CN111549029A - Duck Mx promoter mutant and construction method and application of recombinant plasmid of luciferase reporter gene thereof - Google Patents

Abstract

The invention discloses a method for constructing duck Mx promoter mutants and luciferase reporter gene recombinant plasmids thereof and application thereof, wherein the method comprises the following steps of 1, PCR amplification and recovery of duck TLR7 gene; step 2In Fusion carries out recombination of the vector and the target fragment. Through a gene mutation technology, the ISRE1 sequence AGTTTCGTTTCT on duck Mx gene promoter duMxp on NCBI is subjected to site-directed mutagenesis AGGGTCGGGTCT, and the Mx gene promoter containing ISRE1 mutant is successfully cloned; and the In Fusion seamless cloning method is adopted to insert the In Fusion seamless cloning method into luciferase reporter genes pGL3-Basic Kpn/XhoI, so that a luciferase reporter system containing the Mx gene promoter sequence of the ISRE1 mutant is successfully constructed, and a foundation is laid for further researching the expression regulation mechanism of duck Mx genes and discussing targeted gene therapy.

Description

Duck Mx promoter mutant and construction method and application of recombinant plasmid of luciferase reporter gene thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a duck Mx promoter mutant and a construction method and application of a luciferase reporter gene recombinant plasmid thereof.

Background

Interferon (Interferon, IFN) is an important antiviral active substance, is an important component of the non-specific immune system of the body, and has wide application in anti-infection and tumor clinical treatment of animals and humans. INF is classified into three types: type I IFN-alpha and IFN-beta, wherein IFN-alpha has more than twenty subtypes and IFN-beta has only one subtype. The type I interferon has the effects of inhibiting virus replication, resisting parasites, inhibiting proliferation of various cells, stimulating killing activity of immune cells, participating in immune regulation, resisting tumors and the like; type II only contains IFN-gamma, and only has a subtype, and besides the antiviral and antiproliferative activity, the main biological activity is the immunoregulation function; type III includes IFN-. lambda.1 (IL-29), IFN-. lambda.2 (IL-28a) and IFN-. lambda. (IL-28 b). Research has shown that the major pathway for type i IFNs to exert their antiviral biological functions is via the JAK-STAT signal transduction pathway, i.e. IFN- α/β first acts on type i IFN receptors, activating JAK1 and TYK2, causing tyrosine phosphorylation of STAT proteins, and forming STAT1-STAT2-IRF9 complex, i.e. IFN-stimulated gene factor 3 (ISGF 3), which translocates ISGF3 into the nucleus, binding to the antiviral protein gene promoter containing IFN-stimulated response element (ISRE), resulting in the expression of a series of IFN-induced antiviral protein genes, producing various antiviral proteins, such as protein kinase, 2 ', 5' -oligoadenylate synthetase, Mx protein, etc. Among them, Mx protein is considered as the most specific IFN action index, which can directly inhibit virus replication. Thus, the Mx protein (containing the promoter ISRE) is critical for the biological function of type I IFN.

A promoter is a DNA sequence located upstream of the 5' end of a structural gene, which activates RNA polymerase to bind to a template DNA precisely and with transcription initiation specificity, and contains a conserved sequence required for RNA polymerase specific binding and transcription initiation. The promoter itself is not transcribed, it behaves like a "switch", determining the activity of the gene. However, the promoter itself does not directly control gene activity, but controls gene activity by binding to such a protein called a transcription factor. Transcription factors, like "flags," direct the action of RNA polymerase, which makes RNA copies of a gene. The promoters can be classified into the following three types according to their functions and transcription patterns: inducible promoters, constitutive promoters, and tissue-specific promoters. Promoters contain a core promoter region and regulatory regions. The structure of the promoter affects its affinity for RNA polymerase, and thus the level of gene expression and the efficiency of transcription. Changes (mutations) in the promoter portion of the gene result in a failure to regulate gene expression. Thus, it can be seen that the promoter is an important cis-acting element for regulating the gene of interest. The structure and the regulation mechanism of the promoter are deeply researched, which is helpful for researching the expression characteristics and functions of the gene.

The Luciferase (Luciferase, luc) reporter gene system is a reporter system for detecting the activity of firefly Luciferase (firefly Luciferase) by using luciferin (Luciferase) as a substrate, can sensitively and efficiently detect the expression of genes, and is a detection method for detecting the interaction of transcription factors and target gene promoter region DNA. Among the existing reporter gene systems, the luc detection system is faster and more sensitive than other reporter gene detection systems such as Chloramphenicol Acetyltransferase (CAT), beta-galactosidase (Lac Z) and the like. The method only needs a few minutes to detect 96 samples by adopting the luc system, is convenient for large-flux detection, has the detection capacity as low as 10-20mol, is 100 times more sensitive than CAT, has extremely high sensitivity, is safe and has no radioactivity. The luc reporter gene assay system is widely used in studies on receptor activity, intracellular signaling, mRNA processing, protein folding, protein-protein interaction, and the like.

Disclosure of Invention

The invention aims to provide a duck Mx promoter mutant and a construction method and application of a luciferase reporter gene recombinant plasmid thereof. By means of gene mutation (overlap extension PCR) technology, the ISRE1 sequence (AGTTTCGTTTCT) on duck Mx gene promoter (duMxp) on NCBI is subjected to site-directed mutagenesis (AGGGTCGGGTCT), and the Mx gene promoter containing ISRE1 mutant is successfully cloned; and the In Fusion seamless cloning method is adopted to insert the In Fusion seamless cloning method into luciferase reporter gene pGL3-Basic (Kpn/XhoI), and a luciferase reporter system (pGL3-duMxp-ISRE1-Mut) containing the Mx gene promoter sequence of the ISRE1 mutant is successfully constructed, so that a foundation is laid for further researching the expression regulation mechanism of duck Mx genes and discussing targeted gene therapy.

The specific technical scheme is as follows:

the method for constructing the duck Mx promoter mutant and the luciferase reporter gene recombinant plasmid thereof comprises the following steps:

step 1, PCR amplification and recovery of duck Mx gene

1.1 form

Adopting duck plasmid as template;

1.2 design of primers

According to a duck Mx gene reference sequence (LOC101793492) given by NCBI, designing a PCR primer according to an overlapping extension PCR primer design principle, designing two complementary primers with variant gene bases at the position of an Mx promoter ISRE1 needing mutagenesis, and respectively designing an Mx promoter sequence 5 'primer and a Mx promoter sequence 3' primer;

1.3PCR amplification System and conditions

1.3.1PCR1 amplification System and conditions

In a 50. mu.L reaction system, sterilized deionized water, duMxp-Wild-F (10. mu.M), duMxp-ISRE 1-Mut-R (10uM), duck plasmid and

PCR Supermix, High Fidelity. The PCR1 amplification reaction system is as follows:

PCR1 amplification was performed under the following conditions

1.3.2PCR2 amplification System and conditions

In a 50. mu.L reaction system, sterilized deionized water, duMxp-ISRE 1-Mut-F (10. mu.M), duMxp-Wild-R (10. mu.M), duck plasmid and

PCR Supermix, High Fidelity, PCR2 amplification reaction system was as follows:

PCR2 amplification was performed according to the following conditions:

1.4 overlapping PCR amplification results

And performing overlapping PCR amplification by using the PCR1 amplification product and the PCR2 amplification product as a common template and using the DuMxp-Wild-F and the DuMxp-Wild-R as primers, wherein the amplification system and conditions are the same as 1.3. The obtained PCR product (DuMxp-ISRE1-Mut) was subjected to agarose gel electrophoresis, and the PCR amplification product was recovered by cutting gel, followed by the following fusion reaction.

Step 2, recombining the vector and the target fragment by In Fusion

By using

Seamless Cloning and Assembly Enzyme Mix (Invitrogen cat # A14606) was subjected to pGL3-duMxp-ISRE1-construction of recombinant plasmid for Mut reporter gene. The specific reaction system and conditions are as follows:

reaction conditions are as follows: standing at room temperature for 20 min; after 2-3min on ice, competent cells DH 5. alpha. were transformed in their entirety.

Further, the complementary primers in step 1 are primer duMxp-ISRE 1-Mut-R and primer duMxp-ISRE 1-Mut-F, and the nucleotide sequences thereof are shown in SEQ ID NO: 1-SEQ ID NO: 2, the 5 'primer and the 3' primer of the Mx promoter sequence are a primer duMxp-Wild-F and a primer duMxp-Wild-R, and the nucleotide sequences are shown in SEQ ID NO: 3-SEQ ID NO: 4, respectively.

Further, a positive single colony is selected for sequencing, and the constructed pGL3-duMxp-ISRE1-Mut reporter gene recombinant plasmid has a sequencing result shown in SEQ ID NO: 5, respectively.

The method is applied to the preparation process of the reagent for detecting the biological activity and the regulation mechanism of duck type I interferon.

The method of the invention is applied to the preparation process of the reagent for detecting the biological activity of the type I IFN of the mammal.

Still further, the mammal is a cow, a pig, a human, or the like.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention clones duck Mxp promoter (DuMxp-ISRE1-Mut) containing IFN stimulating response element (ISRE1) mutant, establishes an IFN biological activity detection system based on DuMxp-ISRE1-Mut luciferase reporter gene recombinant plasmid (pGL3-DuMxp-ISRE1-Mut), and can be used for detecting the biological activity and regulation mechanism of duck I-type interferon.

(2) The sequence of ISRE1 in the Mx promoter sequence of duck (5'-AGT T TCG TT TCT-3') is consistent with that of ISRE gene sequence of Mx promoter of mammal (5 '-A/GG TT TCN (1-2) T TTCC/T-3'). Therefore, the IFN biological activity detection system established by the invention not only can be used for detecting the biological activity and the regulation and control mechanism of duck type I interferon, but also can be used for detecting the type I IFN biological activity of mammals such as cows, pigs, people and the like, has wide applicability, and is an important tool for IFN related basic research and application research.

(3) The luciferase reporter gene recombinant plasmid (pGL3-duMxp-ISRE1-Mut) constructed by the invention not only can be used for detecting the influence of different stimulating factors such as IFN and the like on Mx gene expression in immune cells, but also can be used as a platform for screening antiviral infection drugs taking Mx as a target gene, and is a potential target for researching and screening anti-infective drugs.

(4) The invention adopts an In Fusion seamless cloning method to recombine the vector and the target fragment, is not limited by the traditional cloning method, can keep excellent accuracy for various cloning applications no matter the type of the vector or the composition of the inserted fragment, and does not need the subcloning operation of the traditional connection method. Even if the size of the inserted fragment or the number of fragments is increased, the cloning success rate is still high and is faster than that of the traditional ligase method.

(5) The invention adopts for constructing recombinant plasmid

The Seamless Cloning and analysis Enzyme Mix Seamless Enzyme mixing technology can be used for creating a construct with the length of up to 13kb and can be used for high-throughput assembly, 1 to 4 random sequences can be combined into a PCR fragment in a room-temperature reaction and can be cloned to any linear vector simultaneously and directionally, and the advantages of ① rapidness, 15-minute reaction time, ② simplicity, no influence of fragment digestion sites and no need of fragment digestion, ③ high efficiency, more than 95 percent of positive rate, ④ Seamless property, no introduction of additional sequences and the like are achieved.

Drawings

FIG. 1 is a diagram showing the results of agarose gel electrophoresis of PCR products, wherein M: DL5K DNA marker (100, 250, 500, 750, 1000, 2000, 3000, 5000bp), 1: DuMxp-ISRE1-Mut CDS PCR amplification product (2039 bp);

FIG. 2 is the effect of duck RIG-I overexpression on Mx gene expression;

FIG. 3 is the effect of duck RIG-I interference on Mx gene expression.

Detailed Description

The technical solution of the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

PCR amplification and recovery of Mx gene of 1 duck

1.1 form

Duck plasmid is adopted as a template.

1.2 design of primers

According to a duck Mx gene reference sequence (LOC101793492) given by NCBI, PCR primers are designed according to an overlap extension PCR primer design principle, two complementary primers (a primer duMxp-ISRE 1-Mut-R and a primer duMxp-ISRE 1-Mut-F) with variant gene bases are designed at the position of an Mx promoter ISRE1 needing mutagenesis, and an Mx promoter sequence 5 'primer and a 3' primer (a primer duMxp-Wild-F and a primer duMxp-Wild-R) are respectively designed (Table 1).

TABLE 1 primer names and sequences

1.3PCR amplification System and conditions

1.3.1PCR1 amplification System and conditions

As shown in Table 2, in a 50. mu.L reaction system, sterilized deionized water, DuMxp-Wild-F (10. mu.M), DuMxp-ISRE 1-Mut-R (10. mu.M), duck plasmid and

PCR SuperMix，High Fidelity。

TABLE 2PCR amplification reaction System

Injecting: invitrogen, cat no: 12532-016

PCR1 amplification was performed according to the conditions shown in Table 3.

TABLE 3PCR 1 amplification reaction conditions

1.3.2PCR2 amplification System and conditions

As shown in Table 4, in a 50. mu.L reaction system, sterilized deionized water, DuMxp-ISRE 1-Mut-F (10. mu.M), DuMxp-Wild-R (10. mu.M), duck plasmid and

PCR SuperMix，High Fidelity。

TABLE 4 PCR2 amplification reaction System

Injecting: invitrogen, cat no: 12532-016

PCR2 amplification was performed according to the conditions shown in Table 5.

TABLE 5 PCR2 amplification reaction conditions

1.4 overlapping PCR amplification results

And performing overlapping PCR amplification by using the PCR1 amplification product and the PCR2 amplification product as a common template and using the DuMxp-Wild-F and the DuMxp-Wild-R as primers, wherein the amplification system and conditions are the same as 1.3. The PCR product (duMxp-ISRE1-Mut) was subjected to agarose gel electrophoresis, and the results are shown in FIG. 1.

The PCR amplification product was recovered by cutting gel, and the following fusion reaction was carried out.

2In Fusion for recombination of vector and target fragment

By using

Seamless Cloning and Assembly Enzyme Mix (Invitrogen Cat.: A14606) for pGL3-duMxp-ISRE1-Mut reporter gene recombinationAnd (5) constructing a plasmid. The specific reaction system and conditions are as follows:

TABLE 6 DuMxp-ISRE1-Mut and pGL3-Basic fusion reaction systems and conditions

3 analysis of sequencing results

And selecting positive single colonies for sequencing, wherein the constructed pGL3-duMxp-ISRE1-Mut reporter gene recombinant plasmid sequencing result (2039bp) is shown as SEQ ID NO: 5, respectively.

Example 1 Duck RIG-I Gene Regulation of Mx Gene expression in Duck Embryo Fibroblasts (DEFs) study 1 construction of Duck RIG-I overexpression vector plasmid

The duck RIG-I (dRIG-I) gene Open Reading Frame (ORF) is inserted into a pcDNA3.1+ -HA vector by adopting a Clonexpress II One Step Cloning Kit, a pcDNA3.1+ -HA-dRIG-I plasmid is constructed, and a DNA sequencing method is applied for identification.

2 transfection reaction

DEFs were cultured until the fusion degree reached 70% -90%, 2. mu.g/well duck RIG-I overexpression vector plasmid (pcDNA3.1+ -HA-dRIG-I), siRNA (sequences GCAAACCUCCACCUGUCUATT and UAGACAGGUGGAGGUUUGCTT) and blank vector were transfected into DEFs using Lipofectamine 2000(Invitrogen) reagent, respectively, and after 24h transfection, the expression level of Mx gene in DEFs by duck RIG-I gene pair was determined by stimulating DEF (I: C) (500 ng/well).

PCR amplification and recovery of Mx gene of 3 ducks

3.1 form

Duck plasmid is adopted as a template.

3.2 design of primers

According to a duck Mx gene reference sequence (LOC101793492) given by NCBI, PCR primers are designed according to an overlap extension PCR primer design principle, two complementary primers (a primer duMxp-ISRE 1-Mut-R and a primer duMxp-ISRE 1-Mut-F) with variant gene bases are designed at the position of an Mx promoter ISRE1 needing mutagenesis, and an Mx promoter sequence 5 'primer and a 3' primer (a primer duMxp-Wild-F and a primer duMxp-Wild-R) are respectively designed (Table 1).

3.3PCR amplification System and conditions

3.3.1PCR1 amplification System and conditions

As shown in Table 2, in a 50. mu.L reaction system, sterilized deionized water, DuMxp-Wild-F (10. mu.M), DuMxp-ISRE 1-Mut-R (10. mu.M), duck plasmid and

PCR SuperMix，High Fidelity。

PCR1 amplification was performed according to the conditions shown in Table 3.

3.3.2PCR2 amplification System and conditions

As shown in Table 4, in a 50. mu.L reaction system, sterilized deionized water, DuMxp-ISRE 1-Mut-F (10. mu.M), DuMxp-Wild-R (10. mu.M), duck plasmid and

PCR SuperMix，High Fidelity。

PCR2 amplification was performed according to the conditions shown in Table 5.

3.4 overlapping PCR amplification results

And performing overlapping PCR amplification by using the PCR1 amplification product and the PCR2 amplification product as a common template and using the DuMxp-Wild-F and the DuMxp-Wild-R as primers, wherein the amplification system and conditions are the same as 1.3. The PCR product (duMxp-ISRE1-Mut) was subjected to agarose gel electrophoresis, and the results are shown in FIG. 1.

The PCR amplification product was recovered by cutting gel, and the following fusion reaction was carried out.

4In Fusion for recombination of vector and target fragment

By using

A recombinant plasmid pGL 3-dummxp-ISRE 1-Mut reporter gene was constructed by Seamless Cloning and Assembly Enzyme Mix (Invitrogen cat # A14606). Specific reaction systems and conditions are shown in table 6.

5 analysis of sequencing results

And selecting positive single colonies for sequencing, wherein the constructed pGL3-duMxp-ISRE1-Mut reporter gene recombinant plasmid sequencing result (2039bp) is shown as SEQ ID NO: 5, respectively.

6qRT-PCR (quantitative reverse transcription-polymerase chain reaction) for detecting Mx gene expression level of duck in DEFs (defecas)

Primers (qMx-F, AAGACGGTGGGAAGGAG; qMx-R, AGTTGTCGCCGAAGTCA) were designed based on the sequence of SEQ ID NO. 5, and 20. mu.l PCR amplification reaction system was used, including SYBR Green Master Mix 10. mu.l, qMx-F0.4. mu.l, qMx-R0.4. mu.l, cDNA template 2. mu.l, and sterilized deionized water 7.2. mu.l, PCR2 amplification was performed according to the following conditions, including pre-denaturation (95 ℃, 30sec), 40 cycles (95 ℃, 10 sec; 60 ℃, 30sec), duck β -actin as internal reference, relative expression level was 2^-ΔΔCtThe method calculates, and the result is shown in fig. 2 and fig. 3.

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.

Sequence listing

<110> Zhejiang province academy of agricultural sciences

<120> duck Mx promoter mutant and construction method and application of luciferase reporter gene recombinant plasmid thereof

<160>5

<170>SIPOSequenceListing 1.0

<210>1

<211>37

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

ttctctatcg ataggtacca cctcagcagg gaagcac 37

<210>2

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

attgctgagg ccagacccga ccctccat 28

<210>3

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

atggagggtc gggtctggcc tcagcaat 28

<210>4

<211>45

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

cttagatcgc agatctcgag agacacagta actgttctca attgc 45

<210>5

<211>2039

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

ttctctatcg ataggtacca cctcagcagg gaagcaccac ctccctcagc tgccagggct 60

ggttctacct gcattaggga tgcagtgaaa ttgaggaaat ggaggctccc tctctgctca 120

gcagagggta ggagaaacct ggagaagcag agatgatgtg cttcaccctc tagtctcttc 180

tgggcattgc agcatgatga agtggagcca tctcctctct caaaacaagg ttacagaagt 240

gtctgccctt tctcttccag gacatgtgcc ttgaccagca gctctaccct ctgttaacag 300

cctcctgcct ggatgggacc taagtccccc atggtgggca tcacagcaag atgatttgcc 360

agcaaatggt tcatatttag caggtacagt gacatagctg ttctcttcac ttgcttttcc 420

tacagctgct gctcacaggc ttaaatattg tgtgattcaa caaggatcaa tgcatactct 480

tacagtatgt cccatttttt tcttcatcaa aattatttcc aaacatactc tgtacagctg 540

agatttgaat gaaattcagt ttaatctatt tttatacatc ctttattgtg ttgtcttaat 600

ccgtagaatt gtactgccag taggacatgc tggtataagt cattacataa agatgaaacc 660

tgttttcaat gcattatttc ccagtgggtt ttccagtgtg ttaaagatgt tattatgctg 720

attcaggaga aaataggaga acctaaatct ctacaatggg agaaaggaga accctgtcct 780

gttcctgcag agcaagctgc agaatccttc tggtgctgct cagagctcct ctctgaaaca 840

tgcagaggat tgaacaaaac cctttaacca agacaacccc atcggcaatg ctcaaaatta 900

ataacccagt tggtgtatgt gggaacttat tcccttgcag gtctgtctcc tacgtgcctg 960

agggtatgat gaccagtgcc agagggaggc caggacctgc tgcagcccta atagtggtga 1020

cccgggggtg gcatcgggtg ggacagggag ggaggaagat gggctggctc tgaaactgca 1080

acatgccgtt tctggtcagt ctaatctgca ggggaaagtt gggcaatttg agaatcactc 1140

aaaaaaaaaa aaaaatggaa tcgagagtgg ctgataaagg cagcagtcag aaacagagga 1200

actgctaaat aacagagaaa gctgattttg cctaacagct cagaacgcat aaaacaccaa 1260

aaatgggagc aggagagtga cgacagccaa tgactctgag aaacagtgcg gaaacactgg 1320

agacttcacc tgccctgcac tggtgcgcag ccctgagaag tgcctggcag cagcgcaggg 1380

cagcaatggc tcagccctgc cagtatggcc aggttggagg ggcaggcatg gagatgccaa 1440

aagcacacac acacacacac acacaagcct cctgccactt gtacaaattg gggtgctggg 1500

cagcagcaag acatcaattt ctttgatttt ttccctatcc tggtaaacaa tatattcctc 1560

cccgatcctt ttctttacac actagccaac tctcttcctg atatttagca gacagcagaa 1620

acaaaactcc ctgttttccg ctgcagtggg accagagggc aaatggcaga ggtgccaggc 1680

aatattgttg gtgagcgaca gaggtgcaga gtggccactt tggtgagcca gcctattagg 1740

gggagataag gagctgttgc ttgccagcac cccattttgc acattttagg tcctgctgca 1800

gggggaaatc acaggatgag gatggagggt cgggtctggc ctcagcaatg caccaggaag 1860

aaaggtggtt ctggtataaa aaggaaaaag acatcacgtt cctatttcca aagctgttaa 1920

ttgggagcag tttcatttct cccaattctt gtcacagtgt gtaggtggag cctgtacata 1980

aaaaagcacc caaagcaatt gagaacagtt actgtgtctc tcgagatctg cgatctaag 2039

Claims (6)

1. The method for constructing the duck Mx promoter mutant and the luciferase reporter gene recombinant plasmid thereof is characterized by comprising the following steps of:

step 1, PCR amplification and recovery of duck TLR7 gene

1.1 form

Adopting duck plasmid as template;

1.2 design of primers

According to a duck Mx gene reference sequence LOC101793492 given by NCBI, designing a PCR primer according to an overlapping extension PCR primer design principle, designing two complementary primers with variant gene bases at the position of an Mx promoter ISRE1 needing mutagenesis, and respectively designing an Mx promoter sequence 5 'primer and a Mx promoter sequence 3' primer;

1.3PCR amplification System and conditions

1.3.1PCR1 amplification System and conditions

In a 50 mu L reaction system, respectively adding sterilized deionized water, 10 mu M duMxp-Wild-F, 10 mu M duMxp-ISRE 1-Mut-R, duck plasmid and

PCR Supermix, High Fidelity; the 50. mu.l PCR amplification reaction was used as follows: sterilized deionized water 1.0. mu.l, duMxp-Wild-F1.0. mu.l with a concentration of 10. mu.M, duMxp-ISRE 1-Mut-R1.0. mu.l with a concentration of 10. mu.M, duck plasmid 2.0. mu.l,

PCR SuperMix，HighFidelity*45μl；

PCR1 amplification was performed as follows: pre-denaturation: 94 ℃, 2min, 32 cycles: 94 ℃, 30 sec; 60 ℃ for 30 sec; at 68 ℃ for 2 min;

1.3.2PCR2 amplification System and conditions

In a 50 mu L reaction system, respectively adding sterilized deionized water, 10 mu M duMxp-ISRE 1-Mut-F, 10 mu M duMxp-Wild-R, duck plasmid and

PCR Supermix, High Fidelity, using a 50. mu.l PCR2 amplification reaction system was as follows: sterilized deionized water 1.0. mu.l, duMxp-ISRE 1-Mut-F1.0. mu.l at a concentration of 10. mu.M, duMxp-Wild-R1.0. mu.l at a concentration of 10. mu.M, duck plasmid 2.0. mu.l,

PCR SuperMix，HighFidelity*45μl；

PCR2 amplification was performed according to the following conditions: pre-denaturation: 94 ℃, 2min, 32 cycles: 94 ℃, 30 sec; 60 ℃ for 30 sec; 68 ℃ for 30 sec;

1.4 overlapping PCR amplification results

Performing overlapping PCR amplification by using PCR1 amplification products and PCR2 amplification products as common templates and using DuMxp-Wild-F and DuMxp-Wild-R as primers; carrying out agarose gel electrophoresis on the obtained PCR product duMxp-ISRE1-Mut, cutting and recycling PCR amplification products, and carrying out the following fusion reaction;

step 2, recombining the vector and the target fragment by In Fusion

By using

Constructing pGL 3-dummxp-ISRE 1-Mut reporter gene recombinant plasmid by using Seamless Cloning and Assembly Enzyme Mix; the specific reaction system and conditions are as follows: sterilized deionized water 2.0. mu.l, purified product of DuMxp-ISRE1-Mut 2.0. mu.l, pGL3-Basic 1.0. mu.l,

2X Enzyme Mix 5.0. mu.l, total 10. mu.l;

reaction conditions are as follows: standing at room temperature for 20 min; after 2-3min on ice, competent cells DH 5. alpha. were transformed in their entirety.

2. The method for constructing the duck Mx promoter mutant and the luciferase reporter gene recombinant plasmid thereof according to claim 1, wherein the complementary primers in the step 1 are primer duMxp-ISRE 1-Mut-R and primer duMxp-ISRE 1-Mut-F, and the nucleotide sequence of the complementary primers is shown in SEQ ID NO: 1-SEQ ID NO: 2, the 5 'primer and the 3' primer of the Mx promoter sequence are a primer duMxp-Wild-F and a primer duMxp-Wild-R, and the nucleotide sequences are shown in SEQ ID NO: 3-SEQ ID NO: 4, respectively.

3. The method for constructing the duck Mx promoter mutant and the luciferase reporter gene recombinant plasmid thereof according to claim 1, wherein a positive single colony is selected for sequencing, and the sequencing result of the constructed pGL3-duMxp-ISRE1-Mut reporter gene recombinant plasmid is shown as SEQ ID NO: 5, respectively.

4. Use of the method of claim 1 in the preparation of reagents for detecting the biological activity and regulatory mechanisms of duck type i interferon.

5. Use of the method of claim 1 in the preparation of a reagent for the detection of the biological activity of type I IFN in a mammal.

6. The use of claim 5, wherein the mammal is a bovine, porcine, human.

Images