CN110872597A - Construction and identification method of duck TLR7 eukaryotic expression recombinant plasmid vector - Google Patents
Construction and identification method of duck TLR7 eukaryotic expression recombinant plasmid vector Download PDFInfo
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Abstract
The invention discloses a construction and identification method of duck TLR7 eukaryotic expression recombinant plasmid vector, which comprises the following steps of 1, PCR amplification and recovery of duck TLR7 gene, 2, recombination of vector and target fragment by In Fusion, 3, colony PCR detection, adding Kodak sequence and BamHI enzyme cutting site before PCR upstream primer start codon ATG, digesting the obtained PCR product with restriction enzyme to generate viscous end, and recombining the viscous end with vector DNA with complementary viscous end, wherein the cloning method has high efficiency, and can effectively identify the PCR product through sequencing.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a construction and identification method of a duck TLR7 eukaryotic expression recombinant plasmid vector.
Background
Pattern Recognition Receptors (PRRs) are important components of the body's natural immune system, mainly comprise Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), AIM 2-like receptors (ALRs), C-type lectin-like receptors (CLRs) and the like, can recognize pathogen-related molecular patterns (PAMPs), such as microbial flagella, peptidoglycan, lipopolysaccharide, CpG motifs, single-stranded and double-stranded RNAs and the like, and release cytokines (such as IFN- β and the like) with anti-pathogenic microorganism effects, thereby inducing the body to generate anti-pathogenic microorganism natural immune responses and playing an important role in controlling pathogenic microorganism infection and epidemic disease occurrence.
TLRs are an important class of PRRs in the innate immune system discovered and of much interest in recent years, and at least 16 of them have been discovered so far in humans and animals, 10 of which are humans, 12 of mice and 10 of which receptors are all structurally typical type I transmembrane proteins, each of which has similar structural features, including LRRs (leucine-rich repeat motifs) that recognize PAMPs extracellularly, a single transmembrane region and 3 domains of TIR (Toll/IL-1 receptor homology region) associated intracellularly with linker molecules (e.g., MyD88, TIRAP, etc.), and have functions of recognizing viral particles or nucleic acids such as DNA, dsRNA, and ssRNA, inducing cytokine production such as pro-inflammatory factors and type I interferon (IFN-I) and Dendritic Cell (DC) maturation, initiating an adaptive immune response. At present, TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9 which have virus recognition functions are proved in human beings, but PAMPs recognized by different TLRs are different, such as TLR3 recognizes double-stranded RNA virus and double-stranded RNA intermediates generated in the virus replication process, TLR7 and TLR8 recognize single-stranded RNA virus rich in G/U, and TLR9 recognizes unmethylated CpG DNA sequences of DNA virus, and the TLRs have important functions in antiviral natural immune response.
TLR7 is one of the important members of the family of TLRs in mammals, TLR7 activation can produce multiple inflammatory factors [ including up-regulation of IFN- α, IFN- β, interleukin-6 (IL-6), IL-12, tumor necrosis factor- α (TNF- α) ], co-stimulatory molecules (CD40, CD80, CD86), Major Histocompatibility Complex (MHC) IFN- α. however, current studies on TLR7 have begun with limited results demonstrating that chickens have the complete TLR7 gene, coding proteins containing 1047 amino acids and 1 leucine-rich repeat conserved pattern with amino acid sequence homology of only 62% with human TLR7, and ligand recognition pattern of chicken TLR7 is much different from that of human and mouse TLR 7. bioinformatics analysis results show that the gene structure of chicken TLR7 is different from that of human and mouse, exon ii of gene transcription is significantly longer than that of human and mouse, and there is a difference in the gene transcription of chicken TLR 4642, but the gene transcription of chicken TLR7 is a significantly different from that the gene transcription of chicken TLR 4629, which is a similar to date, and the two major TLR 4684 gene transcription of chicken has been reported that the structural difference of chicken TLR 4685, and the two kinds of chicken TLR gene that the gene is similar to cause the structural change of chicken TLR 7.
Since the discovery of TLRs, the research on TLRs immunomodulators aims at regulating the natural immunity and adaptive immunity of organisms and preventing and treating human diseases causes huge adverse reaction and extensive research in the medical field, and lays a foundation for researching and developing novel immunopotentiators and targeted therapeutic drugs by taking TLRs as targets. At present, the safety and efficacy of various TLRs agonists as immunopotentiators and therapeutic agents for human vaccines have been validated and widely used throughout the world. However, the research on the animal TLRs immunomodulator is relatively late, and although researches show that some livestock TLRs ligands have potential immunoregulation effect, can induce DC to be activated to generate a large amount of IL-12 and I-type interferon, and play an important role in natural immune response of organisms, the detailed mechanism of the effect is not clear, and the application in livestock production is rarely reported.
Disclosure of Invention
The invention aims to provide a method for constructing and identifying a duck TLR7 (duckltlr 7) eukaryotic expression recombinant plasmid vector DuckTLR7-pcDNA3.1-HA, which aims to use ① as a delivery vehicle to transfer a target gene TLR7 into a host cell, use ② to carry out mass replication (called cloning) on the target gene TLR7 in the host cell, and lay a foundation for deeply researching the immune regulation function and molecular mechanism of the duck TLR7 and developing a novel immunopotentiator and a targeted therapeutic drug which take the TLR7 as a target, and the specific technical scheme is as follows:
a method for constructing and identifying a duck TLR7 eukaryotic expression recombinant plasmid vector comprises the following steps:
1.1 form
Duck spleen cDNA is adopted as a template.
1.2 design of primers
According to a duck TLR7 reference sequence (XM _005029178.3) given by NCBI, PCR primers are designed according to a primer design principle, a Kodak sequence and a BamHI enzyme cutting site are added before an upstream primer initiation codon ATG, and the primer sequences are shown as SEQ: ID: NO: 1-SEQ: ID: NO: 2 is shown in the specification;
1.3PCR amplification System and conditions
To a 50. mu.L reaction system were added sterilized deionized water 1.0. mu.L, zTLR7-F (10uM) 1.0. mu.L, zTLR7-R (10uM) 1.0. mu.L, respectively,Duck spleen cDNA2.0 μ l and
PCR SuperMix,High Fidelity 45μl。
PCR amplification was performed according to the following conditions: pre-denaturation at 94 ℃ for 2min, and PCR amplification reaction for 32 cycles (94 ℃,30 sec; 62 ℃,30 sec; 68 ℃,3.5 min).
1.4P PCR amplification product is cut and recovered, and the following fusion reaction is performed.
By using
Constructing a recombinant plasmid by using a Seamless Cloning and Assembly Enzyme Mix (Invitrogen Cat: A14606);
Selecting colonies, and carrying out shake culture on an LB liquid culture medium for 3-5 h; PCR amplification was then performed using the specific primers described in step 1. The PCR amplification reaction system is that 1.0 mu L of sterilized deionized water, 1.0 mu L of zTLR7-F (10uM), 1.0 mu L of zTLR7-R10uM, 2.0 mu L of duck spleen cDNA2 and 50 mu L of reaction system are respectively added
PCR SuperMix,HighFidelity 45μl。
The obtained PCR product is subjected to agarose gel electrophoresis, and positive bacteria liquid is selected and sent to the company of Biotechnology engineering (Shanghai) Ltd for sequencing analysis.
Further, in step 2, the specific reaction system is that in a 10. mu.L system, 2.0ul of sterilized deionized water, 2.0ul of purified product of duckTLR7, 2.0ul of purified product of pcDNA3.1-HA (BamHI/EcoRI) (50ng)1.0ul and 10 uL of purified product of duckTLR7 are respectively added
The 2X enzymeMix 5.0 ul. reaction conditions were 20min at room temperature, 2-3min on ice, and then all competent cells DH5 α were transformed.
Further, in step 3, the reaction conditions are 94 ℃ pre-denaturation for 2min, PCR amplification reaction is carried out for 32 cycles, 94 ℃ and 30 sec; 62 ℃ for 30 sec; 68 ℃ for 3.5 min.
Compared with the prior art, the invention has the beneficial effects that:
(1) in the invention, a Kodak sequence and a BamHI restriction site are added before an initiation codon ATG of a PCR upstream primer, so that the obtained PCR product is digested by restriction enzyme to generate a sticky end, and can be recombined with vector DNA with a complementary sticky end.
(2) The pcDNA3.1-HA vector selected by the invention comprises the following components: human Cytomegalovirus (CMV) promoter which can be expressed at high levels in various mammalian cells, forward (+) and reverse (-) multiple cloning sites which can promote cloning, neomycin resistance genes which can be used for selecting stable cell lines, and exosome replication of cell lines latently infected with SV40 or expressing SV40 large T antigens (such as COS-1 and COS-7), thereby enabling high-level stable non-replicative transient expression in most animal cells.
(3) 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.
(4) 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, the advantages of ① rapidness, 15-minute reaction time, ② simplicity, no influence of fragment Enzyme cutting sites and no need of fragment Enzyme cutting, ③ high efficiency, the positive rate of more than 95 percent, ④ Seamless property, no introduction of additional Enzyme and no need of Enzyme cuttingAnd the like.
Drawings
FIG. 1 shows the PCR products subjected to agarose gel electrophoresis, wherein M: DL5K DNA marker (100, 250, 500, 750, 1000, 2000, 3000, 5000bp), 1: zTLR7 CDS PCR amplified product (3270 bp);
FIG. 2 shows the plasmid PCR product subjected to agarose gel electrophoresis, wherein M: DL5K DNA marker (100, 250, 500, 750, 1000, 2000, 3000, 5000bp), duckttlr 71-4: colony PCR (about 3270 bp);
FIG. 3 is a graph of the original sequencing peaks of the duckTLR7-pcDNA3.1-HA recombinant vector fragment;
FIG. 4 is a comparison of DuckTLR7-pcDNA3.1-HA recombinant vector and NCBI provides duck TLR7 sequences.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the accompanying drawings and examples.
PCR amplification and recovery of TLR7 gene of 1 duck
1.1 form
Duck spleen cDNA is adopted as a template.
1.2 design of primers
PCR primers were designed according to the primer design principle based on the reference sequence for duck TLR7 (XM _005029178.3) given by NCBI, and a Kodak sequence and a BamHI cleavage site were added before the start codon ATG of the upstream primer (Table 1).
TABLE 1 primer names and sequences
1.3PCR amplification System and conditions
As shown in Table 2, in a 50. mu.L reaction system, sterilized deionized water, zTLR7-F (10uM), zTLR7-R (10uM), duck spleen cDNA and
PCR SuperMix,High Fidelity。
TABLE 2 PCR amplification reaction System
Injecting: invitrogen, cat no: 12532-016
PCR amplification was performed under the conditions shown in Table 3.
TABLE 3PCR amplification reaction conditions
1.4 PCR amplification results
The PCR product 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.
2 In Fusion for recombination of vector and target fragment
By using
The recombinant plasmid was constructed by using the Seamless Cloning and Assembly Enzyme Mix (Invitrogen cat # A14606), and the reaction system and conditions are shown in Table 4.
TABLE 4 Duck TLR7 and pcDNA3.1 fusion reaction systems and conditions
3 colony PCR detection
Selecting colonies, and carrying out shake culture on an LB liquid culture medium for 3-5 h; then PCR amplification was performed using specific primers (same as step 1). The PCR amplification reaction system and reaction conditions are shown in tables 5 and 6, respectively.
TABLE 5 PCR amplification reaction System
TABLE 6 PCR amplification reaction conditions
The PCR product was subjected to agarose gel electrophoresis, and the results are shown in FIG. 2.
The No. 1 positive bacterial liquid is selected and sent to the company Limited of biological engineering (Shanghai) for sequencing analysis, and the original sequencing peak image of partial fragments is shown in figure 3.
Through MegAlign sequence alignment, the coding region and NCBI provide some sequence differences, but the whole CDS coding of the gene is not affected, no frame shift mutation exists (figure 4), and the duckTLR7-pcDNA3.1-HA recombinant vector is successfully constructed.
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> construction and identification method of duck TLR7 eukaryotic expression recombinant plasmid vector
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<213> Artificial Sequence (Artificial Sequence)
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ttggtaccga gctcggatcc gccaccatgt catgctacac tctcctctc 49
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ctggaacatc gtatgggtaa acagtttcct ggagaaggtt g 41
Claims (3)
1. A method for constructing and identifying a duck TLR7 eukaryotic expression recombinant plasmid vector is characterized by comprising the following steps
Step 1, PCR amplification and recovery of duck TLR7 gene
1.2 form
Adopting duck spleen cDNA as a template;
1.2 design of primers
According to a duck TLR7 reference sequence XM _005029178.3 given by NCBI, a PCR primer is designed according to a primer design principle, a Kodak sequence and a BamHI enzyme cutting site are added in front of an upstream primer initiation codon ATG, and the primer sequence is shown as SEQ: ID: NO: 1-SEQ: ID: NO: 2 is shown in the specification;
1.3PCR amplification System and conditions
Adding sterilized deionized water 1.0 μ L, zTLR 7-F10 uM1.0 μ L, zTLR7-R10uM 1.0 μ L, duck spleen cDNA2.0 μ L and/or buffer solution into 50 μ L reaction system
PCR SuperMix,High Fidelity 45μl；
PCR amplification was performed according to the following conditions: pre-denaturation at 94 ℃ for 2min, PCR amplification reaction for 32 cycles at 94 ℃ for 30 sec; 62 ℃ for 30 sec; 68 deg.C for 3.5 min;
1.4P PCR amplification product is cut and recovered, and the following fusion reaction is carried out;
step 2, recombining the vector and the target fragment by In Fusion
By using
Seamless Cloning and Assembly Enzyme Mix, Invitrogen cat No.: a14606, constructing a recombinant plasmid;
step 3, colony PCR detection
Selecting colonies, and carrying out shake culture on an LB liquid culture medium for 3-5 h; then, PCR amplification is carried out by adopting the specific primer in the step 1; the PCR amplification reaction system is that 1.0 mu L of sterilized deionized water, 1.0 mu L of zTLR 7-F10 uM1.0 mu L of zTLR7-R10uM 1.0 mu L of duck spleen cDNA2.0 mu L and 50 mu L of reaction system are respectively added
PCR SuperMix,High Fidelity45μl；
The obtained PCR product is subjected to agarose gel electrophoresis, and positive bacteria liquid is selected and sent to the company of Biotechnology engineering (Shanghai) Ltd for sequencing analysis.
2. The method for constructing and identifying the eukaryotic expression recombinant plasmid vector of the duck TLR7 according to claim 1, wherein in the step 2, the specific reaction system is that in a 10 mu L system, 2.0ul of sterilized deionized water, 2.0ul of purified product of duckTLR7, pcDNA3.1-HA 50ng 1.0ul and 50 ul of sterilized deionized water are respectively added
2X Enzyme Mix 5.0ul, reaction conditions room temperature 20min, ice placed for 2-3min, then all transformed competent cell DH5 α.
3. The method for constructing and identifying the eukaryotic expression recombinant plasmid vector of the duck TLR7 according to claim 1, wherein in the step 3, the reaction conditions are 94 ℃ for 2min of pre-denaturation, 32 cycles of PCR amplification reaction are carried out, and the reaction temperature is 94 ℃ for 30 sec; 62 ℃ for 30 sec; 68 ℃ for 3.5 min.
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