Preparation method of gene knockout tailless T4 phage as well as product and application of gene knockout tailless T4 phage

Abstract

The invention discloses a preparation method of a gene knockout tailless T4 phage as well as a product and application thereof, and belongs to the technical field of microbial genetic engineering. The method comprises the following steps: knocking out a T4 bacteriophage neck protein gene to construct a tailless T4 bacteriophage; a two-step process of complementary host enlarged culture and common host induced assembly is adopted, chloroform cracking, PEG precipitation enrichment and CsCl density gradient centrifugal purification are combined, the titer of the obtained tailless T4 bacteriophage is high, the titer is not remarkably changed after the tailless T4 bacteriophage is stored at 4 DEG C for 30 days, and the tailless T4 bacteriophage has better size uniformity and lower non-specific binding risk and is suitable for industrial production. The problems that a traditional amber mutation method is insufficient in termination rate and tail bacteriophage impurities are easily generated are thoroughly solved. The preparation method is easy and convenient to operate and controllable in cost, large-scale production can be achieved, and the constructed tailless T4 phage has wide application prospects in the fields of clinical diagnosis, environmental monitoring, food safety and the like.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering and biological agent preparation technology, specifically relating to a method for preparing gene knockout tailless T4 phage, its products, and applications. Background Technology

[0002] Bacteriophages, as viruses that specifically infect bacteria, possess unique advantages such as strong targeting, clear genetic background, and high copy number of capsid proteins, making them valuable for applications in bioassay and biopharmaceutical preparation. T4 phage, due to its strong lytic ability and abundant modifiable sites, has become the model strain for phage display technology. Its head structure contains non-essential proteins Hoc (approximately 155 copies) and Soc (approximately 870 copies), which can serve as highly efficient display vectors for exogenous functional elements. Furthermore, gene knockout of these genes does not affect phage proliferation, providing a natural basis for the construction of high-performance detection probes.

[0003] T4 phages naturally possess a complete head, tail, and tail fiber structure, with the tail being crucial for host infection. However, during probe preparation, labeling, and long-term storage, the tail is prone to breakage or structural damage, leading to probe functional failure and increased batch-to-batch variability. Simultaneously, an intact tail structure increases the risk of non-specific binding, interfering with detection accuracy and limiting its application in highly sensitive detection fields. In contrast, tailless T4 phages, through genetic modification to eliminate the tail structure, not only retain the head's ability to display exogenous proteins but also possess core advantages such as superior size uniformity, strong storage stability, and low risk of non-specific binding, making them a more ideal probe carrier with broad application potential in the detection of target molecules across multiple fields.

[0004] However, existing technologies for preparing tailless T4 phages have significant bottlenecks: they primarily rely on the amber mutagenesis method, requiring double-gene mutation of two genes (10am13am and 17am18am) to achieve the tailless phenotype. However, the termination rate of amber codons cannot reach 100%, and tailed phage impurities are easily generated during the preparation process, resulting in insufficient product purity (usually below 80%), large batch-to-batch variations, and severely affecting the performance of subsequent probes. Furthermore, traditional phage preparation often employs single-host fermentation combined with conventional centrifugation purification processes, which suffers from low preparation efficiency, insufficient product purity, and poor stability. Some technologies improve phage yield through dual-host bacterial systems, but these are not adapted to the assembly characteristics differences caused by the tail-deficient T4 phage, easily leading to incomplete phage particles and high levels of impurities. Other technologies optimize the fermentation process through physical fields or special additives, which can improve potency to some extent, but suffer from high equipment costs, complex operation, and unsuitability for large-scale production.

[0005] At the probe application level, existing phage-based detection probes suffer from limitations such as single functional adaptability and limited signal amplification efficiency. Some probes can only carry a single type of recognition element, failing to simultaneously achieve specific binding and efficient signal transduction. Other probes rely on chemical cross-linking to modify signal molecules, which can easily lead to loss of recognition element activity or detachment of signal molecules, affecting detection sensitivity and stability. Especially in low-concentration target molecule detection scenarios, traditional probes struggle to balance high sensitivity and strong specificity, failing to meet the precise detection needs of fields such as clinical diagnosis and environmental monitoring.

[0006] Existing research on recombinant T4 phages largely focuses on the display of exogenous proteins in tailed phages, lacking a systematic approach for the efficient preparation of tailless T4 phages and failing to fully explore their application potential as vectors for various functional elements. Therefore, developing a tailless T4 phage preparation method that requires only the knockout of a single gene, boasts high efficiency, and exhibits excellent product purity and stability is crucial. Constructing two high-performance detection probes based on this phage, suitable for different application scenarios, is of significant practical importance and industrial value. These probes can achieve efficient binding of signal molecules through two-element assembly, or simplify the preparation process through single-element assembly combined with direct enzymatic modification. Summary of the Invention

[0007] Purpose of the invention: The first objective of this invention is to provide a method for preparing tailless T4 phage particles by knocking out the coding gene of the neck protein of T4 phage, thereby solving the problems of insufficient termination rate, easy generation of tailed phage impurities, and low preparation efficiency, poor purity, and large batch differences in the traditional amber mutation method (such as 10am13am, 17am18am double gene mutation) in the preparation of tailless T4 phage.

[0008] The second objective of this invention is to provide tailless phages obtained by the above preparation method that are morphologically uniform, have excellent storage stability, can be stored at 4°C for 30 days without significant changes in titer, and have a lower risk of nonspecific binding.

[0009] A third objective of this invention is to provide applications of the tailless T4 phage.

[0010] A fourth objective of this invention is to provide a detection probe constructed based on the tailless T4 phage.

[0011] Technical solution: To achieve the above objectives, this invention provides a method for preparing gene knockout tailless T4 phage, comprising the following steps:

[0012] (1) T4 phage was selected as the starting phage;

[0013] (2) Knock out the gene encoding the neck protein in T4 phage;

[0014] (3) Construct a complementary plasmid containing the coding gene of the neck protein, and transform the complementary plasmid into the host cell to obtain a complementary host;

[0015] (4) Infect the T4 phage that has been gene knocked out in step (2) with the complementary host described in step (3) and expand the culture to obtain tailed recombinant T4 phage;

[0016] (5) The tailed recombinant T4 phage described in step (4) is used to infect a normal host without the complementary plasmid, and the tailless phage is induced to assemble. After lysis, enrichment and purification, the tailless T4 phage product is obtained.

[0017] The starting phage mentioned in step (1) includes wild-type T4 phage, T4 phage lacking the hoc gene, T4 phage lacking the soc gene, or T4 phage lacking both the hoc and soc genes.

[0018] In step (2), CRISPR / Cas9 technology is used for gene knockout. Preferably, the gene encoding the neck protein is one or more of the gp13, gp14, and gp15 genes. Since gp13 and gp14 form a protein complex, knocking out either the gp13 or gp14 gene will achieve the goal of obtaining tailless T4 phage.

[0019] The specific steps of the CRISPR / Cas9 technology include: designing a gRNA targeting the gene encoding the neck protein using the CRISPOR online tool, and ligating the gRNA with a linearized vector to construct a recombinant editing plasmid. Preferably, the recombinant editing plasmid also contains left and right homologous arm sequences that are homologous to the gene encoding the neck protein.

[0020] The nucleotide sequences of the sense and antisense strands of the gRNA targeting the gp13 gene are shown in SEQ ID NO. 1~2; the nucleotide sequences of the sense and antisense strands of the gRNA targeting the gp15 gene are shown in SEQ ID NO. 19~20.

[0021] The construction steps of the complementary plasmid in step (3) are as follows: the coding gene of the neck protein is amplified using wild-type T4 phage DNA as a template, inserted into the expression vector, and then point-mutated to obtain a complementary plasmid that avoids being cut by the CRISPR / Cas9 system; preferably, the expression vector is pET-28a plasmid; preferably, the complementary plasmid constructed for the gp13 gene is pET-28a-gp13b plasmid, and the nucleotide sequences of the primers used for point mutation are shown in SEQ ID NO.13~14; preferably, the complementary plasmid constructed for the gp15 gene is pET-28a-gp15b plasmid, and the nucleotide sequences of the primers used for point mutation are shown in SEQ ID NO.29~30.

[0022] In step (4), the MOI value of the complementary host is 0.1; in step (5), the MOI value of the ordinary host is greater than or equal to 1, and the host is allowed to stand at 37°C for 5-15 minutes after infection. Preferably, two infections are performed during induced assembly, and the host is allowed to stand at 37°C for 10 minutes after each infection.

[0023] In step (5), the pyrolysis is performed using chloroform, with the amount of chloroform added being 1 / 40 of the total volume of the pyrolysis system. The pyrolysis is carried out at 37°C for 15-45 min, preferably 30 min at 37°C. The enrichment is performed by precipitation enrichment using PEG8000 and NaCl, with a final concentration of PEG8000 of 4-10%, preferably 4%, and a final concentration of NaCl of 0.5 M. Precipitation is carried out overnight at 4°C. The purification is performed by CsCl density gradient centrifugation, with a CsCl density of 1.2-1.4 g / cm³. 3 The centrifugation conditions were 4℃, 180,000×g for 3h, and the intermediate layer phage components were collected.

[0024] The present invention also includes the gene-knockout tailless T4 phage prepared by the aforementioned preparation method.

[0025] The present invention also includes the application of the gene knockout tailless T4 phage in the detection of target molecules. Preferably, the application includes assembling a specific recognition element and modifying a signal molecule to construct a target molecule detection probe using phage display technology. The target molecule includes Aβ42, Aβ40, Tau protein, APP, BSA or other biomolecules, environmental pollutants or harmful substances in food. Preferably, the specific recognition element is scFv-Hoc or scFv-Soc. Preferably, the signal molecule is horseradish peroxidase, alkaline phosphatase or luminol.

[0026] The present invention also includes a detection probe, which is constructed based on the tailless T4 phage; preferably, the probe displays scFv-Hoc or Soc-Avi recombinant protein on the surface of the tailless T4 phage using phage display technology; preferably, the detection probe further includes biotin modification and / or HRP and / or streptavidin.

[0027] The detection probe is used for sandwich ELISA detection.

[0028] The application scenarios include clinical diagnosis, environmental monitoring, food safety testing, biosensing, drug development, microbial detection, or other scenarios requiring high specificity, high sensitivity detection or targeted combination.

[0029] The tailless T4 phage can be adapted to the needs of different application scenarios by replacing specific recognition elements, signal molecules, or modifying them.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0031] (1) This invention precisely knocks out the gene gp13 or gp15 encoding a single neck protein using CRISPR / Cas9 technology, employs a two-step process of complementary host amplification culture and normal host induction assembly, and uses a PEG precipitation-CsCl density gradient centrifugation purification process. After purification by CsCl density gradient centrifugation, there are no obvious tailed phage impurities, which significantly improves the purity of the product. Moreover, the entire process is controlled at low temperature, which can better preserve the phage activity. The obtained phage titer can be as high as 10¹¹~10¹² PFU / mL, and the titer does not change significantly after 30 days of storage at 4℃. It has better size uniformity and lower risk of non-specific binding. It completely solves the problems of insufficient termination rate and easy generation of tailed phage impurities in the traditional amber mutagenesis method, as well as the defects of tailless phages that cannot proliferate autonomously due to the lack of tail structure, and greatly improves the preparation efficiency.

[0032] (2) The Aβ42 detection probe constructed based on tailless T4 phage of the present invention has high detection sensitivity and a detection limit as low as 0.79 pg / mL, which is up to 19.6 times higher than the detection sensitivity of traditional ELISA; it has strong specificity and no cross-reactivity with Aβ40, Tau protein, APP, BSA, etc. At the same time, it can be extended to the detection of a variety of target molecules by replacing specific elements, and has broad application prospects. Attached Figure Description

[0033] Figure 1The image shows the agarose gel electrophoresis detection of the pTCPLS-13g plasmid described in this embodiment of the invention; M is the molecular weight marker; all lanes are PCR identification products of the pTCPLS-13g plasmid (primers are gRNA-seq-pPT / Gseq-R-pPT, SEQ ID NO. 3~4), showing the target band, proving that the plasmid was successfully constructed.

[0034] Figure 2 The image shows the agarose gel electrophoresis detection of the pTCPLS-13g-LR recombinant plasmid described in this embodiment of the invention; M is the molecular weight marker; all lanes are the identification products of the pTCPLS-13g-LR recombinant plasmid (primers are gRNA-seq-pPT / Gseq-R-pPT, SEQ ID NO. 3~4), and the band size is consistent with the expectation (including the left homologous arm 563bp, the right homologous arm 749bp and the vector fragment), indicating that the left and right homologous arms of the gp13 gene were successfully inserted into the pTCPLS-13g plasmid.

[0035] Figure 3 The image shows the agarose gel electrophoresis detection of the pET-28a-gp13b plasmid described in this embodiment of the invention; M is the molecular weight marker; all lanes are PCR identification products of the pET-28a-gp13b plasmid (primers are 28a universal primers, SEQ ID NO.15~16), and the appearance of specific target bands indicates that the plasmid was successfully constructed.

[0036] Figure 4 This is an electrophoresis diagram of colony PCR identification of BL21 cells after transformation with the dual plasmids (pTCPLS-13g-LR + pET-28a-gp13b) as described in this embodiment of the invention; M is a molecular weight marker; the first four lanes are PCR identification products of the pET-28a-gp13b plasmid in the colony (primers are universal 28a primers, SEQ ID NO.15~16), and the last four lanes are PCR identification products of the pTCPLS-13g-LR plasmid in the colony (primers are gRNA-seq-pPT / Gseq-R-pPT SEQ ID NO.3~4). The simultaneous amplification of specific bands of the two plasmids indicates that the dual plasmids have been successfully co-transformed into BL21 competent cells.

[0037] Figure 5 The hoc described in the embodiments of the present invention - soc - gp13 - T4 phage knockout identification electrophoresis image; M is the molecular weight marker; all lanes in the left image are hoc. - soc - gp13 -The T4 phage PCR identification product (primer gp13-J F / R, SEQ ID NO. 17~18) showed a band of the expected size, with no wild-type band contamination. The right figure shows the wild-type T4 phage PCR identification product (primer gp13-J F / R, SEQ ID NO. 17~18), proving that the gp13 gene has been successfully knocked out.

[0038] Figure 6 The hoc described in the embodiments of the present invention - soc - gp13 - T4 phage gp13 gene sequencing identification image; sequencing results show gp13 gene sequence deletion, consistent with the expected knockout result, confirming hoc... - soc - gp13 - T4 phage was successfully constructed.

[0039] Figure 7 The image shows the agarose gel electrophoresis detection of the pTCPLS-15g plasmid described in this embodiment of the invention; M is the molecular weight marker; all lanes are PCR identification products of the pTCPLS-15g plasmid (primers are gRNA-seq-pPT / Gseq-R-pPT, SEQ ID NO. 3~4), and the bands are consistent with the expected size, indicating that the plasmid was successfully constructed.

[0040] Figure 8 The image shows the agarose gel electrophoresis results of the pTCPLS-15g-LR recombinant plasmid described in this embodiment of the invention; M is the molecular weight marker; all lanes are identification products of the pTCPLS-15g-LR recombinant plasmid (primers are gRNA-seq-pPT / Gseq-R-pPT, SEQ ID NO. 3~4), which meet the design size (including the left homologous arm 681bp, the right homologous arm 543bp and the vector fragment), confirming that the recombinant plasmid has been successfully constructed.

[0041] Figure 9 The image shows the agarose gel electrophoresis detection of the pET-28a-gp15b plasmid described in this embodiment of the invention; M is the molecular weight marker; all lanes are the identification products of the pET-28a-gp15b plasmid (primers are 28a universal primers, SEQ ID NO.15~16), and the bands are single and meet the expected size, proving that the plasmid was successfully constructed.

[0042] Figure 10This is an electrophoresis diagram of colony PCR identification of BL21 cells after transformation with the dual plasmids (pTCPLS-15g-LR + pET-28a-gp15b) as described in this embodiment of the invention; M is the molecular weight marker; all lanes in the left image are PCR identification products of the pTCPLS-15g-LR plasmid in the colony (primers are gRNA-seq-pPT / Gseq-R-pPT, SEQ ID NO. 3~4), and all lanes in the right image are PCR identification products of the pET-28a-gp15b plasmid in the colony (primers are 28a universal primers, SEQ ID NO. 15~16). The detection of the target bands corresponding to the two plasmids confirms that the co-transformation with the dual plasmids was successful.

[0043] Figure 11 The hoc described in the embodiments of the present invention - gp15 - T4 phage knockout identification electrophoresis image; M is the molecular weight marker; all lanes in the left image are hoc. - gp15 - The T4 phage PCR product (primers gp15-J F / R, SEQ ID NO.31~32) shows bands that match the expected size after knockout. The right figure shows the wild-type T4 phage PCR identification product (primers gp15-J F / R, SEQ ID NO.31~32), indicating that the gp15 gene knockout was successful.

[0044] Figure 12 The hoc described in the embodiments of the present invention - gp15 - T4 phage gp15 gene sequencing identification image; sequencing results show gp15 gene sequence deletion, consistent with the expected knockout result, confirming hoc... - gp15 - T4 phage was successfully constructed.

[0045] Figure 13 The tailless hoc described in the embodiments of the present invention - soc - gp13 - T4 phage and tailless hoc - gp15 - Transmission electron microscopy (TEM) image of T4 phage; left image shows tailless hoc - soc - gp13 - T4 phage, the image on the right shows a tailless hoc. - gp15 - T4 phage; both phages exhibited a regular icosahedral head structure, without a tail structure, and were uniform in morphology and well dispersed, confirming that the tailless phage was successfully prepared and structurally intact.

[0046] Figure 14 The tailless hoc described in the embodiments of the present invention - soc - gp13 - T4 phage and tailless hoc - gp15 - SDS-PAGE electrophoresis image of T4 phage; M is the protein molecular weight marker; lanes 1-6 are serial dilutions of Soc. - Hoc - T4 bacteriophage (titer 1.0 × 10⁻⁶) 8 PFU / μL, 7.5×10 7 PFU / μL, 5.0×10 7 PFU / μL, 2.5×10 7 PFU / μL, 1.0×10 7 PFU / μL, 7.5×10 6 PFU / μL), as a titer control; A is a tailless hoc - soc - gp13 - T4 phage, B stands for tailless hoc - gp15 - Both T4 phage and gp23 core protein bands were visible, and titer could be assessed by gp23 protein grayscale value (hoc). - soc - gp13 - The titer of T4 phage was 6 × 10¹¹ PFU / mL, hoc - gp15 - The titer of T4 phage was 10¹² PFU / mL.

[0047] Figure 15 This is a graph verifying the stability of the tailless bacteriophage stored at 4°C according to an embodiment of the present invention; the horizontal axis represents storage time (d), and the vertical axis represents bacteriophage titer (PFU / mL); the left graph shows the tailless hoc - soc - gp13 - T4 phage, the image on the right shows a tailless hoc. - gp15 - T4 phage; the titers of both phages did not change significantly within 30 days of storage at 4℃, indicating their excellent storage stability.

[0048] Figure 16This is an SDS-PAGE electrophoresis image of the purified scFv-Hoc recombinant protein described in this embodiment of the invention; M is the protein molecular weight marker; lanes 1-10 are, in order, lysis buffer, flow-through buffer, washing buffer W2, washing buffer W8, and elution buffers E1-E6. A specific band of about 70 kDa appears in the lane corresponding to the elution buffer, with few impurities, proving that the recombinant protein is purified well.

[0049] Figure 17 This is an SDS-PAGE electrophoresis image of the purified Soc-Avi recombinant protein described in this embodiment of the invention; M is the protein molecular weight marker; lanes 1-14 are, in order, lysis buffer, flow-through buffer, washing buffer W2, washing buffer W8, and elution buffers E1-E10. A specific band of about 15 kDa appears in the lane corresponding to the elution buffer, indicating high purity, which meets the requirements for subsequent experiments.

[0050] Figure 18 This is an indirect ELISA detection graph of the binding ability of T4@scFv@Avi to Aβ42 antigen described in this embodiment of the invention; the vertical axis represents the absorbance value at OD450nm; the horizontal axis, from left to right, represents the assembled T4@scFv@Avi, mAb 2E7 positive control, negative control, and blank control; the results show that T4@scFv@Avi and mAb 2E7 can specifically bind to Aβ42 antigen, and the absorbance values ​​are significantly higher than those of the negative control and blank control, proving that the assembled scFv-Hoc recombinant protein has good antigen-binding activity.

[0051] Figure 19 This is a Western blotting analysis of the binding ability of T4@scFv@B to HRP@SA described in this embodiment of the invention; M is the protein molecular weight marker; lane 1 is the biotinylated T4@scFv@Avi (T4@scFv@B), showing the target protein band; lane 2 is the unbiotinylated T4@scFv@Avi, showing no band, proving that T4@scFv@Avi was successfully biotinylated and can bind to HRP@SA.

[0052] Figure 20 The image shows a transmission electron microscope (TEM) image of the tailless T4 phage probe T4@scFv@HRP described in this embodiment of the invention. The probe is uniformly distributed, with no significant aggregation, and maintains good dispersion and structural integrity.

[0053] Figure 21This is an indirect ELISA detection graph of the binding ability of T4@scFv to Aβ42 antigen described in this embodiment of the invention; the vertical axis is the absorbance value at OD450nm; the horizontal axis, from left to right, represents the assembled T4@scFv, mAb 2E7 positive control, negative control, and blank control; the results show that T4@scFv and mAb 2E7 can specifically bind to Aβ42 antigen, and the absorbance values ​​are significantly higher than those of the negative control and blank control, proving that the probe antigen binding activity is good.

[0054] Figure 22 The image shows a transmission electron microscope (TEM) image of the double-tailed T4 phage probe T4@scFv@HRP described in this embodiment of the invention; the probe is uniformly distributed, with no significant aggregation, and has good structural integrity.

[0055] Figure 23 This is a standard curve for detecting Aβ42 using a sandwich ELISA based on a three-tailed T4 phage probe, as described in this embodiment of the invention. The horizontal axis represents the Aβ42 concentration (pg / mL), and the vertical axis represents the absorbance value at OD450nm. The curve shows good linearity (R² > 0.99), with a detection range of 1.56 pg / mL to 100 pg / mL and a limit of detection (LOD) of 0.86 pg / mL (S / N = 3), which is 18 times more sensitive than traditional methods.

[0056] Figure 24 This is a standard curve for detecting Aβ42 using a sandwich ELISA based on a double-tailed T4 phage probe, as described in this embodiment of the invention. The horizontal axis represents the Aβ42 concentration (pg / mL), and the vertical axis represents the absorbance value at OD450nm. The curve shows good linearity (R² > 0.99), with a detection range of 1.56 pg / mL to 100 pg / mL and a limit of detection (LOD) of 0.79 pg / mL (S / N = 3), which is 19.6 times more sensitive than the traditional method.

[0057] Figure 25 The graph shows the specificity detection results of the sandwich ELISA method based on the three-tailed tailless T4 phage probe described in this embodiment of the invention; the horizontal axis represents the sample type to be tested (Aβ42, APP, Aβ40, Tau, BSA in order), and the vertical axis represents the absorbance value at OD450nm; only the absorbance value of the Aβ42 sample is significantly higher than that of other samples and the blank control, indicating that the method has extremely high specificity.

[0058] Figure 26This is a graph showing the specificity detection results of the sandwich ELISA method based on double-missing tailless T4 phage probes described in this embodiment of the invention; the horizontal axis represents the sample type to be tested (Aβ42, APP, Aβ40, Tau, BSA in order), and the vertical axis represents the absorbance value at OD450nm; only the absorbance value of the Aβ42 sample is significantly higher than that of other samples and the blank control, indicating that the method has extremely high specificity.

[0059] Figure 27 This is a standard curve for detecting Aβ42 using the traditional sandwich ELISA method described in this embodiment of the invention; the horizontal axis represents the Aβ42 concentration (pg / mL), and the vertical axis represents the absorbance value at OD450nm; the curve shows good linearity, with a detection range of 15.62 pg / mL to 500 pg / mL and a detection limit (LOD) of 15.51 pg / mL (S / N=3), and is used for comparison with the method of this invention. Detailed Implementation

[0060] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0061] All DNA sequences, RNA sequences, and primer sequences in this invention were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0062] Example 1: hoc - soc - gp13 - Construction of T4 phage

[0063] CRISPR / Cas9 gRNA (13gRNA F / R, SEQ ID NO.1~2) targeting the gp13 gene of T4 bacteriophage was designed using the CRISPOR online tool (https: / / crispor.gi.ucsc.edu / ). The synthesized 13gRNA F and 13gRNA R were dissolved in pure water to a final concentration of 100 μM and mixed in the following proportions (5 μL each of 13gRNA F and 13gRNA R, 5 μL of 10×T4Polynucleotide Kinase Buffer, and 35 μL of ddH2O). The mixture was then placed in a PCR instrument and subjected to gradient cooling using a program of 95℃ for 5 min, 70℃ for 10 min, 50℃ for 10 min, 30℃ for 10 min, and storage at 4℃ to obtain a small dsDNA fragment of 13gRNA.

[0064] The pTCPLS plasmid (gifted by Professor Le Lu of Army Medical University, Yang L, Wang J, Lu S, Zhong Y, Xiong K, Liu X, Liu B, Wang X, Wang P, Le S. Temperature-dependent carrier state mediated by H-NS promotes the long-term coexistence of Y. pestis and a phage in soil. PLoS Pathog. 2023 Jun 22;19(6):e1011470. doi: 10.1371 / journal.ppat.1011470.) was linearized by restriction endonuclease Sap I. The linearized plasmid was mixed with a dsDNA fragment 13gRNA at a molar ratio of 1:3, and T4 DNA ligase was added and ligated overnight at 16 ℃ to construct the pTCPLS-13g plasmid. The recombinant pTCPLS-13g plasmid was transformed into *E. coli* DH5α competent cells using a heat shock transformation method and cultured overnight at 37°C. The pTCPLS-13g plasmid was extracted using the Fastpure Plasmid Mini Kit (Vazyme) and identified by primers gRNA-seq-pPT / Gseq-R-pPT (SEQ ID NO. 3~4). The results showed the acquisition of the target band, confirming the successful construction of the pTCPLS-13g plasmid (e.g., ...). Figure 1 (As shown).

[0065] Primers gp13-L F / R (SEQ ID NO. 5~6) and gp13-R F / R (SEQ ID NO. 7~8) were designed and synthesized. Wild-type T4 phage DNA (Bacteriophage T4, purchased from Mingzhou Biotechnology, catalog number MP002437, phage DNA extracted using FastPure ViralDNA / RNA Mini Kit V2 (Vazyme)) was used as a template to amplify the left and right homologous arm sequences (left homologous arm 563 bp, right homologous arm 749 bp). The PCR reaction system consisted of 25 μL Phanta Max, 21 μL ddH2O, 1 μL primers, and 2 μL template DNA. The amplification program was 95 ℃ pre-denaturation for 5 min, 35 cycles (95 ℃ 30 s, 58 ℃ 30 s, 72 ℃ 1 min), and 72 ℃ extension for 10 min. The amplification product was ligated with the enzyme-digested pTCPLS-13g plasmid (double digestion with Pst I and Xho I) to construct the pTCPLS-13g-LR recombinant plasmid. This plasmid was transformed into *E. coli* BL21 competent cells via electroporation and cultured overnight at 37°C. The pTCPLS-13g-LR plasmid was extracted using a kit, and PCR amplification was performed using primers gRNA-seq-pPT / Gseq-R-pPT (SEQ ID NO. 3~4) to screen for positive *E. coli* BL21 cells containing the recombinant plasmid. Electrophoresis results showed that the band size was consistent with expectations, indicating successful construction of the recombinant plasmid (e.g., ...). Figure 2 (As shown).

[0066] Primers 28a-13 F / R (SEQ ID NO. 9~10) and 13-28a F / R (SEQ ID NO. 11~12) were designed and synthesized to amplify the gp13 gene and the pET-28a vector using wild-type T4 phage DNA and pET-28a plasmid, respectively, as templates. Homologous recombination was then performed to construct the pET-28a-gp13 plasmid. Point mutations were performed on this plasmid using primer 13b-F / R (SEQ ID NO. 13~14) to prevent cleavage by the CRISPR / Cas9 system, yielding the pET-28a-gp13b plasmid. Identification was performed using universal pET-28a primers (SEQ ID NO. 15~16), and the results showed a specific target band, confirming the successful construction of the plasmid (e.g., ...). Figure 3 (As shown).

[0067] The pTCPLS-13g-LR plasmid and pET-28a-gp13b plasmid were mixed at a 1:1 ratio and co-transformed into BL21 competent cells. The cells were plated on LB agar plates containing gentamicin (20 μg / mL) and kanamycin (50 μg / mL), and cultured overnight at 37°C. Single colonies were picked and identified using primers gRNA-seq-pPT / Gseq-R-pPT (SEQ ID NO. 3~4) and universal 28a primers (SEQ ID NO. 15~16). Electrophoresis results showed simultaneous amplification of specific bands for both plasmids, indicating successful co-transformation (e.g., pTCPLS-13g-LR and pET-28a-gp13b). Figure 4 (As shown).

[0068] Add 300 μL of BL21 (OD600=0.6) containing two plasmids, gentamicin (20 μg / mL), kanamycin (50 μg / mL), arabinose (0.2%), and IPTG (1 mM) to 8 mL of LB semi-solid medium, mix well, and then spread onto antibiotic-free LB solid agar plates; Soc - Hoc - T4 phage (prepared in this laboratory, He L, Liu L, Zhou X, Hu Z, Shen J. Visual Counting of Influenza A Viruses with Magnetic T4 Phage SPR Probe. ACSSens. 2025 Apr 25;10(4):2928-2937. doi: 10.1021 / acssensors.4c03670. Epub 2025Mar 27. PMID: 40150976.) was prepared in 10⁻¹~10⁻ 8 Serial dilution (original phage concentration of 10) 9 PFU / mL), 40 μL / dilution was spotted at 8 sites and incubated overnight at 37℃; single phage plaques were picked and identified by PCR using primers gp13-J F / R (SEQ ID NO.17~18). Positive phage plaques were purified three times, and phage DNA was extracted for sequencing. PCR results showed bands of the expected size (e.g., PFU / mL). Figure 5 As shown), sequencing results revealed a deletion of the gp13 gene sequence (as shown). Figure 6 As shown), confirming the presence of a tail hoc. - soc - gp13 - T4 phage was successfully constructed.

[0069] Example 2: hoc - gp15 - Construction of T4 phage

[0070] CRISPR / Cas9 gRNA (15gRNAF / R, SEQ ID NO.19~20) targeting the T4 phage gp15 gene was designed using the CRISPOR online tool. The synthesized 15gRNA F and 15gRNA R were dissolved in pure water to a final concentration of 100 μM and mixed in the following proportions (5 μL each of 15gRNA F and 15gRNA R, 5 μL of 10×T4 Polynucleotide Kinase Buffer, and 35 μL of ddH2O). Primer annealing was performed using a PCR instrument with gradient cooling to form a small dsDNA fragment of 15gRNA.

[0071] The pTCPLS plasmid was linearized using the restriction endonuclease SapI and ligated with 15g RNA to construct the pTCPLS-15g plasmid. Positive plasmids were identified using primers gRNA-seq-pPT and Gseq-R-pPT (SEQ ID NO. 3~4). Electrophoresis results showed that the bands were consistent with the expected size, indicating successful plasmid construction. Figure 7 (As shown).

[0072] Primers gp15-L F and gp15-L R (SEQ ID NO. 21~22), gp15-R F and gp15-R R (SEQ ID NO. 23~24) were designed and synthesized. Using wild-type T4 phage DNA as a template, the left and right homologous arm sequences (left homologous arm 681 bp, right homologous arm 543 bp) were amplified. The pTCPLS-15g plasmid was linearized using restriction endonucleases Pst I and Xho I, and then ligated with the aforementioned homologous arms to construct the pTCPLS-15g-LR plasmid. Positive plasmids were identified using primers gRNA-seq-pPT and Gseq-R-pPT (SEQ ID NO. 3~4). The results showed that the bands conformed to the designed size, confirming the successful construction of the recombinant plasmid (e.g., ...). Figure 8 (As shown).

[0073] Primers 28a-15 F / R (SEQ ID NO. 25~26) and 15-28a F / R (SEQ ID NO. 27~28) were designed and synthesized to amplify the gp15 gene and pET-28a vector using wild-type T4 phage DNA and pET-28a plasmid, respectively, as templates. Homologous recombination was then performed to construct the pET-28a-gp15 plasmid. Point mutations were performed on this plasmid using primers 15b-F / R (SEQ ID NO. 29~30) to obtain the pET-28a-gp15b plasmid. Identification was performed using universal 28a primers (SEQ ID NO. 15~16). Electrophoresis results showed a single band of the expected size, confirming successful plasmid construction. Figure 9 (As shown).

[0074] The pTCPLS-15g-LR plasmid and the pET-28a-gp15b plasmid were co-transformed into BL21 competent cells. The two plasmids were identified using primers gRNA-seq-pPT / Gseq-R-pPT (SEQ ID NO. 3~4) and universal 28a primers (SEQ ID NO. 15~16). The results showed that the target bands corresponding to both plasmids were detected, confirming successful co-transformation. Figure 10 (As shown).

[0075] Add 300 μL of BL21 (OD600=0.6) containing two plasmids, gentamicin (20 μg / mL), kanamycin (50 μg / mL), arabinose (0.2%), and IPTG (1 mM) to 8 mL of LB semi-solid medium, mix well, and then spread onto antibiotic-free LB solid agar plates; add hoc - T4 phage (prepared in our laboratory, He L, Liu L, Zhou X, Hu Z, Shen J. Visual Counting of Influenza A Viruses with Magnetic T4 Phage SPR Probe. ACSSens. 2025 Apr 25;10(4):2928-2937. doi: 10.1021 / acssensors.4c03670. Epub 2025Mar 27. PMID: 40150976.) was serially diluted 10-fold, and 40 μL / dilution was used for spotting. A single phage plaque was picked and identified by knockout using primers gp15-JF and gp15-JR (SEQ ID NO.31~32). Positive phages were purified in three rounds and identified by PCR and sequencing to obtain hoc phages. - gp15 - T4 phage. PCR identification results showed that the bands conformed to the expected size after knockout (e.g., Figure 11 As shown), sequencing results revealed a deletion of the gp15 gene sequence (as shown). Figure 12 As shown), this indicates that there is a tail hoc. - gp15 - T4 phage was successfully constructed.

[0076] Example 3: Preparation and purification of tailless T4 phage

[0077] 1. Expand the culture of BL21 containing the pET-28a-gp13 plasmid constructed in Example 1 until the OD600 is 0.6, and add the tailed hoc plasmid constructed in Example 1. - soc- gp13 - T4 phage infection (MOI=0.1) was performed, followed by overnight incubation at 37 ℃ and 220 rpm. Cell debris was removed by centrifugation at 8,000×g for 10 min, and the phage pellet was precipitated by centrifugation at 80,000×g for 3 h. The phage was resuspended in Tris-Mg buffer (10 mM Tris-HCl, 50 mM NaCl, 10 mM MgSO4, pH=7.4). The phage titer was roughly determined using the double-layer plate method in BL21 containing pET-28a-gp13 plasmid, and the result was 2.5 × 10⁻⁶. 12 PFU / mL.

[0078] 2. Expand the culture of BL21 competent cells to an OD600 of 0.9, and add the tailed hoc obtained in step 1. - soc - gp13 - Infect with T4 phage (MOI=1), allow to stand at 37 ℃ for 10 min for adsorption, then infect again (MOI=1), and incubate at 37 ℃ and 220 rpm for 2 h; add chloroform (1 / 40 of the total volume), lyse by shaking at 37 ℃ and 150 rpm for 30 min, centrifuge at 8,000×g for 10 min to remove cell debris, and collect the supernatant.

[0079] 3. Add PEG8000 and NaCl to the supernatant to achieve a final concentration of 4% PEG8000 and 0.5M NaCl. Incubate overnight at 4°C to allow precipitation. Centrifuge at 10,000×g for 30 min, discard the supernatant, and resuspend the precipitate in Tris-Mg buffer to obtain tailless phage crude extract. Add the crude extract to a pre-prepared CsCl density gradient centrifuge tube (density range 1.2~1.4 g / cm³), centrifuge at 180,000×g for 3 h at 4°C, and collect the intermediate layer phage fraction. Dialyze the collected fraction with Tris-Mg buffer to desalt and obtain tailless hoc phage. - soc - gp13 - T4 phage finished product.

[0080] 4. Following the same procedure described above, using BL21 containing the pET-28a-gp15 plasmid as the host, prepare and purify tailless hoc cells. - gp15 - T4 phage.

[0081] 5. Transmission electron microscopy revealed that the bacteriophages in the finished product all exhibited a regular icosahedral head structure, without tail structures, and were uniform in morphology, well-dispersed, and free of tailed bacteriophage impurities (such as...). Figure 13 (as shown); using a series of diluted Soc - Hoc -T4 phage (titer 1.0 × 10⁻⁶) 8 PFU / μL, 7.5×10 7 PFU / μL, 5.0×10 7 PFU / μL, 2.5×10 7 PFU / μL, 1.0×10 7 PFU / μL, 7.5×10 6 The titer of tailless T4 phage was assessed using PFU / μL (calculated based on the relative gray value of gp23 protein). Figure 14 As shown), the results indicate that hoc - soc - gp13 - The titer of T4 phage was 6 × 10⁻⁶. 11 PFU / mL, hoc - gp15 - The T4 phage titer was 10. 12 PFU / mL.

[0082] Two types of tailless hockey samples were taken and stored at 4℃ for 1 day, 10 days, 20 days, and 30 days, respectively. - soc - gp13 - T4 and hoc - gp15 - The titer of T4 phage was determined to assess its stability. The results showed that the titers of both tailless phages did not change significantly within 30 days of storage at 4°C, indicating excellent storage stability (e.g., ...). Figure 15 (As shown).

[0083] Example 4: Preparation and purification of recombinant scFv-Hoc and Soc-Avi proteins

[0084] The heavy chain variable region (VH) and light chain variable region (VL) of the anti-Aβ42 specific monoclonal antibody (mAb2E7, prepared in our laboratory, Tian, ​​R., Zhang, Q., Zhu, H. et al. Phage-linked Immunosorbent Assay for Ultrasensitive Detection of Aβ42. J. Anal. Test.(2025). https: / / doi.org / 10.1007 / s41664-025-00399-2) were cloned using primers VH F / R and VL F / R (SEQ ID NO.33~36). The VH and VL regions were then ligated using overlap extension PCR to obtain the scFv gene (SEQ ID NO.37). Using genetic engineering techniques, the scFv gene sequence was integrated into the N-terminus of the Hoc gene, ligated into a linearized pET-28a vector (with Nde I and Xho I restriction sites), and the expression vector pET-28a-scFv-Hoc was constructed (scFv-Hoc gene sequence shown in SEQ ID NO. 38). The Avi-tag gene sequence (SEQ ID NO. 39) was integrated into the C-terminus of the Soc gene, ligated into a linearized pET-28a vector (with Nde I and Xho I restriction sites), and the expression vector pET-28a-Soc-Avi was constructed (Soc-Avi gene sequence shown in SEQ ID NO. 40). High-efficiency expression of the recombinant proteins scFv-Hoc and Soc-Avi was performed in the *E. coli* BL21(DE3) system. The fusion proteins were purified by Ni²⁺-NTA affinity chromatography to obtain high-purity fusion proteins. SDS-PAGE electrophoresis results showed that the scFv-Hoc recombinant protein exhibited a specific band of approximately 70 kDa (e.g., scFv-Hoc). Figure 16 As shown), the Soc-Avi recombinant protein exhibits a specific band of approximately 15 kDa (e.g. Figure 17 As shown in the figure, the presence of fewer contaminating proteins indicates that the two recombinant proteins were purified effectively.

[0085] Example 5: Construction and Identification of Aβ42 Detection Probe

[0086] 1. Construct Aβ42 detection probe 1 (T4@scFv@HRP three-tailed tailless T4 phage probe)

[0087] Take the tailless hoc prepared in Example 3 - soc - gp13 -The T4 phage product was assembled with recombinant proteins scFv-Hoc and Soc-Avi at a molar ratio of 1:1,000:8,000 at 37 °C for 1 h to obtain T4@scFv@Avi. Indirect ELISA analysis showed that T4@scFv@Avi could specifically bind to Aβ42 (as shown in Figure 18). Western blot analysis showed that biotinylated T4@scFv@Avi (T4@scFv@B) could specifically bind to HRP@SA (e.g., ...). Figure 19 As shown in the figure, this proves that the assembly was successful and the assembled protein is fully functional.

[0088] T4@scFv@Avi was biotinylated using the Avi-tagged protein biotinylation kit (BirA method) (Shanghai Beyotimes Biotechnology Co., Ltd.) to obtain T4@scFv@B, which was then bound to HRP@SA (HRP-modified streptavidin) at a molar ratio of 1:1,000 (37℃, 1h) to construct Aβ42 detection probe 1 (triple-tail-free T4 phage probe T4@scFv@HRP). Transmission electron microscopy showed that the triple-tail-free T4 phage probe was uniformly distributed and showed no significant aggregation (e.g., ...). Figure 20 (As shown).

[0089] 2. Construct Aβ42 detection probe 2 (double-empty tailless T4 phage probe T4@scFv@HRP)

[0090] Take the tailless hoc prepared in Example 3 - gp15 - T4 phage was used to assemble the recombinant protein scFv-Hoc using phage display technology to obtain T4@scFv. HRP modification was then performed using the sodium periodate method to construct Aβ42 detection probe 2 (double-empty tailless T4 phage probe T4@scFv@HRP). Indirect ELISA analysis showed that T4@scFv can specifically bind to Aβ42 (e.g., ...). Figure 21 (As shown). Transmission electron microscopy revealed that the double-tailed T4 phage probes were evenly distributed and showed no significant aggregation (as shown). Figure 22 As shown in the figure, this indicates that the probe was successfully constructed.

[0091] Example 6: Sandwich ELISA detection of Aβ42

[0092] 1. Coating: Aβ42 monoclonal antibody 3B6 (prepared in our laboratory; antibody 3B6 is derived from the article Tian, ​​R., Zhang, Q., Zhu, H. et al. Phage-linked Immunosorbent Assay for Ultrasensitive Detection of Aβ42. J. Anal. Test. (2025). https: / / doi.org / 10.1007 / s41664-025-00399-2, which can be paired with mAb 2E7 for sandwich ELISA) was diluted to 1 μg / mL with carbonate coating buffer (0.05 M, pH=9.6), and 100 μL was added to each well of a 96-well microplate and incubated overnight at 4°C.

[0093] 2. Blocking: Discard the coating solution, wash 5 times with PBST (PBS + 0.05% Tween-20), add 200 μL of 5% skim milk blocking solution to each well, and incubate at room temperature for 2 hours;

[0094] 3. Sample addition: Add serially diluted Aβ42 standard (1.56 pg / mL~100 pg / mL, β-Amyloid (1-42), human, purchased from Shanghai Qiangyao Biotechnology Co., Ltd., catalog number 04010011526), ​​100 μL per well, and incubate at room temperature for 1.5 h; after washing 5 times with PBST, add probe 1 (triple-tail-free T4 phage probe T4@scFv@HRP) or probe 2 (double-tail-free T4 phage probe T4@scFv@HRP), 100 μL per well, and incubate at room temperature for 1 h; during detection, the capture antibody first binds to Aβ42, and Aβ42 then specifically binds to scFv-Hoc on probe 1 or probe 2, forming a capture antibody-Aβ42-probe 1 sandwich structure and a capture antibody-Aβ42-probe 2 sandwich structure, respectively;

[0095] 4. Color development and termination: Wash 5 times with PBST, add 100 μL of color development solution (TMB substrate solution) to each well, develop color at room temperature for 15 min, add 50 μL of stop solution, and read the absorbance value at OD450nm using a microplate reader.

[0096] Sensitivity analysis showed that the detection method corresponding to probe 1 (triple-tail-free T4 phage probe T4@scFv@HRP) exhibited good linearity (R² > 0.99), with a limit of detection (LOD) of 0.86 pg / mL (S / N = 3). Figure 23As shown); the detection method corresponding to probe 2 (double-empty tailless T4 phage probe T4@scFv@HRP) showed good linearity (R²>0.99), and the limit of detection (LOD) was 0.79 pg / mL (S / N=3) (as shown). Figure 24 (As shown).

[0097] Specificity analysis results showed that both detection methods based on tailless T4 phage probes only specifically reacted to Aβ42, and showed no cross-reactivity to potential interfering antigens such as APP, Aβ40, Tau, and BSA (e.g., Figure 25 , Figure 26 As shown in the figure, the constructed detection probe has stable performance and high specificity.

[0098] Comparative Example 1

[0099] The neck protein was knocked out using a conventional host containing only the knockout plasmid (BL21 host containing only pTCPLS-13g-LR or pTCPLS-15g-LR plasmids, but not pET-28a-gp13b or pET-28a-gp15b plasmids). The remaining steps were the same as in Example 1 or Example 2. The results showed that all picked single plaques were negative, and no phages with the neck protein knocked out were detected. This indicates that additional plasmids (pET-28a-gp13b or pET-28a-gp15b plasmids) are needed to provide additional neck protein during the knockout process; otherwise, phages with the neck protein gene knocked out cannot form plaques.

[0100] Comparative Example 2

[0101] Tailless T4 phage prepared using the traditional amber mutagenesis method

[0102] 1. Primers 13am-LF / R (SEQ ID NO.41~42) and 13am-RF / R (SEQ ID NO.43~44) were designed and synthesized. Using wild-type T4 phage DNA (Bacteriophage T4, purchased from Mingzhou Biotechnology, catalog number MP002437, phage DNA extracted using FastPure Viral DNA / RNA Mini Kit V2 (Vazyme)) as a template, the left and right homologous arm sequences (left homologous arm 563 bp, right homologous arm 801 bp) were amplified. The amplified product was ligated to the enzyme-digested pTCPLS-13g plasmid (double digested with Pst I and Xho I) constructed in Example 1 to construct the pTCPLS-13am-LR recombinant plasmid. This plasmid was transformed into *E. coli* Trans1-Blue competent cells via electroporation to obtain Trans1-Blue cells containing the pTCPLS-13am-LR recombinant plasmid.

[0103] 2. Add 300 μL of Trans1-Blue (OD600=0.6) containing pTCPLS-13am-LR plasmid, gentamicin (20 μg / mL), and arabinose (0.2%) to 8 mL of LB semi-solid medium, mix well, and then spread onto antibiotic-free LB solid agar plates; Soc - Hoc - T4 phage at 10⁻¹~10⁻ 8 Serial dilution (original concentration 10) 9 (PFU / mL), 40 μL / dilution was spotted at 8 sites and incubated overnight at 37℃; single plaques were picked and identified by PCR and sequencing using primers gp13am-J F / R (SEQ ID NO.45~46). After 3 rounds of purification, tailed hoc cells with amber mutation successfully occurring at the target position of the gp13 gene were obtained. - soc - gp13amT4 phage.

[0104] 3. Expand the culture of Trans1-Blue competent cells to an OD600 of 0.6, and add the tailed hoc cells prepared in step 2. - soc - Infect with gp13amT4 phage (MOI=0.1), incubate overnight at 37 ℃ and 220 rpm; centrifuge at 8,000×g for 10 min to remove cell debris, centrifuge at 80,000×g for 3 h to precipitate the phage, and resuspend the phage in Tris-Mg buffer (10 mM Tris-HCl, 50 mM NaCl, 10 mM MgSO4, pH=7.4).

[0105] 4. Expand the culture of BL21 competent cells to an OD600 of 0.9, then add the tailed hoc obtained in step 3. - soc - bacteriophage infection with gp13amT4 (MOI=1) was followed by static adsorption at 37 ℃ for 10 min, then re-infection (MOI=1), and incubation at 37 ℃ and 220 rpm for 2 h. Tail-free hoc cells were obtained by chloroform lysis, PEG / NaCl precipitation, and CsCl density gradient centrifugation. - soc - gp13amT4 phage.

[0106] The results showed that obvious tailed T4 phage bands appeared during CsCl gradient centrifugation, indicating that the host consumed a portion of its energy production to produce tailed T4 phage.

[0107] Comparative Example 3

[0108] Tailless T4 phages were enriched and purified using conventional purification techniques (ultracentrifugation + CsCl density gradient centrifugation), with the remaining steps consistent with Example 3. TEM results showed that the obtained tailless T4 phages had poor morphology, were deformed, and were not regular icosahedrons.

[0109] Comparative Example 4

[0110] Aβ42 was detected using a conventional sandwich ELISA method (with the same antibody pairs mAb 3B6 and mAb 2E7, without phage probes). The results showed a limit of detection (LOD) of 15.51 pg / mL. Figure 27 As shown, its detection limit is 18 times higher than that of probe 1 (triple-tail-free T4 phage probe T4@scFv@HRP, LOD=0.86pg / mL) and 19.6 times higher than that of probe 2 (double-tail-free T4 phage probe T4@scFv@HRP, LOD=0.79pg / mL), indicating that the detection sensitivity of the probe of the present invention is superior.

[0111] Comparative Example 5

[0112] Based on Soc - Hoc - The tailed T4 phage probe was prepared using the same steps as in Step 1 of Example 5. Then, a sandwich ELISA method for detecting Aβ42 was established based on the tailed T4 phage probe, with the remaining steps consistent with Example 6. This tailed T4 phage probe, due to the presence of a tail sheath and tail filaments, exhibits significant steric hindrance, limiting the formation efficiency of the sandwich complex (capture antibody / Aβ42 / phage probe). Simultaneously, the phage tail sheath and tail filaments readily undergo non-specific adsorption with the capture antibody and impurities in the sample, resulting in an OD450nm greater than 0.3 for blank and negative wells. In contrast, the tailless T4 phage probe of this invention has a regular icosahedral structure, which is more concise, has less steric hindrance, significantly reduces non-specific binding, and allows the background OD450nm to be controlled below 0.1, indicating that the tailless T4 phage probe of this invention performs better.

Claims

1. A method for preparing a gene-knockout tailless T4 phage, characterized in that, Includes the following steps: (1) T4 phage was selected as the starting phage; (2) Knock out the gene encoding the neck protein in T4 phage; (3) Construct a complementary plasmid containing the coding gene of the neck protein, and transform the complementary plasmid into the host cell to obtain a complementary host; (4) Infect the T4 phage that has been gene knocked out in step (2) with the complementary host described in step (3) and expand the culture to obtain tailed recombinant T4 phage; (5) The tailed recombinant T4 phage described in step (4) is used to infect a normal host without the complementary plasmid, and the tailless phage is induced to assemble. After lysis, enrichment and purification, the tailless T4 phage product is obtained.

2. The method for preparing gene knockout tailless T4 phage according to claim 1, characterized in that, The starting phage mentioned in step (1) includes wild-type T4 phage, T4 phage lacking the hoc gene, T4 phage lacking the soc gene, or T4 phage lacking both the hoc and soc genes.

3. The method for preparing gene knockout tailless T4 phage according to claim 1, characterized in that, In step (2), CRISPR / Cas9 technology is used for gene knockout; preferably, the gene encoding the neck protein is one or more of the gp13, gp14 and gp15 genes.

4. The method for preparing gene knockout tailless T4 phage according to claim 3, characterized in that, The specific steps of the CRISPR / Cas9 technology include: designing a gRNA targeting the coding gene of the neck protein using the CRISPOR online tool, and ligating the gRNA with a linearized vector to construct a recombinant editing plasmid. Preferably, the recombinant editing plasmid also contains left and right homologous arm sequences that are homologous to the coding gene of the neck protein.

5. The method for preparing gene knockout tailless T4 phage according to claim 4, characterized in that, The sense and antisense strands of the gRNA nucleotide sequence for the gp13 gene are shown in SEQ ID NO. 1~2; the sense and antisense strands of the gRNA nucleotide sequence for the gp15 gene are shown in SEQ ID NO. 19~20.

6. The method for preparing gene knockout tailless T4 phage according to claim 1, characterized in that, The construction steps of the complementary plasmid in step (3) are as follows: the coding gene of the neck protein is amplified using wild-type T4 phage DNA as a template, inserted into the expression vector, and then point-mutated to obtain a complementary plasmid that avoids being cut by the CRISPR / Cas9 system; preferably, the expression vector is pET-28a plasmid; preferably, the complementary plasmid constructed for the gp13 gene is pET-28a-gp13b plasmid, and the nucleotide sequences of the primers used for point mutation are shown in SEQ ID NO.13~14; preferably, the complementary plasmid constructed for the gp15 gene is pET-28a-gp15b plasmid, and the nucleotide sequences of the primers used for point mutation are shown in SEQ ID NO.29~30.

7. The method for preparing gene knockout tailless T4 phage according to claim 1, characterized in that; In step (5), the MOI value of the infected host is greater than or equal to 1, and after infection, the host is allowed to stand at 37°C for 5-15 minutes for adsorption.

8. The method for preparing gene knockout tailless T4 phage according to claim 1, characterized in that, The pyrolysis in step (5) is performed using chloroform, with the amount of chloroform added being 1 / 40 of the total volume of the pyrolysis system. Pyrolysis is carried out at 37°C for 15-45 min. The enrichment is performed by precipitation enrichment using PEG8000 and NaCl, with a final concentration of PEG8000 of 4-10% and a final concentration of NaCl of 0.5 M. Precipitation is carried out overnight at 4°C. Purification is performed by CsCl density gradient centrifugation, with a CsCl density of 1.2-1.4 g / cm³. 3 The centrifugation conditions were 4℃, 180,000×g for 3h, and the intermediate layer phage components were collected.

9. The gene-knockout tailless T4 phage prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the gene knockout tailless T4 phage according to claim 9 in the detection of target molecules, characterized in that, The application includes assembling specific recognition elements and modifying signal molecules using phage display technology to construct target molecule detection probes; the target molecules include Aβ42, Aβ40, Tau protein, APP, BSA or other biomolecules, environmental pollutants or food hazards; preferably, the specific recognition element is scFv-Hoc or scFv-Soc, and preferably, the signal molecule is horseradish peroxidase, alkaline phosphatase or luminol signal molecule.

11. A detection probe, characterized in that, The detection probe is constructed based on the tailless T4 phage as described in claim 9; preferably, the probe displays scFv-Hoc or Soc-Avi recombinant protein on the surface of the tailless T4 phage using phage display technology; preferably, the detection probe further includes biotin modification and / or HRP and / or streptavidin.

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