KR102129584B1 - Insertion method of target genes in linearized vectors - Google Patents

Abstract

The present invention relates to a method for inserting a target gene into a linear vector. Specifically, the method according to the present invention can not only clone the target gene of the influenza virus having various gene sequences by various subtypes in sequence, but also restriction enzyme treatment and elution performed in the conventional cloning method ( Since processes such as elution and ligation can be omitted, it can be usefully used to easily and economically produce a vector into which a target gene has been inserted.

Description

How to insert a target gene into a linear vector {INSERTION METHOD OF TARGET GENES IN LINEARIZED VECTORS}

The present invention relates to a method of inserting a target gene into a vector, and more particularly, to a method of inserting a target gene of an influenza virus into a linear vector using a PCR reaction.

Gene cloning refers to a technique in which a target gene is coupled to a vector having self-replicating ability and introduced into a host such as E. coli to multiply to make the same gene population in large quantities.

Gene cloning combines a target gene amplified with a polymerase chain reaction (PCR) with a vector having a replication initiation point and a vector having a marker for selection of an antibiotic using DNA ligase, and then introduces it into E. coli cells, This is done by selecting cloned cells by examining resistance.

This conventional cloning method forms the basis of modern biotechnology, and has achieved rapid development after the human genome project was completed. However, the conventional genetic manipulation method has limitations such as cutting using a restriction enzyme having the same recognition site without cutting the inside of the inserted DNA and the vector.

Since the 1990s, interest in gene manipulation technology using sequence-specific gene recombinases that recognize specific base sequences and induce recombination between DNA fragments has increased. In particular, a gateway system for cloning a gene using a gene integrase that recognizes a specific DNA sequence has been developed, and has been used to clone large amounts of genes quickly and easily.

The gateway system is carried out by extracellular genetic manipulation in the same manner as a conventional restriction enzyme-linked reaction, but is a novel cloning method using LR lonase and BP chromase. However, the gateway system clones the target gene into a vector using a conventional genetic manipulation method, and expresses the target gene differently by a homologous recombination method of a specific recombination sequence present in the cloned vector and a chromase that recognizes the sequence. It takes a lot of time and effort in that it is a method of transferring a gene into a vector. In this regard, Korean Patent Registration No. 10-0751587 discloses a DNA subcloning method using homologous recombination methods mediated by bacterial recombinases such as RecE/T and Redα/β.

On the other hand, influenza A virus is composed of nine structural proteins, and has two non-structural proteins having regulatory functions. By using the fragmented nature of the viral genome, multiple re-infection of multiple viruses in a cell can result in genetic reassortment. Influenza A virus is classified into various types of subtypes by different hemagglutinin (HA) and neuraminidase (NA) presented on its surface, and each subtype has different pathological properties. In addition, influenza viruses develop different subtypes at each epidemic due to multiple mutations and evolution. Since influenza virus can be changed particularly aggressively by a single mutation, continuous research on various types of influenza virus is required.

Republic of Korea Patent Registration No. 10-0751587

It is an object of the present invention to provide a method for inserting a target gene into a linear vector.

In order to achieve the above object, the present invention is a PCR reaction solution comprising a target gene of the influenza virus, a forward primer composed of the base sequence represented by the following general formula 1 and a reverse primer composed of the base sequence represented by the general formula 2 Denaturation, annealing and elongation; Adding a linear vector to the denatured, annealed and stretched PCR reaction solution; And denaturing, annealing and stretching the PCR reaction solution to which the linear vector is added, to provide a method of inserting a target gene into the linear vector:

[Formula 1]

5'- X1-Y1 -3'

[Formula 2]

5'- X2-Y2 -3'

In the general formulas 1 and 2, X1 and X2 are polynucleotides composed of base sequences complementary to 15 to 30 base sequences from each of the 5'ends of the linear vector,

Y1 and Y2 are polynucleotides composed of base sequences complementary to 1 to 15 base sequences from the 3'end of the influenza virus target gene.

The method according to the present invention can not only clone the target gene of the influenza virus having various gene sequences by various subtypes independently of the sequence, but also restriction enzyme treatment, elution, and restriction enzyme performed in the conventional cloning method. Since a process such as ligation can be omitted, it can be usefully used to easily and economically produce a vector into which a target gene has been inserted.

Figure 1 is a schematic diagram showing the structure of the pHW2000 vector and the ORF (open reading frame) portion to insert the gene of the influenza A virus in one embodiment of the present invention (p _II CMV: pol II promoter, p _Ih : pol I promoter, t _I : pol I terminator, a _II BGH: human cytomegalovirus and polyadenyl signals, Ctr: conserved terminal region of 8 segments).
2 is a schematic diagram showing the process of producing a pHW2000 vector as a linear vector using a PCR reaction in one embodiment of the present invention.
3 is a picture showing the structure of the linear vector produced in one embodiment of the present invention (A) and a photograph showing the result of confirming the produced linear vector by agarose gel electrophoresis (B).
4 is a schematic diagram showing a method of inserting a target gene of the present invention into a linear vector.
Figure 5 is a photograph showing the results of confirming by agarose gel electrophoresis of the plasmid (A) in which the target gene of the influenza virus is inserted by the method according to the present invention and the product (B) obtained by cutting the plasmid with NCOI restriction enzyme.
6 is a schematic diagram showing the method of inserting a target gene of an influenza virus into a linear vector using the PCR reaction according to the present invention.

Hereinafter, the present invention will be described in detail.

The present invention denaturation, annealing a PCR reaction solution comprising a target gene of the influenza virus, a forward primer composed of the base sequence represented by the following general formula 1 and a reverse primer composed of the base sequence represented by the general formula 2 (annealing) and elongation (elongation); Adding a linear vector to the denatured, annealed and stretched PCR reaction solution; And denaturing, annealing and stretching the PCR reaction solution to which the linear vector is added, to provide a method of inserting a target gene into the linear vector:

[Formula 1]

5'- X1-Y1 -3'

[Formula 2]

5'- X2-Y2 -3'

In the general formulas 1 and 2, X1 and X2 are polynucleotides composed of base sequences complementary to 15 to 30 base sequences from each of the 5'ends of the linear vector,

Y1 and Y2 are polynucleotides composed of base sequences complementary to 1 to 15 base sequences from the 3'end of the influenza virus target gene.

The method of inserting the target gene of the present invention into a linear vector includes a target gene of the influenza virus, a forward primer composed of the base sequence represented by the following general formula 1, and a reverse primer composed of the base sequence represented by the general formula 2 It provides a step of denaturation, annealing and elongation of the PCR reaction solution:

[Formula 1]

5'- X1-Y1 -3'

[Formula 2]

5'- X2-Y2 -3'

In the general formulas 1 and 2, X1 and X2 are polynucleotides composed of base sequences complementary to 15 to 30 base sequences from each of the 5'ends of the linear vector,

Y1 and Y2 are polynucleotides composed of base sequences complementary to 1 to 15 base sequences from the 3'end of the influenza virus target gene.

The influenza virus may be an avian or mammalian host influenza virus. For example, the birds or mammals may include humans, chickens, ducks, pigs, dogs, seals, ferrets, bats, and the like. In addition, the influenza virus may be a type A influenza virus.

In the method according to the present invention, the target gene is PB2 (polymerase basic protein 2), PB1 (polymerase basic protein 1), PA (polymerase acidic protein), HA (hemagglutinin), NP (nucleoprotein), NA (neuraminidase) of influenza virus , M (matrix protein) and NS (nonstructural protein) may be any one or more selected from the group consisting of genes. Specifically, the target gene may be PB2, PB1, PA, HA, NP, NA, M and NS genes of the influenza virus.

The forward primer composed of the nucleotide sequence represented by the general formula 1 can bind to the 3'end of the antisense strand in the double strand constituting the target gene, and is composed of the nucleotide sequence represented by the general formula 2 The reverse primer can bind to the 3'end of the sense strand in the double helix that constitutes the target gene. The term, "anti-sense strand" is a strand that is a template for transcription of mRNA among double-stranded DNA, and refers to a strand having a base sequence complementary to mRNA. In addition, the term, "sense strand (sense strand)" means a strand having the same nucleotide sequence as mRNA, except that uracil (U) is present in place of thymine (T) in double-stranded DNA.

In general formulas 1 and 2, X1 and X2 are 15 to 30, 17 to 30, 18 to 30, 15 to 28, 17 to 28, 18 to 28, 15 from each of the 5'ends of the linear vector. It may be a polynucleotide composed of a base sequence complementary to 26 to 17, 26 to 18 or 26 to 26 base sequences. Specifically, in General Formula 1, X1 is 15 to 30, 17 to 30, 18 to 30, 15 to 28, 17 to 28, 18 to 28, 15 from the 5'end of the linear vector sense strand It may be a polynucleotide composed of a base sequence complementary to 26 to 17, 26 to 18 or 26 to 26 base sequences. On the other hand, X2 in the general formula 2 is 15 to 30, 17 to 30, 18 to 30, 15 to 28, 17 to 28, 18 to 28, 15 to 15 from the 5'end of the linear vector antisense strand It may be a polynucleotide composed of a base sequence complementary to 26, 17 to 26 or 18 to 26 base sequences.

In addition, in the general formulas 1 and 2, Y1 and Y2 are 1 to 15, 1 to 14, 1 to 13, 2 to 15, 2 to 14, or 2 to 13 from the 3'end of the influenza virus target gene. It may be a polynucleotide composed of a nucleotide sequence complementary to the nucleotide sequence of a dog. Specifically, in General Formula 1, Y1 is 1 to 15, 1 to 14, 1 to 13, 2 to 15, 2 to 14, or 2 to 13 from the 3'end of the target gene antisense strand of the influenza virus. It may be a polynucleotide composed of a nucleotide sequence complementary to the nucleotide sequence of a dog. On the other hand, in the general formula 2 Y2 is 1 to 15, 1 to 14, 1 to 13, 2 to 15, 2 to 14 or 2 to 13 from the 3'end of the target gene sense strand of the influenza virus It may be a polynucleotide composed of a base sequence complementary to the base sequence.

In one example, the forward primer consisting of the base sequence represented by the general formula 1 and the reverse primer consisting of the base sequence represented by the general formula 2 is composed of the base sequence of SEQ ID NO: 17 to 32 and SEQ ID NO: 43 to 50 It may be a polynucleotide. Specifically, the primer for the PB2 gene of the influenza virus is a polynucleotide composed of the nucleotide sequence set forth in SEQ ID NOs: 17 and 18, and the primer for the PB1 gene is a polynucleotide composed of the nucleotide sequences set forth in SEQ ID NO: 19 and 20, The primer for the PA gene is a polynucleotide composed of the nucleotide sequence set forth in SEQ ID NO: 21 and 22, the primer for the HA gene is a polynucleotide composed of the nucleotide sequence set forth in SEQ ID NO: 23 and 24, and the primer for the NP gene is It is a polynucleotide consisting of the nucleotide sequence of SEQ ID NOs: 25 and 26, and the primer for the NA gene is a polynucleotide consisting of the nucleotide sequence of SEQ ID NOs: 27, 28 and 43 to 50, and the primer for the M gene is SEQ ID NO: It is a polynucleotide consisting of the nucleotide sequence of 29 and 30, and the primer for the NS gene may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 31 and 32.

In the method according to the present invention, the PCR reaction solution may be prepared according to methods well known in the art. For example, the PCR reaction solution may include a DNA polymerase, dNTP, etc., and buffers and reagents required for the PCR reaction, and the amount of these may be appropriately adjusted by a person skilled in the art as necessary.

As used herein, the term "denaturation (denaturation) step" refers to the process of dissociating into a single strand by exposing the double-stranded DNA to high temperatures. In addition, the term "annealing (annealing) step" means a process in which forward and reverse primers are bound to sense and antisense DNA dissociated into single strands, respectively. Furthermore, the term, "elongation step" refers to a process in which DNA polymerase acts from the 3'end of each of the bound forward and reverse primers to extend nucleotides. The denaturing, annealing and stretching steps may be performed under temperature and time known in the art, and the temperature and time may be appropriately modified as necessary.

In the method according to the invention, an initial denaturation step may be additionally carried out as necessary before the denaturation step, and the temperature and time for the initial denaturation can also be performed as known in the art.

The step of denaturing, annealing and stretching the PCR reaction solution in the above step is 20 to 30 times, 22 to 30 times, 24 to 30 times, 20 to 28 times, 22 to 28 times, 24 to 28 times, 20 to 26 times, It can be repeated 22 to 26 times or 24 to 26 times.

In addition, the method of inserting the target gene of the present invention into a linear vector provides a step of adding a linear vector to the denatured, annealed and stretched PCR reaction solution.

The linear vector is composed of a forward primer composed of the base sequence represented by the following general formula 3 and a base sequence represented by the general formula 4 that complementarily bind to both sides based on the position where the target gene is to be inserted in the expression vector. It can be obtained by PCR reaction using reverse primer:

[Formula 3]

5'- A1-B1 -3'

[Formula 4]

5'- A2-B2 -3'

In the general formulas 3 and 4, A1 and A2 are polynucleotides composed of base sequences complementary to 1 to 15 base sequences from each of the 5'ends of the target gene,

B1 and B2 are polynucleotides composed of base sequences complementary to 35 to 55 base sequences from each of the 3'ends on the basis of the position to insert the target gene in the expression vector.

The forward primer composed of the nucleotide sequence represented by the general formula 3 can be bound to the 3'end of the antisense strand in the double strand constituting the expression vector, and is composed of the nucleotide sequence represented by the general formula 4 The reverse primer can bind to the 3'end of the sense strand in the double helix that constitutes the expression vector.

In the general formulas 3 and 4, A1 and A2 are 1 to 15, 1 to 14, 1 to 13, 2 to 15, 2 to 14, or 2 to 13 nucleotide sequences from the 5'ends of the target gene, respectively. It may be a polynucleotide composed of a base sequence complementary to. Specifically, in the general formula 3, A1 is 1 to 15, 1 to 14, 1 to 13, 2 to 15, 2 to 14, or 2 to 13 base sequences from the 5'end of the target gene sense strand It may be a polynucleotide composed of a base sequence complementary to. On the other hand, in the general formula 3 A2 is 1 to 15, 1 to 14, 1 to 13, 2 to 15, 2 to 14 or 2 to 13 base sequences from the 5'end of the target gene antisense strand It may be a polynucleotide composed of complementary base sequences.

Meanwhile, in the general formulas 3 and 4, B1 and B2 are 35 to 55, 38 to 55, 40 to 55, 35 to 52, 38 to 52, 40 to 52 from each of the 3'ends of the expression vector. , 35 to 50, 38 to 50 or 40 to 50 base sequence may be a polynucleotide consisting of a base sequence complementary to. Specifically, in the general formula 3, B1 is 35 to 55, 38 to 55, 40 to 55, 35 to 52, 38 to 52, 40 to 52 from the 3'end of the antisense strand of the expression vector, It may be a polynucleotide composed of a base sequence complementary to 35 to 50, 38 to 50 or 40 to 50 base sequences. Meanwhile, in the general formula 4, B2 is 35 to 55, 38 to 55, 40 to 55, 35 to 52, 38 to 52, 40 to 52, 35 from the 3'end of the sense strand of the expression vector It may be a polynucleotide consisting of a base sequence complementary to 50, 38 to 50 or 40 to 50 base sequences.

As an example, the forward primer composed of the base sequence represented by the general formula 3 and the reverse primer composed of the base sequence represented by the general formula 4 is composed of the base sequence of SEQ ID NO: 1 to 16 and SEQ ID NO: 35 to 42 It may be a polynucleotide. Specifically, the primer of the vector for the PB2 gene of the influenza virus is a polynucleotide consisting of the nucleotide sequence of SEQ ID NOs: 1 and 2, and the primer of the vector for the PB1 gene consists of the nucleotide sequences of SEQ ID NO: 3 and 4. A polynucleotide, the primer of the vector for the PA gene is composed of a nucleotide sequence of SEQ ID NOs: 5 and 6, and the primer of the vector for a HA gene is composed of the nucleotide sequences of SEQ ID NOs: 7 and 8 Is, the primer of the vector for the NP gene is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9 and 10, the primer of the vector for the NA gene is composed of the nucleotide sequence of SEQ ID NO: 11, 12 and 35 to 42 Polynucleotide, the primer of the vector for the M gene is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 13 and 14, and the primer of the vector for the NS gene is composed of the nucleotide sequence of SEQ ID NO: 15 and 16 Nucleotides.

The PCR reaction to obtain the linear vector can be carried out by conventional methods, and can be appropriately modified as necessary. The obtained linear vector can be added in an appropriate amount by a person skilled in the art.

In addition, the method of inserting the target gene of the present invention into a linear vector provides a step of denaturing, annealing and stretching the PCR reaction solution to which the linear vector is added.

In this step, denaturation, annealing and stretching steps can have the features described above. The denaturation, annealing and stretching steps are 5 to 15 times, 7 to 15 times, 9 to 15 times, 5 to 13 times, 7 to 13 times, 9 to 13 times, 5 to 11 times, 7 to 11 times, or 9 to 9 times. It can be repeated 11 times.

In addition, the method according to the present invention may further include a final elongation step after the denaturation, annealing and stretching steps are completed. At this time, the temperature and time for further stretching can also be performed as is known in the art.

Hereinafter, the present invention will be described in detail by the following examples, provided that the following examples are only for illustrating the present invention, and the present invention is not limited by them. Any thing that has substantially the same configuration and the same operation and effect as the technical idea described in the claims of the present invention is included in the technical scope of the present invention.

Example 1. Preparation of linear vectors

A linear vector was prepared in the following manner to insert all genes of the influenza A virus into the ORF (open reading frame) portion of the pHW2000 vector (FIG. 1).

First, using the pHW2000 vector (Dr. Robert G. Webster, St. Jude Children's Research Hospital) as a template, universal vectors primers were used to obtain linear vectors for each gene of the influenza A virus as PCR products (FIG. 2).

Specifically, the PCR reaction has a proofreading function, and a fusion polymerase (Phusion Polymerase) in HF buffer (New England BioLabs, Germany) was used. PCR reactions include 2 μl of fusion polymerase, 4 μl of 5 pmol forward and reverse primers, 10 μl of 10×HF buffer, 10 μl of 2 mM dNTP, 1 μl of 50 mM MgCl ₂ , and 100 ng of empty pHW2000 vector To the mixture containing, distilled water was added to prepare a total of 100 μl. The sequence and PCR conditions of the primers used are shown in Tables 1 and 2, respectively, and the primers were constructed to include the conserved terminal region (Ctr), uni 12 and uni 13 of the pHW2000 vector.

primer Sequence (5'→3') Sequence number PB2 forward AAACGACCTTGTTTCTACTAATAACCCGGCGGCCCAAAATGCCGACTCG SEQ ID NO: 1 PB2 reverse GACCTGCTTTCGCTCCCCCCCAACTTCGGAGGTCGACCAGTACTCCGGTTA SEQ ID NO: 2 PB1 forward AAATGCCTTGTTTCTACTAATAACCCGGCGGCCCAAAATGCCGACTCG SEQ ID NO: 3 PB1 reverse TGCCTGCTTTCGCTCCCCCCCAACTTCGGAGGTCGACCAGTACTCCGGTTA SEQ ID NO: 4 PA forward AAGTACCTTGTTTCTACTAATAACCCGGCGGCCCAAAATGCCGACTCG SEQ ID NO: 5 PA reverse GTACCTGCTTTCGCTCCCCCCCAACTTCGGAGGTCGACCAGTACTCCGGTTA SEQ ID NO: 6 HA forward AAAACACCCTTGTTTCTACTAATAACCCGGCGGCCCAAAATGCCGACTCG SEQ ID NO: 7 HA reverse CCCCTGCTTTCGCTCCCCCCCAACTTCGGAGGTCGACCAGTACTCCGGTTA SEQ ID NO: 8 NP forward AAAATACCCTTGTTTCTACTAATAACCCGGCGGCCCAAAATGCCGACTCG SEQ ID NO: 9 NP reverse TACCCTGCTTTCGCTCCCCCCCAACTTCGGAGGTCGACCAGTACTCCGGTTA SEQ ID NO: 10 NA forward AAAAAACTCCTTGTTTCTACTAATAACCCGGCGGCCCAAAATGCCGACTCG SEQ ID NO: 11 NA reverse ACTCCTGCTTTCGCTCCCCCCCAACTTCGGAGGTCGACCAGTACTCCGGTTA SEQ ID NO: 12 M forward AAAAACTACCTTGTTTCTACTAATAACCCGGCGGCCCAAAATGCCGACTCG SEQ ID NO: 13 M reverse CTACCTGCTTTCGCTCCCCCCCAACTTCGGAGGTCGACCAGTACTCCGGTTA SEQ ID NO: 14 NS forward AAAACACCCTTGTTTCTACTAATAACCCGGCGGCCCAAAATGCCGACTCG SEQ ID NO: 15 NS reverse CACCCTGCTTTCGCTCCCCCCCAACTTCGGAGGTCGACCAGTACTCCGGTTA SEQ ID NO: 16

Initial denaturation stage 98℃, 30 seconds 1 time Metamorphic stage 98℃, 10 seconds Episode 30 Annealing step 58℃, 30 seconds Elongation step 72℃, 1 minute 30 seconds Final stretching step 72℃, 7 minutes 1 time

Then, the vector present in the PCR product was removed by adding 1 μl (10 units) of DPN1 and reacting for 2 hours. PCR products were purified using the QIAquick® Gel Extraction Kit (Qiagen, USA) according to the manufacturer's protocol. Fig. 3B shows the results of electrophoresis of the purified PCR product on an agarose gel.

As shown in Figure 3B, influenza A virus PB2 (polymerase basic protein 2), PB1 (polymerase basic protein 1), PA (polymerase acidic protein), HA (hemagglutinin), NP (nucleoprotein), NA (neuraminidase), M A linear pHW2000 vector having a size of about 3 kb, capable of cloning (matrix protein) and nonstructural protein (NS) proteins, was identified.

Example 2. Linear cDNA preparation of influenza A virus

Genes encoding each protein of the influenza A virus for cloning in a linear vector were prepared in the following manner.

First, RNA of the influenza A virus was isolated from the A/Puerto Rico/8/1934 (H1N1, PR8) virus by a conventional method. 5 μl of 2 mM DNTP was added to the isolated 38 μl of viral RNA, and 2 μl of uni12-CPEC(G) or uni12-CPEC(A) primers shown in Table 3 below were added to RT-PCR. The mixture was prepared.

primer Sequence (5'→3') Sequence number PB2 1F AAGTTGGGGGGGAGCGAAAGCAGGTC SEQ ID NO: 17 PB2 2341R GGTTATTAGTAGAAACAAGGTCGTTT SEQ ID NO: 18 PB1 1F AAGTTGGGGGGGAGCGAAAGCAGGCA SEQ ID NO: 19 PB1 2341R GGTTATTAGTAGAAACAAGGCATTT SEQ ID NO: 20 PA 1F AAGTTGGGGGGGAGCGAAAGCAGGTAC SEQ ID NO: 21 PA 2341R GGTTATTAGTAGAAACAAGGTACTT SEQ ID NO: 22 HA 1F AAGTTGGGGGGGAGCAAAAGCAGGGG SEQ ID NO: 23 HA 890R GGTTATTAGTAGAAACAAGGGTGTTTT SEQ ID NO: 24 NP 1F AAGTTGGGGGGGAGCAAAAGCAGGGTA SEQ ID NO: 25 NP 1565R GGTTATTAGTAGAAACAAGGGTATTTTT SEQ.ID No. 26 NA 1F AAGTTGGGGGGGAGCAAAAGCAGGAGT SEQ ID NO: 27 NA 1413R GGTTATTAGTAGAAACAAGGAGTTTTTT SEQ.ID No. 28 M 1F AAGTTGGGGGGGAGCAAAAGCAGGTAG SEQ ID NO: 29 M 1027R GGTTATTAGTAGAAACAAGGTAGTTTTT SEQ ID NO: 30 NS 1F AAGTTGGGGGGGAGCAAAAGCAGGGTG SEQ.ID No. 31 NS 890R GGTTATTAGTAGAAACAAGGGTGTTTT SEQ ID NO: 32 uni12-CPEC(G) CGAAGTTGGGGGGGAGCGAAAGCAGG SEQ ID NO: 33 uni12-CPEC(A) CGAAGTTGGGGGGGAGCAAAAGCAGG SEQ ID NO: 34

The mixture was left at 65° C. for 5 minutes and then quickly transferred onto ice. After the mixture cooled, 2 μl of 10×HF buffer and 1 μl of M-MLV reverse transcriptase were added and reacted at 37° C. for 1 hour. For inactivation of the added polymerase, after further reacting at 72°C for 10 minutes, the prepared cDNA was stored until use at -80°C.

Example 3. One-pot PCR cloning (OPPC)

Each gene of the influenza A virus was cloned into a linear vector for each gene as follows.

Specifically, 5 μl of 10×HF buffer, 5 μl of 2 mM dNTP, 1 μl of 50 mM MgCl ₂ , 2 μl of 5 pmol forward and reverse primer, 6 μl of cDNA obtained in Example 2 and 1 μl The fusion polymerase was mixed and PCR reaction was prepared by adding distilled water to a final volume of 45 µl. At this time, primers (SEQ ID NOs: 17 to 32) described in Table 3 were used. In addition, the PCR reaction was performed as described in Table 4 below, after the denaturation-annealing-stretching step was repeated 25 times, the linear vector of 300 ng prepared in Example 1 was added, and this was repeated 10 times. (Figure 4).

Initial denaturation stage 98℃, 30 seconds 1 time Metamorphic stage 98℃, 10 seconds 25 times + 10 times Annealing step 58℃, 30 seconds Elongation step 72℃, 1 minute 30 seconds Final stretching step 72℃, 5 minutes 1 time

After the reaction was completed, 5 µl of the obtained PCR product was taken and transformed into E. coli by a conventional method. Colony PCR was performed using the obtained colonies, and at the same time, plasmids were separated from the colonies, cut with NCOI enzyme, and electrophoresed to confirm whether the cDNA obtained in Example 2 was cloned. At this time, as a control group, each gene of the general A/Puerto Rico/8/1934 (H1N1, PR8) virus was cloned and compared by colony PCR and NCOI enzyme digestion. As a result, the number of colonies obtained and the number of colonies in which cloning was confirmed are shown in Table 5, and a photo of agarose gel electrophoresis of the plasmid cut with the NCOI enzyme is shown in FIG. 5.

gene Length
(nt) Vector sheep
(ng) Linear vector Colony number Number of colonies cloned/total number of selected colonies (% of percentage) PB2 2341+19 300 PB2 75 7/11 (63.6) PB1 2341+19 300 PB1 69 8/11 (72.7) PA 2233+19 300 PA 85 8/11 (72.7) HA 1778+19 300 HA 98 7/11 (63.6) NP 1565+19 300 NP 67 9/11 (81.8) NA 1413+19 300 NA 59 8/11 (72.2) M 1027+19 300 M 68 8/11 (72.2) NS 890+19 300 NS 72 11/11(100)

As shown in Table 5, genes encoding the PB2, PB1, PA, HA, NP, NA, M and NS proteins of the influenza A virus were cloned into linear vectors at a rate of 63% or more. In addition, as shown in Figure 5, by comparing the band size of the prepared plasmid (Fig. 5A) and the band size (Fig. 5B) when the plasmid was cut with NCOI, compared to the control group, PB2, PB1 of influenza A virus, It was confirmed that genes encoding PA, HA, NP, NA, M and NS proteins were cloned.

Example 4. Production of recombinant influenza A virus

Recombinant influenza A virus was produced by the following method using plasmids expressing PB2, PB1, PA, HA, NP, NA, M and NS proteins of influenza A virus, respectively.

First, HEK-293T (ATCC) and MDCK (ATCC) cell lines were prepared by culturing in a conventional manner, and the prepared HEK-293T and MDCK cells were mixed at a volume ratio of 3:1 and co-cultured in a 6-well plate. Here, 1 µl of plasmids expressing PB2, PB1, PA, HA, NP, NA, M and NS proteins of the influenza A virus obtained in Example 3 were taken and co-transfected. Specifically, 1 ng of plasmid expressing 8 kinds of protein was mixed with 182 μl of opti-MEM and 18 μl of TransIT-LT1 transfection preparation, and allowed to stand at room temperature for 45 minutes. Thereafter, 800 μl of opti-MEM medium was added to the mixture, and the co-cultured HEK-293T and MDCK cells were treated. After 18 hours of transfection, the plate was replaced with 1 ml opti-MEM medium containing 1% antibiotic, and 24 hours after transfection, 1 ml opti-MEM containing 1 μg/ml TPCK-trypsin. Medium was added. After culturing it for 48 hours, the supernatant of the culture was taken and injected into a 10-day-old SPF (specific pathogen free) embryonated chicken egg, which was cultured for 48 to 72 hours. The HA test was performed to confirm the growth of the recombinant virus, and RT-PCR was performed to confirm the sequence of the virus.

As a result, it was possible to produce recombinant influenza A virus using plasmids expressing PB2, PB1, PA, HA, NP, NA, M and NS proteins, respectively, prepared by the method according to the present invention. The sequence was identical to the base sequence used in the preparation of the plasmid.

Example 5. OPPC using various subtypes of influenza A virus

OPPC was performed as follows using various subtypes of influenza A virus. First, A/duck/Korea/436/2014(W436, H1N8), A/duck/Korea/369/2008(W369, H2N3), A/duck/Korea/538/2016(W538, H3N8), A/duck /Korea/137/2006(W137, H4N4), A/duck/Korea/562/2016(W562, H5N3), A/duck/Korea/502/2015(W502, H6N2), A/duck/Korea/557/ 2016(W557, H7N7), A/duck/Korea/563/2016(W563, H8N6), A/duck/Korea/233/2007(W233, H9N2), A/duck/Korea/530/2016(W530, H10N4 ), A/duck/Korea/552/2016(W552, H11N9), A/duck/Korea/373/2008(W373, H12N5), A/Environment/Korea/W468/2015 (W468, H5N8) or A/Korea /CNH1/2016 (CNH1, H1N1) virus was isolated from feces of domestic wild migratory birds. The isolated viruses were used to prepare plasmids expressing PB2, PB1, PA, HA, NP, NA, M and NS proteins, respectively, under the same conditions and methods as described above. At this time, the primers described in Tables 1 and 3 above were used for the production of the linear vector for the subtypes of the HA and NA genes, and only for the N3, N6, N7, and N9 subtypes of the NA gene, Table 6 below (for linear vectors) Primers) and 7 (primers for viral genes) were used.

primer Sequence (5'→3') Sequence number NA N3 forward AAAAAGCACCTTGTTTCTACTAATAACCCGGCGGCCCAAAATGCCGACTCG SEQ ID NO: 35 NA N3 reverse GCACCTGCTTTCGCTCCCCCCCAACTTCGGAGGTCGACCAGTACTCCGGTTA SEQ ID NO: 36 NA N6 forward AAAACACCCTTGTTTCTACTAATAACCCGGCGGCCCAAAATGCCGACTCG SEQ.ID No. 37 NA N6 reverse CATTTTCACCCTGCTTTCGCTCCCCCCCAACTTCGGAGGTCGACCAGTACTCCGGTTA SEQ ID NO: 38 NA N7 forward AAAACACCCTTGTTTCTACTAATAACCCGGCGGCCCAAAATGCCGACTCG SEQ ID NO: 39 NA N7 reverse CATTCTCAATCACCCTGCTTTCGCTCCCCCCCAACTTCGGAGGTCGACCAGTACTCCGGTTA SEQ ID NO: 40 NA N9 forward AAGACCCTTGTTTCTACTAATAACCCGGCGGCCCAAAATGCCGACTCG SEQ.ID No. 41 NA N9 reverse GACCCTGCTTTCGCTCCCCCCCAACTTCGGAGGTCGACCAGTACTCCGGTTA SEQ.ID No. 42

primer Sequence (5'→3') Sequence number NA N3 1F AAGTTGGGGGGGAGCAAAAGCAGGTGC SEQ.ID No. 43 NA N3 1420R GGTTATTAGTAGAAACAAGGAGCTTTTT SEQ.ID No. 44 NA N6 1F AAGTTGGGGGGGAGCAAAAGCAGGGTGAAAATG SEQ.ID No. 45 NA N6 890R GGTTATTAGTAGAAACAAGGGTGTTTT SEQ.ID No. 46 NA N7 1F AAGTTGGGGGGGAGCAAAAGCAGGGTGATTGAGAATG SEQ.ID No. 47 NA N7 890R GGTTATTAGTAGAAACAAGGGTGTTTT SEQ.ID No. 48 NA N9 1F AAGTTGGGGGGGAGCAAAAGCAGGGTC SEQ.ID No. 49 NA N9 1473R GGTTATTAGTAGAAACAAGGGTCTT SEQ.ID No. 50

In Table 8 below, it was confirmed whether the genes expressing PB2, PB1, PA, HA, NP, NA, M, and NS proteins were cloned into the prepared plasmid, and whether these recombinant plasmids could be used to produce recombinant influenza A virus. Shown.

virus Subtype Check cloning and virus production PB2 PB1 PA HA NP NA M NS w436 H1N8 + + + + + + + + W369 H2N3 + + + + + + + + w538 H3N8 + + + + + + + + W137 H4N4 + + + + + + + + w562 H5N3 + + + + + + + + w502 H6N2 + + + + + + + + w524 H7N1 + + + + + + + + W557 H7N7 + + + + + + + + w563 H8N6 + + + + + + + + W233 H9N2 + + + + + + + + W530 H10N4 + + + + + + + + w552 H11N9 + + + + + + + + W373 H12N5 + + + + + + + + W452 H5N8 + + + + + + + + CNH1 H1N1 + + + + + + + +

As shown in Table 8, it was possible to economically clone genes of various subtypes of influenza A virus by the method according to the present invention, and the rest of each plasmid and A/Puerto Rico/8/34 virus prepared from the above 7 It was confirmed that a recombinant influenza A virus was produced using a dog gene plasmid.

Therefore, it was confirmed that the method according to the present invention can be used to clone viral genes easily and economically.

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> INSERTION METHOD OF TARGET GENES IN LINEARIZED VECTORS <130> DP-2018-0221-KR <160> 50 <170> KoPatentIn 3.0 <210> 1 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> PB2 forward <400> 1 aaacgacctt gtttctacta ataacccggc ggcccaaaat gccgactcg 49 <210> 2 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> PB2 reverse <400> 2 gacctgcttt cgctcccccc caacttcgga ggtcgaccag tactccggtt a 51 <210> 3 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> PB1 forward <400> 3 aaatgccttg tttctactaa taacccggcg gcccaaaatg ccgactcg 48 <210> 4 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> PB1 reverse <400> 4 tgcctgcttt cgctcccccc caacttcgga ggtcgaccag tactccggtt a 51 <210> 5 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> PA forward <400> 5 aagtaccttg tttctactaa taacccggcg gcccaaaatg ccgactcg 48 <210> 6 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> PA reverse <400> 6 gtacctgctt tcgctccccc ccaacttcgg aggtcgacca gtactccggt ta 52 <210> 7 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> HA forward <400> 7 aaaacaccct tgtttctact aataacccgg cggcccaaaa tgccgactcg 50 <210> 8 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> HA reverse <400> 8 cccctgcttt cgctcccccc caacttcgga ggtcgaccag tactccggtt a 51 <210> 9 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> NP forward <400> 9 aaaataccct tgtttctact aataacccgg cggcccaaaa tgccgactcg 50 <210> 10 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> NP reverse <400> 10 taccctgctt tcgctccccc ccaacttcgg aggtcgacca gtactccggt ta 52 <210> 11 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> NA forward <400> 11 aaaaaactcc ttgtttctac taataacccg gcggcccaaa atgccgactc g 51 <210> 12 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> NA reverse <400> 12 actcctgctt tcgctccccc ccaacttcgg aggtcgacca gtactccggt ta 52 <210> 13 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> M forward <400> 13 aaaaactacc ttgtttctac taataacccg gcggcccaaa atgccgactc g 51 <210> 14 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> M reverse <400> 14 ctacctgctt tcgctccccc ccaacttcgg aggtcgacca gtactccggt ta 52 <210> 15 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> NS forward <400> 15 aaaacaccct tgtttctact aataacccgg cggcccaaaa tgccgactcg 50 <210> 16 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> NS reverse <400> 16 caccctgctt tcgctccccc ccaacttcgg aggtcgacca gtactccggt ta 52 <210> 17 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PB2 1F <400> 17 aagttggggg ggagcgaaag caggtc 26 <210> 18 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PB2 2341R <400> 18 ggttattagt agaaacaagg tcgttt 26 <210> 19 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PB1 1F <400> 19 aagttggggg ggagcgaaag caggca 26 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PB1 2341R <400> 20 ggttattagt agaaacaagg cattt 25 <210> 21 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PA 1F <400> 21 aagttggggg ggagcgaaag caggtac 27 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PA 2341R <400> 22 ggttattagt agaaacaagg tactt 25 <210> 23 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> HA 1F <400> 23 aagttggggg ggagcaaaag cagggg 26 <210> 24 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> HA 890R <400> 24 ggttattagt agaaacaagg gtgtttt 27 <210> 25 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NP 1F <400> 25 aagttggggg ggagcaaaag cagggta 27 <210> 26 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> NP 1565R <400> 26 ggttattagt agaaacaagg gtattttt 28 <210> 27 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NA 1F <400> 27 aagttggggg ggagcaaaag caggagt 27 <210> 28 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> NA 1413R <400> 28 ggttattagt agaaacaagg agtttttt 28 <210> 29 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> M 1F <400> 29 aagttggggg ggagcaaaag caggtag 27 <210> 30 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> M 1027R <400> 30 ggttattagt agaaacaagg tagttttt 28 <210> 31 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NS 1F <400> 31 aagttggggg ggagcaaaag cagggtg 27 <210> 32 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NS 890R <400> 32 ggttattagt agaaacaagg gtgtttt 27 <210> 33 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> uni12-CPEC(G) <400> 33 cgaagttggg ggggagcgaa agcagg 26 <210> 34 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> uni12-CPEC(A) <400> 34 cgaagttggg ggggagcaaa agcagg 26 <210> 35 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> NA N3 forward <400> 35 aaaaagcacc ttgtttctac taataacccg gcggcccaaa atgccgactc g 51 <210> 36 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> NA N3 reverse <400> 36 gcacctgctt tcgctccccc ccaacttcgg aggtcgacca gtactccggt ta 52 <210> 37 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> NA N6 forward <400> 37 aaaacaccct tgtttctact aataacccgg cggcccaaaa tgccgactcg 50 <210> 38 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> NA N6 reverse <400> 38 cattttcacc ctgctttcgc tcccccccaa cttcggaggt cgaccagtac tccggtta 58 <210> 39 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> NA N7 forward <400> 39 aaaacaccct tgtttctact aataacccgg cggcccaaaa tgccgactcg 50 <210> 40 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> NA N7 reverse <400> 40 cattctcaat caccctgctt tcgctccccc ccaacttcgg aggtcgacca gtactccggt 60 ta 62 <210> 41 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> NA N9 forward <400> 41 aagacccttg tttctactaa taacccggcg gcccaaaatg ccgactcg 48 <210> 42 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> NA N9 reverse <400> 42 gaccctgctt tcgctccccc ccaacttcgg aggtcgacca gtactccggt ta 52 <210> 43 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NA N3 1F <400> 43 aagttggggg ggagcaaaag caggtgc 27 <210> 44 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> NA N3 1420R <400> 44 ggttattagt agaaacaagg agcttttt 28 <210> 45 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> NA N6 1F <400> 45 aagttggggg ggagcaaaag cagggtgaaa atg 33 <210> 46 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NA N6 890R <400> 46 ggttattagt agaaacaagg gtgtttt 27 <210> 47 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> NA N7 1F <400> 47 aagttggggg ggagcaaaag cagggtgatt gagaatg 37 <210> 48 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NA N7 890R <400> 48 ggttattagt agaaacaagg gtgtttt 27 <210> 49 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NA N9 1F <400> 49 aagttggggg ggagcaaaag cagggtc 27 <210> 50 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> NA N9 1473R <400> 50 ggttattagt agaaacaagg gtctt 25

Claims (8)

1) Denaturation, annealing and elongation of a PCR reaction solution comprising a primer consisting of a target gene of influenza A virus, a nucleotide sequence of SEQ ID NOs: 17 to 32 and SEQ ID NOs: 43 to 50 step;
2) adding a linear vector of pHW2000 having a cleavage map shown in FIG. 1 to the modified, annealed and stretched PCR reaction solution of step 1); And
3) the purpose of the step of denaturing, annealing and stretching the PCR reaction solution to which the pHW2000 linear vector of step 2) is added using primers consisting of the nucleotide sequences of SEQ ID NOs: 17 to 32 and SEQ ID NOs: 43 to 50 To insert a gene into a linear vector:
The influenza A virus genes include PB2 (polymerase basic protein 2), PB1 (polymerase basic protein 1), PA (polymerase acidic protein), HA (hemagglutinin), NP (nucleoprotein), NA (neuraminidase), and M (matrix protein). And a non-structural protein (NS) gene.
The primer for the PB2 gene is a polynucleotide composed of the nucleotide sequence set forth in SEQ ID NO: 17 and 18, the primer for the PB1 gene is a polynucleotide composed of the nucleotide sequence set forth in SEQ ID NO: 19 and 20, and the primer for the PA gene is It is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 21 and 22, the primer for the HA gene is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 23 and 24, and the primer for the NP gene is shown in SEQ ID NO: 25 and 26 It is a polynucleotide consisting of the nucleotide sequence described, the primer for the NA gene is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 27, 28 and 43 to 50, the primer for the M gene is shown in SEQ ID NO: 29 and 30 It is a polynucleotide consisting of a sequence, and the primer for the NS gene is a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NOs: 31 and 32,
Among the linear vectors of pHW2000, the linear vector for the PB2 gene is amplified with a primer consisting of the nucleotide sequence shown in SEQ ID NOS: 1 and 2, and the linear vector for the PB1 gene consists of the nucleotide sequence shown in SEQ ID NO: 3 and 4. Amplified with a primer, the linear vector for the PA gene is amplified with a primer consisting of the nucleotide sequence set forth in SEQ ID NOs: 5 and 6, and the linear vector for the HA gene is a primer consisting of the nucleotide sequence shown in SEQ ID NOS: 7 and 8. Amplified, the linear vector for the NP gene is amplified with a primer consisting of the nucleotide sequences set forth in SEQ ID NOs: 9 and 10, and the linear vector for the NA gene consists of the nucleotide sequences set forth in SEQ ID NOs: 11, 12 and 35 to 42 Amplified with a primer, the linear vector for the M gene is amplified with a primer consisting of the nucleotide sequence of SEQ ID NOs: 13 and 14, and the linear vector for the NS gene is a primer consisting of the nucleotide sequences of SEQ ID NOs: 15 and 16. Is amplified.
delete The method of claim 1, wherein the step of denaturing, annealing, and stretching the PCR reaction solution of step 1) is repeated 20 to 30 times, wherein the target gene is inserted into a linear vector.
The method of claim 1, wherein the step of denaturing, annealing, and stretching the PCR reaction solution of step 3) is repeated 5 to 15 times, wherein the target gene is inserted into a linear vector.
delete delete delete The method of claim 1, further comprising a final elongation step.

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