CN113999866B

CN113999866B - Phagemid vector for high-density display and application thereof

Info

Publication number: CN113999866B
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: 2023-09-05
Anticipated expiration: 2041-11-04
Also published as: CN113999866A

Abstract

The invention belongs to the technical field of biology, and relates to a phagemid vector for high-density display, which comprises application of the vector in construction of a random polypeptide library. According to the invention, a phagemid vector pComb-pVIII capable of being used for high-density display of foreign proteins is constructed based on a phagemid pComb3xss displayed by pIII through a molecular cloning technology. The carrier displays the foreign protein at the N end of the protein pVIII of the filamentous phage, connects the two through a flexible connecting arm GGGSS, and regulates the expression of the downstream foreign protein gene by a lactose promoter and an operon. The vector introduces a restriction nucleic acid cleavage site incapable of cleaving a random polypeptide coding sequence, thereby avoiding the loss of random polypeptide library diversity. The invention also relates to application of the phagemid vector in construction of random polypeptide libraries. The phage display random polypeptide library constructed based on the phagemid has the advantages of low idle rate, large library capacity, high display density and the like, and has good application value in the aspect of screening 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 phagemid vector for high-density display, which comprises application of the vector in construction of phage display random polypeptide library.

Background

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

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

The random polypeptide library based on phage display has wide application in the aspects of screening polypeptides with biological functions, protein/ligand interaction analysis and the like. The random polypeptide library is typically comprised of random polypeptides of 5-20 amino acids, each encoded by degenerate codon NNK (N stands for deoxynucleotide A, T, C or G; K stands for T or G), which may be any of the 20 natural amino acids, and minimizes the occurrence of stop codons. 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 when a polypeptide library is constructed, the theoretical library capacity of the polypeptide library is reduced, and the acquisition of functional polypeptides is affected. At present, cloning sites (restriction enzyme sites) of phage display systems (phagemids or phage vectors) which have been reported and commercialized at home and abroad mostly contain NNK sequence features. Therefore, constructing the phagemid vector containing the NNK sequence-free characteristic cloning site for high-density display has important significance for guaranteeing the theoretical diversity (library capacity) of the polypeptide library and improving the panning success rate of the functional polypeptide.

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 high-density polypeptide display, and application of related vectors in construction of random polypeptide libraries.

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

(1) Synthesizing a gene fragment 1 containing a phage pVIII protein signal peptide and Kpn I cleavage sites by means of oligonucleotide chain synthesis and annealing extension, and inserting the gene fragment into pComb3xss by means of Sac I and Mfe I cleavage sites to replace the original Omp A signal peptide (named pComb-1);

(2) Synthesizing a gene fragment 2 containing phage pVIII protein and Xho I cleavage site by means of oligonucleotide chain synthesis and annealing extension, inserting into pComb-1 by means of Spe I and Afl II cleavage site, and replacing original phage pIII protein (named pComb-pVIII);

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

(4) Phagemids containing random polypeptide genes were transferred into ER2738 e.coli by electrotransformation and amplified into phage display random polypeptide libraries with the aid of helper phage M13KO 7.

The phagemid vector of the invention 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) Kpn I and Xho I cleavage sites used in phagemids do not recognize the nucleotide sequence (NNK) encoding the random polypeptide, thereby avoiding the theoretical loss of random polypeptide diversity; (2) The larger interval fragments (1686 bp) among the enzyme cutting sites enable the enzyme-cut phagemid and the non-enzyme-cut phagemid to be easy to distinguish, and reduce the probability of empty load of the transformant; (3) The smaller genome (-2920 bp) after ligation ensures high electrotransformation efficiency; (4) The phagemid regulates the expression of downstream exogenous protein genes through lactose promoters and operators, reduces the genetic preference of escherichia coli for expressing different polypeptides, and simultaneously induces the high-level expression of exogenous 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 gene map of a phagemid, A being pComb3xss and B being pComb-pVIII;

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

FIG. 3 is an electrophoretogram of phage display random loop 8, 9, 10 peptide library construction, A is pComb-pVIII, B-D is random loop 8, 9, 10 peptide gene, respectively;

FIG. 4A-C is an electrophoretogram of colony PCR after construction of phage display random circular 8, 9, 10 peptide libraries, respectively.

Detailed Description

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

main experimental materials:

pComb3xss phagemid was given by dr.

The main reagent comprises:

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 Co., UK), taq DNA polymerase, DL 5,000 DNA Marker, DNA fragment purification kit (Japanese Takara Co., ltd.), plasmid extraction kit, gel recovery kit (Omega Co., USA), ER2738 electric competent cells (Lucigen Co., USA), polyethylene glycol 8000 (PEG) (Amresco Co., USA), yeast extract, tryptone (Thermo Fisher Co., USA), D- (+) -glucose, isopropyl-. Beta. -D-thiogalactoside, ampicillin hydrochloride, kanamycin, tetracycline (Sigma Co., USA)

The main reagent formula comprises:

1. LB medium: each liter contains 10g tryptone, 5g yeast extract, 5g NaCl. Autoclaving and storing at room temperature;

2. SB medium: each liter contains 32g tryptone, 20g yeast extract, 5g NaCl. Autoclaving and storing at room temperature;

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

4. ampicillin stock solution (Amp): dissolved in sterilized ddH at a concentration of 100mg/mL ₂ O, passing through 0.22 μm filter membrane, and storing at-20deg.C;

5. kanamycin stock (Kan): dissolved in sterilized ddH at a concentration of 10mg/mL ₂ O, passing through 0.22 μm filter membrane, and storing at-20deg.C;

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

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

Embodiment one: construction of pComb-pVIII phagemid

By an oligonucleotide strand annealing extension method, an M13 phage pVIII signal peptide gene (fragment 1) containing Kpn I cleavage site is synthesized by using primers SEQ ID NO 1 and SEQ ID NO 2, an M13 phage pVIII protein gene (fragment 2) containing Xho I cleavage site is synthesized by using primers SEQ ID NO 3 and SEQ ID NO 4, and then pComb3xss phagemids (genetic map see FIG. 1-A) are sequentially inserted to construct pComb-pVIII phagemids (genetic map see FIG. 1-B). The primer sequences are shown in Table 1.

TABLE 1 primer for construction of pComb-pVIII phagemid

1. Electric conversion

(1) Taking out the prepared ER2738 electric competence from a refrigerator at the temperature of minus 80 ℃, taking out an electric stun cup with the length of 0.1cm for pre-cooling, immediately inserting the electric stun cup into ice, waiting for 5min to melt the competence, and setting the condition of an electrotometer to be 1.8KV and 4ms in advance;

(2) Slowly adding the DNA to be converted into the frozen ER2728 electric competence in an ultra-clean bench, and gently stirring and uniformly mixing;

(3) Slowly adding the competence mixed with the connection product into a electric shock cup to perform electric shock;

(4) Immediately after the end of the electric shock, 950. Mu.L of SOC medium preheated at 37℃was added and resuscitated in a shaker at 37℃at 250rpm for 1h.

2. Synthesis and insertion of pVIII Signal peptide Gene containing Kpn I cleavage site

(1) Annealing:

the reaction system was incubated for 20min at 95℃with 50. Mu.L, and then slowly annealed to room temperature.

(2) Extension:

200 mu L of the reaction system is divided into 4 200 mu L of centrifuge tubes, 50 mu L/tube is used for extension at 25 ℃ for 15min, the Klenow fragment is inactivated after incubation at 75 ℃ for 20min, the extension product is purified by a DNA fragment purification kit according to the description step, and the DNA concentration is measured by a Nanodrop One ultraviolet spectrophotometer.

(3) Cleavage of fragment 1:

the reaction system is 100 mu L in total, and after being evenly mixed, the mixture is divided into 2 200 mu L centrifuge tubes, 50 mu L/tube and reacted for 1h at 37 ℃, and the DNA fragment purification kit purifies enzyme digestion products.

(4) Cleavage of pComb3 xss:

the reaction system is 250 mu L in total, the mixture is split into 5 200 mu L centrifuge tubes, 50 mu L/tube is reacted for 1h at 37 ℃, the target fragment is separated by 1% agarose gel electrophoresis, and the carrier fragment after enzyme digestion is recovered by the gel recovery kit (the electrophoresis result is shown in figure 2-A).

(5) Ligation of fragment 1 with pComb3xss vector after cleavage:

the reaction system was ligated overnight at 16℃with 10. Mu.L, then T4 DNA ligase was inactivated at 65℃for 15min, and the ligation product was purified using a DNA fragment purification kit, and the DNA concentration was measured using a Nanodrop One ultraviolet spectrophotometer.

(6) Electric conversion:

mu.L of the purified ligation product was taken and subjected to electrotransformation according to step 1. After resuscitating, 100 mu L of bacterial liquid is taken, the bacterial liquid is diluted by proper times by LB culture liquid, then is coated on LB-Amp solid culture medium, is cultured for 12 hours at 37 ℃, single clone is selected, is cultured overnight in 3mL of LB-Amp culture liquid, bacterial liquid is collected, phagemid is extracted by a plasmid extraction kit according to the description step, DNA concentration is measured by a Nanodrop One ultraviolet spectrophotometer, and the phagemid is sequenced. The phagemid after the first step of engineering was designated pComb-1.

3. Synthesis and insertion of pVIII protein Gene containing Xho I cleavage 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 the primer 2 (2);

(2) Extension: the system and the steps are the same as those of the step (2);

(3) Cleavage of fragment 2: the restriction enzymes are Spe I and Afl II, and the system and the steps are the same as those of 2 (3);

(4) Cleavage of pComb-1 phagemid: the restriction enzymes used are Spe I and Afl II, the system and the steps are the same as those of 2 (4) (the electrophoresis results are shown in FIG. 2-B);

(5) Ligation of fragment 2 with pComb-1 phagemid after cleavage: the system and the steps are the same as those of the step (2) and (5); the ligated phagemid was designated pComb-pVIII (SEQ ID NO 5);

(6) Electric conversion: the step is the same as 2 (6)

Embodiment two: construction of phage display random circular 8, 9, 10 peptide libraries

Random 8, 9 and 10 peptide genes encoded by NNK are synthesized by an oligonucleotide chain annealing extension method by using primers SEQ ID NO 6 and SEQ ID NO 7-9 respectively, and are inserted into pComb-pVIII phagemid after being digested by Xho I and Kpn I, so that the random polypeptide sequence is positioned between the N end of phage pVIII protein and 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 circular 8, 9, 10 peptide genes

(1) Annealing

The reaction system was incubated for 20min at 95℃with 50. Mu.L, and slowly annealed to room temperature.

(2) Extension: the system and the procedure are the same as in example one 2 (2)

(3) Cleavage of random circular 8, 9, 10 peptide genes: 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 completion of the cleavage, 1. Mu.L of the extension and cleavage products were subjected to 8% non-polyacrylamide gel electrophoresis, and the cleavage efficiency was confirmed (see FIG. 3-B-D for electrophoresis results).

(4) Cleavage of pComb-pVIII:

the reaction system is 1mL, the mixture is packaged in 200 mu L centrifuge tubes after being mixed evenly, 50 mu L/tube is incubated for 1h at 37 ℃, the target fragment is separated by 0.7% agarose gel electrophoresis (the result is shown in figure 3-A), and the fragment after enzyme digestion is recovered by the gel recovery kit.

(5) Before formal warehouse establishment, the optimal connection proportion of random circular 8, 9, 10 peptide genes and phagemid after enzyme digestion is optimized. The ligation was performed by setting the molar ratio of the random circular 8, 9, 10 peptide genes to phagemid at 1:1, 1:3, 1:5, and 1 treatment without inserted fragment (CK), and the reaction system and steps were the same as in example one 2 (5). After ligation, 2. Mu.L of the purified ligation product was subjected to electrotransformation according to step 1. After resuscitating, diluting with LB culture solution to a proper multiple, taking 100 mu L of LB-Amp coated plates, culturing at 37 ℃ for 12 hours, calculating the colony number on the plates, and calculating the conversion rate according to the following formula: conversion (cfu/. Mu.g) = (10 x dilution x colony count)/DNA mass. The results of the experiments are shown in Table 3, and the subsequent experiments were performed by selecting the ligation ratio (1:3) with the highest conversion.

Table 3 test connection results

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

846 mu L of the reaction system is fully and evenly mixed, the mixture is split into 200 mu L of centrifuge tubes, 20 mu L/tube is incubated for 12h at 16 ℃, T4 DNA ligase is inactivated by incubation for 15min at 65 ℃, the connection product is purified by a DNA fragment purification kit, and the DNA concentration is measured by a Nanodrop One ultraviolet spectrophotometer.

(7) Electric conversion: the conversion and reservoir capacity of phage display random circular 8, 9, 10 peptide libraries were calculated as per example two step 1 (5) procedure, using 50 ng/time electrotransformation of the purified ligation products into ER2738 electrocompetent cells, as shown in Table 4.

TABLE 4 conversion and reservoir Capacity of phage display random circular 8, 9, 10 peptide reservoirs

2. Amplification of phage libraries

After all the bacterial solutions in the previous step are uniformly mixed, the bacterial solutions are added into 4 conical flasks containing 400mL SB-Amp (50 mug/mL) -Tet (20 mug/mL) -glucose (20 mM) culture solution in equal quantity according to the proportion of 1:40, and the mixture is cultured at 37 ℃ under shaking at 250rpm until the mixture reaches OD ₆₀₀ Adding helper phage M13KO7 to give complex number (helper phage number/E.coli number) > 20, standing at 37deg.C for 1 hr, centrifuging bacterial solution 6000g at 4deg.C for 15min, discarding supernatant, re-suspending the precipitate, adding equal amount into 4 conical flasks containing 400mL SB-Kan (50 μg/mL) -Amp (50 μg/mL) -IPTG (0.1 mM) culture solution, shaking culture at 37deg.C 250rpm for 12 hr, centrifuging bacterial solution 14000g at 4deg.C for 15min, pouring supernatant into new 250mL centrifuge tube, adding 1/4 volume of 20% PEG/2.5M NaCl, standing at 4deg.C for more than 4 hr to give phage sufficient precipitate, centrifuging at 14000g at 4deg.C for 30min, discarding supernatant, re-suspending each precipitate with 400 μL sterilizing PBS, mixing, packaging into 400 μL tubes, adding equal volume of glycerol, mixing, measuring titer according to example two steps 3, storing phage at-80deg.C to give phage display random peptide library of 8, 10.10×10, 10 titer, 10% random peptide library, and random peptide library ¹³ 、4.5×10 ¹³ And 1.0X10 ¹⁴ pfu/mL。

3. Determination of phage titer

(1) Taking out the stored ER2738 escherichia coli from the temperature of minus 80 ℃, dipping a bacterial liquid by an inoculating loop after thawing, drawing lines on an LB/Amp (50 mug/mL) plate, and culturing overnight at 37 ℃;

(2) Single colonies were picked with an inoculating loop in 3mL LB-Amp (50. Mu.g/mL) medium and shake-cultured at 37℃at 250rpm to OD ₆₀₀ ＝0.5；

(3) After diluting phage used for titer determination with LB culture solution by a proper multiple, 10. Mu.L was taken, added to 100. Mu.L of ER2738 E.coli of step (2), left standing at 37℃for 30min, the bacterial solution was spread on LB-Amp plates, cultured at 37℃for 12h, the colony count on the plates was calculated, and phage titer was calculated according to the following formula: phage titer (pfu/mL) =100×dilution x colony count

4. After the electrotransformation is finished, 24 single colonies are respectively picked from phage display random circular eight, nine and decapeptide libraries for colony PCR. Primers SEQ ID NOs 10 and 11 used for colony PCR are shown in Table 5, the system is as follows:

TABLE 5 primers for plasmid PCR

Preparing a reaction system according to a proportion, subpackaging the reaction system into a 200 mu L centrifuge tube, placing the centrifuge tube on ice, dipping a single bacterial colony by using a sterilized toothpick, slightly stirring the sterilized toothpick in the centrifuge tube, and then placing the sterilized toothpick on a PCR instrument for amplification for 35 cycles, wherein the reaction conditions are as follows:

after the PCR reaction was completed, 5. Mu. LPCR was used as a product, and the size of the amplified fragment was detected by 1% agarose gel electrophoresis to determine the empty rate. From the position of the binding of the upstream and downstream primers to the vector, it can be deduced that: if the vector is empty, the colony PCR fragment is 2085bp; if the vector was successfully ligated with the insert, the colony PCR fragment was 467bp. The experimental results are shown in FIG. 4, which is attributed to the larger spacer (1686 bp) between the cleavage sites of pComb-pVIII phagemid, and no empty load phenomenon exists in the constructed phage display random polypeptide library.

According to the phage display system established by the invention, through two enzyme cleavage sites of Xho I and Kpn I, which cannot identify NNK, the exogenous protein is displayed on the phage pVIII protein in a high density, so that the random polypeptide diversity is prevented from being theoretically lost, and meanwhile, the influence of the phage vector-based pVIII display system on phage assembly efficiency is overcome. The constructed phage random cyclic polypeptide library has no empty load phenomenon, high conversion rate, large library capacity and high display density, and has good practical application value.

Claims

1. A phagemid vector pComb-pVIII has a DNA sequence shown in SEQ ID NO 5.

2. Use of the phagemid vector pComb-pVIII according to claim 1 for the construction of a library of phage display random polypeptides.