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CN-122012559-A - Preparation method of gene knockout tailless T4 phage, product and application thereof

CN122012559ACN 122012559 ACN122012559 ACN 122012559ACN-122012559-A

Abstract

The invention discloses a preparation method of a tailless T4 bacteriophage with a gene knockout function, a product and application thereof, and belongs to the technical field of microbial genetic engineering. The invention constructs tailless T4 phage by knocking out neck protein genes of T4 phage, adopts two steps of complementary host expansion culture and common host induction assembly, combines chloroform cracking, PEG precipitation enrichment and CsCl density gradient centrifugal purification, and has the advantages of high titer of the obtained tailless T4 phage, no obvious change of 30d titer in 4 ℃ storage, better size uniformity and lower non-specific binding risk, and thoroughly solves the problems of insufficient termination rate and easy generation of tailed phage impurities in the traditional amber mutation method. The preparation method is simple and convenient to operate, the cost is controllable, the large-scale production can be realized, and the constructed tailless T4 bacteriophage has wide application prospect in the fields of clinical diagnosis, environmental monitoring, food safety and the like.

Inventors

  • ZHOU XIN
  • TIAN RUNBO

Assignees

  • 河北医科大学第一医院

Dates

Publication Date
20260512
Application Date
20260210

Claims (11)

  1. 1. The preparation method of the tailless T4 bacteriophage with the gene knocked out is characterized by comprising the following steps: (1) Selecting a T4 bacteriophage as a starting bacteriophage; (2) Knocking out a coding gene of neck protein in the T4 phage; (3) Constructing a complementary plasmid containing a coding gene of neck protein, and transforming the complementary plasmid into a host cell to obtain a complementary host; (4) Infecting the complementary host in the step (3) with the T4 phage knocked out in the step (2), and performing amplification culture to obtain a tailed recombinant T4 phage; (5) Infecting the tail recombinant T4 phage in the step (4) into a common host without the complementary plasmid, inducing assembly of the tail-free phage, and obtaining a tail-free T4 phage finished product after splitting, enriching and purifying.
  2. 2. The method for preparing a tail-free T4 bacteriophage with a knocked-out gene according to claim 1, wherein the starting phage in the step (1) comprises a wild-type T4 phage, a T4 phage with a deleted hoc gene, a T4 phage with a deleted soc gene or a T4 phage with both a deleted hoc gene and a soc gene.
  3. 3. The method for preparing a gene knockout tailless T4 bacteriophage according to claim 1, wherein the gene knockout is performed in the step (2) by using CRISPR/Cas9 technology, and preferably, the coding gene of the neck protein is one or more of gp13 gene, gp14 gene and gp15 gene.
  4. 4. The method for preparing the gene knockout tailless T4 bacteriophage according to claim 3, wherein the CRISPR/Cas9 technology specifically comprises the steps of designing gRNA of a coding gene for the neck protein by utilizing CRISPOR on-line tool, and connecting the gRNA with a linearization vector to construct a recombinant editing plasmid, wherein the recombinant editing plasmid preferably further comprises left and right homologous arm sequences homologous to the coding gene of the neck protein.
  5. 5. The method for preparing the gene knockout tailless T4 bacteriophage of claim 4, wherein the sense strand and the antisense strand of the gRNA nucleotide sequence of the gp13 gene are shown in SEQ ID NO. 1-2, and the sense strand and the antisense strand of the gRNA nucleotide sequence of the gp15 gene are shown in SEQ ID NO. 19-20.
  6. 6. The preparation method of the gene knockout tailless T4 phage according to claim 1, wherein the construction step of the complementary plasmid in the step (3) is characterized in that a wild type T4 phage DNA is taken as a template to amplify a coding gene of neck protein, the coding gene is inserted into an expression vector and then subjected to point mutation to obtain the complementary plasmid which is prevented from being cut by a CRISPR/Cas9 system, preferably, the expression vector is a pET-28a plasmid, preferably, the complementary plasmid constructed aiming at gp13 gene is a pET-28a-gp13b plasmid, the nucleotide sequence of a primer used for point mutation is shown as SEQ ID NO. 13-14, preferably, the complementary plasmid constructed aiming at gp15 gene is a pET-28a-gp15b plasmid, and the nucleotide sequence of the primer used for point mutation is shown as SEQ ID NO. 29-30.
  7. 7. The preparation method of the gene knockout tailless T4 phage, which is disclosed in claim 1, is characterized in that in the step (5), the MOI value of infected common hosts is greater than or equal to 1, and the infected common hosts are subjected to standing adsorption at 37 ℃ for 5-15min.
  8. 8. The preparation method of the gene knockout tailless T4 phage of claim 1, wherein in the step (5), chloroform is adopted for pyrolysis, the addition amount of chloroform is 1/40,37 ℃ for pyrolysis for 15-45 min, the enrichment is carried out by adopting PEG8000 and NaCl for precipitation enrichment, the final concentration of PEG8000 is 4-10%, the final concentration of NaCl is 0.5M, the precipitation is overnight at 4 ℃, csCl density gradient centrifugation is adopted for purification, the CsCl density is 1.2-1.4 g/cm 3 , the centrifugation condition is 4 ℃ and 180,000Xg is adopted for centrifugation for 3h, and the intermediate layer phage component is collected.
  9. 9. The tailless T4 bacteriophage with gene knockout produced by the production method according to any one of claims 1 to 8.
  10. 10. The use of the knockout tailless T4 bacteriophage of claim 9 in detection of a target molecule, wherein the use comprises assembling a specific recognition element by phage display technology and modifying a signal molecule to construct a target molecule detection probe, wherein the target molecule comprises aβ42, aβ40, tau protein, APP, BSA or other biological molecules, environmental pollutants or food harmful substances, preferably, the specific recognition element is scFv-Hoc or scFv-Soc, preferably, the signal molecule is horseradish peroxidase, alkaline phosphatase or luminol signal molecule.
  11. 11. A detection probe which is constructed based on the tailless T4 bacteriophage of claim 9, preferably, the detection probe displays scFv-Hoc or Soc-Avi recombinant protein on the surface of the tailless T4 bacteriophage by phage display technology, and preferably, the detection probe further comprises biotin modification and/or HRP and/or streptavidin.

Description

Preparation method of gene knockout tailless T4 phage, product and application thereof Technical Field The invention belongs to the technical field of microbial genetic engineering and biological preparation, and particularly relates to a preparation method of a gene knockout tailless T4 bacteriophage, a product and application thereof. Background The phage serving as a virus for specifically infecting bacteria has the unique advantages of strong targeting, clear genetic background and high coat protein copy number, and has important application value in the fields of biological detection, biological preparation and the like. The T4 phage has strong splitting ability and rich modifiable sites, becomes a model strain of phage display technology, has a head structure containing nonessential proteins Hoc (about 155 copies) and Soc (about 870 copies), can be used as a high-efficiency display carrier of an exogenous functional element, does not influence phage proliferation after being knocked out, and provides a natural basis for constructing a high-performance detection probe. The T4 phage naturally has complete structures of head, tail and tail fibers, wherein the tail is the key of infection of a host, but the tail is easy to break or damage in structure in the processes of probe preparation, marking and long-term storage, so that the functions of the probe are invalid and the batch difference is increased, and meanwhile, the complete tail structure can increase the risk of nonspecific binding to interfere with the detection accuracy, so that the application of the T4 phage in the field of high-sensitivity detection is limited. In contrast, the tailless T4 phage has the tail structure deleted by genetic modification, not only retains the exogenous protein display capability of the head, but also has the core advantages of excellent size uniformity, strong storage stability, low risk of non-specific binding and the like, becomes a more ideal probe carrier, and has wide application potential in multi-field target molecule detection. However, the existing technology for preparing tailless T4 phage has obvious bottleneck that the technology mainly depends on an amber mutation method, double gene mutation is needed to be carried out on two genes (10 am13am and 17am18 am) to realize a tailless phenotype, but the termination rate of amber codons can not reach 100%, and impurities of the tailless phage are easy to generate in the preparation process, so that the purity of the product is insufficient (usually lower than 80%), the batch difference is large, and the performance of a subsequent probe is seriously influenced. In addition, the traditional phage preparation mostly adopts single host fermentation combined with conventional centrifugal purification technology, so that the problems of low preparation efficiency, insufficient product purity, poor stability and the like exist, part of technologies are used for improving phage yield through a double-host bacterial system, but the situation that phage particles are incomplete and the content of impurity proteins is high easily occurs due to assembly characteristic difference of unadapted tail-free T4 phage caused by tail deletion is easy to occur, and other technologies are used for optimizing fermentation process through physical fields or special additives, so that the titer can be improved to a certain extent, but the defects of high equipment cost, complex operation and inapplicability to large-scale production exist. In the aspect of probe application, the existing phage-based detection probes have the problems of single function suitability, limited signal amplification efficiency and the like, wherein part of probes can only carry a single type of recognition element and cannot realize specific binding and efficient signal transmission at the same time, and part of probes are used for modifying signal molecules in a chemical crosslinking mode, so that the activity of the recognition element is lost or the signal molecules fall off easily, and the detection sensitivity and stability are affected. Especially in the low concentration target molecule detection scene, traditional probe is difficult to compromise high sensitivity and strong specificity, can't satisfy the accurate detection demand in fields such as clinical diagnosis, environmental monitoring. The existing recombinant T4 phage related research focuses on the exogenous protein display of the tailed phage, a systematic scheme is not formed aiming at the efficient preparation technology of the tailless T4 phage, and the application potential of the recombinant T4 phage as a vector of a multi-type functional element is not fully explored. Therefore, the development of the tailless T4 phage preparation method which only needs to knock out a single gene, has high preparation efficiency and excellent product purity and stability, and the construction of two high-performance detection probes adapting to