Thermoresistant helper phage TR-3 and its application

Abstract

The present invention provides a high temperature resistant helper phage TR-3 and its application, and relates to the field of biotechnology. The high temperature resistant helper phage TR-3 provided by the present invention is a mutant of helper phage M13KO7 obtained by multiple rounds of high temperature treatment (98°C), infection and amplification. TR-3 survives stably under 98°C conditions, has high infection efficiency, and retains the ability to package pIII fusion display phagemids with normal efficiency and high-quality ssDNA extraction. Therefore, it can be used for phage ssDNA preparation, phage display library construction and separation of stable protein variants.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a high temperature resistant helper phage TR-3 and application thereof. Background Art

[0002] There are two types of vectors commonly used for phage display: phage vectors and phagemid vectors. Phagemid vectors are the most commonly used because they have the advantages of small genome, easy operation, large insertable fragments, high transformation efficiency, and more stable recombinants. Phagemids only contain part of the genetic information of phages, so both library construction and library display require helper phages to provide the proteases and coat proteins required for replication and packaging of the phagemid DNA of the host cell.

[0003] Helper phage is a mutant of a filamentous phage with extremely low DNA replication efficiency. Its genomic DNA has all the functional genes of M13 phage, but the IG region is defective, so its pII protein cannot effectively recognize this sequence, and the DNA of the helper phage itself cannot replicate in a single strand, so it can provide pII protein and packaging protein for the phagemid. The inactivation of the IG region of the helper phage also makes its own genes unable to be expressed. When the phagemid containing the recombinant gene is transfected into Escherichia coli and then superinfected with the helper phage, the pII protein synthesized by the helper phage will preferentially recognize the normal gene spacer on the phagemid, initiate rolling circle replication, and produce ssDNA. The ssDNA packaged by the coat protein of the daughter phage produced in this way is mainly from the DNA of the phagemid containing the recombinant gene, and at the same time displays the fusion protein of the pIII or pVIII encoded by the phagemid and the exogenous peptide segment and the wild-type pIII or pVIII protein encoded by the helper phage, ensuring that the recombinant phage can infect, assemble and proliferate normally.

[0004] There are two main categories of helper phages constructed through different mechanisms, namely helper phages containing full-length gIII, helper phages with gIII deletion or gIII defect. Helper phages containing full-length gIII mainly include M13KO7, R408 and VCSM13. M13KO7 is a mutant of M13 phage, carrying a plasmid replication origin, kanamycin resistance gene and mutant gene II of G6125T. Studies have shown that the efficient production of progeny phage particles and the yield of single-stranded DNA are closely related to the cell growth temperature. Higher temperatures will promote rapid cell growth, cause potential cell lysis and partial phage inactivation, and even lose the ability of phage to infect cells. Ultimately, only low-yield and poorly stable phage particles can be obtained, affecting the yield of phage single-stranded DNA and the titer of phage display antibody library. For example, compared with culturing at 37°C, culturing CJ236 cells at 25°C can significantly improve the yield and purity of ssDNA, which may be related to the low tolerance of the helper phage M13K07 itself to temperature. It can be seen that there is a difference between the suitable temperature of the helper phage and the suitable temperature of the cells. Therefore, improving the tolerance of the helper phage is of great significance for phage display.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] One of the purposes of the present invention is to provide a high temperature resistant helper phage TR-3 to at least solve one of the technical problems existing in the prior art.

[0007] A second object of the present invention is to provide a method for preparing the above-mentioned high temperature resistant helper phage TR-3.

[0008] The third object of the present invention is to provide an application of the above-mentioned high temperature resistant helper phage TR-3.

[0009] A fourth object of the present invention is to provide a phage display system.

[0010] A fifth object of the present invention is to provide a test kit.

[0011] A sixth object of the present invention is to provide an application of the above-mentioned phage display system.

[0012] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0013] A thermostable helper phage TR-3, wherein the amino acid sequence of the thermostable helper phage TR-3 differs from that of the helper phage M13KO7 only in that:

[0014] The pVIII of the thermostable helper phage TR-3 has a V6A mutation;

[0015] The thermostable helper phage TR-3 had G287R and S378G mutations in pIII; and

[0016] The pIV of the thermostable helper phage TR-3 underwent I87N mutation.

[0017] A method for preparing a thermostable helper phage TR-3, wherein the helper phage M13KO7 is modified as follows:

[0018] The pVIII of helper phage M13KO7 was subjected to V6A mutation;

[0019] The pill of the helper phage M13KO7 was mutated to G287R and S378G; and

[0020] The pIV of helper phage M13KO7 was subjected to I87N mutation.

[0021] Application of the above-mentioned high temperature resistant helper phage TR-3 in phage display.

[0022] The application of the above-mentioned high temperature resistant helper phage TR-3 in the preparation of ssDNA.

[0023] The application of the above-mentioned high temperature resistant helper phage TR-3 in preparing antibody library.

[0024] The application of the above-mentioned high temperature resistant helper phage TR-3 in constructing a phage display library.

[0025] A phage display system containing the above-mentioned high temperature resistant helper phage TR-3.

[0026] Furthermore, the phage display system also includes a phagemid.

[0027] A kit containing the phage display system.

[0028] Application of the above phage display system in preparing ssDNA or antibody library.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The high-temperature-resistant helper phage TR-3 provided by the present invention is a mutant of the helper phage M13KO7 obtained through multiple rounds of high-temperature treatment (98°C), infection and amplification. TR-3 survives stably under 98°C conditions, has high infection efficiency, and retains the ability to package pIII fusion display phagemids with normal efficiency and high-quality ssDNA extraction. Therefore, it can be used for phage ssDNA preparation, phage display library construction and separation of stable protein variants. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 The high temperature resistant helper phage TR-3 provided in Example 3 of the present invention is used to extract ssDNA. DETAILED DESCRIPTION

[0033] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] In order to solve the problem of low yield and poor purity of phage display technology products caused by poor tolerance of helper phage in the prior art, the inventors screened and obtained the high-temperature resistant helper phage TR-3 provided by the present invention through high-temperature stress. It was found that the amino acid sequence of the high-temperature resistant helper phage TR-3 was different from that of the helper phage M13KO7 only in that: pVIII of the high-temperature resistant helper phage TR-3 had a V6A mutation, pIII had a G287R and S378G mutations, and pIV had an I87N mutation.

[0035] The pVIII protein sequence of the thermostable helper phage TR-3 is shown in SEQ ID NO.1, the pIII protein sequence is shown in SEQ ID NO.2, and the pIV protein sequence is shown in SEQ ID NO.3.

[0036] The TR-3 provided by the present invention can stably survive at 98°C, has high infection efficiency, and retains the ability to package pIII fusion display phagemids with normal efficiency and high-quality ssDNA extraction, thereby overcoming the defect of poor tolerance of current helper phages.

[0037] The high temperature resistant helper phage TR-3 of the present invention can be widely used in phage display technology, and can be specifically used in the preparation of ssDNA, the preparation of antibody libraries, etc. The phage display system containing TR-3 and phagemid can adapt to a wide range of cell culture temperatures and perform efficient production.

[0038] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0039] Example 1: High temperature resistant M13KO7 assisted phage screening

[0040] 1. Bioscreening: Take 2×10 10 The mother M13KO7 helper phage (purchased from NEB Biolab, USA, catalog number: N0315S) was incubated at 98°C for 30 minutes. After centrifugation at 12000g for 10 minutes, 100 μL of supernatant was taken and infected with 1 mL of NEB5alphaF' bacterial solution (OD value was 0.8), and cultured in a 3D shaker for 1 hour; part of the infected bacterial solution was plated on 2YT / Kan25 and placed in a 37°C biochemical incubator overnight. The remaining bacterial solution was transferred to 35 mL of 2YT / Kan25, cultured in a shaker overnight, and phages were collected the next day to form a heat-resistant helper phage library for each round. Repeat the above steps until the R3 round of high-temperature-resistant helper phages are enriched.

[0041] 2. Optimize the enriched clones by helper phage production: Randomly select 5 clones from the 2YT / Kan25 plate in the R3 round of screening, named TR-1 to TR-5, culture them overnight in 35 mL of 2YT / Kan25 medium, and collect helper phage the next day. Take 2×10 10 The helper phage was incubated at 98°C for 30 min, centrifuged at 12000g for 10 min, and 100 μL of the supernatant was taken to infect 1 mL of NEB5alpaF' bacterial solution (OD 600 value is 0.8); after culturing for 1 hour, 20 μL of bacterial solution was then taken for appropriate dilution and plated on LB / Tet10 and LB / Kan25 plates for overnight culture. The next day, the number of clones on the LB / Kan25 plate was divided by the number of clones on the LB / Tet10 plate to obtain the infection efficiency of the clone (Table 1). The remaining bacterial solution was transferred to 35 mL 2YT / Kan25 medium for overnight culture. The helper phage was collected the next day; the OD260 ratio was determined (when the OD260 of the helper phage was 2, the concentration of the helper phage was determined to be 1×10 13 pfu / mL) and calculated the yield (Table 1). The top three clones in yield, TR-1, TR-3, and TR-4, were selected for further experiments.

[0042] Table 1. Infection efficiency and helper phage production of R3 round clones

[0043]

[0044] 3. Optimization by superinfection efficiency and antibody library phage yield: 10 phages were selected from the prepared natural alpaca antibody library (LibVHH) 8 phages were used to infect 1 mL of NEB5alphaF' (OD value was 0.8) and cultured in a shaking incubator at 37°C for 1 hour. 10 TR-1, TR-3, and TR-4 helper phages were superinfected separately and then placed in a 37°C shaker for 1 hour. Subsequently, 20L of bacterial solution was diluted to an appropriate multiple and plated on LB / Carb50 and LB / Carb50 / Kan25 plates for overnight culture. The next day, the number of clones on the LB / Carb50 / Kan25 plate was divided by the number of clones on the LB / Carb50 plate to obtain the superinfection efficiency of the clone (Table 2). The remaining bacterial solution was transferred to 35mL of 2YT / Carb50 / Kan25 for overnight culture. The antibody library phages were collected the next day and the yield was tested (Table 2). The results showed that TR-3 had a higher superinfection efficiency and antibody library yield, so clone TR-3 was selected as our high-temperature resistant M13KO7 helper phage.

[0045] Table 2. Superinfection efficiency of selected clones and antibody library phage yield

[0046]

[0047] Example 2: Application of TR-3 helper phage in antibody library preparation

[0048] 1. Preparation of the full human antibody library: RNA was extracted from human peripheral blood lymphocytes and reverse transcribed into cDNA, and the Fab fragment of the antibody was obtained by nested PCR amplification; the Fab gene fragment obtained by PCR amplification was connected to the phagemid p3short through the Gibson Assembly reaction; after the connection product was purified and recovered, 1 μg was electrotransferred to 100 μL of super competent cells SS320 containing TR-3 helper phage or parent M13KO7 helper phage, and then activated in 5mL SOC medium for 1hr. Take 20 μL of bacterial solution for appropriate dilution, drop plate on LB / Carb50 culture plate, culture overnight, count the number of clones on the next day, and obtain the antibody library capacity (Table 3). The remaining bacterial solution was transferred to 500mL of 2YT / Carb50 / Kan25, placed in a 37°C shaker for overnight culture, and the antibody library phage was collected on the next day to detect the yield (Table 3). The results showed that the antibody library prepared using super competent cells containing TR-3 helper phage (TR-3 library) and the antibody library prepared using super competent cells containing parent M13KO7 helper phage (M13KO7 library) had equivalent library capacities, but the phage yield of the TR-3 library was significantly higher than that of the M13KO7 library.

[0049] 2. Antibody library titer detection: 10 samples were taken from TR-3 library and M13KO7 library respectively. 8 phages were infected with 1 mL NEB5alphaF' (OD value was 0.8) and cultured at 37°C in a shaker for 1 hr; then 20 μL of the bacterial solution was diluted at an appropriate multiple and plated on an LB / Carb50 culture plate for overnight culture. The number of clones was counted the next day to obtain the antibody library titer (Table 3). The results showed that the phage titer of the TR-3 library was significantly higher than that of the M13KO7 library.

[0050] Table 3. Storage capacity, yield and titer of antibody library

[0051]

[0052] Example 3: Application of TR-3 helper phage in ssDNA preparation

[0053] Phagemid containing Herceptin Fab (4D5 Fab-short P3) was transferred into CJ236 cells, activated for 1 hour, and infected with TR-3 helper phage for 1 hour (1×10 12 pfu / mL), transferred to 2YT / carb50 / kan25 medium, cultured overnight at 37°C, centrifuged at 6000g for 10 min, and removed the cells. The phage particles in the supernatant were precipitated with PEG8000 / NaCl and resuspended with 1mL phosphate buffered saline (PBS). The phage ssDNA was extracted using the M-13DNA Isolation kit (Omega Bio-tek, Norcross, GA, USA), quantified using a Nanodrop1000 spectrophotometer, and analyzed by agarose gel electrophoresis. The experimental results showed that ssDNA could be obtained from Escherichia coli infected with the thermostable TR-3 helper phage after being cultured at 37°C. The identification results of 1% agarose electrophoresis showed that ( Figure 1 ), the obtained ssDNA had a single band of the same size as expected, about 2000 bp, and no nonspecific bands were visible to the naked eye.

[0054] Example 4: Gene sequencing of TR-3 helper phage

[0055] The gene of TR-3 helper phage was extracted by PCR amplification and sent to a third party for gene sequencing. The obtained sequence was compared with the parent, and multiple amino acid differences were found in the pVIII, pIII, and pIV gene regions (Table 4). The mutated amino acids are marked in bold and underlined.

[0056] Table 4. Amino acid differences between TR-3 and the parent M13KO7 helper phage

[0057]

[0058]

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A thermostable helper phage TR-3, It is characterized in that Compared with the amino acid sequence of helper phage M13KO7: The pVIII of the thermostable helper phage TR-3 has a V6A mutation; The thermostable helper phage TR-3 had G287R and S378G mutations in pIII; and The pIV of the thermostable helper phage TR-3 has an I87N mutation. The pVIII protein sequence of the thermostable helper phage TR-3 is shown in SEQ ID NO.1, the pIII protein sequence is shown in SEQ ID NO.2, and the pIV protein sequence is shown in SEQ ID NO.

3.

2. A method for preparing a thermostable helper phage TR-3, It is characterized in that The helper phage M13KO7 was transformed as follows: The pVIII of helper phage M13KO7 was subjected to V6A mutation; The pill of the helper phage M13KO7 was mutated to G287R and S378G; and The pIV of helper phage M13KO7 was mutated to I87N. The pVIII protein sequence of the thermostable helper phage TR-3 is shown in SEQ ID NO.1, the pIII protein sequence is shown in SEQ ID NO.2, and the pIV protein sequence is shown in SEQ ID NO.

3.

3. Use of the high temperature resistant helper phage TR-3 according to claim 1 in phage display.

4. Use of the thermostable helper phage TR-3 according to claim 1 in preparing ssDNA.

5. Use of the thermostable helper phage TR-3 according to claim 1 in preparing an antibody library.

6. Use of the thermostable helper phage TR-3 according to claim 1 in constructing a phage display library.

7. A phage display system comprising the thermostable helper phage TR-3 according to claim 1.

8. The phage display system according to claim 7, It is characterized in that The phage display system also includes a phagemid.

9. A kit comprising the phage display system according to claim 7 or 8.

10. Use of the phage display system according to claim 7 or 8 in preparing ssDNA or antibody library.

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