WO2019128836A1 - 一种改进的启动子及其应用 - Google Patents

一种改进的启动子及其应用 Download PDF

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WO2019128836A1
WO2019128836A1 PCT/CN2018/122309 CN2018122309W WO2019128836A1 WO 2019128836 A1 WO2019128836 A1 WO 2019128836A1 CN 2018122309 W CN2018122309 W CN 2018122309W WO 2019128836 A1 WO2019128836 A1 WO 2019128836A1
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vector
seq
region
promoter
lacz
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PCT/CN2018/122309
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French (fr)
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薛高旭
齐甜铭
冯爱华
谢正立
贾延凯
吴昕
孙中平
廖国娟
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苏州金唯智生物科技有限公司
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Priority to EP18893487.1A priority Critical patent/EP3733849A4/en
Publication of WO2019128836A1 publication Critical patent/WO2019128836A1/zh
Priority to US16/914,266 priority patent/US20210032636A1/en

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/55—Vector systems having a special element relevant for transcription from bacteria

Definitions

  • the present application belongs to the field of genetic engineering, and relates to an improved promoter and application thereof, in particular to an improved promoter, a cloning vector carrying the improved promoter, a host cell carrying the cloning vector and application thereof .
  • PCR technology is a major breakthrough in the field of molecular biology and genetic engineering.
  • a PCR product usually a plasmid
  • Common and relatively simple cloning methods include TA cloning and blunt end ligation.
  • the PCR product amplified by the Taq enzyme contains a dAMP tail and can be linked to a T-terminal-containing vector (T-vector) under the action of a T4 ligase, which is a TA clone.
  • High-fidelity DNA polymerase usually contains 3'-5' exonuclease activity, and the PCR products amplified by them are blunt ends, and these fragments are ligated with the blunt-ended vector under the action of T4 ligase. End connection.
  • the common feature of these two methods is that it is not necessary to treat the PCR product with a special enzyme in advance, but directly connected to the carrier, which has the characteristics of simple and easy operation.
  • the vector based on the principle of blue-white spot screening has the following problems in cloning: (1) due to the use of a strong promoter, it is possible to activate transcription of a foreign gene in a large amount, Translation, resulting in some structurally complex foreign gene transcription or translation products are toxic to the host and unable to clone; (2) Excision of the exonuclease activity of the restriction enzyme residues and repeated freezing of the restriction enzyme vector The long-term preservation of the vector and the restriction enzyme linearization vector made the prepared vector lack 1-2 bases at the restriction site, and the frameshift mutation of the LacZ ⁇ gene was introduced, so that the clone containing no foreign gene was frameshifted by the LacZ ⁇ gene.
  • any DNA sequence that is capable of independently binding to a transcription factor and initiating transcription can be referred to as a promoter.
  • the regions that can be recognized by the sigma factor have very conserved sequence characteristics.
  • two sequences at about 10 nt and 35 nt upstream of the transcription start site (+1) (called the -10 region and the -35 region) have a decisive role in the recognition of the sigma factor, so these two sequences are also It is called a narrow promoter or a core promoter.
  • the upstream sequence of the -35 region may also have an effect on the intensity of transcription, which is called UPelement.
  • the technical problem to be solved by the present application is to overcome the inability of the cloning vector prepared in the prior art to clone or to generate a large number of false positive or false negative clones, thereby providing an improved promoter and its use.
  • the application provides an improved promoter obtained by mutating a nucleic acid sequence between the -35 region to the -10 region in a promoter region to an endonuclease recognition site.
  • the change in the number of nucleic acids between the -35 region and the -10 region in the prokaryote affects the transcriptional activity of the gene, and the nucleic acid sequence between the -35 region and the -10 region of the promoter region is mutated, thereby enabling It is recognized by the endonuclease, and when the clone is cloned, the vector is first prepared as a linearized vector, and the foreign gene is ligated to the linearized vector, so that the activity of the gene regulated by the promoter is decreased, the expression amount is decreased, and the function is further exerted.
  • the endonuclease recognition site refers to a site which can be recognized by any endonuclease, which does not limit the endonuclease, and the selection of the endonuclease is mainly based on the experimental operation by those skilled in the art. Convenience, mutations of one or a few bases can be successfully mutated.
  • the improved promoter is obtained by mutating a nucleic acid sequence between the -35 region to the -10 region in the promoter region of ⁇ -galactosidase to an endonuclease recognition site.
  • the nucleic acid sequence between the -35 region and the -10 region of the strong promoter region is mutated to a recognition endonuclease recognition site, but it is After being cut into a linearized vector and inserted into the exogenous fragment, the ⁇ -galactosidase strong promoter is significantly reduced in activity due to the insertion of the foreign DNA fragment, and the expression level of the lacZ ⁇ gene is significantly decreased, thereby allowing the recombinant plasmid to be contained.
  • the colony is white, so that it can overcome the problem that the transcription of the foreign gene or the translation product may be toxic to the host due to the presence of a strong promoter in the blue-white-screened vector, and the vector may be prevented from being deleted at the restriction site.
  • 1-2 bp leads to a defect in the lacZ ⁇ gene frameshift mutation producing a false positive clone, which can eliminate the false negative phenomenon of the blue spot due to the small foreign DNA fragment and the insertion of the foreign DNA does not change the reading frame of the lacZ ⁇ gene. .
  • nucleic acid sequence between the -35 region and the -10 region in the promoter region of the ⁇ -galactosidase is represented by SEQ ID NO. 1-2, and the SEQ ID NO. 1-2
  • the nucleic acid sequences shown are as follows:
  • SEQ ID NO. 1 5'-TTTACACTTTATGCTTCCGGCTCGTATGTT-3';
  • SEQ ID NO. 2 5'-CTTTATGCTTCCGGCTCG-3';
  • RNA polymerase II is usually in the -35 region to the -10 region, both of which are important: RNA polymerase and bases in the -35 and -10 sequences and in the DNA backbone
  • the phosphate group is in contact; the activity of the promoter leaving the common sequence is also weak; and the Applicant has found that by mutating the sequence in the -35 region to the -10 region, especially the sequence shown in SEQ ID NO. Insertion of the foreign gene resulted in a significant decrease in the expression level of the lacZ ⁇ gene.
  • the endonuclease can be selected as needed, and different endonuclease recognition sites can be selected according to the sequence of the mutated promoter region.
  • the present application is selected from, but not limited to, Any one or a combination of at least two of EcoRV, AleI, BamHI, XhoI or PmlI.
  • nucleic acid sequence between the -35 region and the -10 region of the improved promoter is shown in SEQ ID NO. 3-14, and the nucleic acid sequences shown in SEQ ID NO. 3-14 are as follows:
  • SEQ ID NO. 3 5'-GATATCGCTTCCGGCTCG-3';
  • SEQ ID NO. 4 5'-CTTGATATCTCCGGCTCG'-3';
  • SEQ ID NO. 5 5'-CTTTATGATATCGGCTCG-3';
  • SEQ ID NO. 6 5'-CTTTATGCTGATATCTCG-3';
  • SEQ ID NO. 7 5'-CTTTATGCTTCCGATATC-3';
  • SEQ ID NO. 8 5'-CTTTCACCTTCGTGCTCG-3';
  • SEQ ID NO. 9 5'-CTCGAGGATATCGGATCC-3';
  • SEQ ID NO. 10 5'-CACGTGGCTTCCGGCTCG-3';
  • SEQ ID NO. 11 5'-CTTCACGTGTCCGGCTCG-3';
  • SEQ ID NO. 12 5'-CTTTATCACGTGGGCTCG-3';
  • SEQ ID NO. 13 5'-CTTTATGCTCACGTGTCG-3';
  • SEQ ID NO. 14 5'-CTTTATGCTTCCCACGTG-3'.
  • the application provides a vector comprising the improved promoter of the first aspect.
  • the vector further comprises a gene of interest operably linked between the endonuclease recognition sites of the improved promoter;
  • the vector can be selected as needed by a person skilled in the art, and the selection of the vector does not affect the function of the promoter, and the vector can be a cloning vector and/or an expression vector, and the cloning vector is used for cloning.
  • the application provides a host cell, the host cell comprising the vector of the second aspect.
  • the host cell is E. coli, which encodes only the C-terminal omega fragment of ⁇ -galactosidase.
  • the lacZ ⁇ gene of the cloning vector encodes a ⁇ -galactosidase (lacZ) N-terminal ⁇ fragment
  • the Escherichia coli only encodes a ⁇ -galactosidase C-terminal ⁇ fragment
  • the host and plasmid-encoded fragments are None of the galactosidase activities, but when they are present, the ⁇ fragment and the ⁇ fragment can form the colorless compound X-gal (5-bromo-4-) by ⁇ -complementing to form an enzymatically active ⁇ -galactosidase.
  • Chloro-3-indole- ⁇ -D-galactoside is cleaved into galactose and dark blue material 5-bromo-4-indigo, and 5-bromo-4-indigo can make the entire colony blue.
  • the expression level of lacZ ⁇ is significantly decreased, and a large amount of ⁇ -galactosidase having enzymatic activity cannot be efficiently formed by ⁇ -complementation. It eventually causes the colonies to appear white.
  • the present application provides a method for preparing a carrier according to the second aspect, comprising the steps of:
  • step (2) cyclizing the product of step (1) by Gibson recombination to obtain a vector with a promoter
  • the target gene is ligated to the linearized vector of the step (3) to obtain the vector.
  • nucleic acid sequence of the primer of step (1) is shown in SEQ ID NO. 15-38.
  • nucleic acid sequence shown in SEQ ID NO. 2 is mutated to SEQ ID NO. a nucleic acid sequence as shown in 4;
  • nucleic acid sequence shown in SEQ ID NO. 2 is mutated to SEQ ID NO. a nucleic acid sequence as shown in 5;
  • nucleic acid sequence shown in SEQ ID NO. 2 is mutated to SEQ ID NO. a nucleic acid sequence as shown in 6;
  • nucleic acid sequence shown in SEQ ID NO. 2 is mutated to SEQ ID NO. a nucleic acid sequence as shown in 7;
  • nucleic acid sequence shown in SEQ ID NO. 2 is mutated to SEQ ID NO. a nucleic acid sequence as shown in 8;
  • nucleic acid sequence shown in SEQ ID NO. 2 is mutated to SEQ ID NO. a nucleic acid sequence as shown in 9;
  • nucleic acid sequence shown by SEQ ID NO. 2 is mutated to the SEQ ID NO. Nucleic acid sequence;
  • nucleic acid sequence shown by SEQ ID NO. 2 is mutated to the SEQ ID NO. Nucleic acid sequence;
  • nucleic acid sequence shown by SEQ ID NO. 2 is mutated to the SEQ ID NO. Nucleic acid sequence;
  • nucleic acid sequence shown by SEQ ID NO. 2 is mutated to the SEQ ID NO. Nucleic acid sequence;
  • nucleic acid sequence shown in SEQ ID NO. 2 is mutated to the SEQ ID NO. Nucleic acid sequence.
  • step (3) the linearization described in step (3) is obtained by endonuclease digestion and/or PCR amplification.
  • step (1) also includes codon optimization of the gene that regulates expression.
  • the gene that regulates expression is the lacZ gene, and the nucleic acid sequence thereof is shown in SEQ ID NO. 39, and the nucleic acid sequence shown in SEQ ID NO. 39 is as follows:
  • the lacZ gene is codon-optimized, and the codon-optimized nucleic acid sequence is shown in SEQ ID NO. 40, and the nucleic acid sequence shown in SEQ ID NO. 40 is as follows:
  • the present application provides a method for preparing a protein of interest, comprising:
  • the host cell of the third aspect is cultured under conditions suitable for expression of the protein of interest to obtain the protein of interest; wherein the vector in the host cell is an expression vector, and the protein of interest is the object Gene-encoded protein.
  • the present application provides a kit comprising the improved promoter of the first aspect, the vector of the second aspect, or the host cell of the third aspect.
  • the present application mutates the nucleic acid sequence between the -35 region and the -10 region of the promoter region, thereby being recognized by the endonuclease.
  • the vector is first prepared as a linearized vector, and then the foreign source is The gene is linked to the linearized vector, so that the activity of the promoter is significantly decreased, and the amount of the gene regulated by the gene is significantly decreased, thereby functioning;
  • the present application is capable of overcoming the problem that the transcription or translation product of a foreign gene may be toxic to the host due to the presence of a strong promoter in the blue-white-selected vector, and the vector may be prevented from being deleted at the restriction site.
  • -2bp causes a defect in the lacZ ⁇ gene frameshift mutation to produce a false positive clone, which can eliminate the fact that the foreign DNA fragment is small and the insertion of the foreign DNA does not change the reading frame of the lacZ ⁇ gene, causing the plate to be a false negative phenomenon of the blue spot;
  • the cloning vector described in the present application has a simple construction method, is easy to operate, and has high efficiency, and can complete the construction of the cloning vector in a short time.
  • Example 1 is an electrophoresis pattern of colony PCR identification of Example 2 of the present application, wherein the DNA marker size is 0.1 kb, 0.25 kb, 0.5 kb, 0.75 kb, 1 kb, 1.5 kb, 2 kb, 3 kb, 5 kb;
  • Example 2 is an electrophoresis pattern of colony PCR identification of Example 4 of the present application, wherein the DNA marker size is 0.1 kb, 0.25 kb, 0.5 kb, 0.75 kb, 1 kb, 1.5 kb, 2 kb, 3 kb, 5 kb;
  • Fig. 3 is an electrophoresis pattern of colony PCR identification of Example 5 of the present application, wherein the DNA marker size is 0.1 kb, 0.25 kb, 0.5 kb, 0.75 kb, 1 kb, 1.5 kb, 2 kb, 3 kb, 5 kb.
  • LacZ gene a gene widely used in gene expression regulation studies.
  • the encoded ⁇ -galactosidase ( ⁇ -gal) is a tetramer composed of 4 subunits that catalyze the hydrolysis of lactose.
  • Beta-gal It is relatively stable.
  • X-Gal is used as a substrate, it is blue, which is easy to detect and observe.
  • the many advantages of LacZ gene make it a commonly used marker gene in genetic engineering experiments, such as screening for transformation strains, ⁇ - Galactosidase color reaction selection method, ie, blue-white screening;
  • LacZ ⁇ gene encodes a ⁇ -galactosidase (lacZ) N-terminal ⁇ fragment, which can form a colorless compound X-gal (5-bromo-4-chloro) by ⁇ -complementation to form an enzymatically active ⁇ -galactosidase.
  • lacZ ⁇ -galactosidase
  • X-gal 5-bromo-4-chloro
  • -3- ⁇ - ⁇ -D-galactoside cleavage into galactose and dark blue substance 5-bromo-4-indigo;
  • An endonuclease an enzyme that produces an oligonucleotide by hydrolyzing a phosphodiester bond inside a molecular chain in a nuclease;
  • PCR technology polymerase chain reaction, which uses DNA to become single-stranded when denatured at 95°C in vitro, and combines with single-stranded base pairing when the temperature is low (usually around 60°C).
  • the temperature is up to the DNA polymerase optimum reaction temperature (about 72 ° C), and the DNA polymerase synthesizes a complementary strand along the direction of phosphoric acid to the five carbon sugar (5'-3').
  • the PCR-based PCR instrument is actually a temperature-controlled device that can be well controlled between denaturation temperature, refolding temperature, and extension temperature.
  • the codon-optimized lacZ ⁇ gene includes the following steps:
  • the lacZ ⁇ gene of pUC57 (SEQ ID NO. 39) was codon optimized using codon optimization software (Codon optimization software, developed by Suzhou Jinweizhi Biotechnology Co., Ltd.), and the optimized lacZ ⁇ gene was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.
  • the nucleotide sequence is shown in SEQ ID No. 39, and is specifically as follows:
  • lacZ ⁇ gene (SEQ ID NO. 39):
  • a method for constructing a high copy cloning vector comprises the following specific steps:
  • the pUC57 plasmid having kanamycin resistance was used as a template, and the PCR amplification reaction was carried out using SEQ ID NO. 41-42 as a primer.
  • the specific sequence is as follows:
  • SEQ ID NO. 41 forward primer: ATGCAGGCTCGGTTCCAGCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCC;
  • SEQ ID NO. 42 reverse primer: AGCACCATTTGCAGCGATGCCGCCTAATTAAGCCAGCCCCGACACCCGCCAACAC;
  • the PCR reaction solution obtained in the step (1) is subjected to 1% agarose gel electrophoresis, and then the gel is recovered and purified to obtain a PCR amplification product;
  • reaction conditions for the ligation are: 50 ° C for 1 h;
  • primers F1-EcoRV, R1-EcoRV, F2-EcoRV, R2-EcoRV, F3-EcoRV, R3-EcoRV, F4-EcoRV, R4-EcoRV , F5-EcoRV, R5-EcoRV, F6-AleI, R6-AleI, F7-BamHI-XhoI, R7-BamHI-XhoI (SEQ ID NO. 15-SEQ ID NO. 28) are primers for PCR amplification reaction, specifically The sequence is as follows in Table 4:
  • the PCR reaction solution obtained in the step (1) is subjected to 1% agarose gel electrophoresis, and then the gel is recovered and purified to obtain a PCR amplification product, and then Gibson is separately used.
  • the Master Mix kit performs the ligation reaction, and the ligation reaction system is shown in Table 5 below:
  • Ligation reaction conditions ligation reaction at 50 ° C for 1 hour;
  • Step II construct the correct plasmids pUC57-lacZ-Mu-1, pUC57-lacZ-Mu-2, pUC57-lacZ-Mu-3, pUC57-lacZ-Mu-4, pUC57-lacZ-Mu-5
  • Enzyme digestion with EcoRV restriction enzyme pUC57-lacZ-Mu-6 was digested with AleI restriction enzyme
  • pUC57-lacZ-Mu-7 was digested with BamHI and XhoI restriction enzymes, and the digested product was subjected to 1% agarose gel. After electrophoresis, the gel is recovered and purified;
  • SEQ ID NO. 43 TTCATACAGCAGGCTATGTTTAGG;
  • SEQ ID NO. 44 CCTAAACATAGCCTGCTGTATGAA;
  • SEQ ID NO. 45 TAAGCCGATACTGTATTTTTTATCCATAGCTGTTTCCTGTGTGAAATT;
  • SEQ ID NO. 46 AATTTCACACAGGAAACAGCTATGGATAAAAAATACAGTATCGGCTTA;
  • PCR amplification was carried out by using ⁇ DNA as a template and F- ⁇ DNA-200bp+R- ⁇ DNA-200bp as a primer, and the nucleotide sequence of the primer F- ⁇ DNA-200bp and R- ⁇ DNA-200bp was as SEQ ID NO. 47-SEQ ID NO. 48, as follows:
  • SEQ ID NO. 47 (F- ⁇ DNA-200 bp): AATGGTCAGGATCCGTTGAATGGGCGGATGCTAATTACTATCTCCCG;
  • SEQ ID NO. 48 (R- ⁇ DNA-200 bp): TGAAGACACTCTCGAGTTATGCTCTATAAAGTAGGCATAAACACCCAGC;
  • the PCR reaction system is shown in Table 1, and the PCR amplification procedure is shown in Table 6:
  • the PCR reaction solution obtained in the step (3) is subjected to 1% agarose gel electrophoresis, and then the gel is recovered and purified to obtain a PCR amplification product, and then the purified PCR amplification product is digested with BamHI and XhoI, and the enzyme is digested.
  • the reaction system is shown in Table 7 below:
  • Digestion reaction conditions digestion at 37 ° C for 1 hour, the digestion product was recovered and purified using Axygen purification kit;
  • Ligation reaction conditions ligation reaction at 22 ° C for 1 hour;
  • a method for constructing a pUC57-lacZ-Mu-2 plasmid comprising the steps of:
  • PCR was carried out using F1-del+R1-del, F2-del+R2-del, F3-del+R3-del as primers, respectively.
  • the nucleotide sequences of the primers F1-del, R1-del, F2-del, R2-del, F3-del, and R3-del are as shown in SEQ ID NO. 49-SEQ ID NO. details as follows:
  • SEQ ID NO. 49 (F1-del): ATACGAGCCGGAGAATCAAGTGTAAAGCCTGGGGTGCCTAAT;
  • SEQ ID NO. 50 (R1-del): GCTTTACACTTGATTCTCCGGCTCGTATGTTGTGTGGAATTG;
  • SEQ ID NO. 51 (F2-del): TACGAGCCGGAGATTCAAGTGTAAAGCCTGGGGTGCCTAATG;
  • SEQ ID NO. 52 (R2-del): GGCTTTACACTTGAATCTCCGGCTCGTATGTTGTGTGGAATTG;
  • SEQ ID NO. 53 (F3-del): ATACGAGCCGGAGATCAAGTGTAAAGCCTGGGGTGCCTAATG;
  • SEQ ID NO. 54 (R4-del): GGCTTTACACTTGATCTCCGGCTCGTATGTTGTGTGGAATTG;
  • the PCR reaction system is shown in Table 1 of Example 2, and the PCR amplification procedure is shown in Table 2;
  • the PCR reaction solution obtained in the step (1) is subjected to 1% agarose gel electrophoresis, and then the gel is recovered and purified to obtain a PCR amplification product, and then Gibson is separately used.
  • the Master Mix (NEB) kit performs a ligation reaction, and the ligation reaction system is shown in Table 5 of Example 2;
  • Ligation reaction conditions ligation reaction at 50 ° C for 1 hour.
  • the EcoR V cleavage site of pUC57-lacZ-Mu-2A was mutated to GATTC, ie the sequence was mutated from 5'-CTTGATATCTCCGGCTCG-3' to 5'-CTTGATTCTCCGGCTCG-3', and EcoR V of pUC57-lacZ-Mu-2B
  • the cleavage site was mutated to GAATC, ie the sequence was mutated from 5'-CTTGATATCTCCGGCTCG-3' to 5'-CTTGAATCTCCGGCTCG-3'
  • the EcoR V cleavage site of pUC57-lacZ-Mu-2C was mutated to GATC, ie the sequence was 5 '-CTTGATATCTCCGGCTCG-3' mutation to 5'-CTTGATCTCCGGCTCG-3';
  • step (3) The correct pUC57-lacZ-Mu-2A, pUC57-lacZ-Mu-2B, pUC57-lacZ-Mu-2C plasmids in step (3) were transformed into Top10F' competent cells, respectively, and finally coated with IPTG and X- The kanamycin-resistant LB plate of gal was cultured overnight at 37 ° C. The colonies of the three plates were found to be blue on the next day, and 5 monoclonal clones were picked from each plate for Sanger sequencing. The sequencing results showed that all clones were The sequence is correct.
  • the linearized vector lacking 1 base can still express lacZ ⁇ after ligation, so that the colony is blue, that is, the vector of the present application is digested by endonuclease without being cut at one end of the restriction site. Or a deletion of 1 base at both ends occurs and a false positive clone is produced.
  • a method for constructing a low copy cloning vector comprises the following specific steps:
  • SEQ ID NO. 55 forward primer: ATGCAGGCTCGGTTCCAGCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCC;
  • SEQ ID NO. 56 reverse primer: AGCACCATTTGCAGCGATGCCGCCTAATTAAGCCAGCCCCGAGTAGCTAGACAGG;
  • the PCR reaction system is shown in Table 1 of Example 2, and the reaction conditions are as shown in Table 2 of Example 2;
  • the PCR reaction solution obtained in the step (1) is subjected to 1% agarose gel electrophoresis, and then the gel is recovered and purified to obtain a PCR amplification product;
  • reaction conditions for the ligation are: 50 ° C for 1 h;
  • primers F1-EcoRV, R1-EcoRV, F2-EcoRV, R2-EcoRV, F3-EcoRV, R3-EcoRV, F4-EcoRV, R4-EcoRV , F5-EcoRV, R5-EcoRV, F6-AleI, R6-AleI, F7-BamHI-XhoI, R7-BamHI-XhoI (SEQ ID NO. 15-SEQ ID NO. 28) are primers for PCR amplification reaction, specifically The sequence is shown in Table 4 of Example 2, the specific PCR reaction system is shown in Table 1 of Example 2, and the reaction conditions are as shown in Table 2 of Example 2;
  • the PCR reaction solution obtained in the step (1) is subjected to 1% agarose gel electrophoresis, and then the gel is recovered and purified to obtain a PCR amplification product, which is then used separately.
  • the Master Mix kit performs a ligation reaction, and the ligation reaction system is shown in Table 5 of Example 2;
  • Ligation reaction conditions ligation reaction at 50 ° C for 1 hour;
  • Step II construct the correct plasmids pCK-lacZ-Mu-1, pCK-lacZ-Mu-2, pCK-lacZ-Mu-3, pCK-lacZ-Mu-4, pCK-lacZ-Mu-5
  • Enzyme digestion with EcoRV restriction enzyme pCK-lacZ-Mu-6 was digested with AleI restriction enzyme
  • pCK-lacZ-Mu-7 was digested with BamHI and XhoI restriction enzymes, and the digested product was subjected to 1% agarose gel. After electrophoresis, the gel is recovered and purified;
  • the PCR reaction system is shown in Table 1 of Example 2, and the PCR amplification procedure is shown in Table 6;
  • the PCR reaction solution obtained in the step (3) is subjected to 1% agarose gel electrophoresis, and then the gel is recovered and purified to obtain a PCR amplification product, and then the purified PCR amplification product is digested with BamHI and XhoI, and the enzyme is digested.
  • the reaction system is shown in Table 7 of Example 2;
  • Digestion reaction conditions digestion at 37 ° C for 1 hour, the digestion product was recovered and purified using Axygen purification kit;
  • step (2) The 24, 48 bp fragment formed by annealing in step (2) and the pCK-lacZ-Mu-1, pCK-lacZ-Mu-2, pCK-lacZ-Mu-3, which were digested with the step (1), respectively.
  • the pCK-lacZ-Mu-4, pCK-lacZ-Mu-5, pCK-lacZ-Mu-6 vector is subjected to a ligation reaction, and the purified product of the step (4) and the pCK-lacZ purified by the step (1) are purified.
  • -Mu-7 carrier is subjected to a ligation reaction, and the ligation reaction system is as shown in Table 8 of Example 2;
  • Ligation reaction conditions ligation reaction at 22 ° C for 1 hour;
  • a method for constructing a single copy cloning vector includes the following specific steps:
  • SEQ ID NO. 57 forward primer: ATGCAGGCTCGGTTCCAGCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCC;
  • SEQ ID NO. 58 (reverse primer): AGCACCATTTGCAGCGATGCCGCCTAATTAAGCCAGCCCCGACACCCGCCAACAC;
  • the PCR reaction solution obtained in the step (1) is subjected to 1% agarose gel electrophoresis, and then the gel is recovered and purified to obtain a PCR amplification product;
  • reaction conditions for the ligation are: 50 ° C for 1 h;
  • the successful pCC1-lacZ plasmid was constructed as a template in step I), and the primer pCC1-lacZ plasmid was used as a template, and primers F1-PmlI+R1-PmlI, F2-PmlI+R2-PmlI, F3-PmlI+R3- PmlI, F4-PmlI+R4-PmlI, F5-PmlI+R5-PmlI, F7-BamHI-XhoI+R7-BamHI-XhoI (SEQ ID NO.29-SEQ ID NO.38, SEQ ID NO.27-SEQ ID NO.28) PCR amplification reaction for the primers, the specific sequence is shown in Table 13:
  • Ligation reaction conditions ligation reaction at 50 ° C for 1 hour;
  • pCC1-lacZ-Mu-1 mutantation of 5'-CTTTATGCTTCCGGCTCG-3' to 5'-CACGTGGCTTCCGGCTCG-3', plasmid from F1-PmlI+R1 -PmlI primer construction
  • pCC1-lacZ-Mu-2 mutantation of 5'-CTTTATGCTTCCGGCTCG-3' to 5'-CTTCACGTGTCCGGCTCG-3', plasmid constructed from F2-PmlI+R2-PmlI primer
  • pCC1-lacZ-Mu -3 mutantation of 5'-CTTTATGCTTCCGGCTCG-3' to 5'-CTTTATCACGTGGGCTCG-3', plasmid constructed from F3-PmlI+R3-PmlI primer
  • pCC1-lacZ-Mu-4 will be 5'-CTTTATGCTTCCGGCTCG-3
  • Step II construct the correct plasmids pCC1-lacZ-Mu-1, pCC1-lacZ-Mu-2, pCC1-lacZ-Mu-3, pCC1-lacZ-Mu-4, pCC1-lacZ-Mu-5
  • the enzyme was digested with PmlI restriction enzyme
  • pCC1-lacZ-Mu-6 was digested with BamHI and XhoI restriction enzymes, and the digested product was subjected to 1% agarose gel electrophoresis, and then the gel was recovered and purified.
  • the PCR reaction system is shown in Table 1, and the PCR amplification procedure is shown in Table 6;
  • the PCR reaction solution obtained in the step (3) is subjected to 1% agarose gel electrophoresis, and then the gel is recovered and purified to obtain a PCR amplification product, and then the purified PCR amplification product is digested with BamHI and XhoI, and the enzyme is digested.
  • the reaction system is shown in Table 7 of Example 2;
  • Digestion reaction conditions digestion at 37 ° C for 1 hour, the digestion product was recovered and purified using Axygen purification kit;
  • Ligation reaction conditions ligation reaction at 22 ° C for 1 hour;
  • the present application cleaves a sequence between the -35 region and the -10 region of the strong promoter region of ⁇ -galactosidase to form a site capable of being digested by an endonuclease, when cloning
  • the linearized vector is prepared by digestion or PCR with a suitable endonuclease, and then the foreign gene is ligated to the linearized vector, so that the ⁇ -galactosidase strong promoter is inserted by the foreign DNA fragment.
  • the activity is significantly decreased, and the expression level of the lacZ ⁇ gene is significantly decreased, thereby making the colony containing the recombinant plasmid white.
  • This application overcomes the common method of starting the foreign gene by the strong promoter in the blue-white screening vector by the above method. Transcription or translation products may be toxic to the host, leading to the problem of cloning, and it is possible to avoid the defect that the vector lacks 1-2 bp at the cleavage site, resulting in a hap-positive clone of the lacZ ⁇ gene frameshift mutation, and can eliminate the foreign DNA fragment Smaller and insertion of foreign DNA did not alter the reading frame of the lacZ ⁇ gene, causing the plate to be a false negative phenomenon of blue spots.

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Abstract

一种改进的启动子及其应用，改进是将启动子区域中的-35区至-10区之间的核酸序列突变为核酸内切酶识别位点。该改进用于克服强启动子下外源基因的转录或翻译产物可能对宿主有毒性而导致无法克隆的问题，以及避免蓝白斑筛选时的假阳性和假阴性现象。

Description

一种改进的启动子及其应用 技术领域
本申请属于基因工程领域，涉及一种改进的启动子及其应用，具体涉及一种改进的启动子、带有所述改进启动子的克隆载体，带有所述克隆载体的宿主细胞及其应用。
背景技术
PCR技术的发明是分子生物学与基因工程领域的重大突破。PCR技术诞生之后，将PCR产物克隆入载体(通常为质粒)的技术也获得发展。常用且相对简单的克隆方法包括TA克隆和平末端连接。由Taq酶扩增出的PCR产物含有dAMP尾巴，在T4连接酶的作用下，可以和含有T末端的载体(T载体)连接，这就是TA克隆。而高保真DNA聚合酶通常含有3’-5’核酸外切酶活性，它们扩增出的PCR产物为平末端，将这些片段与切成平末端的载体在T4连接酶的作用下连接就是平末端连接。这两种方法的共同特点是不需要事先用特殊的酶对PCR产物进行处理，而是直接连入载体，具有简单易操作的特性。
目前商业化的T载体和可以用于平末端克隆的载体通常是基于蓝白斑筛选的原理，蓝白斑筛选是最常用的将空载体和有插入片段的载体相分开的一种筛选方案。在这种方法中，报告基因LacZα作为蓝白斑筛选的标记基因，然而基于蓝白斑筛选原理的载体在克隆时有以下问题：(1)由于使用强启动子，能够大量启动外源基因的转录、翻译，导致了某些结构复杂的外源基因转录或翻译产物对宿主有毒性而无法克隆；(2)酶切载体时由于限制性内切酶残留的外切酶活性、酶切载体的反复冻融以及酶切线性化载体的长期保存等因素使得制备的载体在酶切位点缺失1-2碱基，导LacZα基因的移码突变，使得不含外源基因的克隆由于LacZα基因的移码突变而显白色，导致产生大量假阳性克隆；(3)在克隆外源DNA小片段并且外源DNA的插入没有改变lacZα基因的读码框时会造成平板都是蓝斑的假阴性现象；(4)载体在平末端克隆大于2kb的外源DNA片段时，白斑会很少，而蓝斑却很多，而且本来就很少的白斑还可能与蓝斑长在一起，使得白斑单克隆极少，挑选足够数量的阳性克隆会很困难。此外，蓝白斑筛选还需要用到X-gal和IPTG等昂贵且有毒性的化学物质。
任何一段能够独立与转录因子结合并起始转录的DNA序列都可以被称为启动子。在启动子中，能够被σ因子识别的区域具有非常保守的序列特征。其中，在转录起始位点(+1)上游大约10nt以及35nt处的两段序列(称为-10区与-35区)对于σ因子的识别具有决定性的作用，因此这两段序列也被称作狭义启动子或核心启动子。除这段核心启动子区域之外，-35区的上游序列也可能对转录的强度产生影响，这些序列被称作UP元件(UPelement)。
发明内容
因此，本申请要解决的技术问题在于克服现有技术中制备的克隆载体无法克隆，或产生大量假阳性或假阴性克隆，从而提供一种改进的启动子及其应用。
为达此目的，本申请采用以下技术方案：
一方面，本申请提供一种改进的启动子，所述改进的启动子为将启动子区域中的-35区至-10区之间的核酸序列突变为核酸内切酶识别位点而得到。
本申请中，原核生物中-35区至-10区之间核酸数目的变动会影响基因转录活性的高低，将启动子区域的-35区至-10区之间的核酸序列进行突变，从而能够被核酸内切酶识别，克隆时，先将载体制备为线性化载体，再将外源基因与线性化载体连接，使得启动子调控表达的基因活性下降，表达量下降，进而发挥功能。
所述核酸内切酶识别位点指的是能够被任意的核酸内切酶识别的位点，其对核酸内切酶不作限制，其核酸内切酶的选择主要是基于本领域技术人员实验操作的方便性，突变1个或几个碱基就能够突变成功即可。
根据本申请，所述改进的启动子为将β-半乳糖苷酶的启动子区域中的-35区至-10区之间的核酸序列突变为核酸内切酶识别位点而得到。
本申请中，对于β-半乳糖苷酶的启动子，将其强启动子区域-35区至-10区之间的核酸序列突变为能够被识别的核酸内切酶识别位点，但其被剪切成线性化载体，插入外源片段后，使得β-半乳糖苷酶强启动子由于外源DNA片段的插入使得活性明显下降，进而使得lacZα基因的表达量显著下降，进而使得含有重组质粒的菌落显白色，这样就能够克服由于采用蓝白筛选的载体存在强启动子启动外源基因的转录或翻译产物可能对宿主有毒性而导致无法克隆的问题，可以避免载体在酶切位点缺失1-2bp导致lacZα基因移码突变产生假阳性克隆的缺陷，可以消除由于外源DNA片段较小并且外源DNA的插入没有改变lacZα基因的读码框，造成平板都是蓝斑的假阴性现象。
根据本申请，所述β-半乳糖苷酶的启动子区域中的-35区至-10区之间的核酸序列如SEQ ID NO.1-2所示，所述SEQ ID NO.1-2所示的核酸序列如下：
SEQ ID NO.1：5’-TTTACACTTTATGCTTCCGGCTCGTATGTT-3’；
SEQ ID NO.2：5’-CTTTATGCTTCCGGCTCG-3’；
本申请中，RNA聚合酶II的结合部位通常在-35区至-10区，这两个部位都很重要：RNA聚合酶能和-35和-10序列中的碱基和DNA主链中的磷酸基相接触；离开共同顺序较远的启动子的活性亦较弱；而申请人发现，通过突变-35区至-10区中的序列，尤其是SEQ ID NO.2所示的序列，能够插入外源基因，使得lacZα基因的表达量显著下降。
根据本申请，所述核酸内切酶本领域技术人员可以根据需要进行选择，可以根据突变的启动子区域的序列的不同从而选择不同的核酸内切酶识别位点，本申请选自但不限于EcoRV、AleI、BamHI、XhoI或PmlI中的任意一种或至少两种的组合。
根据本申请，所述改进的启动子的-35区至-10区之间的核酸序列如SEQ ID NO.3-14所示，所述SEQ ID NO.3-14所示的核酸序列如下：
SEQ ID NO.3：5’-GATATCGCTTCCGGCTCG-3’；
SEQ ID NO.4：5’-CTTGATATCTCCGGCTCG’-3’；
SEQ ID NO.5：5’-CTTTATGATATCGGCTCG-3’；
SEQ ID NO.6：5’-CTTTATGCTGATATCTCG-3’；
SEQ ID NO.7：5’-CTTTATGCTTCCGATATC-3’；
SEQ ID NO.8：5’-CTTTCACCTTCGTGCTCG-3’；
SEQ ID NO.9：5’-CTCGAGGATATCGGATCC-3’；
SEQ ID NO.10：5’-CACGTGGCTTCCGGCTCG-3’；
SEQ ID NO.11：5’-CTTCACGTGTCCGGCTCG-3’；
SEQ ID NO.12：5’-CTTTATCACGTGGGCTCG-3’；
SEQ ID NO.13：5’-CTTTATGCTCACGTGTCG-3’；
SEQ ID NO.14：5’-CTTTATGCTTCCCACGTG-3’.
第二方面，本申请提供一种载体，包括如第一方面所述的改进的启动子。
根据本申请，所述载体还包括目的基因，所述目的基因可操作地连接在所述改进的启动子的核酸内切酶识别位点之间；
本申请中，所述载体本领域技术人员可以根据需要进行选择，载体的选择不会对启动子的功能造成影响，所述载体可以是克隆载体和/或表达载体，所述克隆载体用于克隆所述目的蛋白，所述表达载体用于表达所述目的蛋白，所述启动子在克隆载体或是表达载体上都能发挥功能，优选为克隆载体，所述克隆载体例如可以选择高拷贝克隆载体pUC57、低拷贝克隆载体pCK或单拷贝克隆载体，其都可以带上本申请启动子，从而进行后续试验，对载体本身并不造成影响，带上本申请启动子的载体仍然是高拷贝克隆载体、低拷贝克隆载体或单拷贝克隆载体。
第三方面，本申请提供一种宿主细胞，所述宿主细胞包括如第二方面所述的载体。
根据本申请，所述宿主细胞为大肠杆菌，所述大肠杆菌仅编码β-半乳糖苷酶C端ω片段。
本申请中，所述克隆载体的lacZα基因编码β-半乳糖苷酶(lacZ)N端α片段，所述大 肠杆菌仅编码β-半乳糖苷酶C端ω片段，宿主和质粒编码的片段虽都没有半乳糖苷酶活性，但它们同时存在时，α片段与ω片段可通过α-互补形成具有酶活性的β-半乳糖苷酶可以将无色化合物X-gal(5-溴-4-氯-3-吲哚-β-D-半乳糖苷)切割成半乳糖和深蓝色的物质5-溴-4-靛蓝，5-溴-4-靛蓝可使整个菌落显蓝色。而当外源DNA插入到本申请的克隆载体的β-半乳糖苷酶启动子区域后，使得lacZα的表达量显著下降，不能有效通过α-互补形成大量具有酶活性的β-半乳糖苷酶，最终导致菌落显白色。
第四方面，本申请提供一种如第二方面所述的载体的制备方法，包括如下步骤：
(1)根据要突变的核酸内切酶识别位点设计引物，以原启动子及其调控表达的基因为模板，进行PCR扩增，获得带有改进的启动子的产物；
(2)用Gibson重组的方法将步骤(1)的产物进行环化，得到带有启动子的载体；
(3)将步骤(2)所述载体进行线性化；以及
(4)将目的基因与步骤(3)所述线性化载体连接，得到所述载体。
根据本申请，步骤(1)所述引物的核酸序列如SEQ ID NO.15-38所示。
本申请中，通过SEQ ID NO.15-16所述的核酸序列的引物对对pUC57-lacZ或pCK-lacZ进行PCR扩增构建得到的质粒中，SEQ ID NO.2所示的核酸序列突变为SEQ ID NO.3所示的核酸序列；
通过SEQ ID NO.17-18所述的核酸序列的引物对对pUC57-lacZ或pCK-lacZ进行PCR扩增构建得到的质粒中，SEQ ID NO.2所示的核酸序列突变为SEQ ID NO.4所示的核酸序列；
通过SEQ ID NO.19-20所述的核酸序列的引物对对pUC57-lacZ或pCK-lacZ进行PCR扩增构建得到的质粒中，SEQ ID NO.2所示的核酸序列突变为SEQ ID NO.5所示的核酸序列；
通过SEQ ID NO.21-22所述的核酸序列的引物对对pUC57-lacZ或pCK-lacZ进行PCR扩增构建得到的质粒中，SEQ ID NO.2所示的核酸序列突变为SEQ ID NO.6所示的核酸序列；
通过SEQ ID NO.23-24所述的核酸序列的引物对对pUC57-lacZ或pCK-lacZ进行PCR扩增构建得到的质粒中，SEQ ID NO.2所示的核酸序列突变为SEQ ID NO.7所示的核酸序列；
通过SEQ ID NO.25-26所述的核酸序列的引物对对pUC57-lacZ或pCK-lacZ进行PCR扩增构建得到的质粒中，SEQ ID NO.2所示的核酸序列突变为SEQ ID NO.8所示的核酸序列；
通过SEQ ID NO.27-28所述的核酸序列的引物对对pUC57-lacZ或pCK-lacZ进行PCR扩增构建得到的质粒中，SEQ ID NO.2所示的核酸序列突变为SEQ ID NO.9所示的核酸序列；
通过SEQ ID NO.29-30所述的核酸序列的引物对对pCC1-lacZ进行PCR扩增构建得到的质粒中，SEQ ID NO.2所示的核酸序列突变为SEQ ID NO.10所示的核酸序列；
通过SEQ ID NO.31-32所述的核酸序列的引物对对pCC1-lacZ进行PCR扩增构建得到的质粒中，SEQ ID NO.2所示的核酸序列突变为SEQ ID NO.11所示的核酸序列；
通过SEQ ID NO.33-34所述的核酸序列的引物对对pCC1-lacZ进行PCR扩增构建得到的质粒中，SEQ ID NO.2所示的核酸序列突变为SEQ ID NO.12所示的核酸序列；
通过SEQ ID NO.35-36所述的核酸序列的引物对对pCC1-lacZ进行PCR扩增构建得到的质粒中，SEQ ID NO.2所示的核酸序列突变为SEQ ID NO.13所示的核酸序列；
通过SEQ ID NO.37-38所述的核酸序列的引物对对pCC1-lacZ进行PCR扩增构建得到的质粒中，SEQ ID NO.2所示的核酸序列突变为SEQ ID NO.14所示的核酸序列。
根据本申请，步骤(3)所述线性化为核酸内切酶酶切和/或PCR扩增获得。
根据本申请，步骤(1)之前还包括将调控表达的基因进行密码子优化。
根据本申请，所述调控表达的基因为lacZ基因，其核酸序列如SEQ ID NO.39所示，所述SEQ ID NO.39所示的核酸序列如下：
Figure PCTCN2018122309-appb-000001
根据本申请，所述lacZ基因进行密码子优化，其密码子优化后的核酸序列如SEQ ID NO.40所示，所述SEQ ID NO.40所示的核酸序列如下：
Figure PCTCN2018122309-appb-000002
第五方面，本申请提供一种制备目的蛋白的方法，包括：
在适于所述目的蛋白表达的条件下，培养第三方面所述的宿主细胞，以便获得所述目的蛋白；其中，所述宿主细胞中的载体为表达载体，所述目的蛋白是所述目的基因编码的蛋白。
第六方面，本申请提供一种试剂盒，所述试剂盒包括第一方面所述的改进的启动子、第 二方面所述的载体或第三方面所述的宿主细胞。
与现有技术相比，本申请具有如下有益效果：
(1)本申请将启动子区域的-35区至-10区之间的核酸序列进行突变，从而能够被核酸内切酶识别，克隆时，先将载体制备为线性化载体，再将外源基因与线性化载体连接，使得启动子活性显著下降，其调控的基因表达量显著下降，进而发挥功能；
(2)本申请对β-半乳糖苷酶的启动子的效果明显，将其强启动子区域-35区至-10区之间的核酸序列突变为能够被识别的核酸内切酶识别位点，当其被酶切成线性化载体，插入外源片段后，使得β-半乳糖苷酶强启动子由于外源DNA片段的插入使得活性明显下降，进而使得lacZα基因的表达量显著下降，进而使得含有重组质粒的菌落显白色；
(3)本申请就能够克服由于采用蓝白筛选的载体存在强启动子启动外源基因的转录或翻译产物可能对宿主有毒性而导致无法克隆的问题，可以避免载体在酶切位点缺失1-2bp导致lacZα基因移码突变产生假阳性克隆的缺陷，可以消除由于外源DNA片段较小并且外源DNA的插入没有改变lacZα基因的读码框，造成平板都是蓝斑的假阴性现象；
(4)本申请所述的克隆载体的构建方法简单，易操作，效率高，可以在短时间内完成所述克隆载体的构建。
附图说明
图1为本申请实施例2的菌落PCR鉴定的电泳图，其中，DNA marker大小为0.1kb、0.25kb、0.5kb、0.75kb、1kb、1.5kb、2kb、3kb、5kb；
图2为本申请实施例4的菌落PCR鉴定的电泳图，其中，DNA marker大小为0.1kb、0.25kb、0.5kb、0.75kb、1kb、1.5kb、2kb、3kb、5kb；
图3为本申请实施例5的菌落PCR鉴定的电泳图，其中，DNA marker大小为0.1kb、0.25kb、0.5kb、0.75kb、1kb、1.5kb、2kb、3kb、5kb。
具体实施方式
为更进一步阐述本申请所采取的技术手段及其效果，以下通过具体实施方式来进一步说明本申请的技术方案，但本申请并非局限在实施例范围内。
本申请用到了遗传工程和分子生物学领域使用的常规技术和方法，一般性参考文献提供了本领域技术人员已知的定义和方法。但是，本领域的技术人员可以在本申请所记载的技术方案的基础上，采用本领域其它常规的方法、实验方案和试剂，而不限于本申请具体实施例的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常 规产品。
术语解释：
LacZ基因：广泛用于基因表达调控研究中的一种基因，编码的β一半乳糖苷酶(简称β-gal)是由4个亚基组成的四聚体，可催化乳糖的水解.Beta-gal比较稳定，用X-Gal为底物进行染色时，呈蓝色，便于检测和观察，LacZ基因的诸多优点使它成为基因工程实验中的一个常用标记基因，比如常用于转化菌株筛选，β-半乳糖苷酶显色反应选择法，即，蓝白筛选；
LacZα基因：编码β-半乳糖苷酶(lacZ)N端α片段，可以通过α-互补形成具有酶活性的β-半乳糖苷酶可以将无色化合物X-gal(5-溴-4-氯-3-吲哚-β-D-半乳糖苷)切割成半乳糖和深蓝色的物质5-溴-4-靛蓝；
核酸内切酶：在核酸水解酶中，为可水解分子链内部磷酸二酯键生成寡核苷酸的酶；
PCR技术：聚合酶链式反应，是利用DNA在体外摄氏95°高温时变性会变成单链，低温(经常是60℃左右)时引物与单链按碱基互补配对的原则结合，再调温度至DNA聚合酶最适反应温度(72℃左右)，DNA聚合酶沿着磷酸到五碳糖(5′-3′)的方向合成互补链。基于聚合酶制造的PCR仪实际就是一个温控设备，能在变性温度，复性温度，延伸温度之间很好地进行控制。
材料：
Figure PCTCN2018122309-appb-000003
实施例1：密码子优化lacZα基因
密码子优化lacZα基因，包括如下步骤：
将pUC57的lacZα基因(SEQ ID NO.39)使用密码子优化软件(Codon optimization软件，由苏州金唯智生物科技有限公司开发)进行密码子优化，优化的lacZα基因由苏州金唯智生物科技有限公司合成，核苷酸序列如SEQ ID No.39所示，具体如下：
lacZα基因(SEQ ID NO.39)：
Figure PCTCN2018122309-appb-000004
优化后的lacZα基因(SEQ ID NO.40)：
Figure PCTCN2018122309-appb-000005
实施例2：高拷贝克隆载体的构建
高拷贝克隆载体的构建方法，包括如下具体步骤：
I)采用实施例1中优化后的lacZα基因替换pUC57(卡那霉素抗性)的lacZα基因，具体如下：
(1)以具有卡那霉素抗性的pUC57质粒为模板，以SEQ ID NO.41-42为引物进行PCR扩增反应，具体序列如下：
SEQ ID NO.41(正向引物)：ATGCAGGCTCGGTTCCAGCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCC；
SEQ ID NO.42(反向引物)：AGCACCATTTGCAGCGATGCCGCCTAATTAAGCCAGCCCCGACACCCGCCAACAC；
PCR反应体系如下表1所示：
表1
模板 约50ng，0.5μL
正向引物 10pM，0.5μL
反向引物 10pM，0.5μL
dNTP 5mM each，0.5μL
5×PCR缓冲液 10μL
pfu DNA聚合酶 5U/μL，0.5μL
H 2O 37.5μL
；
其中，一组以水为样本的阴性对照；
反应条件如下表2所示：
表2
Figure PCTCN2018122309-appb-000006
(2)将步骤(1)获得的PCR反应液经1％琼脂糖凝胶电泳后切胶回收纯化得到PCR扩增产物；
(3)使用Gibson
Figure PCTCN2018122309-appb-000007
Master Mix试剂盒将步骤(2)所得的PCR纯化产物与实施例1获得的密码子优化后的lacZα基因进行连接反应，连接反应体系如下表3所示：
表3
Figure PCTCN2018122309-appb-000008
连接反应条件为：50℃连接反1h；
(4)将上述步骤(3)中获得的连接产物转化Top10F’感受态细胞，最终涂布含IPTG及X-gal的卡那霉素抗性的LB平板并37℃培养过夜，次日挑取蓝色单克隆并进行Sanger测序，保留测序正确的质粒，并将质粒命名为pUC57-lacZ；
II)将pUC57-lacZ质粒的β-半乳糖苷酶启动子区域的-35区与-10区之间的序列5’-CTTTATGCTTCCGGCTCG-3’突变为能够被核酸内切酶识别的序列，具体如下：
(1)以步骤I)构建成功的pUC57-lacZ质粒为模板，以引物F1-EcoRV、R1-EcoRV、F2-EcoRV、R2-EcoRV、F3-EcoRV、R3-EcoRV、F4-EcoRV、R4-EcoRV、F5-EcoRV、R5-EcoRV、F6-AleI、R6-AleI、F7-BamHI-XhoI、R7-BamHI-XhoI(SEQ ID NO.15-SEQ ID NO.28)为引物 进行PCR扩增反应，具体序列如下表4：
表4
Figure PCTCN2018122309-appb-000009
具体的PCR反应体系如表1所示，反应条件如表2所示；
(2)将步骤(1)获得的PCR反应液经1％琼脂糖凝胶电泳后切胶回收纯化得到PCR扩增产物，然后分别使用Gibson
Figure PCTCN2018122309-appb-000010
Master Mix试剂盒进行连接反应，连接反应体系如下表5所示：
表5
Figure PCTCN2018122309-appb-000011
Figure PCTCN2018122309-appb-000012
连接反应条件：50℃连接反应1小时；
(3)将步骤(2)中获得的连接产物分别转化Top10F’感受态细胞，最终涂布含IPTG及X-gal的卡那霉素抗性的LB平板并37℃培养过夜，次日挑取蓝色单克隆进行Sanger测序，保留测序正确的质粒构建，分别命名为pUC57-lacZ-Mu-1(将5’-CTTTATGCTTCCGGCTCG-3’突变为5’-GATATCGCTTCCGGCTCG-3’，质粒由F1-EcoRV+R1-EcoRV引物构建)、pUC57-lacZ-Mu-2(将5’-CTTTATGCTTCCGGCTCG-3’突变为5’-CTTGATATCTCCGGCTCG-3’，质粒由F2-EcoRV+R2-EcoRV引物构建)、pUC57-lacZ-Mu-3(将5’-CTTTATGCTTCCGGCTCG-3’突变为5’-CTTTATGATATCGGCTCG-3’，质粒由F3-EcoRV+R3-EcoRV引物构建)、pUC57-lacZ-Mu-4(将5’-CTTTATGCTTCCGGCTCG-3’突变为5’-CTTTATGCTGATATCTCG-3’，质粒由F4-EcoRV+R4-EcoRV引物构建)、pUC57-lacZ-Mu-5(将5’-CTTTATGCTTCCGGCTCG-3’突变为5’-CTTTATGCTTCCGATATC-3’，质粒由F5-EcoRV+R5-EcoRV引物构建)、pUC57-lacZ-Mu-6(将5’-CTTTATGCTTCCGGCTCG-3’突变为5’-CTTTCACCTTCGTGCTCG-3’，质粒由F6-AleI+R6-AleI引物构建)、pUC57-lacZ-Mu-7(将5’-CTTTATGCTTCCGGCTCG-3’突变为5’-CTCGAGGATATCGGATCC-3’，质粒由F7-BamHI-XhoI+R7-BamHI-XhoI引物构建)。
III)载体克隆实验
(1)将步骤II)构建正确的质粒pUC57-lacZ-Mu-1、pUC57-lacZ-Mu-2、pUC57-lacZ-Mu-3、pUC57-lacZ-Mu-4、pUC57-lacZ-Mu-5用EcoRV限制酶进行酶切，pUC57-lacZ-Mu-6使用AleI限制酶进行酶切，pUC57-lacZ-Mu-7用BamHI和XhoI限制酶进行酶切，酶切产物经1％琼脂糖凝胶电泳后切胶回收纯化；
(2)合成反向互补的24、48bp引物，并退火形成双链DNA，反向互补的24、48bp引物核苷酸序列如SEQ ID NO.43-SEQ ID NO.46所示，具体如下：
SEQ ID NO.43：TTCATACAGCAGGCTATGTTTAGG；
SEQ ID NO.44：CCTAAACATAGCCTGCTGTATGAA；
SEQ ID NO.45：TAAGCCGATACTGTATTTTTTATCCATAGCTGTTTCCTGTGTGAAATT；
SEQ ID NO.46：AATTTCACACAGGAAACAGCTATGGATAAAAAATACAGTATCGGCTTA；
(3)以λDNA为模板，F-λDNA-200bp+R-λDNA-200bp为引物进行PCR扩增，所述引物F-λDNA-200bp、R-λDNA-200bp的核苷酸序列如SEQ ID NO.47-SEQ ID NO.48所示，具体如下：
SEQ ID NO.47(F-λDNA-200bp)：AATGGTCAGGATCCGTTGAATGGGCGGATGCTAATTACTATCTCCCG；
SEQ ID NO.48(R-λDNA-200bp)：TGAAGAACCTCGAGTTATGCTCTATAAAGTAGGCATAAACACCCAGC；
PCR反应体系如表1所示，PCR扩增程序如表6所示：
表6
Figure PCTCN2018122309-appb-000013
(4)将步骤(3)获得的PCR反应液经1％琼脂糖凝胶电泳后切胶回收纯化得到PCR扩增产物，然后使用BamHI和XhoI对纯化的PCR扩增产物进行酶切，酶切反应体系如下表7所示：
表7
PCR扩增产物 约900ng，12μL
BamHI 1μL
XhoI 1μL
10×buffer 2μL
灭菌去离子H 2O 4μL
酶切反应条件：37℃酶切1小时，酶切产物使用Axygen纯化试剂盒进行回收纯化；
(5)将步骤(2)退火形成的24、48bp的片段分别与步骤(1)酶切纯化的pUC57-lacZ-Mu-1、pUC57-lacZ-Mu-2、pUC57-lacZ-Mu-3、pUC57-lacZ-Mu-4、pUC57-lacZ-Mu-5、pUC57-lacZ-Mu-6载体进行连接反应，将步骤(4)纯化的酶切产物与步骤(1)酶切纯化的pUC57-lacZ-Mu-7载体进行连接反应，连接反应体系如下表8所示：
表8
外源DNA 约90ng，3μL
酶切后的载体 约30ng，1μL
10×缓冲液 1μL
T4 DNA连接酶 1μL
灭菌去离子H 2O 4μL
连接反应条件：22℃连接反应1小时；
(6)将上述步骤(5)中获得的连接产物分别转化Top10F’感受态细胞，最终涂布含IPTG及X-gal的卡那霉素抗性的LB平板并37℃培养过夜，次日从克隆约200bp(pUC57-lacZ-Mu-7 载体)DNA片段的平板上挑取24个白色单克隆进行菌落PCR鉴定，PCR反应体系如表9所示：
表9 PCR反应体系
菌液模板 3μL
F-λDNA-200bp 10pM，0.5μL
R-λDNA-200bp 10pM，0.5μL
dNTP 5mM each，0.5μL
10×Taq buffer 5μL
Taq DNA聚合酶 5U/μL，0.5μL
H 2O 40μL
PCR扩增程序如下表10所示：
表10
Figure PCTCN2018122309-appb-000014
PCR鉴定结果如图1所示。
图1结果显示所有克隆均为阳性克隆，从24个菌检阳性的克隆中随机挑选12个克隆，同时，从克隆24、48bp外源DNA片段的平板上分别挑取12个白色单克隆分别进行Sanger测序，测序结果显示所有克隆的序列均正确，实验结果表明，本申请的载体可以用于克隆不小于24bp的外源DNA。
实施例3三种突变型pUC57-lacZ-Mu-2质粒的构建
pUC57-lacZ-Mu-2质粒构建的方法，包括如下步骤：
(1)以实施例2中构建的质粒pUC57-lacZ-Mu-2为模板，分别以F1-del+R1-del、F2-del+R2-del、F3-del+R3-del为引物进行PCR扩增反应，所述引物F1-del、R1-del、F2-del、R2-del、F3-del、R3-del的核苷酸序列如SEQ ID NO.49-SEQ ID NO.54所示，具体如下：
SEQ ID NO.49(F1-del)：ATACGAGCCGGAGAATCAAGTGTAAAGCCTGGGGTGCCTAAT；
SEQ ID NO.50(R1-del)：GCTTTACACTTGATTCTCCGGCTCGTATGTTGTGTGGAATTG；
SEQ ID NO.51(F2-del)：TACGAGCCGGAGATTCAAGTGTAAAGCCTGGGGTGCCTAATG；
SEQ ID NO.52(R2-del)：GGCTTTACACTTGAATCTCCGGCTCGTATGTTGTGTGGAATTG；
SEQ ID NO.53(F3-del)：ATACGAGCCGGAGATCAAGTGTAAAGCCTGGGGTGCCTAATG；
SEQ ID NO.54(R4-del)：GGCTTTACACTTGATCTCCGGCTCGTATGTTGTGTGGAATTG；
PCR反应体系如实施例2中表1所示，PCR扩增程序如表2所示；
(2)将步骤(1)获得的PCR反应液经1％琼脂糖凝胶电泳后切胶回收纯化得到PCR扩增产物，然后分别使用Gibson
Figure PCTCN2018122309-appb-000015
Master Mix(NEB)试剂盒进行连接反应，连接反应体系如实施例2中表5所示；
连接反应条件：50℃连接反应1小时。
(3)将步骤(2)中获得的连接产物分别转化Top10F’感受态细胞，最终涂布含IPTG及X-gal的卡那霉素抗性的LB平板并37℃培养过夜，次日挑取蓝色单克隆进行Sanger测序，保留测序正确的质粒，最终获得三种突变型的pUC57-lacZ-Mu-2质粒，分别命名为pUC57-lacZ-Mu-2A、pUC57-lacZ-Mu-2B、pUC57-lacZ-Mu-2C；
其中，pUC57-lacZ-Mu-2A的EcoR V酶切位点突变为GATTC，即序列由5’-CTTGATATCTCCGGCTCG-3’突变为5’-CTTGATTCTCCGGCTCG-3’、pUC57-lacZ-Mu-2B的EcoR V酶切位点突变为GAATC，即序列由5’-CTTGATATCTCCGGCTCG-3’突变为5’-CTTGAATCTCCGGCTCG-3’、pUC57-lacZ-Mu-2C的EcoR V酶切位点突变为GATC，即序列由5’-CTTGATATCTCCGGCTCG-3’突变为5’-CTTGATCTCCGGCTCG-3’；
(4)将步骤(3)正确的pUC57-lacZ-Mu-2A、pUC57-lacZ-Mu-2B、pUC57-lacZ-Mu-2C质粒分别转化Top10F’感受态细胞，最终涂布含IPTG及X-gal的卡那霉素抗性的LB平板并37℃培养过夜，次日发现3个平板的菌落均为蓝色，每个平板分别挑取5个单克隆进行Sanger测序，测序结果显示所有克隆的序列均正确。
实验结果表明：三种突变型pUC57-lacZ-Mu-2质粒(pUC57-lacZ-Mu-2A、pUC57-lacZ-Mu-2B、pUC57-lacZ-Mu-2C)的β-半乳糖苷酶启动子仍有活性，在IPTG的诱导条件下，能够表达lacZα使菌落显蓝色。同时，实验结果说明：经过EcoRV酶切线性化的pUC57-lacZ-Mu-2载体在酶切位点一端(pUC57-lacZ-Mu-2A、pUC57-lacZ-Mu-2B质粒)或两 端(pUC57-lacZ-Mu-2C质粒)缺失1个碱基的线性化载体自连后仍能够表达lacZα使菌落显蓝色，即本申请的载体经过核酸内切酶酶切不会因为酶切位点一端或两端缺失1个碱基发生自连而产生假阳性克隆。
实施例4低拷贝克隆载体的构建及功能验证
低拷贝克隆载体的构建方法，包括如下具体步骤：
I)采用实施例1中优化后的lacZα基因替换pCK质粒的lacZα基因，具体如下：
(1)以pCK质粒为模板，以SEQ ID NO.55-56为引物进行PCR扩增反应，具体序列如下：
SEQ ID NO.55(正向引物)：ATGCAGGCTCGGTTCCAGCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCC；
SEQ ID NO.56(反向引物)：AGCACCATTTGCAGCGATGCCGCCTAATTAAGCCAGCCCCGAGTAGCTAGACAGG；
PCR反应体系如实施例2的表1所示，反应条件如是实施例2表2所示；
(2)将步骤(1)获得的PCR反应液经1％琼脂糖凝胶电泳后切胶回收纯化得到PCR扩增产物；
(3)使用Gibson
Figure PCTCN2018122309-appb-000016
Master Mix试剂盒将步骤(2)所得的PCR纯化产物与实施例1获得的密码子优化后的lacZα基因进行连接反应，连接反应体系如实施例2的表3所示；
连接反应条件为：50℃连接反1h；
(4)将上述步骤(3)中获得的连接产物转化Top10F’感受态细胞，最终涂布含IPTG及X-gal的卡那霉素抗性的LB平板并37℃培养过夜，次日挑取蓝色单克隆并进行Sanger测序，保留测序正确的质粒，并将质粒命名为pCK-lacZ；
II)将pCK-lacZ质粒的β-半乳糖苷酶启动子区域的-35区与-10区之间的序列5’-CTTTATGCTTCCGGCTCG-3’突变为能够被核酸内切酶识别的序列，具体如下：
(1)以步骤I)构建成功的pCK-lacZ质粒为模板，以引物F1-EcoRV、R1-EcoRV、F2-EcoRV、R2-EcoRV、F3-EcoRV、R3-EcoRV、F4-EcoRV、R4-EcoRV、F5-EcoRV、R5-EcoRV、F6-AleI、R6-AleI、F7-BamHI-XhoI、R7-BamHI-XhoI(SEQ ID NO.15-SEQ ID NO.28)为引物进行PCR扩增反应，具体序列如实施例2的表4所示，具体的PCR反应体系如实施例2的表1所示，反应条件如实施例2的表2所示；
(2)将步骤(1)获得的PCR反应液经1％琼脂糖凝胶电泳后切胶回收纯化得到PCR扩 增产物，然后分别使用
Figure PCTCN2018122309-appb-000017
Master Mix试剂盒进行连接反应，连接反应体系如实施例2的表5所示；
连接反应条件：50℃连接反应1小时；
(3)将步骤(2)中获得的连接产物分别转化Top10F’感受态细胞，最终涂布含IPTG及X-gal的卡那霉素抗性的LB平板并37℃培养过夜，次日挑取蓝色单克隆进行Sanger测序，保留测序正确的质粒构建，分别命名为pCK-lacZ-Mu-1(将5’-CTTTATGCTTCCGGCTCG-3’突变为5’-GATATCGCTTCCGGCTCG-3’，质粒由F1-EcoRV+R1-EcoRV引物构建)、pCK-lacZ-Mu-2(将5’-CTTTATGCTTCCGGCTCG-3’突变为5’-CTTGATATCTCCGGCTCG-3’，质粒由F2-EcoRV+R2-EcoRV引物构建)、pCK-lacZ-Mu-3(将5’-CTTTATGCTTCCGGCTCG-3’突变为5’-CTTTATGATATCGGCTCG-3’，质粒由F3-EcoRV+R3-EcoRV引物构建)、pCK-lacZ-Mu-4(将5’-CTTTATGCTTCCGGCTCG-3’突变为5’-CTTTATGCTGATATCTCG-3’，质粒由F4-EcoRV+R4-EcoRV引物构建)、pCK-lacZ-Mu-5(将5’-CTTTATGCTTCCGGCTCG-3’突变为5’-CTTTATGCTTCCGATATC-3’，质粒由F5-EcoRV+R5-EcoRV引物构建)、pCK-lacZ-Mu-6(将5’-CTTTATGCTTCCGGCTCG-3’突变为5’-CTTTCACCTTCGTGCTCG-3’，质粒由F6-AleI+R6-AleI引物构建)、pCK-lacZ-Mu-7(将5’-CTTTATGCTTCCGGCTCG-3’突变为5’-CTCGAGGATATCGGATCC-3’，质粒由F7-BamHI-XhoI+R7-BamHI-XhoI引物构建)。
III)载体克隆实验
(1)将步骤II)构建正确的质粒pCK-lacZ-Mu-1、pCK-lacZ-Mu-2、pCK-lacZ-Mu-3、pCK-lacZ-Mu-4、pCK-lacZ-Mu-5用EcoRV限制酶进行酶切，pCK-lacZ-Mu-6使用AleI限制酶进行酶切，pCK-lacZ-Mu-7用BamHI和XhoI限制酶进行酶切，酶切产物经1％琼脂糖凝胶电泳后切胶回收纯化；
(2)合成反向互补的24、48bp引物，并退火形成双链DNA，反向互补的24、48bp引物核苷酸序列如实施例2的SEQ ID NO.43-SEQ ID NO.46所示；
(3)以λDNA为模板，F-λDNA-200bp+R-λDNA-200bp为引物进行PCR扩增，所述引物F-λDNA-200bp、R-λDNA-200bp的核苷酸序列如实施例2的SEQ ID NO.47-SEQ ID NO.48所示；
PCR反应体系如实施例2的表1所示，PCR扩增程序如表6所示；
(4)将步骤(3)获得的PCR反应液经1％琼脂糖凝胶电泳后切胶回收纯化得到PCR扩增产物，然后使用BamHI和XhoI对纯化的PCR扩增产物进行酶切，酶切反应体系如实施例 2表7所示；
酶切反应条件：37℃酶切1小时，酶切产物使用Axygen纯化试剂盒进行回收纯化；
(5)将步骤(2)退火形成的24、48bp的片段分别与步骤(1)酶切纯化的pCK-lacZ-Mu-1、pCK-lacZ-Mu-2、pCK-lacZ-Mu-3、pCK-lacZ-Mu-4、pCK-lacZ-Mu-5、pCK-lacZ-Mu-6载体进行连接反应，将步骤(4)纯化的酶切产物与步骤(1)酶切纯化的pCK-lacZ-Mu-7载体进行连接反应，连接反应体系如实施例2表8所示；
连接反应条件：22℃连接反应1小时；
(6)将上述步骤(5)中获得的连接产物分别转化Top10F’感受态细胞，最终涂布含IPTG及X-gal的卡那霉素抗性的LB平板并37℃培养过夜，次日从克隆约200bp(pCK-lacZ-Mu-7载体)DNA片段的平板上挑取12个白色单克隆进行菌落PCR鉴定，PCR反应体系如表9所示；
PCR扩增程序如表10所示。
图2结果显示所有克隆均为阳性克隆，其它平板分别挑取12个白色单克隆连同菌检均阳性的12个单克隆分别进行Sanger测序，测序结果显示所有克隆的序列均正确，实验结果表明，本申请的载体可以用于克隆不小于24bp的外源DNA。
实施例5单拷贝克隆载体的构建及功能验证
单拷贝克隆载体的构建方法，包括如下具体步骤：
I)采用实施例1中优化后的lacZα基因替换pCC1质粒的lacZα基因，具体如下：
(1)以pCC1质粒为模板，以SEQ ID NO.57-58为引物进行PCR扩增反应，具体序列如下：
SEQ ID NO.57(正向引物)：ATGCAGGCTCGGTTCCAGCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCC；
SEQ ID NO.58(反向引物)：AGCACCATTTGCAGCGATGCCGCCTAATTAAGCCAGCCCCGACACCCGCCAACAC；
PCR反应体系如实施例2的表1所示，反应条件如下表11所示：
表11
Figure PCTCN2018122309-appb-000018
Figure PCTCN2018122309-appb-000019
(2)将步骤(1)获得的PCR反应液经1％琼脂糖凝胶电泳后切胶回收纯化得到PCR扩增产物；
(3)使用Gibson
Figure PCTCN2018122309-appb-000020
Master Mix试剂盒将步骤(3)所得的PCR纯化产物与实施例1获得的密码子优化后的lacZα基因进行连接反应，连接反应体系如实施例2的表12所示：
表12
Figure PCTCN2018122309-appb-000021
连接反应条件为：50℃连接反1h；
(4)将上述步骤(3)中获得的连接产物转化Top10F’感受态细胞，最终涂布含IPTG及X-gal的氯霉素抗性的LB平板并37℃培养过夜，次日挑取蓝色单克隆并进行Sanger测序，保留测序正确的质粒，并将质粒命名为pCC1-lacZ；
II)将pCC1-lacZ质粒的β-半乳糖苷酶启动子区域的-35区与-10区之间的序列5’-CTTTATGCTTCCGGCTCG-3’突变为能够被核酸内切酶识别的序列，具体如下：
(1)以步骤I)构建成功的pCC1-lacZ质粒为模板，以引物pCC1-lacZ质粒为模板，以引物F1-PmlI+R1-PmlI、F2-PmlI+R2-PmlI、F3-PmlI+R3-PmlI、F4-PmlI+R4-PmlI、F5-PmlI+R5-PmlI、F7-BamHI-XhoI+R7-BamHI-XhoI(SEQ ID NO.29-SEQ ID NO.38、SEQ ID NO.27-SEQ ID NO.28)为引物进行PCR扩增反应，具体序列如表13所示：
表13
Figure PCTCN2018122309-appb-000022
Figure PCTCN2018122309-appb-000023
具体的PCR反应体系如实施例2的表1所示，反应条件如表11所示；
(2)将步骤(1)获得的PCR反应液经1％琼脂糖凝胶电泳后切胶回收纯化得到PCR扩增产物，然后分别使用Gibson
Figure PCTCN2018122309-appb-000024
Master Mix试剂盒进行连接反应，连接反应体系如实施例2的表14所示：
表14
Figure PCTCN2018122309-appb-000025
连接反应条件：50℃连接反应1小时；
(3)将步骤(2)中获得的连接产物分别转化Top10F’感受态细胞，最终涂布含IPTG及X-gal的氯霉素抗性的LB平板并37℃培养过夜，次日挑取蓝色单克隆进行Sanger测序，保留测序正确的质粒构建，分别命名为pCC1-lacZ-Mu-1(将5’-CTTTATGCTTCCGGCTCG-3’突变为5’-CACGTGGCTTCCGGCTCG-3’，质粒由F1-PmlI+R1-PmlI引物构建)、pCC1-lacZ-Mu-2(将5’-CTTTATGCTTCCGGCTCG-3’突变为5’-CTTCACGTGTCCGGCTCG-3’，质粒由F2-PmlI+R2-PmlI引物构建)、pCC1-lacZ-Mu-3(将5’-CTTTATGCTTCCGGCTCG-3’突变为5’-CTTTATCACGTGGGCTCG-3’，质粒由F3-PmlI+R3-PmlI引物构建)、pCC1-lacZ-Mu-4(将5’-CTTTATGCTTCCGGCTCG-3’突变为5’-CTTTATGCTCACGTGTCG-3’，质粒由F4-PmlI+R4-PmlI引物构建)、pCC1-lacZ-Mu-5(将5’-CTTTATGCTTCCGGCTCG-3’突变为5’-CTTTATGCTTCCCACGTG-3’，质粒由F5-PmlI+R5-PmlI引物构建)、pCC1-lacZ-Mu-6(将5’-CTTTATGCTTCCGGCTCG-3’突变为5’-CTCGAGGATATCGGATCC-3’，质粒由F7-BamHI-XhoI+R7-BamHI-XhoI引物构建)。
III)载体克隆实验
(1)将步骤II)构建正确的质粒pCC1-lacZ-Mu-1、pCC1-lacZ-Mu-2、pCC1-lacZ-Mu-3、pCC1-lacZ-Mu-4、pCC1-lacZ-Mu-5用PmlI限制酶进行酶切，pCC1-lacZ-Mu-6使用BamHI和XhoI限制酶进行酶切，酶切产物经1％琼脂糖凝胶电泳后切胶回收纯化；
(2)合成反向互补的24、48bp引物，并退火形成双链DNA，反向互补的24、48bp引物核苷酸序列如实施例2的SEQ ID NO.43-SEQ ID NO.46所示；
(3)以λDNA为模板，F-λDNA-200bp+R-λDNA-200bp为引物进行PCR扩增，所述引物F-λDNA-200bp、R-λDNA-200bp的核苷酸序列如实施例2的SEQ ID NO.47-SEQ ID NO.48所示；
PCR反应体系如表1所示，PCR扩增程序如表6所示；
(4)将步骤(3)获得的PCR反应液经1％琼脂糖凝胶电泳后切胶回收纯化得到PCR扩增产物，然后使用BamHI和XhoI对纯化的PCR扩增产物进行酶切，酶切反应体系如实施例2表7所示；
酶切反应条件：37℃酶切1小时，酶切产物使用Axygen纯化试剂盒进行回收纯化；
(5)将步骤(2)退火形成的24、48bp的片段分别与步骤(1)酶切纯化的pCC1-lacZ-Mu-1、pCC1-lacZ-Mu-2、pCC1-lacZ-Mu-3、pCC1-lacZ-Mu-4、pCC1-lacZ-Mu-5载体进行连接反应，将步骤(4)纯化的酶切产物与步骤(1)酶切纯化的pCC1-lacZ-Mu-6载体进行连接反应，连接反应体系如实施例2表7所示；
连接反应条件：22℃连接反应1小时；
(6)将上述步骤(5)中获得的连接产物分别转化Top10F’感受态细胞，最终涂布含IPTG及X-gal的氯霉素抗性的LB平板并37℃培养过夜，次日从克隆约200bp(pCC1-lacZ-Mu-6载体)DNA片段的平板上挑取24个白色单克隆进行菌落PCR鉴定，PCR反应体系如表9所示；
PCR扩增程序如表10所示，结果如图3所示。
图3电泳结果显示所有克隆均为阳性克隆，从24个菌检阳性的克隆中随机挑选12个克隆，同时，从克隆24、48bp外源DNA片段的平板上分别挑取12个白色单克隆分别进行Sanger测序，测序结果显示所有克隆的序列均正确，实验结果表明，本申请的载体可以用于克隆不小于24bp的外源DNA。
综上所述，本申请通过对β-半乳糖苷酶强启动子区域的-35区与-10区之间的序列进行突变形成能够被核酸内切酶识别酶切的位点，当克隆时，先用合适的核酸内切酶将载体酶切或PCR的方法制备线性化载体，然后将外源基因与线性化载体连接，使得β-半乳糖苷酶强启动 子由于外源DNA片段的插入使得活性明显下降，进而使得lacZα基因的表达量显著下降，进而使得含有重组质粒的菌落显白色；本申请通过上述方法克服了常见的由于采用蓝白筛选的载体存在强启动子启动外源基因的转录或翻译产物可能对宿主有毒性而导致无法克隆的问题，而且可以避免载体在酶切位点缺失1-2bp导致lacZα基因移码突变产生假阳性克隆的缺陷，并且可以消除由于外源DNA片段较小并且外源DNA的插入没有改变lacZα基因的读码框，造成平板都是蓝斑的假阴性现象。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (15)

  1. 一种改进的启动子，其为将启动子区域中的-35区至-10区之间的核酸序列突变为核酸内切酶识别位点而得到。
  2. 根据权利要求1所述的改进的启动子，其中，所述改进的启动子为将β-半乳糖苷酶的启动子区域中的-35区至-10区之间的核酸序列突变为核酸内切酶识别位点而得到。
  3. 根据权利要求2所述的改进的启动子，其中，所述β-半乳糖苷酶的启动子区域中的-35区至-10区之间的核酸序列如SEQ ID NO.1-2所示。
  4. 根据权利要求2所述的改进的启动子，其中，所述核酸内切酶为EcoRV、AleI、BamHI、XhoI或PmlI中的任意一种或至少两种的组合。
  5. 根据权利要求2所述的改进的启动子，其中，所述改进的启动子的-35区至-10区之间的核酸序列如SEQ ID NO.3-14所示。
  6. 一种载体，其包括如权利要求1-5中任一项所述的改进的启动子。
  7. 根据权利要求6所述的载体，其中，所述载体还包括目的基因，所述目的基因可操作地连接在所述改进的启动子的核酸内切酶识别位点之间。
  8. 根据权利要求7所述的载体，其中，所述载体为克隆载体和/或表达载体，优选为克隆载体。
  9. 一种宿主细胞，其包括如权利要求6-8中任一项所述的载体。
  10. 根据权利要求9所述的宿主细胞，其中，所述宿主细胞为大肠杆菌。
  11. 根据权利要求10所述的宿主细胞，其中，所述大肠杆菌仅编码β-半乳糖苷酶C端ω片段。
  12. 一种如权利要求6-8中任一项所述的载体的制备方法，其包括如下步骤：
    (1)根据要突变的核酸内切酶识别位点设计引物，以原启动子及其调控表达的基因为模板，进行PCR扩增，获得带有改进的启动子的产物；
    (2)用Gibson重组的方法将步骤(1)的产物进行环化，得到带有启动子的载体；
    (3)将步骤(2)所述载体进行线性化；以及
    (4)将目的基因与步骤(3)所述线性化载体连接，得到所述载体。
  13. 根据权利要求12所述的制备方法，其中，步骤(1)所述引物的核酸序列如SEQ ID NO.15-38所示；
    优选地，步骤(3)所述线性化为核酸内切酶酶切和/或PCR扩增获得；
    优选地，步骤(1)之前还包括将调控表达的基因进行密码子优化；
    优选地，所述调控表达的基因为lacZ基因，其核酸序列如SEQ ID NO.39所示；
    优选地，所述lacZ基因进行密码子优化，其密码子优化后的核酸序列如SEQ ID NO.40所示。
  14. 一种制备目的蛋白的方法，其包括：
    在适于所述目的蛋白表达的条件下，培养权利要求9-11中任一项所述的宿主细胞，以便获得所述目的蛋白；
    其中，所述宿主细胞中的载体为表达载体，所述目的蛋白是所述目的基因编码的蛋白。
  15. 一种试剂盒，其包括权利要求1-5中任一项所述的改进的启动子、权利要求6-8中任一项所述的载体或权利要求9-11中任一项所述的宿主细胞。
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