CN116676274A - Self-deactivatable phage, preparation method and application thereof - Google Patents
Self-deactivatable phage, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a self-deactivatable phage and a preparation method and application thereof. Bacillus cereus bacteriophage DeltaTail DK1, accession number: GDMCC No:62951-B1. The phage preparation can be used for preventing and controlling pollution bacteria and pathogenic bacteria pollution in food industry and breeding industry and clinically treating pathogenic bacteria infection. According to the preparation method, recognition and splitting of the host are carried out based on phage, the tail protein coding genes in the genome are not relied on, the tail protein coding genes in the phage genome are knocked out, a host expressed by the plasmid auxiliary tail protein coding genes is used as a first infected progeny producer in the phage production process, and activity assurance of progeny of the phage preparation after the first infection is realized. In practical application, the killing object does not carry tail gene encoding plasmid, and active offspring cannot be synthesized due to the deletion of tail protein encoding after infection, so that phage self-inactivation is realized.
Description
Technical field:
the invention belongs to the field of bioengineering, and particularly relates to a self-deactivatable phage for preparing food industry, breeding industry and clinic, and a preparation method and application thereof.
Background
The phage is used as a virus of bacteria, has high specificity, and can effectively avoid the killing of non-pathogenic bacteria or probiotics by means of conventional sterilization methods such as physics or chemistry. At present, phage prevention and control technology is mature gradually, and the applied fields comprise food safety, livestock breeding, clinical treatment and the like. Meanwhile, the phage artificially modified based on the genetic engineering means has excellent antibacterial performance in clinic, and patients infected by multi-drug resistant mycobacterium are effectively cured, so that the phage preparation is expected to replace antibacterial drugs to become a new antibacterial means.
In order to effectively interfere the antibacterial activity of phage under external conditions, phage with good stability is usually selected in the phage preparation process; however, since phage genetic diversity is abundant, a large number of phage-encoding genes lack sufficient experimental evidence or have no homology to known functional proteins, resulting in about half to two-thirds of phage-encoding genes being genes of unknown function, and phages carrying a large number of unknown genes are risky to use for a long period of time in humans and environments and retain activity. Meanwhile, since phages are live viruses, they can be propagated in an antibacterial process, producing live progeny phages, which results in easy retention of the phages for growth time in application environments, it is currently known that in agriculture and food environments, active phages can be retained for more than 4 months. The use of large doses of phage and long-lasting properties can cause problems including: 1. promoting the appearance of phage tolerant bacteria and affecting the long-term use effect of phage; 2. contamination of the production environment and laboratory of the inspection sector can lead to false negative results in the detection of microorganisms after infection in the food sector or clinically. More importantly, a large amount of conditional pathogenic bacteria such as bacillus cereus exist in the environment, so that pollution can be caused in the food industry, and diseases can be clinically caused; however, non-pathogenic bacillus cereus can be used as an environmental heavy metal treatment bacterium or a plant growth promoting bacterium in the planting industry. Therefore, a more controllable phage antibacterial means needs to be developed, so that the self-inactivation of the phage after use can be ensured, the influence on non-pathogenic bacteria in the environment after use of the phage is reduced, the antibacterial function of the phage in food safety, livestock breeding and clinical treatment is ensured, and meanwhile, the influence on the use of functional bacteria in the environment is avoided. Therefore, how to efficiently remove active phage in application scenarios after phage preparation completes bacterial killing is a problem to be solved.
Constructing phage with a defect of encoding tail protein structural gene based on biosynthesis and molecular genetics, and assembling the phage into a complete phage structure at a protein level by expressing the protein of the tail gene in a host by a plasmid during phage preparation, so as to ensure the antibacterial activity of phage preparations; when the antibacterial function is actually exerted, the killing object does not carry the plasmid for encoding the tail protein structural gene, so that active phage filial generation cannot be synthesized, the continuous transmission capability of phage in the environment is stopped, and a foundation is laid for further realizing the safe and efficient application of phage.
The invention comprises the following steps:
the invention aims to provide a self-deactivatable phage which retains the specific host lysis capability but also has the self-inactivation capability and avoids the increase of the concentration of active phage after bacterial removal is completed, and a preparation method and application thereof.
The self-deactivatable phage of the present invention, bacillus cereus bacteriophage ΔTail DK1, was deposited at the Guangdong province microbiological bacterial collection center (GDMCC) at 2022, 11, 07, accession number: GDMCC No:62951-B1, accession number: guangzhou city first middle road No. 100 college No. 59 building 5.
The invention also provides a preparation method of the Bacillus cereus bacteriophage delta Tail DK1, which is obtained by knocking out the Tail gene of phage DK1.
Preferably, the tail gene knockout of the phage DK1 is to take the parent phage DK1 as a target to perform in vivo homologous recombination, CRISPR-Cas gene editing, in vitro gene synthesis and other methods to knock out the tail structural gene of the phage.
Preferably, adding crRNA targeting DK1 tail gene and inserting the tail gene which is synonymously mutated in the crRNA recognition region so that the tail gene cannot be recognized by the crRNA into the plasmid pcrF11-erm to obtain plasmid pcrF11-erm-1; the plasmids pcrF11-erm-1 and pXCR6-S are sequentially and directly transformed into the same host bacillus cereus 233-1 to obtain a recombinant host carrying two plasmids;
mixing phage DK1 with a recombinant host, and obtaining single plaques by a double-layer agar method respectively, wherein xylose with the final concentration of 3g/L is required to be added into agar, selecting a plate in which the single plaque morphology can be observed, and carrying out plaque PCR to identify a gene knockout result, so as to successfully obtain a Tail gene knockout mutant strain DeltaTail DK, namely Bacillus cereus bacteriophage DeltaTail DK1.
Preferably, the plaque PCR identification adopts the primers of Tail-TF1 and Tail-TR1, the length of delta Tail DK amplified fragment which is knocked out successfully is 1,786bp, and the amplified length of wild DK1 is 4,117bp;
Tail-TF1：AGAATATTCGTATAGCTCAAACAGATAATGATGGTATTTC；
Tail-TR1：CTTACCAGTGTTATCTCCACCACAACTATTGATAG。
the invention also provides application of Bacillus cereus bacteriophage delta Tail DK1 in removing pathogenic bacteria or pollutant bacteria preparations.
Preferably, the bacillus cereus is applied to the preparation of efficiently preventing, controlling and removing the polluted bacteria and pathogenic bacteria in the food industry, the breeding industry or the clinic, such as bacillus cereus.
Preferably, bacillus cereus bacteriophage ΔTail DK1 is used in bacterial detection.
The present invention also provides an antibacterial agent containing Bacillus cereus bacteriophage ΔTail DK1 as an active ingredient.
The antibacterial agent can be a food additive or a food production environment disinfectant.
According to the invention, recognition and splitting of the host based on phage are carried out without depending on tail protein coding genes in genome, tail protein coding genes in phage genome are knocked out, and a host expressed by the tail protein coding genes is assisted by plasmid in phage production process as a first infected progeny producer, so that activity assurance of progeny of the phage preparation after first infection is realized. In practical application, the killing object does not carry tail gene encoding plasmid, and active offspring cannot be synthesized due to the deletion of tail protein encoding after infection, so that phage self-inactivation is realized.
The beneficial effects of the invention are as follows:
(1) The phage prepared by the invention has good pathogenic bacteria clearing effect, and retains the specific host splitting ability;
(2) The phage prepared in the invention has self-inactivating capability, and avoids the increase of the concentration of active phage after the bacterial removal is completed;
(3) The preparation method is suitable for the same operation with different phages as objects, so as to be applied to the efficient removal of pathogenic bacteria and the self-inactivation of phage preparations in the food industry, the breeding industry and the clinic.
Bacillus cereus bacteriophage DeltaTail DK1 (hereinafter referred to as inactivated phage DeltaTail DK) deposited at the Guangdong province microorganism strain collection (GDMCC) on month 07 of 2022 under accession number: GDMCC No:62951-B1.
Drawings
FIG. 1 is a schematic illustration of the preparation method of the present invention.
FIG. 2 is a schematic diagram of a method for knocking out a phage tail gene in the preparation method of the present invention.
FIG. 3 shows the result of knocking out the tail gene of phage in the preparation method of the present invention.
FIG. 4 detection of lytic activity of phage preparations prepared in the present invention.
FIG. 5 is a graph showing the evaluation results of the phage preparation prepared in the present invention on the bacterial-removing ability.
FIG. 6 shows the results of activity detection of phage preparations prepared in the present invention after completion of bacterial removal.
FIG. 7 is a transmission electron microscope morphology map of wild-type phage and progeny phage morphology released after lysing bacteria from inactivated phage.
Detailed Description
In order to more clearly demonstrate the technical scheme, objects and advantages of the present invention, the technical scheme of the present invention is described in detail below with reference to the specific embodiments and the accompanying drawings.
The phage DK1 (GenBank: MK 284526) used for verifying the feasibility of the technical scheme is separated by the laboratory in the early work, the constructed Tail gene knockout strain DeltaTail DK1 is obtained by knocking out Tail genes by DK1, the Tail gene knockout strain is named Bacillus cereus bacteriophage DeltaTail DK1 (hereinafter called as inactivated phage DeltaTail DK), and the phage is preserved in the Guangdong province microorganism strain preservation center (GDMCC) on the 11 th month 07 of 2022, and the preservation number is: GDMCC No:62951-B1. The plasmid pHT304-PDK1-GFP used for expressing tail protein is obtained by modifying pHT304 (which is also commercially available) taught by agricultural university of China Sun Ming in the early stage of the experiment, and the modification method is that a promoter PDK1 (100 bp upstream from the head protein of phage DK 1) and a green fluorescent protein gene are inserted between EcoRI and SalI on the plasmid, so that the plasmid has the capability of expressing exogenous proteins in bacillus cereus. The gene knockout plasmids pcrF11-erm and pXCR6-S used in the invention are obtained by teaching the complimentary pcrF11 and pXCR6 (purchased through commercial paths) from Jiang Nada science Liu Long and are transformed in the early stage of the laboratory, and the transformation method is that the plasmid pHT304 plasmid erythromycin resistance gene erm is inserted into the pcrF11 to obtain the plasmid pcrF11-erm with an erythromycin resistance marker; the plasmid pXCR6 has a plasmid length of about 14kb, is not easy to be transformed into bacillus cereus, is deleted by redundant sequences in the early stage, and retains a Cas12a expression frame and a crRNA expression frame on the plasmid to obtain a plasmid with a plasmid length of about 12kb so as to realize efficient transfer into bacillus cereus.
Tryptone soy broth (TSB medium), technical agar, syringe, 0.22 μm microporous filter membrane for the test of the invention were purchased from the CycloKai company; 2 XPimerSTAR was purchased from Takara; hieffPlus Multi One Step Cloning Kit from assist organisms, primer synthesis and one generation of sequencing services were provided by Huada.
Example 1 knockout of phage tail Gene
According to the preparation method of the present invention (FIG. 1), phage DK1 was selected for method validation. According to the CRISPR-Cas12a gene knockout method (fig. 2), the required crRNA and fragment amplification primers (table 1) are designed, wherein crRNA targeting DK1 tail gene and tail gene inserted into crRNA recognition region to make synonymous mutation unrecognizable by crRNA (synthesis of helper phage active progeny expressing tail protein) are added into plasmid pcrF11-erm to obtain plasmid pcrF11-erm-1; and the plasmids pcrF11-erm-1 and pXCR6-S are sequentially and directly transformed into the same host bacillus cereus 233-1 to obtain a recombinant host carrying the two plasmids.
Will have a titer of about 10 8 PFU/mL phage DK1 was diluted in gradient, mixed with recombinant hosts, and individual plaques were obtained by double-layer agar method, respectively, wherein xylose was added to the agar to a final concentration of 3g/L (Cas 12a expression was induced to achieve gene knockout). A dilution plate in which a single plaque morphology is observed is selected, a plaque PCR is performed to identify gene knockout results, primers of Tail-TF1 and Tail-TR1 are adopted, the length of a delta Tail DK amplified fragment which is knocked out successfully is 1,786bp, and the amplification length of a wild DK1 is 4,117bp. The PCR identification shows that the Tail gene knockout mutant strain DeltaTail DK (figure 3) is successfully obtained, the PCR product is sent to a large gene for first generation sequencing, the result is confirmed to be correct, the result is named as Bacillus cereus bacteriophage DeltaTail DK1 (hereinafter called as inactivated phage DeltaTail DK), and the phage is preserved in the Guangdong province microorganism strain preservation center (GDMCC) on the 11 th month 07 of 2022, and the preservation number is: GDMCC No:62951-B1.
TABLE 1 primers used in experiments
Example 2 preparation of self-inactivating phages
Host strain Bacillus cereus 233-1 used to carry both the recombinant plasmids pcrF11-erm-1 and pXCR6-S was used to prepare a ΔTail DK1 suspension by taking 1mL of host bacteria, centrifuging to remove the supernatant, adding 1mL of TSB to resuspension, centrifuging again to remove the supernatant, repeating 2 times to wash the residual antibiotics in the bacteria, suspending the washed bacteria in 1mL of TSB, and adding 500. Mu.L each of the bacterial solution and the self-inactivating phage ΔTail DK to 50mL of TSB medium (containing 1mM calcium chloride), shaking and culturing overnight at 25℃at 100 rpm. The culture broth was centrifuged at 10,000g at 4℃for 20min, and the supernatant was obtained by filtration through a 0.22. Mu.M filter and stored at 4 ℃.
Example 3 self-inactivating phage lysis experiments
The ability to lyse the self-inactivating phage ΔTail DK was determined by plaque assay. Bacillus cereus 233-1 to be tested is inoculated into a culture medium for overnight culture, 100 mu L of the culture medium is taken out and added into 4mL of melted upper agar, and the mixture is poured onto a TSB solid plate after uniform mixing, and the mixture is stood for solidification. After phage were diluted in gradient, 4. Mu.L of phage suspension was taken for each dilution, spotted on agar, and left to stand for liquid absorption. The plaques were observed by resting at 37℃for 4-6h, if clear plaques were present, indicating that the phage could lyse the bacteria, and conversely, indicating that the phage could not lyse. The results indicate that the self-inactivating phage Δtail DK maintains the lytic ability to the host bacteria (fig. 4), while single plaques cannot be formed at low titers like wild-type strain DK1 due to the self-inactivating property.
Example 4 comparison of the antibacterial Capacity of self-inactivating phages with non-self-inactivating phages
The antibacterial ability of wild DK1 and self-inactivating phage ΔTail DK was compared by bacterial clean-up experiments. The logarithmic phase bacillus cereus 233-1 bacteria were diluted and added to three conical flasks containing 45mL of TSB with a final bacterial concentration of 1X 10 5 CFU/mL; 500. Mu.L each was taken to determine the initial bacterial load and defined as 0h, after which the phage was diluted and 5mL phage were added to the Erlenmeyer flasks, respectively, to a final concentration of 10 6 CFU/mL causes moi=10. After mixing, 1mL of the initial phage amount was measured by filtration, defined as 0h, the flask was left to stand at a constant temperature of 25 ℃ for 3h, and then sampled and measured, viable bacteria were calculated by colony counting, viable phage titer was calculated by plaque counting, and the above experiment was repeated three times to determine the bacterial removal capacity of phage preparations after host changes. The results show (fig. 5) that the self-inactivating phage Δtail DK can effectively kill bacteria, reducing the number of viable bacteria; meanwhile, after bacterial removal was completed, wild DK1 active phage titres increased and Δtail DK decreased (fig. 6), i.e., self-inactivation was achieved.
Example 5 self-inactivating phage lysates after completion of bacterial body observations by transmission electron microscopy
The self-inactivating phage ΔTail DK was mixed with Bacillus cereus 233-1 at MOI=0.1, added to 3mL TSB (containing 1mM calcium chloride), cultured with shaking at 37℃and 200rpm for 3 hours, and the supernatant suspension was obtained by filtration with a 0.22. Mu.M filter, while the wild DK1 suspension was subjected to a synchronous operation. Dropping the suspension on a copper mesh, naturally precipitating for 15min, sucking excessive liquid from the side face by using filter paper, adding a drop of 2% phosphotungstic acid on the copper mesh to dye the phage for 10min, sucking the dyeing liquid by using filter paper, and observing the shape of the phage by using an electron microscope after the sample is dried. The transmission electron microscopy results showed that wild DK1 had a complete phage structure (FIG. 7A), and that the self-inactivating phage ΔTail DK exhibited two structures, either the head structure alone (FIG. 7B) or the head structure plus Tail structure lacking an appendage protein (FIG. 7C), both of which lacked structural proteins encoded by the knocked-out Tail gene (white arrows in FIG. 7A) compared to wild phage DK1.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that variations and modifications can be made without departing from the spirit of the invention, including similar operations to replace other bacteria or phage, which are within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1.Bacillus cereus bacteriophage DeltaTail DK1, accession number: GDMCC No:62951-B1.
2. A preparation method of a self-inactivating phage is characterized in that the phage is obtained by knocking out tail genes of the phage.
3. The preparation method of claim 2, wherein the tail gene knockout of phage DK1 is performed by in vivo homologous recombination, CRISPR-Cas gene editing or in vitro gene synthesis using parent phage DK1 as a target.
4. The preparation method according to claim 2, wherein the plasmid pcrF11-erm-1 is obtained by adding crRNA targeting the DK1 tail gene to the plasmid pcrF11-erm and inserting the tail gene which is synonymously mutated in the crRNA recognition region so that it cannot be recognized by the crRNA; the plasmids pcrF11-erm-1 and pXCR6-S are sequentially and directly transformed into the same host bacillus cereus 233-1 to obtain a recombinant host carrying two plasmids;
mixing phage DK1 with recombinant host, obtaining single plaque by double-layer agar method, adding xylose with final concentration of 3g/L, selecting plate with single plaque form observed, and performing plaque PCR to identify gene knockout result to obtain Tail gene knockout mutant strain DeltaTail DK, namely Bacillus cereus
bacteriophageΔTail DK1。
5. The method of claim 4, wherein the plaque PCR assay is performed using primers
The length of the delta Tail DK amplified fragment which is knocked out successfully is 1,786bp, and the amplification length of the wild DK1 is 4,117bp;
Tail-TF1：AGAATATTCGTATAGCTCAAACAGATAATGATGGTATTTC；
Tail-TR1：CTTACCAGTGTTATCTCCACCACAACTATTGATAG。
6. use of Bacillus cereus bacteriophage ΔTail DK1 as defined in claim 1 for the removal of pathogenic or contaminating bacteria.
7. The use according to claim 6, wherein the use is in the food industry, farming or clinical setting for the efficient control and removal of contaminating and pathogenic bacteria, preferably bacillus cereus.
8. The use according to claim 6, characterized by the use of Bacillus cereus bacteriophage ΔTail DK1 in bacterial detection.
9. An antibacterial agent comprising Bacillus cereus bacteriophage ΔTail dk1 as defined in claim 1 as an active ingredient.
10. The antimicrobial agent of claim 9, wherein the antimicrobial agent is a food additive or a food production environment disinfectant.
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