-3,. p)<0.05，**p<0.01，***p <0.001)。

FIGS. 10A-10C evaluate the distribution of BCP1, SC, and TB2 phages for three components of finely separated blood; wherein, FIG. 10A is the distribution rule of BCP1 bacteriophage in blood detected by in vivo experiment; FIG. 10B is a graph showing the distribution of SC phage in blood in vivo; fig. 10C shows the distribution of TB2 phage in blood (n-3) measured in vivo.

FIG. 11 shows that BCP and SC short peptides affect the binding of phage to blood cells in vitro, 1X 10⁹BCP1 phage was incubated with 1ml of rat whole blood for 1.5h at 37 ℃ in vitro to assess the binding of phage to different blood cells in the presence (500. mu.g/ml) and absence of BCP or SC short peptide.

Fig. 12 shows the in vitro antibacterial results of the four phages BCP1, SC, BGL and BCP1-BGL at an MOI of 10 (n ═ 3).

Fig. 13 shows the levels of endotoxin released during in vitro antibacterial treatment of BCP1, SC, BGL, and BCP1-BGL four phages (n ═ 3).

Fig. 14A and 14B show the mean SEM of the number of 4 phages in blood and abdominal cavity over time (n-3); wherein, FIG. 14A shows the same amounts (1X 10) of four phages BCP1, SC, BGL and BCP1-BGL¹¹) Tail vein injection of rats, number of blood phages (mean ± SEM) at different time points; FIG. 14B shows four phages BCP1, SC, BGL and BCP1-BGLEqual amount (1X 10)¹¹) Tail vein injection of rats, number of phages in the abdominal cavity at different time points (mean ± SEM, n ═ 3).

FIGS. 15A and 15B show the in vivo antibacterial effect of BCP 1-BGL; wherein, FIGS. 15A and 15B are each the same amount (1X 10)¹¹) BCP1, SC, BGL and BCP1-BGL four phages were injected into rats in tail vein respectively, and infected bacteria were injected into the abdominal cavity 18h later (1 × 10)⁸) After 5h, the number of bacteria in the ascites and liver (mean ± s.e.m., n ═ 3, × p)<0.01，***p<0.001)。

Figure 16 shows the IFN- γ content in the plasma of rats treated with different combinations (mean ± s.e.m., n ═ 3,. p <0.05,. p < 0.01).

Fig. 17A and 17B show the ALT (17A) and AST (17B) content in the plasma of rats treated in different combinations (mean ± s.e.m., n ═ 3, × <0.01, × < 0.001).

Fig. 18 shows graphs demonstrating the effect of cellular infiltration by HE staining rat liver tissue sections after different combination treatments.

Detailed Description

The present invention will be more readily understood by reference to the following detailed description of the preferred embodiments thereof and the examples included therein. However, before describing the polypeptides, compounds, compositions, and methods involved in the present invention, it is to be understood that the present invention is not limited to use with a particular polypeptide, protein, drug, or other material, nor with a particular cell type, host cell, conditions, and methods, etc. Of course, changes may be made in that regard, and such a wide variety of modifications will become apparent to those skilled in the art. Also, it is to be understood that the terminology used herein is for the purpose of describing particular devices only, and is not intended to be limiting. It is also to be understood that the terms "a" and "an" as used in the specification and claims may be one or more, depending on the context in which the term is used. Thus, for example, reference to "a cell" can mean that at least one cell is utilized.

Described herein is a method for using phage display libraries to screen for displayed peptides that improve the extended blood residence time. As used herein, "phage display library" refers to a collection of genetically engineered phage that are displayed on their surface by the expression of a series of polypeptides. A "displayed peptide" consists of a contiguous sequence of amino acids that includes a protein displayed on the surface of a bacteriophage. The term "polypeptide" refers to a chain of at least three amino acids linked together by peptide bonds, which chain may be linear, branched, circular, or a combination thereof.

The study object of the phage library is a large number of displayed peptides expressing different amino acids by genetic engineering methods, and after using the object, phage particles in the study object are collected and identified. Herein, the "subject" refers to a mammal such as a mouse, a rabbit, a human, etc. Phage particles are typically collected from one or more organ tissues, cells, blood, urine, or other various body fluids, and in a preferred embodiment, phage particles are injected into an animal through the tail vein and residual phage particles are collected from the blood circulation over a period of time, and it is those phage particles that contain polypeptides that extend the circulation time in the blood.

The peptide sequences expressed on the surfaces of phage particles collected from experimental animals can be isolated by solid medium plates, i.e., in cilia positive bacteria, phage particles can be propagated in vitro on biological plates. The bacteria are not lysed by the phage, but instead secrete multiple copies of the phage to display the inserted peptide. The amino acid sequence of the inserted display peptide is determined by the DNA sequence of the phage genome corresponding to the inserted peptide.

In the context of the present invention, the polypeptide analogue refers to other short peptides with time-delay function obtained by phage screening, or polypeptides obtained by adding, deleting, changing the sequence or replacing amino acids of the polypeptide sequence shown in SEQ ID NO. 1.

The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

Example 1: screening of specific delayed short peptides

In this example, the library used to screen for specific delayed short peptides was provided by New England Biolabs, a heptapeptide library containing disulfide bonds (ph.d).^TM-C7C). The random fragments in this library are flanked by cysteine residues that can be oxidized during phage assembly to form disulfide bonds, thus forming a cyclic peptide that interacts with the target. This library contained over two billion clones. The random peptide in the library was at the amino terminus of the small coat protein pIII, so five copies were expressed per phage particle. And Ph.D.^TMThe position of the phage expression random sequence in the C7C library was preceded by alanine-cysteine. A short linker sequence (glycine-serine) is contained between the random peptide and the pIII protein. (Ph.D.^TM-C7C phage peptide display kit, http:// www.neb.com/nebecom/products E8120. asp).

Will be 1 × 10¹¹Each phage was dissolved in 300. mu.l of physiological saline, caudal vein was injected into 150g SD rats, and whole blood was collected in vivo after 48 hours and spread on LB plates containing X-gal (5-bromo-4-chloro-3-indoyl-b-D-galactoside) and IPTG (isopropyl-b-D-thiogalacside), and 300 plaques were obtained from 6 rats. All of them were picked up and amplified to 1X 10¹¹And continuously carrying out the second round of in vivo screening on the phages, carrying out the third round of screening on 500 plaques obtained after 96 hours by mixed amplification, and prolonging the time to 120 hours.

FIG. 1 shows the number of phages in whole blood of rats after 48h of three rounds of screening, and it can be seen that the number of phages in each round of the same time point is greatly increased as the screening is carried out, and the phages in the rats of 48h in the third round can reach 3.5X 10⁴And (4) respectively.

Randomly selecting 30 plaques obtained from the second round and the third round of screening for sequencing to obtain a display peptide sequence. After the series of tests such as time delay capability and the like, the following three phage display peptides are finally selected:

BCP1：CNARGDMHC(SEQ ID NO:1)；

BCP2：CIVRGDNVC(SEQ ID NO:2)；

BCP6：CVPRGDMHC(SEQ ID NO:3)。

example 2: delay capability test

Respectively taking 1 × 10¹¹Phage carrying the displayed peptides BCP1, BCP2 and BCP6 obtained in example 1 were mixed with random phage SC (displayed peptide sequence: CNATLPHQC, SEQ ID NO:4), injected into rat body at tail vein, and blood was plated at 0h (3min) and 72h, respectively, to detect phage.

FIG. 2 shows the number of phages at 0h and 72 h. As can be seen, the number of the four phages was similar at 0h, but the SC phages were not available at 72h, the proportion of BCP1 was the highest, and the numbers of BCP6 and BCP2 were the next. It can be seen that the phages carrying the three displayed peptides obtained in example 1 all had a significant ability to prolong the circulation time of the phages in the blood of rats.

To further determine the time-delayed effect of BCP1, we injected equal amounts of BCP1 and SC phage into rats separately and measured the amount of phage in the blood at different time points, and the results are shown in fig. 3.

Meanwhile, BCP1 was compared with the white spot phage REW (display peptide sequence: CTARSPWIC, SEQ ID NO:5) 1:1 were mixed and injected into the same rat, and the ratio of both in the blood was measured at different time points, and the results are shown in FIG. 4.

The results in fig. 3 show that the number of SC phages decreased faster over time relative to BCP1 phages, which were not detected at 48h, but BCP1 phages were still detectable at 144h, further confirming the delayed ability of BCP1 phages in blood.

The results in FIG. 4 show that the ratio of BCP1/REW reaches 100 at 12h and exceeds 1000 at 36h, thus excluding the influence factor of animal individual difference, the bacteriophage still has better ability to prolong blood circulation time. .

Example 3: verification that the time-delay function of the phage has sequence specificity

During the screening process, we found that phages containing the RGD sequence appeared more and more frequently with the enrichment of phages round by round, so to determine the importance of RGD in the phage-displayed peptide sequence obtained in example 1, we constructed three BCP1 phages, specifically as follows:

TB 1: CRNHDMGAC (SEQ ID NO:6, scrambled BCP1 sequence);

TB 2: CNAAGAMHC (SEQ ID NO:7, RGD mutated to AGA);

TB 3: CAARGDAAC (SEQ ID NO:8, RGD retained, the remaining four amino acids were mutated to A).

Then 1 × 10¹¹Individual TB1, TB2, TB3 and BCP1, SC phages were injected into different rats respectively or into the same rat after mixing with REW 1:1, and the amount of phages in blood was measured at different time points, and the results are shown in fig. 5.

The results in fig. 5 show that the blood residence time of TB1 and TB2 is comparable to SC, barely detectable in the blood at 48h, suggesting that it has lost its time-delay capability; TB3 also partially lost its latency capability.

The results in FIG. 6 show that the three mutant phages and BCP1 were similar to the REW ratio at 0h, while after 48h, the remaining phages, except BCP1, were similar to the REW ratio in vivo and no longer have the ability to delay.

The above results show that RGD is necessary for the time-delayed function of phage in blood, but the light with RGD is not enough to support its time-delayed function.

Example 4: the delayed function of the phage is not achieved by the resistance to the complement system

Plasma obtained by separating whole blood was incubated at 56 ℃ or 37 ℃ for 10minutes (treatment at 56 ℃ can inactivate the complement system in blood). Then 1 × 10⁹The individual BCP1 and SC phages were incubated separately in a mixture with the pre-treated plasma and the number of phages was measured after different periods of time, the results are shown in FIG. 7.

The results of FIG. 7 show that the number of phages decreased in a time-gradient in the state where the complement system was not inactivated, and that this rate of decrease was slowed down when the complement system was inactivated, suggesting that the complement system in the blood caused the inactivation of phages; more critically, however, the reduction in the number of BCP1 and SC phages did not show a significant difference, suggesting that the delayed function of BCP1 phage was not achieved by resistance to the complement system.

Example 5: the phage can delay time by interacting with blood cells

Respectively mixing 1 × 10⁸The amount of phage in the blood was measured by tail vein injection of BCP1 and SC phage previously bound to PBC in blood cells and equal amounts of unbound BCP1 and SC phage into rats, respectively, and blood was drawn at different time points, and the results are shown in FIG. 8.

Further, in order to examine which component of blood the BCP1 phage binds to, 1X 10 phage, respectively¹¹BCP1, SC and TB2 phages were injected into rats, and 1ml of blood was taken at different time points with 100. mu.l of an anticoagulant CPD (16 mM citric acid, 90mM sodium citrate, 16mM NaH)₂PO₄142mM dextrose, pH 7.4), left at room temperature for 15min, centrifuged at 25 ℃ at 2000g for 10min, and the number of phages in the two fractions was determined by coarse separation of blood cells from plasma, the results being shown in FIGS. 9A-9C.

The results in fig. 8 show that both SC and BCP1 phages after binding to blood cells have improved delayed ability in blood and prolonged blood retention time of BCP1 relative to phages not bound to blood cells.

FIGS. 9A, 9B and 9C show the numbers of SC, BCP1 and TB2 phages in plasma and blood cells at different time points, respectively, the number of BCP1 phages in plasma was 10 times higher than the phages bound to blood cells at 0h, and was nearly the same at 24h, but the number of phages bound to blood cells was already much higher than in plasma at 36h and continued up to 72 h.

The above results suggest that the delayed ability of BCP1 phage is achieved due to binding to blood cells.

Example 6: the phage interacts with blood platelet in blood cell to delay time

After injection of BCP1 phage into rats, 1ml of blood was taken from the heart at different time points, mixed with 100. mu.l of CPD, left to stand at room temperature for 15min, and centrifuged at 200g for 20 min. The sample is divided into three layers, wherein the first layer is a platelet enrichment region, the second layer is a leukocyte enrichment region and the third layer is a red blood cell enrichment region from top to bottom. The leukocyte layer and the erythrocyte layer can directly detect the titer which represents the phage binding amount. Platelets were obtained by centrifugation of 2000g of the first layer of supernatant for 10 min.

Further, the three components are more accurately obtained by flow sorting. Will be 1 × 10¹¹After tail vein injection of BCP1, SC or TB2 phage into rats, 1ml of blood was taken from the heart at various time points, mixed with 100. mu.l of CPD, left to stand at room temperature for 15min, and centrifuged at 200g for 20 min. The sample is divided into three layers, wherein the first layer is a platelet enrichment region, the second layer is a leukocyte enrichment region and the third layer is a red blood cell enrichment region from top to bottom. Adding 0.2M apyrase into the supernatant platelet layer to prevent platelet activation, and centrifuging at 2000g and 4 deg.C for 10min to enrich platelets. And removing the red blood cells by adding red blood cell lysate into the white blood cells in the middle layer, and then further enriching and recovering. The three Cell fractions were resuspended in PBS containing 1% FBS, CD16/32 antibody was added, and after standing on ice for 10min, labeled FITC-anti-rat CD45, PE-anti-rat Erythroid Cell and PerCP/Cy5.5-anti-mouse/rat CD42d antibody were separately recovered by sorting. The enriched and sorted cells were recovered by centrifugation at 2000g and 4 ℃ for 5min and the number of phages was measured, and the results are shown in FIG. 10, where PLT represents platelets, WBC is white blood cells and RBC is red blood cells.

The results in fig. 10A show that BCP1 phage binds more platelets to leukocytes than erythrocytes at different time points, while the results in fig. 10B and 10C show that SC and TB2 bind to blood cells in greatly reduced amounts, respectively.

Example 7: BCP1 phage interacts with platelets via display peptides

1×10⁹A single BCP1 phage was mixed with 1ml rat blood in vitro, and synthetic BCP short peptide (SEQ ID NO: ACNARGDMHCG, SEQ: 9) or SC short peptide (SEQ ID NO: ACNATLPHQCG, SEQ: 10) was added to the other two groups, and after incubation for 1.5h at 37 ℃, the amount of phage binding in the three cell fractions of blood was measured by flow sorting, and the results are shown in FIG. 11.

The results in fig. 11 show that the addition of the short BCP1 peptide significantly reduced the binding of BCP1 phage to platelets and leukocytes relative to the short SC peptide, demonstrating that the BCP1 phage was bound to platelets or leukocytes by a specific display peptide.

Example 8: time-delay antibacterial phage BCP1-BGL

The PCR product containing BglII R gene (bactericidal gene, BglII site 5 '-AGATCT-3' capable of specifically recognizing and cutting host DNA) is cut by restriction enzymes EcoR I and Hind III, the recovered fragment is connected with M13KE vector (purchased from New England Biolabs) at 16 ℃ overnight, whether BCP1 nucleic acid sequence is contained between the cutting sites of Eag I and Kpn I on M13KE vector is determined, the recombinant phage containing BCP1 nucleic acid sequence is BCP1-BGL, and the non-contained one is BGL.

Wherein, the PCR product containing BglII R gene adopts the following primers:

forward direction: 5'-CCCAAGCTTAAATTAGACCGCACTTACATAGGCG-3', respectively;

and (3) reversing: 5'-CCGGAATTCTTAATATGTCACGATTGTTCCTCTTTTCC-3', respectively;

using PMRB1 plasmid as a template, obtained by the following PCR conditions:

the middle three steps are repeated for 30 cycles.

The recombinant phage BCP1-BGL was transferred into ER2738 competent cells containing PBM1 plasmid (containing BglII M gene, which can methylate BglII site of host DNA and thus not be cut by BglII protein) for amplification and purification of phage for subsequent experiments. Wherein the PMRB1 plasmid and the PBM1 plasmid are both taught by Armin research of Vienna university.

Example 9: in vitro antibacterial ability of antibacterial phages BGL and BCP1-BGL

Escherichia coli MC4100F' cultured at OD600 to 0.2 was further cultured with BCP1, SC, BGL and BCP1-BGL at different ratios of MOI (number of Escherichia coli/number of phages) in LB medium containing 0.003mol/L IPTG while detecting the number of Escherichia coli in the culture broth at different time points with PBS as a control to verify the in vitro antibacterial ability of the 4 phages, the results of which are shown in FIG. 12.

The results in fig. 12 show that after 4h of culture at MOI of 10, the number of escherichia coli did not increase for the BGL and BCP1-BGL groups relative to the other groups, indicating that the constructed bacteriophages BGL and BCP1-BGL were antibacterial.

Example 10: in vitro antibacterial endotoxin release level of antibacterial phage BGL and BCP1-BGL

Coli MC4100F' cultured at OD600 to 0.2 was cultured in LB medium containing 0.003mol/L IPTG with PBS as a control, with four phages of BCP1, SC, BGL and BCP1-BGL added under the condition of MOI of 10. Detection by endotoxin kit (ToxinSensor)^TMChromogenic LAL endogenous Assay Kit), the Endotoxin content in the culture supernatant was measured at 0, 1, 2 and 4 hours, respectively, and the results are shown in fig. 13.

The results in fig. 13 show that at an MOI of 10, infection with the four phages did not result in an increased release of endotoxin relative to the negative control, indicating that the four phages were safe.

Example 11: in vivo delay capability of detecting antibacterial phage BGL and BCP1-BGL

Will be 1 × 10¹¹Respectively injecting BCP1, SC, BGL and BCP1-BGL phages into a rat body through tail veins, taking blood at different time points, and detecting the content of the phages in the blood; meanwhile, 10ml of physiological saline is injected into the abdominal cavity of a rat at each time point, and the quantity of the phage in the ascites is recovered and detected. The detection results are shown in fig. 14A and 14B.

FIGS. 14A and 14B show the number of phages in blood and ascites at different time points, respectively, of the four phages, and it can be seen that the phages containing BCP1-BGL of BCP1 still had a longer delay time in blood and ascites than the phages SC and BGL, indicating that the delay function of BCP1 was not destroyed by the addition of BGL.

Example 12: in vivo antibacterial ability detection of BGL and BCP1-BGL

Will be 1 × 10¹¹BCP1, SC, BGL and BCP1-BGL phages were injected into rats through tail vein, PBS was used as a control, and 1X 10 was injected into abdominal cavity after 18h⁸Coli MC4100F', 2. mu.M IPTG was injected 1h later. After 5h, the abdominal cavity was washed with 10ml of physiological saline, and ascites was recovered. Another 1g of liver was resuspended in 1ml of physiological saline and ground thoroughly. The number of E.coli in ascites and liver were measured, respectively, and the results are shown in FIGS. 15A and 15B.

The results in FIG. 15A and FIG. 15B show that BCP1-BGL phage treated group, which had both time-lapse and antibacterial abilities, had minimal amounts of E.coli in ascites and liver, indicating that they had antibacterial ability.

Example 13: detecting the effect of BCP1-BGL treatment on IFN-gamma in rat blood after bacterial infection

Will be 1 × 10¹¹BCP1, SC, BGL and BCP1-BGL phages were injected into rats through tail vein, PBS was used as a control, and 1X 10 was injected into abdominal cavity after 18h⁸Coli MC4100F' and rat injected with PBS to SC and BCP1-BGL phage as control, 2 μ M IPTG was injected after 1h, and IFN- γ content in rat plasma was detected by ELISA kit after 5h, as shown in FIG. 16.

The results in FIG. 16 show that BCP1-BGL treatment significantly reduced IFN-. gamma.levels in rat blood after bacterial infection, indicating that it has some antibacterial effect.

Example 14: effect of BCP1-BGL treatment on liver function in rats after infection with bacteria

Will be 1 × 10¹¹BCP1, SC, BGL and BCP1-BGL phages are respectively injected into a rat body through tail veins, and PBS is used as a control; intraperitoneal injection is carried out for 18h and the injection is 1X 10⁸Coli MC4100F' with PBS phage injection as control; injection of 2. mu.M IPTG 1h later; the plasma was assayed for ALT and AST content after 5h by an automated biochemical analyzer, and the results are shown in fig. 17A and 17B.

Another 1g of liver tissue was paraffin-coated and approximately 5mm sections were H & E stained, and the results are shown in FIG. 18.

The results in FIGS. 17A and 17B show that the BCP1-BGL treated group, which had both time-lapse and antibacterial capabilities, showed less elevation of ALT and AST values in blood relative to the remaining three phages after infection with bacteria, indicating less liver function destruction in the rats of this group.

The results of the liver tissue sections of fig. 18 show the same results, indicating that BCP1-BGL has some antibacterial activity in vivo.

Although the above examples describe the polypeptide for increasing the blood circulation time of bacteriophage of the present invention, the bacteriophage carrying the polypeptide, the time-lapse antibacterial bacteriophage carrying both the polypeptide and the antibacterial protein BGL, and the mechanism of action thereof in detail. However, those skilled in the art will readily appreciate that appropriate modifications thereof are possible within the scope of the technical disclosure of the present invention. For example, appropriate modifications, including deletion, addition, substitution, change of order, etc., are made to the polypeptide sequence of the present invention, and thus such modifications are also within the scope of the present invention. Furthermore, it goes without saying that the nucleotide sequences encoding these amino acid sequences also fall within the scope of the present invention.

Sequence listing

<110> university of southern China's science

<120> a polypeptide for prolonging the blood circulation time of bacteriophage

<130> 181042

<141> 2018-05-24

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9

<212> PRT

<213> phage

<400> 1

Cys Asn Ala Arg Gly Asp Met His Cys

1 5

<210> 2

<211> 9

<212> PRT

<213> phage

<400> 2

Cys Ile Val Arg Gly Asp Asn Val Cys

1 5

<210> 3

<211> 9

<212> PRT

<213> phage

<400> 3

Cys Val Pro Arg Gly Asp Met His Cys

1 5

<210> 4

<211> 9

<212> PRT

<213> phage

<400> 4

Cys Asn Ala Thr Leu Pro His Gln Cys

1 5

<210> 5

<211> 9

<212> PRT

<213> phage

<400> 5

Cys Thr Ala Arg Ser Pro Trp Ile Cys

1 5

<210> 6

<211> 9

<212> PRT

<213> phage

<400> 6

Cys Arg Asn His Asp Met Gly Ala Cys

1 5

<210> 7

<211> 9

<212> PRT

<213> phage

<400> 7

Cys Asn Ala Ala Gly Ala Met His Cys

1 5

<210> 8

<211> 9

<212> PRT

<213> phage

<400> 8

Cys Ala Ala Arg Gly Asp Ala Ala Cys

1 5

<210> 9

<211> 11

<212> PRT

<213> phage

<400> 9

Ala Cys Asn Ala Arg Gly Asp Met His Cys Gly

1 5 10

<210> 10

<211> 11

<212> PRT

<213> phage

<400> 10

Ala Cys Asn Ala Thr Leu Pro His Gln Cys Gly

1 5 10

Claims

1. A polypeptide for prolonging the blood circulation time of a bacteriophage, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO 1, 2 or 3.

2. A nucleotide sequence encoding the amino acid sequence of claim 1.

3. Use of the polypeptide according to claim 1 for the preparation of a bacteriophage having the function of prolonging the circulation time of blood.

4. A bacteriophage carrying the polypeptide of claim 1.

5. A bacteriophage antibacterial agent comprising a time-lapse antibacterial bacteriophage carrying at least one of the polypeptide of claim 1 and an antibacterial protein BglII.

6. A time-lapse antibacterial phage carrying the polypeptide for prolonging the blood circulation time of phage according to claim 1 and a nucleotide sequence corresponding to antibacterial protein BglII.

7. Use of a bacteriophage according to claim 6 for the preparation of a bacteriophage antibacterial medicament, wherein the bacteriophage carries at least one of the polypeptide according to claim 1 and a nucleotide sequence corresponding to the antibacterial protein BglII.