WO2019227266A1 - Polypeptide for prolonging blood circulation time of phage - Google Patents

Abstract

Provided is a polypeptide for prolonging the blood circulation time of a phage, wherein the polypeptide contains the amino acid sequences as shown in SEQ ID NO: 1, 2 or 3. Also provided are a phage which carries the polypeptide and has the function of prolonging a blood circulation time, a method for screening the polypeptide, an antimicrobial drug containing the phage, and the use of such polypeptides and phages.

Description

Polypeptide for extending blood circulation time of phage Technical field

The invention belongs to the field of biomedicine, and in particular relates to a polypeptide that prolongs the blood circulation time of phage.

Background technique

Phage therapy is a treatment method that uses phage to fight bacterial infections and has been used in clinical practice since the early 20th century. For example, in 1919, d'Herelle tried to use phage to treat avian influenza (Salmonella), rabbit dysentery (Shigella), and bovine hemorrhagic septicemia (Pasteurium), which was the first attempt to use phage therapy. In 1921, Richard Bruynoghe and Joseph Maisin used bacteriophage to treat human skin diseases caused by S. aureus. In 1932, at the request of the British government, d’ Herelle reached the endemic area of Indian cholera to try to use bacteriophage for prevention and control, and quickly controlled the spread of the epidemic, while effectively curbing the second outbreak of the epidemic. In the same year, some researchers in Eastern Europe obtained scientific doses of phage treatment through a large number of animal and human experiments. In 1934, American scientists reported successful treatment of enterococcal infection by phage. From 1930 to 1939, many researchers and companies have commercialized phage therapy. For example, Parker Davis and Eli Lilly in the United States began to produce phage preparations for treating Staphylococcus and E. coli. .

However, with the advent of antibiotics such as penicillin during the Second World War, scientists in the United States and Western Europe basically abandoned in-depth research on phage therapy and began to invest in the torrent of antibiotic research. From 1950 to 1980, few research articles on phage therapy were published. Since 1980, the problem of antibiotic resistance has become increasingly serious. In fact, antibiotic resistance is now seriously affecting global public health. According to statistics, two million people in the United States are affected by antibiotic-resistant infections each year, which directly causes the death of about 23,000 people each year. Therefore, the development of a new antibacterial therapy has become increasingly urgent. As a bacterial virus, bacteriophage has incomparable advantages in antibiotics in the treatment of bacterial infections, especially multi-drug-resistant bacteria. Increasing global resistance.

Despite the huge potential to address the worsening crisis of antibiotic resistance, phage therapy currently has multiple limiting factors. Among them, the main problem hindering its clinical application is that the phage is rapidly cleared in the blood circulation after administration, and the short blood retention time prevents it from effectively entering the target site for treatment. Therefore, the engineering treatment of phages that prolongs the blood circulation time is the focus of research. It will be a more effective phage therapy to treat existing bacterial infections or prevent bacterial reinfection. In this regard, the strategies reported in related studies to extend the blood circulation time of phages include: through naturally occurring mutations, chemical modification of phage surfaces with polyethylene glycol, and avoiding complement-mediated inactivation by changing phage capsid proteins Wait. Nonetheless, the above schemes still have their limitations. The application of more innovative strategies to obtain long-term circulating phages with better antibacterial effects remains to be broken.

Phage display as a powerful technique has been used to screen peptide sequences with certain characteristics. Usually phage libraries (including phage displaying> 109 different sequence polypeptides) are panned in vivo or in vitro to quickly obtain polypeptides with specific interactions, such as material interactions, tissue targeting, organ targeting, and barrier penetration Role, etc. 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.

Summary of the Invention

The inventors of the present application used New England Biolabs' phage display peptide library Ph.D. ^TM -C7C to perform a series of screenings, and finally obtained a short peptide with the function of extending the blood circulation time of the phage.

Therefore, in a first aspect of the present invention, a polypeptide that prolongs the blood circulation time of a phage is provided.

According to the present invention, the polypeptide that prolongs the blood circulation time of the phage contains the amino acid sequence shown in SEQ ID NO: 1, 2 or 3 or an analog thereof.

According to a second aspect of the present invention, use of the polypeptide for extending the blood circulation time of a phage is provided.

According to the present invention, the polypeptide that prolongs the blood circulation time of the phage can be used to prepare a phage that has the function of extending the blood circulation time.

According to a third aspect of the present invention, a phage having a function of extending blood circulation time is provided.

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

According to a fourth aspect of the present invention, the application of the phage with the function of extending blood circulation time is provided.

According to the present invention, the phage with the function of extending blood circulation time can be used for preparing phage antibacterial drugs.

According to a fifth aspect of the present invention, a pharmaceutical composition is provided.

According to the present invention, the pharmaceutical composition includes at least one of the polypeptides that prolong the blood circulation time of phage, and at least one pharmaceutically active protein, drug, or material that is effective in treating a disease.

According to a sixth aspect of the present invention, a phage antibacterial drug is provided.

According to the present invention, the phage antibacterial drug comprises the phage having the function of extending blood circulation time.

According to a seventh aspect of the present invention, a time-delayed antibacterial phage is provided.

According to the present invention, the delayed antibacterial phage carries the polypeptide that prolongs the blood circulation time of the phage and the nucleotide sequence corresponding to the antibacterial protein BglII.

According to an eighth aspect of the present invention, a method for screening a polypeptide having the ability to extend the circulating time of a phage in vivo is provided, including the following steps:

a The phage bank is injected into the animal through the tail vein;

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

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

d. Repeat steps a to c above at least twice;

e. Monocloning the recovered phage, and then amplifying and verifying the time-delay ability of the recovered phage, and then sequencing, to obtain a polypeptide with the ability to extend the cycle time of phage in vivo.

In the present invention, a polypeptide capable of significantly prolonging the circulation time of the phage is screened by phage display technology, and it proves that the function has sequence specificity, and its principle of action is achieved by binding with platelets; the phage displaying the polypeptide is genetically engineered. It is engineered to express the antibacterial protein BglII at the same time, to obtain a phage with a delayed function and antibacterial activity, which has good antibacterial activity in vitro and in vivo in rats.

The polypeptide for prolonging the circulation time of phages in the present invention, phages with the function of extending blood circulation time, and phages with both time-delay and antibacterial functions are of great significance for phage therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the screening process of delayed phage BCP1, the enrichment of long-cycle phage during in vivo screening, and the mean and SEM of the number of phages per milliliter of blood after three rounds of tail vein injection into the rat 48 h. (Standard error) (n = 3, ** p <0.01, *** p <0.001).

Figure 2 shows the comparison of the in vivo time-delay ability of long-cycle phage monoclonals. The same amount (1 × 10 ¹¹ ) of three BCP phages and random phage SC were mixed and injected into rats. 30 phages were randomly selected at 0 and 72 h. The number of repetitions of the four phages was determined by sequencing (n = 3).

Figure 3 shows the long-cycle characteristics of delayed phages. The equivalent (1 × 10 ¹¹ ) BCP1 and SC phages were injected into different rats through the tail vein. The mean and SEM of the number of phages per milliliter of blood at different time points.

Figure 4 shows the mean and SEM (n≥3, * p <0.05, ** p <0.01, *) of the number of two types of phage per ml of blood at different time points in rats injected with tail veins mixed with BCP1 and REW phage. ** p <0.001).

Figure 5 shows the sequence specificity of delayed phages. Equal amounts (1 × 10 ¹¹ ) of different phage BCP1, SC and three BCP1 mutant phages were injected into the tail vein of different rats, and the number of phages per milliliter of blood at different time points was measured. Mean and SEM.

Figure 6 shows the BCP1 and three BCP1 mutant phages mixed with REW phages at a 1: 1 ratio. The rats were injected into the tail vein separately. = 3, *** p <0.001).

Figure 7 shows the complement system-mediated inactivation of phages. BCP1 and SC phages (1 × 10 ⁹ ) were co-incubated with plasma treated at 37 ° C or 56 ° C in advance, and the average number of phages in plasma at different time points was measured. And SEM (n = 3).

Figure 8 shows the preliminary verification of the binding of BCP1 phage to blood cells. Equal amounts (1 × 10 ⁸ ) of BCP1 and SC phages previously bound to PBC and unbound BCP1 and SC phages were injected into the rat through the tail vein and tested at different time points. Number of phage in blood (mean ± SEM, n = 3, ** p <0.01, *** p <0.001).

Figures 9A-9C show the distribution of different phages in plasma and blood cells over time; of which, Figures 9A, 9B, and 9C correspond to the equivalent (1 × 10 ¹¹ ) SC, BCP1, and TB2 phages injected into rats, respectively. Distribution of phage in plasma and PBC at different time points in vivo (n = 3, * p <0.05, ** p <0.01, *** p <0.001).

Figures 10A-10C show the distribution of BCP1, SC, and TB2 phages by finely separating the three components of blood; Figure 10A shows the distribution of BCP1 phages in blood in vivo experiments; Figure 10B shows the in vivo experiments of SC phages in blood Figure 10C shows the distribution of TB2 bacteriophage in blood (n = 3).

Figure 11 shows the effect of BCP and SC short peptides on the in vitro binding of phages to blood cells in vitro. 1 × 10 ⁹ BCP1 phages were incubated with 1 ml of rat whole blood for 1.5 h at 37 ° C in vitro. 500 μg / ml) and the absence of short peptides in the binding of phage to different blood cells.

FIG. 12 shows the in vitro antibacterial results of four phages of BCP1, SC, BGL, and BCP1-BGL with a MOI of 10 (n = 3).

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

Figures 14A and 14B show the average SEM (n = 3) of the number of four phages in the blood and abdominal cavity over time; of which, Figure 11A is an equivalent (1 × 10 ¹¹ ) The number of blood phages (mean ± SEM) at different time points was injected into the tail vein of the rats; Figure 11B shows that four phages of BCP1, SC, BGL and BCP1-BGL were equated (1 × 10 ¹¹ ) into the tail vein. Rats, the number of phage in the abdominal cavity at different time points (mean ± SEM, n = 3).

Figures 15A and 15B show the antibacterial effect of BCP1-BGL in vivo; of which, Figures 15A and 15B are equivalent (1 × 10 ¹¹ ) BCP1, SC, BGL, and BCP1-BGL four phages injected into the tail vein of rats, respectively, 18h Bacteria were then infected by intraperitoneal injection (1 × 10 ⁸ ), and the number of bacteria in ascites and liver after 5 hours (mean ± SEM, n = 3, ** p <0.01, *** p <0.001).

Figure 16 shows the content of IFN-γ in rat plasma treated with different combinations (mean ± S.E.M., n = 3, * p <0.05, ** p <0.01).

17A and 17B are the contents of ALT (17A) and AST (17B) in rat plasma treated with different combinations (mean ± S.E.M., n = 3, ** p <0.01, *** p <0.001).

Figure 18 shows HE staining of rat liver tissue sections showing different cell infiltration effects after treatment with different combinations.

Detailed ways

The invention will be more easily understood by reference to the following detailed description of the preferred embodiments thereof and the examples contained therein. However, before describing the polypeptides, compounds, composites and methods involved in the present invention, it is necessary to understand that the present invention is not only applicable to a certain polypeptide, protein, drug or other material, nor is it only applicable to a certain polypeptide. Cell type, host cell, conditions and methods, etc. Of course, in that case, there may be changes, and such a large number of modifications and changes will appear in this mature technology. Also, be aware of terminology used only to describe specialized equipment here, these limitations are not intended to be intentional. It is also to be understood that the use of "a" in the specification and claims may be one or more, depending on the context in which the word is used. Thus, for example, reference to "a cell" may refer to at least one cell being utilized.

The present invention describes a method that uses a phage display library to screen display peptides that increase blood retention. As used herein, a "phage display library" refers to a collection of genetically engineered phage displayed on the surface by expressing a series of polypeptides. A "display peptide" consists of a continuous sequence of amino acids that includes proteins displayed on the surface of a phage. The term "polypeptide" refers to a chain consisting of at least three amino acids connected by peptide bonds. This chain can be linear, branched, circular, or a combination thereof.

The research object of phage library is to use genetic engineering to express a large number of display peptides of different amino acids. After using this object, collect and identify phage particles in the research object. Here, the "subject" refers to mammals, such as mice, rabbits, and humans. Phage particles are usually collected from one or more organ tissues, cells, blood, urine, or other various body fluids. In one preferred solution, the phage particles are injected into the animal through the tail vein, and after a period of time Residual phage particles are collected from the blood circulation. It is those phage particles containing peptides that can extend the circulation time in the blood.

The peptide sequences expressed on the surface of phage particles collected from experimental animals can be separated by solid medium plates, that is, in cili-positive bacteria, phage particles can be propagated in vitro on biological plates. 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 corresponding to the inserted peptide in the phage genome.

In the context of the present invention, the polypeptide analog refers to other short peptides having a time-delay function obtained by phage screening, or obtained by adding, deleting, changing the order or replacing the amino acid of the polypeptide sequence shown in SEQ ID NO: 1. Peptide.

The following further describes the present invention in combination with specific embodiments. It should be understood that the following examples are only used to illustrate the present invention but not to limit the scope of the present invention.

Example 1: Screening of specific delayed short peptides

In this example, the library used for screening specific time-lapse peptides was provided by New England Biolabs as a heptapeptide library (Ph.D. ^TM -C7C) containing disulfide bonds. The random fragments in this library are flanked by cysteine residues, which can be oxidized during phage assembly to form disulfide bonds, thus forming a cyclic peptide to interact with the target. This library contains more than two billion clones. The random peptides in the library are at the amino terminus of the small coat protein pIII, so each phage particle expresses five copies. The position of the phage-expressing random sequence in the Ph.D. ^TM- C7C library was preceded by alanine-cysteine. A short linker (glycine-glycine-glycine-serine) is contained between the random peptide and the pIII protein. (Ph.D. ^TM -C7C phage peptide display kit, http://www.neb.com/nebecomm/products/productE8120.asp).

1 × 10 ¹¹ phages were dissolved in 300 μl of physiological saline, and 150 g of SD rats were injected into the tail vein. Whole blood was taken from the body 48 hours later, and spread on X-gal (5-bromo-4-chloro-3-indolyl) -bD-galactoside) and IPTG (isopropyl-bD-thiogalactoside) LB plate, 6 rats obtained 300 plaques. All of them were picked and amplified, and 1 × 10 ¹¹ phages were subjected to the second round of in vivo screening. The 500 plaques obtained after 96 hours were mixed and amplified for the third round of screening, and the time was extended to 120 hours.

Figure 1 shows the number of phage in the whole blood of rats after 48 hours of three rounds of screening. It can be seen that with the progress of the screening, the number of phages in each round of the same time point has increased significantly. It reaches 3.5 × 10 ⁴ pieces.

Thirty randomly selected plaques from the second and third rounds of screening were sequenced to obtain display peptide sequences. After passing a series of tests such as time delay ability, 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 ability test

Take 1 × 10 ¹¹ phages carrying the display peptides BCP1, BCP2, and BCP6 obtained in Example 1, mix them with random phage SC (display peptide sequence: CNATLPHQC, SEQ ID NO: 4), and inject them into the tail vein of rats. Blood samples were taken at 0h (3min) and 72h to detect phage.

Figure 2 shows the number of phages at 0h and 72h. It can be seen from the figure that the number of the four kinds of phages was similar at 0h, but SC phages were gone at 72h, and BCP1 accounted for the highest proportion, followed by BCP6 and BCP2. It can be seen that the phages carrying the three display peptides obtained in Example 1 all have the ability to significantly extend the circulation time of phages in the blood of rats.

In order to further determine the time-delay effect of BCP1, we injected the same amount of BCP1 and SC phages into rats, and detected the number of phages in blood at different time points. The results are shown in Figure 3.

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

The results in Figure 3 show that compared to BCP1 phage, the number of SC phages decreases faster with time, and is not detected at 48 hours, while BCP1 phage can still be detected at 144 hours, further confirming the BCP1 phage in the blood. Delay ability.

The results in Figure 4 show that the ratio of BCP1 / REW reached 100 at 12 h and exceeded 1000 at 36 h. It can be seen that the influence factors of individual differences in animals are excluded, and the phage still has a good ability to extend the blood circulation time. .

Example 3: Verifying the phage's time-delay function is sequence-specific

During the screening process, we found that with the round-by-round enrichment of phages, the frequency of phages containing RGD sequences continued to increase. Therefore, in order to determine the importance of RGD in the phage display peptide sequence obtained in Example 1, we constructed Three BCP1 phages were identified as follows:

TB1: CRNHDMGAC (SEQ ID NO: 6, disrupts the BCP1 sequence);

TB2: CNAAGAMHC (SEQ ID NO: 7, mutation of RGD to AGA);

TB3: CAARGDAAC (SEQ ID NO: 8, RGD is retained, and the remaining four amino acids are mutated to A).

Then 1 × 10 ¹¹ TB1, TB2, TB3 and BCP1, SC phages were injected into different rats, or mixed with REW 1: 1 and injected into the same rats to detect phage in blood at different time points. The results are shown in Figure 5.

The results in Figure 5 show that the blood retention time of TB1 and TB2 is equivalent to that of SC, and it is almost undetectable in the blood at 48h, implying that it has lost the delay ability; and TB3 also partially lost the delay ability.

The results in Figure 6 show that the ratio of the three mutant phages and BCP1 to REW was similar at 0h, and after 48h, except for BCP1, the rest of the phages were similar to REW in vivo and no longer had the ability to delay.

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

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

The whole blood plasma was incubated at 56 ° C or 37 ° C for 10 minutes (treatment at 56 ° C can inactivate the complement system in the blood). Subsequently, 1 × 10 ⁹ BCP1 and SC phages were respectively mixed with pre-treated plasma and incubated, and the number of phages after different time was detected. The results are shown in FIG. 7.

The results in Figure 7 show that in a state where the complement system is not inactivated, the number of phages decreases with time gradient, and when the complement system is inactivated, this decline rate slows down, suggesting that the complement system in the blood caused the inactivation of the phage. But more importantly, the reduction in the number of BCP1 and SC phages did not show a significant difference, suggesting that the delayed function of BCP1 phages was not achieved by the anti-complement system.

Example 5: Bacteriophage achieves time-delay by interacting with blood cells

1 × 10 ⁸ BCP1 and SC phages that were bound to blood cell PBC in advance and the same amount of unbound BCP1 and SC phages were injected into the rat tail vein respectively. Blood was taken at different time points to detect the number of phage in the blood. The results are as follows Figure 8 shows.

Further, in order to detect which component of BCP1 phage is bound to the blood, 1 × 10 ¹¹ BCP1, SC, and TB2 phages were injected into rats, and 1 ml of blood and 100 μl of anticoagulant CPD (16 mM citric acid) were taken at different time points. , 90 mM sodium citrate, 16 mM NaH ₂ PO ₄ , 142 mM dextrose, pH 7.4) were mixed, left at room temperature for 15 minutes, centrifuged at 2000C for 10 minutes at 25 ° C, blood cells and plasma were roughly separated, and the number of phage in the two parts was detected. Results As shown in Figures 9A-9C.

The results in Figure 8 show that compared to unbound blood cells, the time delay ability of SC and BCP1 phages bound to blood cells has been improved, and the blood retention time of BCP1 has been extended longer.

Figures 9A, 9B, and 9C respectively show the numbers of SC, BCP1, and TB2 phages in plasma and blood cells at different time points. At 0h, the number of BCP1 phages in plasma is 10 times higher than that of blood-bound phages, and close to 24h. The same, but the number of phages bound to blood cells was much higher than that in plasma at 36h, and continued to 72h.

The above results suggest that the time delay ability of BCP1 phage is achieved by binding to blood cells.

Example 6: Bacteriophage achieves time delay by interacting with platelets in blood cells

After injecting BCP1 phage into rats, 1 ml of blood was taken from the heart at different time points, mixed with 100 μl CPD, left at room temperature for 15 minutes, and centrifuged at 200g for 20 minutes. The sample is divided into three layers. From top to bottom, the first layer is a platelet-rich region, the second layer is a leukocyte-rich region, and the third layer is a red-cell-rich region. The white blood cell layer and the red blood cell layer can directly detect the titer representing the number of phage binding. Platelets were obtained by centrifuging the first layer of the supernatant at 2000g for 10 minutes.

Further, three components can be obtained more accurately by flow sorting. After injecting 1 × 10 ¹¹ BCP1, SC or TB2 phages into the rat tail, 1 ml of blood was taken from the heart at different time points, mixed with 100 μl CPD, left at room temperature for 15 minutes, and centrifuged at 200g for 20 minutes. The sample is divided into three layers. From top to bottom, the first layer is a platelet-rich region, the second layer is a leukocyte-rich region, and the third layer is a red-cell-rich region. 0.2M adenosine triphosphate diphosphatase was added to the supernatant platelet layer to prevent platelet activation, and platelets were enriched by centrifugation at 2000g and 4 ° C for 10min. The middle layer of white blood cells is further enriched and recovered by adding red blood cell lysate to remove red blood cells. The three cell components were resuspended in PBS containing 1% FBS, CD16 / 32 antibody was added, and after standing on ice for 10 minutes, FITC-anti-rat CD45, PE-anti-rat Erythroid Cell, and PerCP / Cy5.5 were labeled, respectively. -anti-mouse / rat CD42d antibody was sorted and recovered. Centrifuge at 2000g and 4 ° C for 5min to recover the enriched and sorted cells and check the number of phage. The results are shown in Figure 10, where PLT represents platelets, WBC is white blood cells, and RBC is red blood cells.

The results of FIG. 10A show that BCP1 phage binds more to platelets and white blood cells than red blood cells at different time points, while the results of FIGS. 10B and 10C show that the amount of SC and TB2 bound to blood cells is greatly reduced.

Example 7: BCP1 phage interacts with platelets via display peptide

1 × 10 ⁹ BCP1 phages were mixed with 1 ml of rat blood in vitro, and the other two groups were added with a synthetic BCP short peptide (sequence: ACNARGDMHCG, SEQ ID NO: 9) or SC short peptide (sequence: ACNATLPHQCG, SEQ ID NO: 10) After incubation at 37 ° C for 1.5 hours, the number of phage binding in the three cell components of blood was detected by flow sorting. The results are shown in FIG. 11.

The results in FIG. 11 show that compared with the SC short peptide, the addition of the BCP1 short peptide significantly reduced the binding of BCP1 phage to platelets and leukocytes, demonstrating that BCP1 phage binds to platelets or leukocytes through specific display peptides.

Example 8: Delayed antibacterial phage BCP1-BGL

The restriction enzymes EcoR I and Hind III were used to digest the PCR product containing the BglIIR gene (bactericidal gene, which specifically recognizes and cleaves the host cell's BglII site 5'-AGATCT-3 '), and then recovered. The fragment was ligated with M13KE vector (purchased from New England Biolabs) at 16 ° C overnight, and it was determined whether the BCP1 nucleic acid sequence was included between the digestion sites of Eag I and Kpn I on the M13KE vector. The recombinant phage containing the BCP1 nucleic acid sequence was BCP1-BGL. , BGL is not included.

Among them, the PCR product containing the BglIIR gene is using the following primers:

Forward: 5'-CCCAAGCTTAAATTAGACCGCACTTACATAGGCG-3 ';

Reverse: 5'-CCGGAATTCTTAATATGTCACGATTGTTCCTCTTTTCC-3 ';

The PMRB1 plasmid was used as a template and obtained by the following PCR conditions:

In the middle three steps, repeat 30 cycles.

The recombinant phage BCP1-BGL is transformed into ER2738 competent cells containing the PBM1 plasmid (containing the BglII gene, which can methylate the BglII site of the host DNA so as not to be cleaved by the BglII protein). For subsequent experiments. Among them, PMRB1 plasmid and PBM1 plasmid are all by Professor Armin Resch of the University of Vienna.

Example 9: In vitro antibacterial capacity of antibacterial phage BGL and BCP1-BGL

E. coli MC4100F 'cultured with OD600 to 0.2 and BCP1, SC, BGL, and BCP1-BGL with different ratios of MOI (number of E. coli / number of phage) continued to be cultured in LB medium containing 0.003mol / L IPTG. At the same time, using PBS as a control, the number of E. coli in the culture solution was detected at different time points to verify the in vitro antibacterial ability of the four phages. The results are shown in FIG. 12.

The results in FIG. 12 show that the number of E. coli in the BGL and BCP1-BGL groups did not show a constant increase compared to the other groups after 4 h of culture with MOI = 10, indicating that the constructed phages BGL and BCP1-BGL It has antibacterial activity.

Example 10: In vitro antibacterial release of endotoxin by antibacterial phage BGL and BCP1-BGL

E. coli MC4100F 'cultured at OD600 to 0.2 under the condition of MOI = 10, adding four phages BCP1, SC, BGL, and BCP1-BGL, and continued to culture in LB medium containing 0.003mol / L IPTG, while using PBS as comparison. Endotoxin detection kit (ToxinSensor ^™ Chromogenic LAL Endotoxin Assay Kit) was used to detect the content of endotoxin in the culture supernatant at 0, 1, 2 and 4 hours, respectively, and the results are shown in FIG. 13.

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

Example 11: Detecting the in vivo delay ability of antibacterial phage BGL and BCP1-BGL

1 × 10 ¹¹ BCP1, SC, BGL, and BCP1-BGL phages were injected into rats by tail vein, blood was taken at different time points, and the content of phage in the blood was measured; at the same time, 10 ml of physiological saline was injected into the rats at each time point. The peritoneal cavity of rats was recovered and the number of bacteriophages was detected. The test results are shown in Figures 14A and 14B.

Figures 14A and 14B are the numbers of blood and ascites phages at different time points of the four types of phages. It can be seen that compared to SC and BGL phages, BCP1-BGL phages containing BCP1 still have a longer delay in blood and ascites. Time, indicating that the addition of BGL did not destroy the delay function of BCP1.

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

1 × 10 ¹¹ BCP1, SC, BGL and BCP1-BGL bacteriophages were injected into the tail vein of rats respectively, while PBS was used as a control, and 1 × 10 ⁸ Escherichia coli MC4100F ′ was intraperitoneally injected 18 hours later, and 2 μM was injected 1 hour later. IPTG. After 5 hours, the abdominal cavity was washed with 10 ml of physiological saline, and ascites was recovered. Another 1 g of the liver was resuspended and fully ground in 1 ml of physiological saline. The amounts of E. coli in ascites and liver were measured separately, and the results are shown in Figs. 15A and 15B.

The results of FIGS. 15A and 15B show that the BCP1-BGL phage-treated group, which has both time-delay and antibacterial ability, has the least amount of E. coli in ascites and liver, which indicates that it has antibacterial ability.

Example 13: Detecting the effect of BCP1-BGL treatment on IFN-γ in blood of rats after bacterial infection

1 × 10 ¹¹ BCP1, SC, BGL and BCP1-BGL bacteriophages were injected into rats by tail vein, and PBS was used as a control. After 18 hours, 1 × 10 ⁸ E. coli MC4100F ′ was injected intraperitoneally. SC and BCP1-BGL phage rats were used as controls. 2 μM of IPTG was injected 1 hour later, and the IFN-γ content in rat plasma was detected by an ELISA kit 5 hours later. The results are shown in FIG. 16.

The results in Figure 16 show that BCP1-BGL treatment can significantly reduce the IFN-γ content in the blood of rats after bacterial infection, indicating that it has a certain antibacterial effect.

Example 14: Effects of BCP1-BGL treatment on liver function in rats after bacterial infection

1 × 10 ¹¹ BCP1, SC, BGL and BCP1-BGL bacteriophages were injected into rats by tail vein, and PBS was used as a control. After 18 hours, 1 × 10 ⁸ E. coli MC4100F ′ was injected intraperitoneally, and PBS was injected at the same time. Bacteriophage was used as a control; 2 μM of IPTG was injected after 1 h; 5 hours later, the contents of ALT and AST in plasma were detected by an automatic biochemical analyzer. The results are shown in FIGS. 17A and 17B.

Another 1 g of liver tissue was coated with paraffin, and sections of about 5 mm were subjected to H & E staining. The results are shown in FIG. 18.

The results of FIGS. 17A and 17B show that the BCP1-BGL treatment group with both time-delay and antibacterial ability has less increase in ALT and AST values in the blood compared to the remaining three phages after infection with bacteria, indicating that the liver function of the rats in this group There is less damage.

The liver tissue section results in FIG. 18 show the same results, indicating that BCP1-BGL has a certain antibacterial activity in vivo.

Although the above embodiments have described in detail the polypeptide of the present invention which prolongs the blood circulation time of phage, the phage carrying the polypeptide, the delayed antibacterial phage carrying both the polypeptide and the antibacterial protein BGL, and the mechanism of action thereof. However, those skilled in the art can easily understand that it is possible to appropriately change it within the scope of the technical content disclosed by the present invention. For example, appropriate modifications are made to the polypeptide sequence of the present invention, including deletions, additions, substitutions, and changes in order. Therefore, these modifications also fall within the scope of the present invention. In addition, the nucleotide sequences encoding these amino acid sequences also undoubtedly fall within the scope of the present invention.

Claims (9)

1. A polypeptide that prolongs the blood circulation time of a phage, wherein the polypeptide contains the amino acid sequence shown in SEQ ID NO: 1, 2 or 3 or an analog thereof.
2. A nucleotide sequence encoding the amino acid sequence or the analogue thereof according to claim 1.
3. The use of the polypeptide according to claim 1, characterized in that it is used for preparing a phage having a function of extending blood circulation time.
4. A phage, wherein the phage carries the polypeptide of claim 1.
5. The use of phage according to claim 4, characterized in that it is used for preparing phage antibacterial drugs.
6. A pharmaceutical composition, characterized in that it comprises at least one polypeptide according to claim 1 and at least one pharmaceutically active protein, medicament or material effective for treating a disease.
7. A phage antibacterial drug, comprising the phage according to claim 4.
8. A prolonged antibacterial phage, characterized in that the prolonged antibacterial phage carries the polypeptide that prolongs the blood circulation time of the phage and the nucleotide sequence corresponding to the antibacterial protein BglII.
9. A method for screening a polypeptide having the ability to extend the circulating time of a phage in vivo, which comprises the following steps:

   a The phage bank is injected into the animal through the tail vein;

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

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

   d. Repeat steps a to c above at least twice;

   e. Monocloning the recovered phage, and then amplifying and verifying the time-delay ability of the recovered phage, and then sequencing, to obtain a polypeptide with the ability to extend the cycle time of phage in vivo.

Images