CN113999866A

CN113999866A - Phagemid vector for high-density display and application thereof

Info

Publication number: CN113999866A
Application number: CN202111298003.4A
Authority: CN
Inventors: 华修德; 丁园; 尤天阳; 王鸣华
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2021-11-04
Filing date: 2021-11-04
Publication date: 2022-02-01
Anticipated expiration: 2041-11-04
Also published as: CN113999866B

Abstract

The invention belongs to the technical field of biology, and relates to a high-density displayed phagemid vector, which comprises an application of the vector in random polypeptide library construction. The invention constructs a phagemid vector pComb-pVIII which can be used for high-density display of foreign protein by a molecular cloning technology based on the phagemid pComb3xss displayed by pIII. The carrier displays the exogenous protein at the N end of the filamentous phage pVIII protein, the two are connected through a flexible connecting arm GGGSS, and the expression of the downstream exogenous protein gene is regulated and controlled by a lactose promoter and an operon. The vector introduces restriction endonuclease sites which can not cut random polypeptide coding sequences, and avoids the loss of diversity of random polypeptide libraries. The invention also relates to application of the phagemid vector in random polypeptide library construction. The phage display random polypeptide library constructed based on the phagemid has the advantages of low no-load rate, large library capacity, high display density and the like, and has good application value in the aspect of screening of polypeptides with biological functions.

Description

Phagemid vector for high-density display and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a high-density displayed phagemid vector, which comprises an application of the vector in construction of a phage display random polypeptide library.

Background

The Phage Display Technology (Phage Display Technology) was invented by Smith in 1985, and the principle is to insert a foreign protein gene into a coat protein gene of a filamentous bacteriophage without affecting the normal function of the bacteriophage, thereby expressing the foreign protein on the coat protein of the filamentous bacteriophage. The filamentous phage has definite genetic information and simple genome, is easy to transform by the conventional molecular cloning technology, and can conveniently construct a large-capacity phage display foreign protein library.

Filamentous phages contain five coat proteins and are commonly used for foreign protein display are the pVIII and pIII proteins. The pVIII protein has 50 amino acids in total, about 2700 copies, is a main constituent protein of a phage coat and can be used for displaying foreign proteins at high density; the pIII protein has 406 amino acids and 5 copies, is positioned at one end of the phage, is responsible for the infection of the phage to escherichia coli, and can be used for displaying foreign proteins at low density. High density display differs from low density display in that low density display facilitates screening for high affinity ligands, whereas high density display is more advantageous when screening for ligands with low probability. Phage display technologies can be divided into phage display systems and phagemid display systems, depending on the type of vector used. In the process of assembling the phage vector, the coat protein is completely composed of a foreign protein-coat protein chimeric protein, and the display mode may have great influence on the infection or assembly capacity of the phage; in the assembling process of the phagemid vector, the coat protein consists of the exogenous protein-coat protein chimeric protein and the coat protein provided by the helper phage, 1-5 copies of the exogenous protein can be displayed when the pIII protein is used for displaying, and dozens to hundreds of copies of the exogenous protein (depending on the expression level of the chimeric protein) can be displayed when the pVIII protein is used for displaying, so that the defects of the phage vector can be avoided.

The random polypeptide library based on phage display has wide application in the aspects of screening of polypeptide with biological function, protein/ligand interaction analysis and the like. The random polypeptide library is typically composed of random polypeptides comprising 5-20 amino acids, each amino acid being encoded by the degenerate codon NNK (N represents deoxynucleotide A, T, C or G; K represents T or G), and can be any of the 20 natural amino acids, with a minimal occurrence of a terminator. The continuous NNK sequence can be cut by a large number of restriction endonucleases, so that a large number of random polypeptide sequences are lost due to enzyme digestion during the construction of a polypeptide library, the theoretical library capacity of the polypeptide library is reduced, and the acquisition of functional polypeptides is influenced. At present, the cloning sites (restriction enzyme sites) of the phage display systems (phagemids or phage vectors) reported and commercialized at home and abroad mostly contain NNK sequence characteristics. Therefore, the construction of the phagemid vector containing the high-density display of the characteristic cloning site without the NNK sequence has important significance for ensuring the theoretical diversity (library capacity) of a polypeptide library and improving the panning success rate of functional polypeptides.

Disclosure of Invention

The invention aims to provide a phagemid vector which does not contain NNK sequence characteristic cloning sites and can be used for displaying polypeptides at high density, and application of related vectors in random polypeptide library construction.

The purpose of the invention is realized by the following technical scheme:

(1) synthesizing a gene fragment 1 containing a phage pVIII protein signal peptide and a Kpn I enzyme cutting site by means of oligonucleotide chain synthesis and annealing extension, inserting the gene fragment into pComb3xss through Sac I and Mfe I enzyme cutting sites, and replacing the original Omp A signal peptide (named as pComb-1);

(2) synthesizing a gene fragment 2 containing a phage pVIII protein and an Xho I enzyme cutting site by means of oligonucleotide chain synthesis and annealing extension, inserting the gene fragment into pComb-1 through Spe I and Afl II enzyme cutting sites, and replacing the original phage pIII protein (named as pComb-pVIII);

(3) synthesizing a double-chain fragment containing Kpn I and Xho I restriction enzyme sites and a random polypeptide coding sequence (NNK) by means of oligonucleotide chain synthesis and annealing extension, and inserting the double-chain fragment into the constructed pComb-pVIII phagemid through the Kpn I and Xho I restriction enzyme sites;

(4) the phagemid containing the random polypeptide gene is transferred into ER2738 escherichia coli by means of electrotransformation, and is amplified into a phage display random polypeptide library with the help of a helper phage M13KO 7.

The phagemid vector can be used for constructing a phage display random polypeptide library, and the constructed library can be used for screening polypeptides with biological functions.

The invention has the following beneficial effects: (1) the Kpn I and Xho I restriction sites used by the phagemid do not recognize the nucleotide sequence (NNK) encoding the random polypeptide, thereby avoiding the loss of the random polypeptide diversity theory; (2) the large interval fragment (1686bp) between the enzyme cutting sites enables the phagemid which is cut by enzyme and is not cut by enzyme to be easily distinguished, and reduces the probability of no-load of a transformant; (3) the small genome (-2920 bp) after ligation ensures high electrotransformation efficiency; (4) the phagemid regulates the expression of downstream foreign protein genes through a lactose promoter and an operon, reduces the genetic preference of escherichia coli for expressing different polypeptides, and simultaneously induces the high-level expression of foreign proteins; (5) the success rate of obtaining the target protein can be improved through high-density display based on the pVIII protein.

Drawings

FIG. 1 is a genetic map of a phagemid, A is pComb3xss and B is pComb-pVIII;

FIG. 2 is an electrophoretogram during construction of pComb-pVIII phagemid, wherein A is pComb3xss and B is pComb-1;

FIG. 3 is an electrophoretogram during construction of phage display

random circular

8, 9, 10 peptide libraries, A is pComb-pVIII, B-D are random circular 8, 9, 10 peptide genes, respectively;

FIGS. 4A-C are electrophoresis images of colony PCR after phage display

random circular

8, 9, 10 peptide library construction, respectively.

Detailed Description

The experimental materials, main reagents and formula used in the embodiment of the invention are as follows:

the main experimental materials:

the pComb3xss phagemid was donated by Dr.

The main reagents are as follows:

sac I, Mfe I, Spe I, Afl II, Kpn I, Xho I, T4 DNA ligase, 10mM dNTPs, DNA polymerase I (Klenow) large fragment, helper phage M13KO7 (NeB, UK), Taq DNA polymerase, DL 5,000 DNA Marker, DNA fragment purification kit (Takara, Japan), plasmid extraction kit, gel recovery kit (Omega, USA), ER2738 electrocompetent cell (Lucigen, USA), polyethylene glycol 8000(PEG) (Amresco, USA), yeast extract, tryptone (Thermo Fisher, USA), D- (+) -glucose, isopropyl-beta-D-thiogalactoside, ampicillin hydrochloride, kanamycin, tetracycline (Sigma, USA)

The main reagent formula is as follows:

1. LB culture medium: each liter contains 10g of tryptone, 5g of yeast extract and 5g of NaCl. Sterilizing under high pressure, and storing at room temperature;

2. SB medium: each liter contains 32g of tryptone, 20g of yeast extract and 5g of NaCl. Sterilizing under high pressure, and storing at room temperature;

3. tetracycline stock solution (Tet): dissolving in 50% ethanol at a concentration of 20mg/mL, and storing at-20 deg.C in dark;

4. ampicillin stock solution (Amp): dissolved in sterile ddH at a concentration of 100mg/mL₂Filtering with 0.22 μm filter membrane in O, and storing at-20 deg.C;

5. kanamycin stock (Kan): dissolved in sterile ddH at a concentration of 10mg/mL₂Filtering with 0.22 μm filter membrane in O, and storing at-20 deg.C;

6. PEG/NaCl: 20% (w/v) PEG-8000, 2.5M NaCl, autoclaving, storing at room temperature;

7. TE buffer: 10mM Tris-HCl, 1mM EDTA, adjusted to pH 8.0 and stored at room temperature.

The first embodiment is as follows: construction of pComb-pVIII phagemid

The method of oligonucleotide chain annealing extension is used for synthesizing M13 bacteriophage pVIII signal peptide gene (fragment 1) containing Kpn I restriction site by using primers of SEQ ID NO 1 and SEQ ID NO 2, synthesizing M13 bacteriophage pVIII protein gene (fragment 2) containing Xho I restriction site by using primers of SEQ ID NO 3 and SEQ ID NO 4, and sequentially inserting the genes into pComb3xss phagemid (the gene map is shown in figure 1-A) to construct pComb-pVIII phagemid (the gene map is shown in figure 1-B). The primer sequences are shown in Table 1.

TABLE 1 primers used for construction of pComb-pVIII phagemids

1. Electric conversion

(1) Taking out the prepared ER2738 electric shock cup from a refrigerator at-80 ℃ to be in an electric accepting state, taking out the electric shock cup with the precooled thickness of 0.1cm from the refrigerator at-20 ℃, immediately inserting the electric shock cup on ice, waiting for 5min to melt the accepting state, and setting the conditions of an electric rotating instrument to be 1.8KV and 4ms in advance;

(2) slowly adding the DNA to be transformed into a thawed ER2728 electrocompetent state in a super clean bench, and gently stirring and uniformly mixing;

(3) slowly adding competence uniformly mixed with the connecting product into an electric shock cup for electric shock;

(4) after the shock was completed, 950. mu.L of 37 ℃ preheated SOC medium was immediately added and thawed at 250rpm in a 37 ℃ shaker for 1 h.

2. Synthesis and insertion of pVIII signal peptide gene containing Kpn I enzyme cutting site

(1) Annealing:

the reaction was incubated at 95 ℃ for 20min for a total of 50. mu.L, and then slowly annealed to room temperature.

(2) Extension:

the reaction system is 200 mu L in total, the reaction system is respectively arranged in 4 200 mu L centrifuge tubes, 50 mu L/tube, the Klenow fragment is extended for 15min at 25 ℃, the Klenow fragment is inactivated after 20min of incubation at 75 ℃, the extension product is purified by a DNA fragment purification kit according to the steps of the instruction, and the DNA concentration is measured by a Nanodrop One ultraviolet spectrophotometer.

(3) Cleavage of fragment 1:

the total volume of the reaction system is 100 mu L, the mixture is split and loaded into 2 centrifugal tubes of 200 mu L after being mixed evenly, the reaction is carried out for 1h at 37 ℃ in each tube, and the DNA fragment purification kit purifies the enzyme digestion product.

(4) Cleavage of pComb3 xss:

the reaction system is 250 mu L in total, the mixture is split and loaded into 5 centrifugal tubes of 200 mu L after being mixed evenly, each centrifugal tube of 50 mu L is reacted for 1h at 37 ℃, target fragments are separated by electrophoresis of 1% agarose gel, and the carrier fragments after enzyme digestion are recovered by a gel recovery kit (the electrophoresis result is shown in figure 2-A).

(5) And (3) connecting the fragment 1 after enzyme digestion with a pComb3xss vector:

the reaction system is 10 mu L in total, the reaction system is connected at 16 ℃ overnight, T4 DNA ligase is inactivated at 65 ℃ for 15min, a DNA fragment purification kit purifies a connection product, and a Nanodrop One ultraviolet spectrophotometer determines the DNA concentration.

(6) And (3) electric conversion:

mu.L of the purified ligation product was taken and electrotransformed as in step 1. After recovery, 100 mu L of bacterial liquid is taken, diluted by an appropriate multiple by LB culture solution, coated on an LB-Amp solid culture medium, cultured for 12h at 37 ℃, monoclonal is selected, cultured in 3mL of LB-Amp culture solution overnight, the bacterial liquid is collected, phagemids are extracted by a plasmid extraction kit according to the steps of the instruction, the DNA concentration is measured by a Nanodrop One ultraviolet spectrophotometer, and the phagemids are sequenced. The phagemid after the first step of engineering was named pComb-1.

3. Synthesis and insertion of pVIII protein gene containing Xho I enzyme cutting site

(1) Annealing: the primers are SEQ ID NO 3 and SEQ ID NO 4, and the system and the steps are the same as those of 2 (2);

(2) extension: the system and the steps are the same as 2 and 2;

(3) and (3) enzyme digestion of the fragment 2: the restriction enzymes used are Spe I and Afl II, and the system and the steps are the same as those in the step 2 (3);

(4) digestion of pComb-1 phagemid: the restriction enzymes used were Spe I and Afl II, and the system and procedure were the same as those in 2(4) (see FIG. 2-B for electrophoresis results);

(5) and (3) connecting the digested fragment 2 with the pComb-1 phagemid: the system and the steps are the same as 2 and 5; the ligated phagemid was named pComb-pVIII (SEQ ID NO 5);

(6) and (3) electric conversion: the steps are the same as those of 2 and 6

Example two: construction of phage display

random circular

8, 9, 10 peptide library

Random

8, 9 and 10 peptide genes coded by NNK are respectively synthesized by using primers SEQ ID NO 6 and SEQ ID NO 7-9 through an oligonucleotide chain annealing extension method, and are inserted into a pComb-pVIII phagemid after being digested by Xho I and Kpn I, so that a random polypeptide sequence is positioned between the N end of a phage pVIII protein and a signal peptide thereof. The primers used are shown in Table 2, and the specific steps are as follows:

TABLE 2 primers used in construction of phage display

random circular

8, 9, 10 peptide libraries

1. Synthesis and insertion of random cyclic 8, 9, 10 peptide genes

(1) Annealing

The reaction system was incubated at 95 ℃ for 20min for a total of 50. mu.L, and slowly annealed to room temperature.

(2) Extension: the system and steps are the same as those of the first 2(2) and the second 2

(3) Random cyclic 8, 9 and 10 peptide gene enzyme digestion: the restriction enzymes used were Kpn I and Xho I, and the system and procedure were the same as in example one 2 (3). After the enzyme digestion is finished, taking 1 mu L of extension and enzyme digestion products to perform 8% non-polyacrylamide gel electrophoresis, and verifying the enzyme digestion efficiency (the electrophoresis result is shown in figures 3-B-D).

(4) Cleavage of pComb-pVIII:

the total volume of the reaction system is 1mL, the reaction system is mixed evenly and then is loaded into a 200 mu L centrifuge tube, 50 mu L/tube, the incubation is carried out for 1h at 37 ℃, the target fragment is separated through 0.7% agarose gel electrophoresis (the result is shown in figure 3-A), and the gel recovery kit recovers the fragment after enzyme digestion.

(5) Before the library is formally built, the optimal connection ratio of random cyclic 8, 9 and 10 peptide genes and phagemids after enzyme digestion is optimized. Three treatments of

random circular

8, 9 and 10 peptide genes and phagemid molar ratios of 1: 1, 1: 3 and 1: 5 respectively after enzyme digestion and 1 treatment without adding inserted fragments (CK) are set for connection reaction, and the reaction system and the steps are the same as those of the first 2 and the second 5 of the embodiment. After the ligation was completed, 2. mu.L of the purified ligation product was subjected to electroporation according to step 1. After the recovery is finished, diluting the solution by a proper multiple with LB culture solution, taking 100 mu L of a plate coated with LB-Amp, culturing the plate at 37 ℃ for 12h, calculating the colony number on the plate, and calculating the conversion rate according to the following formula: conversion (cfu/. mu.g) — (10 × dilution × colony count)/DNA mass. The results of the experiments are shown in Table 3, and the ligation ratio (1: 3) with the highest conversion was selected for subsequent experiments.

TABLE 3 test connection results

(6) Random ligation of circular 8, 9, 10 peptide genes and pComb-pVIII after cleavage:

the reaction system is 846 mu L in total, the reaction system is fully and evenly mixed and then is subpackaged in a 200 mu L centrifuge tube, 20 mu L/tube, the T4 DNA ligase is inactivated after the incubation is carried out for 12h at the temperature of 16 ℃, the T4 DNA ligase is inactivated after the incubation is carried out for 15min at the temperature of 65 ℃, a ligation product is purified by a DNA fragment purification kit, and the DNA concentration is measured by a Nanodrop One ultraviolet spectrophotometer.

(7) And (3) electric conversion: the purified ligation products were electroporated into ER2738 electrocompetent cells at 50 ng/time as in example step 1, and the conversion and library capacity of phage display

random circular

8, 9, 10 peptide libraries were calculated as in example step 1(5), and the results are shown in Table 4.

TABLE 4 conversion and library Capacity of phage display

random circular

8, 9, 10 peptide libraries

2. Amplification of phage libraries

Mixing all the bacterial solutions, adding into 4 conical flasks containing 400mL SB-Amp (50. mu.g/mL) -Tet (20. mu.g/mL) -glucose (20mM) culture solution at a ratio of 1: 40, and shake culturing at 37 deg.C and 250rpm to OD₆₀₀0.5, adding helper phage M13KO7 to ensure that the infection complex number (helper phage number/escherichia coli number) is more than 20, standing and infecting for 1h at 37 ℃, then centrifuging 6000g of bacterial liquid at 4 ℃ for 15min, discarding the supernatant, adding the precipitate into 4 conical flasks containing 400mL of SB-Kan (50 mu g/mL) -Amp (50 mu g/mL) -IPTG (0.1mM) culture solution in equal amount after resuspension, shaking and culturing for 12h at 37 ℃ at 250rpm, centrifuging 14000g of bacterial liquid at 4 ℃ for 15min, pouring the supernatant into a new 250mL centrifugal tube, adding 1/4 volume of 20% PEG/2.5M NaCl, standing and more than 4h at 4 ℃, fully precipitating the phage, centrifuging at 14000g at 4 ℃ for 30min, discarding the supernatant, sterilizing 400 mu L of PBS in each centrifugal tube, mixing, subpackaging into 400 mu L of each tube, adding glycerol with equal volume, uniformly mixing, the titer is determined according to the two steps 3 of the embodiment, and the pVIII display phage display random circular 8, 9 and 10 peptide libraries are stored at the temperature of-80 ℃, and the titer is 2.6 multiplied by 10 respectively¹³、4.5×10¹³And 1.0X 10¹⁴pfu/mL。

3. Determination of phage titer

(1) Taking out the stored ER2738 escherichia coli from-80 ℃, dipping bacterial liquid by using an inoculating loop after the escherichia coli is thawed, drawing lines on an LB/Amp (50 mu g/mL) plate, and culturing overnight at 37 ℃;

(2) single colonies were picked with an inoculating loop and cultured in 3mL LB-Amp (50. mu.g/mL) medium at 37 ℃ with shaking at 250rpm to OD₆₀₀＝0.5；

(3) Diluting phage for titer determination with LB culture medium by a proper multiple, taking 10 μ L, adding into 100 μ L of ER2738 Escherichia coli of step (2), standing at 37 deg.C for 30min, coating the bacterial liquid on LB-Amp plate, culturing at 37 deg.C for 12h, calculating colony number on the plate, and calculating phage titer according to the following formula: phage titer (pfu/mL) 100 Xdilution times colony number

4. After the electrotransformation is finished, 24 single colonies are respectively picked from phage display random circular eight-, nine-and ten-peptide libraries for colony PCR. Primers

SEQ ID NO

10 and 11 used for colony PCR are shown in Table 5, and the system is as follows:

TABLE 5 primers for the bacterial particle PCR

Preparing a reaction system according to a certain proportion, subpackaging in a 200 mu L centrifugal tube, placing the centrifugal tube on ice, dipping a single bacterium with a sterilizing toothpick, gently stirring in the centrifugal tube, then placing on a PCR instrument for amplification, and carrying out 35 cycles in total, wherein the reaction conditions are as follows:

after the PCR reaction is finished, taking 5 mu LPCR products, detecting the size of the amplified fragment by 1% agarose gel electrophoresis, and judging the no-load rate. The position of the upstream primer and the downstream primer bound to the carrier can be deduced: if the carrier is no-load, the colony PCR fragment is 2085 bp; if the vector and the insert are successfully connected, the colony PCR fragment is 467 bp. The experimental results are shown in FIG. 4, and due to the larger interval fragment (1686bp) between the cleavage sites of the pComb-pVIII phagemid enzyme, the constructed phage display random polypeptide library has no idling phenomenon.

According to the phage display system established by the invention, exogenous protein is displayed on phage pVIII protein at high density through Xho I and Kpn I enzyme cutting sites which cannot identify NNK, so that loss of random polypeptide diversity theory is avoided, and influence of the pVIII display system based on a phage vector on phage assembly efficiency is overcome. The constructed phage random cyclic polypeptide library has no idle load phenomenon, high conversion rate, large library capacity, high display density and good practical application value.

Claims

1. A phagemid vector pComb-pVIII, the DNA sequence of which is shown in SEQ ID NO 5.

2. The phagemid vector pComb-pVIII of claim 1 comprising Kpn I and Xho I restriction endonuclease cleavage sites through which an exogenous degenerate polypeptide nucleic acid sequence (NNK) n is introduced into the phagemid vector, which is not cleaved by Kpn I and Xho I, avoiding loss of diversity of the random polypeptide library due to cleavage.

3. The phagemid vector pComb-pVIII of claim 1 wherein the maximum separation between the cleavage sites Kpn I and Xho I is 1686bp, which allows easy discrimination and purification of the digested phagemid vector and reduces the probability of empty load of transformants.

4. The phagemid vector pComb-pVIII of claim 1 wherein the genome of the recombinant phagemid vector is small, about 2920bp, after introduction of the exogenous degenerate polypeptide nucleic acid sequence, ensuring high electrotransformation efficiency.

5. Use of the phagemid vector pComb-pVIII of claim 1 for the construction of a phage display random polypeptide library.