CN115820567A - High-temperature-resistant helper phage TR-3 and application thereof - Google Patents

Abstract

The invention provides a high-temperature-resistant helper phage TR‑3 and its application, relating to the field of biotechnology. 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 at 98°C and 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 isolation of stable protein variants.

Description

High temperature resistant helper phage TR-3 and its application

technical field

The invention relates to the field of biotechnology, in particular to a high-temperature-resistant helper phage TR-3 and its application.

Background technique

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

Helper phage is a mutant type of 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. Helper phage itself The DNA can not be single-stranded replication, so it can provide pII protein and packaging protein for phagemid. The inactivation of the IG region of the helper phage also renders its own genes unable to express. When the phagemid containing the recombinant gene is transfected into E. coli and superinfected with helper phage, the pII protein synthesized by the helper phage will preferentially recognize the normal intergenic region on the phagemid, initiate rolling circle replication, and produce ssDNA , the ssDNA packaged by the progeny phage coat protein produced in this way is mainly from the DNA of the phagemid containing the recombinant gene, and at the same time display the fusion protein of pIII or pVIII encoded by the phagemid and the foreign peptide and the helper phage code The wild-type pIII or pVIII protein ensures that the recombinant phage can infect, assemble and proliferate normally.

There are two main categories of helper phages constructed by different mechanisms, namely, helper phages containing full-length gIII, and helper phages with gIII deletion or gIII deficiency. Helper phages containing full-length gIII mainly include M13KO7, R408 and VCSM13. M13KO7 is a mutant of M13 phage with a plasmid origin of replication, kanamycin resistance gene, and mutant gene II of G6125T. Previous studies have shown that the high-efficiency production of progeny phage particles and the yield of single-stranded DNA are closely related to cell growth temperature. Higher temperatures will promote rapid cell growth, resulting in potential cell lysis and inactivation of some phages, and even loss of phage-infected cells. In the end, only low-yield and poorly stable phage particles can be obtained, which affects the yield of phage single-stranded DNA and the titer of the phage-displayed antibody library. For example, cultivating CJ236 cells at 25°C can significantly improve the yield and purity of ssDNA compared with 37°C, which may be related to the lower temperature tolerance of helper phage M13K07 itself. It can be seen that the helper phage There is a difference between the optimum temperature and the optimum temperature of the cells. Therefore, improving the tolerance of helper phage is of great significance for phage display.

In view of this, the present invention is proposed.

Contents of the invention

One of the objectives of the present invention is to provide a high temperature resistant helper phage TR-3, so as to at least solve one of the technical problems in the prior art.

The second object of the present invention is to provide a method for preparing the above-mentioned thermostable helper phage TR-3.

The third object of the present invention is to provide the application of the above-mentioned thermostable helper phage TR-3.

The fourth object of the present invention is to provide a phage display system.

The fifth object of the present invention is to provide a kit.

The sixth object of the present invention is to provide the application of the above phage display system.

In order to realize the above-mentioned purpose of the present invention, special adopt following technical scheme:

A high-temperature-resistant helper phage TR-3, the amino acid sequence of the high-temperature-resistant helper phage TR-3 differs from the amino acid sequence of the helper phage M13KO7 only in that:

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

G287R and S378G mutations occurred in pIII of thermostable helper phage TR-3; and

I87N mutation occurred in the pIV of thermostable helper phage TR-3.

A method for preparing a high-temperature-resistant helper phage TR-3, wherein the helper phage M13KO7 is modified as follows:

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

G287R and S378G mutations were performed on the pill of helper phage M13KO7; and

The pIV of helper phage M13KO7 was mutated with I87N.

Application of the above-mentioned thermostable helper phage TR-3 in phage display.

Application of the above-mentioned thermostable helper phage TR-3 in the preparation of ssDNA.

Application of the above-mentioned high-temperature resistant helper phage TR-3 in the preparation of an antibody library.

Application of the above-mentioned thermostable helper phage TR-3 in constructing a phage display library.

A phage display system containing the above-mentioned thermostable helper phage TR-3.

Further, the phage display system also includes phagemids.

A kit containing the above phage display system.

Application of the above-mentioned phage display system in the preparation of ssDNA or antibody library.

Compared with the prior art, the beneficial effects of the present invention are as follows:

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 at 98°C and 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 isolation of stable protein variants.

Description of drawings

In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the specific implementation or description of the prior art. Obviously, the accompanying drawings in the following description The drawings show some implementations of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative work.

Fig. 1 is the extraction of ssDNA by the thermostable helper phage TR-3 provided in Example 3 of the present invention.

Detailed ways

The technical solutions 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, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

In order to solve the problem of low yield and poor purity of phage display technology products in the prior art due to the poor tolerance of helper phages, the inventor obtained the high-temperature-resistant helper phage TR-3 provided by the present invention through screening under high temperature stress. After testing It was found that the amino acid sequence of the thermostable helper phage TR-3 differs from that of the helper phage M13KO7 only in that pVIII of the thermostable helper phage TR-3 has the V6A mutation, pIII has the G287R and S378G mutations, and pIV An I87N mutation occurred.

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.

The TR-3 provided by the present invention survives stably 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, which can overcome the defect of poor tolerance of the current helper phages .

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

Below by embodiment the present invention will be further described. Unless otherwise specified, the materials in the examples were prepared according to existing methods, or were directly purchased from the market.

Example 1: High temperature resistant M13KO7 assisted phage screening

1. Biological panning: Take 2×10 ¹⁰ female parental M13KO7 helper phages (purchased from NEB Biolab, USA, item number: N0315S) and incubate at 98°C for 30 minutes. After centrifugation at 12000g for 10min, take 100μL supernatant, infect 1mL NEB5alphaF' bacterial solution (OD value 0.8), and culture it in a 3D shaker for 1hr; °C biochemical incubator overnight. The remaining bacterial liquid was transferred to 35mL 2YT/Kan25, placed in a shaker and cultured overnight, and the phages were collected the next day to form a heat-resistant auxiliary phage library for each round. Repeat the above steps until the R3 round of high temperature resistant helper phages are enriched.

2. Optimizing the enriched clones based on the production of auxiliary phage: randomly select 5 clones from the 2YT/Kan25 plate screened in the R3 round, named TR-1 to TR-5, and culture them overnight in 35 mL of 2YT/Kan25 medium , and helper phages were collected the next day. Take 2×10 ¹⁰ helper phages from each clone, incubate at 98°C for 30 minutes, centrifuge at 12000g for 10 minutes, take 100 μL of supernatant, and infect 1 mL of NEB5alpaF’ bacterial solution (OD ₆₀₀ value is 0.8); after culturing for 1 hour, then Take 20 μL of the bacterial solution to dilute the appropriate multiple, drop the plate on the LB/Tet10 and LB/Kan25 culture plates, cultivate overnight, and divide the number of clones on the LB/Kan25 culture plate by the clones on the LB/Tet10 culture plate on the next day The infection efficiency of clones was obtained (Table 1). The remaining bacterial liquid was transferred to 35mL 2YT/Kan25 medium for overnight culture, and the helper phage was collected the next day; by measuring the OD260 ratio (when the OD260 of the helper phage=2, the concentration of the helper phage was determined to be 1×10 ¹³ pfu/mL), and the yield was calculated (Table 1). Select the top three TR-1, TR-3, and TR-4 clones with the highest yield to continue the experiment.

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

3. Optimizing by superinfection efficiency and antibody library phage yield: Take ¹⁰⁸ phages from the prepared natural alpaca antibody library (LibVHH), infect 1mL NEB5alphaF' (OD value 0.8), and culture in a shaker at 37°C 1 hr; then use 10 ¹⁰ TR-1, TR-3, and TR-4 helper phages to perform superinfection respectively, and then culture on a shaker at 37° C. for 1 hr. Then take 20L of the bacterial solution to dilute the appropriate multiple, drop the plate on the LB/Carb50 and LB/Carb50/Kan25 culture plates, cultivate overnight, and divide the number of clones on the LB/Carb50/Kan25 culture plate by LB/Carb50 the next day The number of clones on the culture plate was used to obtain the superinfection efficiency of the clones (Table 2). The remaining bacterial liquid was transferred to 35 mL of 2YT/Carb50/Kan25 for overnight culture, and the antibody library phages were collected the next day to detect the yield (Table 2). The results showed that the superinfection efficiency and antibody library yield of TR-3 were higher, so the clone TR-3 was selected as our thermostable M13KO7 helper phage.

Table 2. Superinfection efficiency and antibody library phage yield of preferred clones

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

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

2. Antibody library titer detection: Take 10 ⁸ phages from TR-3 library and M13KO7 library respectively, infect 1mL NEB5alphaF' (OD value 0.8), place in a shaking table at 37°C for 1hr; Dilute the appropriate multiple, drop the plate on the LB/Carb50 culture plate, culture overnight, count the number of clones the next day, and obtain the titer of the antibody library (Table 3). The results show that the phage titer of the TR-3 library is significantly higher than that of M13KO7 library.

Table 3. Storage capacity, yield and titer of the antibody library

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

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

Example 4: Gene sequencing of TR-3 helper phage

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

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

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. scope.

Claims (10)

1. A high temperature resistant helper phage TR-3, characterized in that, compared with the amino acid sequence of the helper phage M13KO7: The pVIII of the thermostable helper phage TR-3 has a V6A mutation; G287R and S378G mutations occurred in pIII of thermostable helper phage TR-3; and I87N mutation occurred in the pIV of thermostable helper phage TR-3. 2. A method for preparing a high temperature resistant helper phage TR-3, characterized in that the helper phage M13KO7 is transformed as follows: The pVIII of helper phage M13KO7 was subjected to V6A mutation; G287R and S378G mutations were performed on the pill of helper phage M13KO7; and The pIV of helper phage M13KO7 was mutated with I87N. 3. The application of the high temperature resistant helper phage TR-3 according to claim 1 in phage display. 4. The application of the high temperature resistant helper phage TR-3 according to claim 1 in the preparation of ssDNA. 5. The application of the high temperature resistant helper phage TR-3 according to claim 1 in the preparation of antibody library. 6. The application of the high temperature resistant helper phage TR-3 according to claim 1 in the construction of a phage display library. 7. A phage display system containing the high temperature resistant helper phage TR-3 according to claim 1. 8. The phage display system according to claim 6, characterized in that, the phage display system further comprises phagemids. 9. A kit containing the phage display system according to claim 7 or 8. 10. The application of the phage display system according to claim 7 or 8 in the preparation of ssDNA or antibody library.

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