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WO2021184766A1 - 一种基因表达组件及其构建的克隆载体和应用 - Google Patents

一种基因表达组件及其构建的克隆载体和应用 Download PDF

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WO2021184766A1
WO2021184766A1 PCT/CN2020/125162 CN2020125162W WO2021184766A1 WO 2021184766 A1 WO2021184766 A1 WO 2021184766A1 CN 2020125162 W CN2020125162 W CN 2020125162W WO 2021184766 A1 WO2021184766 A1 WO 2021184766A1
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gene
cloning vector
gene expression
cloning
host cell
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PCT/CN2020/125162
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English (en)
French (fr)
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薛高旭
夏立军
陈业
方其
张艳
吴昕
廖国娟
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苏州金唯智生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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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)
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    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01022—Alpha-galactosidase (3.2.1.22)
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/34—Vector systems having a special element relevant for transcription being a transcription initiation element

Definitions

  • This application belongs to the technical field of molecular biology and genetic engineering, and relates to a gene expression component and its constructed cloning vector and application, in particular to a gene expression component encoding ⁇ -galactosidase ⁇ peptide and ⁇ peptide, and a construction Cloning vector and its construction method and application.
  • PCR is an important technology in the fields of molecular biology and genetic engineering, and experimenters often need to clone PCR products into vectors.
  • cloning vectors are mainly based on the principle of blue-white spot screening, and the blue-white spot screening method brings great convenience to picking positive clones.
  • the vector that can be used for blue-white spot screening contains the lacZ' gene, which can encode the ⁇ peptide chain, and contains a multiple cloning site (MCS) for cloning foreign DNA in the lacZ' gene.
  • MCS multiple cloning site
  • the existing vector with blue-white spot screening function is only suitable for ⁇ -galactosidase-deficient genetically engineered bacteria.
  • the gene encoding ⁇ -galactosidase in the chromosome of this engineered bacteria is mutated, and the coding product of the mutant gene ( ⁇ peptide) Compared with wild-type ⁇ -galactosidase, it lacks 146 amino acids in the N segment, so it has no biological activity.
  • the ⁇ -galactosidase-deficient strain cannot encode active ⁇ -galactosidase alone, when the bacterial cell contains a plasmid with the lacZ' gene, the ⁇ peptide encoded by the plasmid lacZ' gene and the strain expressed Omega peptides are complemented to obtain a complementary product with ⁇ -galactosidase activity, so that X-gal produces blue substances.
  • This phenomenon is called ⁇ -complementation.
  • the above is the phenotype produced by the strain carrying the empty vector.
  • the reading frame of the ⁇ peptide is destroyed, and the recombinant plasmid will no longer express the ⁇ peptide.
  • the ⁇ -galactosidase deficient strain no ⁇ -complementation will be produced, and no ⁇ -complementation will be produced.
  • It is a product with ⁇ -galactosidase activity, so it cannot decompose X-gal in the medium to produce blue color, and the culture phenotype presents a white colony.
  • the pUC series vectors are commonly used blue-white screening vectors.
  • the existing vectors with blue-white spot screening function such as the widely used pUC series vectors
  • each E. coli contains a lower The number of genome copies results in low expression of ⁇ peptide. When the strain is transformed into a plasmid expressing ⁇ peptide, the ⁇ -galactosidase content formed is low.
  • This application provides a gene expression module and its constructed cloning vector and application.
  • the cloning vector has the function of screening blue and white spots and is suitable for almost all types of E. coli.
  • the blue spots develop fast and can be used in large quantities without IPTG induction.
  • the expression of proteins or polypeptides with ⁇ -galactosidase activity has important significance and broad application prospects.
  • the present application provides a gene expression module that encodes ⁇ -galactosidase ⁇ peptide and ⁇ peptide;
  • the gene expression component includes a constitutive promoter, and the LacZ ⁇ gene and LacZ ⁇ gene that the constitutive promoter promotes expression.
  • a constitutive promoter is used to regulate the expression of LacZ ⁇ gene and LacZ ⁇ gene, so that LacZ ⁇ gene and LacZ ⁇ gene can be continuously and stably expressed in host cells, and the ⁇ -galactosidase ⁇ peptide and ⁇ peptide obtained by transcription and translation pass
  • the ⁇ -complementation effect produces a substance with ⁇ -galactosidase activity, which decomposes X-gal into blue 5-bromo-4-indigo, achieving the technical effect of producing ⁇ -galactosidase without IPTG induction.
  • the scope of application of host cells is expanded, and positive clones can be screened for blue and white spots in any type of E. coli.
  • the LacZ ⁇ gene contains multiple cloning sites
  • the multiple cloning site in the LacZ ⁇ gene is used to insert foreign DNA, and the ribosome binding site is set between the LacZ ⁇ gene and the LacZ ⁇ gene to ensure that the foreign DNA is inserted or not inserted. All ⁇ peptides can be translated normally.
  • the constitutive promoter includes a resistance gene promoter, preferably a bla promoter (ie, Amp resistance gene promoter).
  • a resistance gene promoter preferably a bla promoter (ie, Amp resistance gene promoter).
  • the LacZ ⁇ gene includes the nucleic acid sequence shown in SEQ ID NO:1;
  • the LacZ ⁇ gene includes the nucleic acid sequence shown in SEQ ID NO: 2;
  • the wild-type LacZ ⁇ gene and LacZ ⁇ gene are used as optimization targets.
  • the codon preference of E. coli the most frequently used codon is selected, the coding sequence is redesigned, and the coding sequence is further optimized to optimize the second order of 5'UTR.
  • Level structure, the LacZ ⁇ gene shown in SEQ ID NO:1 and the LacZ ⁇ gene shown in SEQ ID NO:2 are obtained, which significantly increases the expression of ⁇ peptide and ⁇ peptide, enhances the color rendering effect and color rendering speed, and shortens Color development time.
  • the gene expression component includes the nucleic acid sequence shown in SEQ ID NO: 3;
  • the present application provides a cloning vector, which includes the gene expression component described in the first aspect.
  • the cloning vector also includes an E. coli replicon, preferably a pBR322 replicon.
  • the pBR322 replicon includes the nucleic acid sequence shown in SEQ ID NO: 4;
  • the cloning vector also includes an antibiotic resistance gene.
  • the antibiotic resistance gene includes a kanamycin resistance gene.
  • the kanamycin resistance gene includes the nucleic acid sequence shown in SEQ ID NO: 5;
  • the cloning vector includes the nucleic acid sequence shown in SEQ ID NO: 6;
  • the 146th to 3127 bp reverse complementary sequence of the cloning vector is the coding sequence of ⁇ -galactosidase ⁇ peptide
  • the 3128 to 3176 bp reverse complementary sequence is the ribosome binding site (RBS) sequence
  • the 3177 ⁇ 3542bp reverse complementary sequence is the coding sequence of ⁇ -galactosidase ⁇ peptide
  • the 3445 ⁇ 3533bp is the multiple cloning site (MCS) sequence
  • the 3543 ⁇ 3692bp The reverse complementary sequence of the bla promoter sequence, the 3935-4617 bp is the pBR322 replicon sequence, and the 4712-5527 bp reverse complementary sequence is the coding sequence of Kanamycin (Kan).
  • the constructed cloning vector pWizard plasmid adopts a constitutive promoter to regulate the expression of LacZ ⁇ gene and LacZ ⁇ gene, so that LacZ ⁇ gene and LacZ ⁇ gene can be continuously and stably expressed in host cells, without IPTG induction.
  • Lactosidase-active substances can be screened for positive clones by blue and white spots in any type of E. coli; at the same time, the cloning vector pWizard plasmid is a high-copy plasmid, therefore, the LacZ ⁇ gene and LacZ ⁇ gene have a high copy number in the host bacteria.
  • Significantly increase the expression of ⁇ peptide and ⁇ peptide by the host cell form a large number of enzymes with ⁇ -galactosidase activity, enhance the color development effect and color development speed, and shorten the color development time.
  • this application provides a method for constructing the cloning vector of the second aspect, and the method for constructing includes:
  • the closed circular double-stranded DNA is transformed into competent cells, a single clone is picked, cultured, and the plasmid is extracted for sequencing identification, and the cloning vector is obtained, which is named pWizard plasmid.
  • the gene expression module, pBR322 replicon and the nucleic acid molecule of the kanamycin resistance gene have a 30bp homologous recombination arm, which is beneficial to be connected to a circular DNA by a homologous recombination method.
  • the reaction temperature may be, for example, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C. °C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C or 60°C, preferably 50°C.
  • reaction time may be 0.5h, 0.6h, 0.7h, 0.8h, 0.9h or 1h, for example.
  • the molar ratio of the nucleic acid molecules of the gene expression module, pBR322 replicon and kanamycin resistance gene is 1:1:1.
  • this application provides a method for constructing the cloning vector described in the second aspect, and the method for constructing includes:
  • the closed circular double-stranded DNA is transformed into competent cells, a single clone is picked, cultured, and the plasmid is extracted for sequencing identification, and the cloning vector is obtained, which is named pWizard plasmid.
  • the present application provides a recombinant vector, which is the cloning vector described in the second aspect in which a foreign gene is inserted.
  • the foreign gene is inserted into the multiple cloning site of the cloning vector described in the second aspect.
  • the present application provides a host cell that integrates the gene expression component described in the first aspect into the genome of the host cell, and/or the host cell includes the cloning vector described in the second aspect and/ Or the recombinant vector described in the fourth aspect.
  • the host cell includes ⁇ -galactosidase wild-type genetically engineered bacteria and/or ⁇ -galactosidase-deficient genetically engineered bacteria, including but not limited to Top10, DH5 ⁇ , STBL2 or STBL3 Escherichia coli.
  • the present application provides a kit comprising the gene expression component described in the first aspect, the cloning vector described in the second aspect, the recombinant vector described in the fourth aspect, or the recombinant vector described in the fifth aspect. Any one or a combination of at least two of the above-mentioned host cells.
  • this application provides a method for cloning an exogenous gene, the method comprising:
  • the foreign gene is inserted into the multiple cloning site of the cloning vector described in the second aspect, is introduced into the host cell, and after culture, a positive clone expressing the foreign gene is obtained by screening according to blue and white spots.
  • the method further includes the step of separating and purifying the protein from the culture supernatant of the positive clone or the bacterial cell of the positive clone.
  • the present application provides a gene expression module according to the first aspect, the cloning vector according to the second aspect, the recombinant vector according to the fourth aspect, the host cell according to the fifth aspect, or the sixth aspect The application of the kit in protein preparation.
  • the pWizard plasmid of the present application uses a constitutive promoter to regulate the expression of LacZ ⁇ gene and LacZ ⁇ gene, so that LacZ ⁇ gene and LacZ ⁇ gene can be continuously and stably expressed in host cells, without the need for IPTG induction to produce ⁇ -galactoside Enzymatically active substances can be coated and cultured on a plate that does not contain IPTG and only contains X-gal, which expands the scope of application of host cells, and can screen positive clones for blue and white spots in any type of E. coli;
  • This application has performed codon optimization on the wild-type LacZ ⁇ gene and LacZ ⁇ gene, and the constructed pWizard plasmid has a much higher copy number of the LacZ ⁇ gene and LacZ ⁇ gene in the host bacteria than the genome copy number, which significantly improves the host cell’s response to the ⁇ peptide
  • the expression level of ⁇ and ⁇ peptides increases the expression level of enzymes with ⁇ -galactosidase activity through ⁇ -complementation, enhances the color development effect and color development speed, and shortens the color development time.
  • Figure 1 is a plasmid map of pWizard
  • Figure 2 is the transformation of pUC57 plasmid into DH5 ⁇ strain, and coating the plate containing IPTG and X-gal;
  • Figure 3 shows the transformation of Top10F' strain with pUC57 plasmid and coating on a plate containing IPTG and X-gal;
  • Figure 4 shows the transformation of STBL3 strain with pUC57 plasmid and coating of plates containing IPTG and X-gal;
  • Figure 5 shows the pWizard plasmid transforming the DH5 ⁇ strain and coating the plate containing X-gal;
  • Figure 6 is the transformation of Top10F' strain with pWizard plasmid and coating on X-gal plate;
  • Figure 7 is the transformation of STBL3 strain with pWizard plasmid and coating the plate containing X-gal;
  • Figure 8 is the transformation of STBL3 strain with pET-30a plasmid and coating the plate containing IPTG and X-gal;
  • Figure 9 is the blunt-end cloning of the pWizard plasmid, the exogenous DNA is transformed into the DH5 ⁇ strain, and the plate containing X-gal is coated;
  • Figure 10 is a pWizard plasmid blunt end cloned exogenous DNA transformed STBL3 strain, coated with X-gal plate;
  • Figure 11 shows the bacterial test results of blunt-ended clones of foreign DNA.
  • Lanes 1-8 are DH5 ⁇ bacterial test results
  • lanes 9-16 are STBL3 bacterial test results
  • lane M is DNA ladder.
  • the band sizes are 100bp, 250bp, 500bp, 750bp, 1kb, 1.5kb, 2kb, 3kb and 5kb;
  • Figure 12 is the cloned exogenous DNA of the sticky end of pWizard plasmid transforming the DH5 ⁇ strain and coating the plate containing X-gal;
  • Figure 13 is the transformation of STBL3 strain with foreign DNA cloned from the sticky end of pWizard plasmid and coated with X-gal plate;
  • Figure 14 shows the results of bacterial detection of foreign DNA clones with sticky ends.
  • Lanes 1-8 are the results of DH5 ⁇ bacterial detection
  • lanes 9-16 are the results of STBL3 bacterial detection
  • lane M is DNA ladder.
  • the band sizes are 100bp, 250bp, 500bp, 750bp, 1kb, 1.5kb, 2kb, 3kb and 5kb.
  • LacZ gene a gene widely used in the study of gene expression regulation.
  • the encoded ⁇ -galactosidase ( ⁇ -gal) is a tetramer composed of 4 subunits, which can catalyze the hydrolysis of lactose and stabilize ⁇ -gal It has good properties and is blue when stained with X-Gal as a substrate, which is convenient for detection and observation.
  • the many advantages of LacZ gene make it a commonly used marker gene in genetic engineering experiments. For example, it is often used in the screening of transformed strains.
  • Galactosidase color reaction selection method that is, blue and white selection;
  • LacZ ⁇ gene encodes the N-terminal ⁇ fragment of ⁇ -galactosidase (lacZ), which can form an enzyme with ⁇ -galactosidase activity through ⁇ -complementation.
  • lacZ ⁇ -galactosidase
  • the colorless compound X-gal (5-bromo-4-chloro -3-indole- ⁇ -D-galactoside) cleaved into galactose and the dark blue substance 5-bromo-4-indigo;
  • Endonuclease an enzyme that hydrolyzes the phosphodiester bond in the nucleic acid molecule chain to generate oligonucleotides
  • PCR technology the polymerase chain reaction, the DNA is denatured to single-stranded at a high temperature of 95°C in vitro, and the primers and single-strands are combined in accordance with the principle of base complementary pairing at low temperatures (about 60°C), and then the temperature is adjusted to the DNA polymerase The most suitable reaction temperature (about 72°C), DNA polymerase synthesizes complementary strands along the direction of phosphoric acid to five carbon sugars (5'-3'); PCR machine based on polymerase is a temperature-controlled device that can denature The temperature change is well controlled between temperature, refolding temperature and extension temperature.
  • the reaction system is SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 three gene fragments each 2 ⁇ L, sterilized deionized water 4 ⁇ L, Gibson Master Mix 10 ⁇ L (contains T5 exonuclease, Phusion DNA polymerase and Taq DNA ligase), the reaction conditions are 50 °C for 1 hour, assembled to form a circular cloning vector, named pWizard plasmid;
  • reaction product is transformed into DH5 ⁇ competent cells and coated with Kan resistance plate;
  • the plasmid that is correctly sequenced is the pWizard plasmid, and the length of the plasmid is 5695bp.
  • the positions of the main elements in the pWizard plasmid are as follows: From the 5'end, the reverse complement sequence of 146 ⁇ 3127bp is LacZ ⁇ gene, which encodes ⁇ peptide, and the reverse complementary sequence of 3128 ⁇ 3176bp is RBS.
  • the 3177 ⁇ 3542bp reverse complementary sequence is LacZ ⁇ gene, which encodes ⁇ peptide
  • the 3445 ⁇ 3533bp is the multiple cloning site (MCS) sequence
  • the 3543 ⁇ 3692bp reverse complementary sequence It is the bla promoter
  • the 3935-4617 bp is the pBR322 replicon sequence
  • the 4712-5527 bp reverse complementary sequence is the Kan gene sequence.
  • Figure 2 shows the transformation of pUC57 plasmid into DH5 ⁇ strain and coating with IPTG and X-gal plates
  • Figure 3 shows the transformation of pUC57 plasmid into Top10F' strain and coating with IPTG.
  • Figure 4 is the pUC57 plasmid transformed STBL3 strain, coated with IPTG and X-gal plate
  • Figure 5 is pWizard plasmid transformed DH5 ⁇ strain, coated with X-gal plate
  • Figure 6 is pWizard plasmid transformed Top10F' Strains, coated plates containing X-gal
  • Figure 7 shows the pWizard plasmid transformed STBL3 strain, coated plates containing X-gal
  • Figure 8 shows pET-30a plasmid transformed STBL3 strain, coated plates containing IPTG and X-gal.
  • F- ⁇ DNA-Blunt (SEQ ID NO: 7): ACGTTTGAGCAGAATAACCATGTGG;
  • R- ⁇ DNA-Blunt (SEQ ID NO: 8): AAATAACGTTCTCCACCGACCTCTG;
  • Nanodrop2000 determines the concentration of the purified digested product to be about 20ng/ ⁇ L;
  • Figure 9 shows the blunt-end cloning of pWizard plasmid and exogenous DNA transforming the DH5 ⁇ strain and coating the plate containing X-gal.
  • Figure 10 shows the blunt-end cloning of pWizard plasmid. DNA was transformed into STBL3 strain and spread on a plate containing X-gal.
  • the pWizard plasmid as a cloning vector can form obvious blue and white spots without the induction of IPTG, indicating that the pWizard plasmid is suitable for STBL3 and other strains with no LacZ gene mutations, and positive clones can be screened by blue and white spots.
  • JJ-F-pWizard (SEQ ID NO: 9): CTTCTTCGCTGTTGCGCCAGCTCGC;
  • JJ-R-pWizard (SEQ ID NO: 10): GTATCCGCTCATGAGACAATAACCC.
  • F- ⁇ DNA-Sticky (SEQ ID NO: 11):
  • R- ⁇ DNA-Sticky (SEQ ID NO: 12):
  • Nanodrop2000 determines the concentration of the purified digested product to be about 20ng/ ⁇ L;
  • Nanodrop2000 determines the concentration of the purified digested product to be about 15ng/ ⁇ L;
  • Figure 12 shows the cohesive end cloning of pWizard plasmid and exogenous DNA transforming the DH5 ⁇ strain and coating the plate containing X-gal.
  • Figure 13 shows the cohesive end cloning of pWizard plasmid. DNA was transformed into STBL3 strain and spread on a plate containing X-gal.
  • this application uses a constitutive promoter to regulate the expression of LacZ ⁇ and LacZ ⁇ genes, so that LacZ ⁇ and LacZ ⁇ genes can be continuously and stably expressed in host cells.
  • the constructed cloning vector has the function of screening blue and white spots, which is almost suitable for All types of Escherichia coli have fast blue spots and can express a large number of proteins or peptides with ⁇ -galactosidase activity without IPTG induction, which has important significance and wide application prospects.

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Abstract

一种基因表达组件及其构建的克隆载体和应用，所述基因表达组件编码β-半乳糖苷酶α肽和ω肽；所述基因表达组件包括组成型启动子，和所述组成型启动子启动表达的LacZα基因和LacZω基因。构建的pWizard质粒不需要IPTG诱导便可以在宿主细胞中产生具有β-半乳糖苷酶活性的物质，扩大了宿主细胞的适用范围，所述pWizard质粒属于高拷贝质粒，因此，LacZα基因和LacZω基因在宿主菌中的拷贝数高，显著提高了宿主细胞对具有β-半乳糖苷酶活性的酶的表达量，增强了显色效果和显色速度，缩短了显色时间。

Description

一种基因表达组件及其构建的克隆载体和应用 技术领域
本申请属于分子生物学和基因工程技术领域，涉及一种基因表达组件及其构建的克隆载体和应用，尤其涉及一种编码β-半乳糖苷酶α肽和ω肽的基因表达组件、构建的克隆载体及其构建方法和应用。
背景技术
PCR是分子生物学和基因工程领域的重要技术，实验人员经常需要将PCR产物克隆到载体中。目前常用的克隆载体主要基于蓝白斑筛选原理，蓝白斑筛选方法为挑取阳性克隆带来了极大的方便。能够用于蓝白斑筛选的载体含有lacZ’基因，该基因能够编码α肽链，在lacZ’基因中含有一个用于克隆外源DNA的多克隆位点(MCS)。现有的具有蓝白斑筛选功能的载体仅适用于β-半乳糖苷酶缺陷型基因工程菌，这种工程菌的染色体中编码β-半乳糖苷酶的基因发生突变，该突变基因的编码产物(ω肽)与野生型β-半乳糖苷酶相比缺少N段146个氨基酸，因此不具有生物学活性。尽管该β-半乳糖苷酶缺陷型菌株无法单独编码有活性的β-半乳糖苷酶，但是当菌体中含有带lacZ’基因的质粒后，质粒lacZ’基因编码的α肽和菌株表达的ω肽发生互补，得到具有β-半乳糖苷酶活性的互补产物，从而使X-gal生成蓝色物质，这种现象称为α-互补。以上是携带空载体的菌株产生的表型。当外源DNA连接入lacZ’载体的MCS后，破坏α肽读码框，重组质粒将不再表达α肽，将它导入β-半乳糖苷酶缺陷型菌株后不产生α-互补，无法产生有β-半乳糖苷酶活性的产物，因而无法分解培养基中的X-gal产生蓝色，培养表型呈现白色菌落。pUC系列载体即为常用的蓝白斑筛选载体。
然而，现有的具有蓝白斑筛选功能的载体，譬如使用非常广泛的pUC系列载体，存在以下缺点：第一，菌株适用范围具有局限性，该种载体仅适用于β-半乳糖苷酶缺陷型基因工程菌，但是在实际应用中经常使用β-半乳糖苷酶未突变的大肠杆菌，如STBL2、STBL3菌株，无法使用蓝白斑表型筛选阳性克隆；第二，每个大肠杆菌含有较低的基因组拷贝数，导致ω肽表达量很低，当菌株转化表达α肽的质粒后，形成的β-半乳糖苷酶含量低，菌株需要较长的时间才会形成明显的蓝斑，显色效率低，影响生产效率和大规模工业化应用；第三，该种载体需要使用含有IPTG的平板，但是IPTG价格昂贵且对人体有毒性，成本较高，安全性低。
因此，有必要提供一种适用菌株广泛的具有蓝白斑筛选功能的质粒，使得蓝斑显色快、并且不需要IPTG诱导。
发明内容
本申请提供了一种基因表达组件及其构建的克隆载体和应用，所述克隆载体具有蓝白斑筛选功能，几乎适用于所有类型的大肠杆菌，蓝斑显色快，不需要IPTG诱导就能够大量表达具有β-半乳糖苷酶活性的蛋白或多肽，具有重要意义和广泛的应用前景。
第一方面，本申请提供了一种基因表达组件，所述基因表达组件编码β-半乳糖苷酶α肽和ω肽；
所述基因表达组件包括组成型启动子，和所述组成型启动子启动表达的LacZα基因和 LacZω基因。
本申请中，采用组成型启动子调控LacZα基因和LacZω基因的表达，使得LacZα基因和LacZω基因可以在宿主细胞中持续稳定地表达，转录翻译得到的β-半乳糖苷酶α肽和ω肽通过α-互补作用产生具有β-半乳糖苷酶活性的物质，将X-gal分解为蓝色5-溴-4-靛蓝，实现了不需要IPTG诱导便产生β-半乳糖苷酶的技术效果，扩大了宿主细胞的适用范围，可以在任何类型的大肠杆菌进行蓝白斑筛选阳性克隆。
优选地，所述LacZα基因中含有多克隆位点；
所述LacZα基因和LacZω基因之间具有核糖体结合位点。
本申请中，LacZα基因中的多克隆位点用于插入外源DNA，而在LacZα基因和LacZω基因之间设置核糖体结合位点，则保证了在插入或未插入外源DNA的情况下，ω肽均能够正常翻译。
优选地，所述组成型启动子包括抗性基因启动子，优选为bla启动子(即Amp抗性基因启动子)。
优选地，所述LacZα基因包括SEQ ID NO:1所示的核酸序列；
SEQ ID NO:1：
Figure PCTCN2020125162-appb-000001
优选地，所述LacZω基因包括SEQ ID NO:2所示的核酸序列；
SEQ ID NO:2：
Figure PCTCN2020125162-appb-000002
Figure PCTCN2020125162-appb-000003
本申请中，以野生型LacZα基因和LacZω基因作为优化对象，按照大肠杆菌的密码子偏好性，选择使用频率最高的密码子，重新设计编码序列，并进一步优化编码序列，优化5’UTR的二级结构，得到SEQ ID NO:1所示的LacZα基因和SEQ ID NO:2所示的LacZω基因，显著提高了α肽和ω肽的表达量，增强了显色效果和显色速度，缩短了显色时间。
优选地，所述基因表达组件包括SEQ ID NO:3所示的核酸序列；
SEQ ID NO:3：
Figure PCTCN2020125162-appb-000004
Figure PCTCN2020125162-appb-000005
第二方面，本申请提供了一种克隆载体，所述克隆载体包括第一方面所述的基因表达组件。
优选地，所述克隆载体还包括大肠杆菌复制子，优选为pBR322复制子。
优选地，所述pBR322复制子包括SEQ ID NO:4所示的核酸序列；
SEQ ID NO:4：
Figure PCTCN2020125162-appb-000006
优选地，所述克隆载体还包括抗生素抗性基因。
优选地，所述抗生素抗性基因包括卡那霉素抗性基因。
优选地，所述卡那霉素抗性基因包括SEQ ID NO:5所示的核酸序列；
SEQ ID NO:5：
Figure PCTCN2020125162-appb-000007
Figure PCTCN2020125162-appb-000008
优选地，所述克隆载体包括SEQ ID NO:6所示的核酸序列；
SEQ ID NO:6：
Figure PCTCN2020125162-appb-000009
Figure PCTCN2020125162-appb-000010
本申请中，自5’端起，克隆载体的第146～3127bp的反向互补序列为β-半乳糖苷酶ω肽的编码序列，第3128～3176bp的反向互补序列为核糖体结合位点(RBS)序列，起始lacZω翻译，第3177～3542bp的反向互补序列为β-半乳糖苷酶α肽的编码序列，第3445～3533bp为多克隆位点(MCS)序列，第3543～3692bp的反向互补序列为bla启动子序列，第3935～4617bp为pBR322复制子序列，第4712～5527bp的反向互补序列为卡那霉素(Kan)的编码序列。
本申请中，构建的克隆载体pWizard质粒采用组成型启动子调控LacZα基因和LacZω基因的表达，使得LacZα基因和LacZω基因可以在宿主细胞中持续稳定地表达，不需要IPTG诱导便产生具有β-半乳糖苷酶活性的物质，可以在任何类型的大肠杆菌中通过蓝白斑筛选阳性克隆；同时，所述克隆载体pWizard质粒属于高拷贝质粒，因此，LacZα基因和LacZω基因在宿主菌中的拷贝数高，显著提高了宿主细胞对α肽和ω肽的表达量，形成大量的具有β-半乳糖苷酶活性的酶，增强了显色效果和显色速度，缩短了显色时间。
第三方面，本申请提供了一种第二方面所述的克隆载体的构建方法，所述构建方法包括：
将基因表达组件、pBR322复制子和卡那霉素抗性基因的核酸分子混合，加入核酸外切酶、DNA聚合酶和DNA连接酶，40～60℃反应0.5～1h，得到闭合的环状双链DNA；
将所述闭合的环状双链DNA转化感受态细胞，挑取单克隆培养后抽提质粒进行测序鉴定，得到所述克隆载体，命名为pWizard质粒。
本申请中，基因表达组件、pBR322复制子和卡那霉素抗性基因的核酸分子之间具有30bp的同源重组臂，有利于通过同源重组方法连接成为环状DNA。
优选地，所述反应温度例如可以是40℃、41℃、42℃、43℃、44℃、45℃、46℃、47℃、48℃、49℃、50℃、51℃、52℃、53℃、54℃、55℃、56℃、57℃、58℃、59℃或60℃，优选为50℃。
优选地，所述反应时间例如可以是0.5h、0.6h、0.7h、0.8h、0.9h或1h。
优选地，所述基因表达组件、pBR322复制子和卡那霉素抗性基因的核酸分子的摩尔比为1:1:1。
作为优选技术方案，本申请提供了一种第二方面所述的克隆载体的构建方法，所述构建方法包括：
将基因表达组件、pBR322复制子和卡那霉素抗性基因的核酸分子按照摩尔比为1:1:1混合，加入T5核酸外切酶、Phusion DNA聚合酶和Taq DNA连接酶，50℃反应0.5～1h，得到闭合的环状双链DNA；
将所述闭合的环状双链DNA转化感受态细胞，挑取单克隆培养后抽提质粒进行测序鉴定，得到所述克隆载体，命名为pWizard质粒。
第四方面，本申请提供了一种重组载体，所述重组载体为插入有外源基因的第二方面所述的克隆载体。
优选地，所述外源基因插入第二方面所述的克隆载体的多克隆位点。
第五方面，本申请提供了一种宿主细胞，所述宿主细胞的基因组中整合有第一方面所述的基因表达组件，和/或所述宿主细胞包括第二方面所述的克隆载体和/或第四方面所述的重组载体。
优选地，所述宿主细胞包括β-半乳糖苷酶野生型基因工程菌和/或β-半乳糖苷酶缺陷型基因工程菌，包括但不限于Top10、DH5α、STBL2或STBL3大肠杆菌。
第六方面，本申请提供了一种试剂盒，所述试剂盒包括第一方面所述的基因表达组件、第二方面所述的克隆载体、第四方面所述的重组载体或第五方面所述的宿主细胞中的任意一种或至少两种的组合。
第七方面，本申请提供了一种外源基因克隆方法，所述方法包括：
将外源基因插入第二方面所述的克隆载体的多克隆位点，导入宿主细胞，培养后根据蓝白斑筛选获得表达外源基因的阳性克隆。
优选地，所述方法还包括从阳性克隆培养上清或阳性克隆菌体中分离纯化得到蛋白质的步骤。
第八方面，本申请提供了一种第一方面所述的基因表达组件、第二方面所述的克隆载体、第四方面所述的重组载体、第五方面所述的宿主细胞或第六方面所述的试剂盒在蛋白质制备中的应用。
与现有技术相比，本申请具有如下有益效果：
(1)本申请的pWizard质粒采用组成型启动子调控LacZα基因和LacZω基因的表达，使得LacZα基因和LacZω基因可以在宿主细胞中持续稳定地表达，不需要IPTG诱导便产生具有β-半乳糖苷酶活性的物质，可以涂布培养于不含IPTG仅含X-gal的平板中，扩大了宿主 细胞的适用范围，可以在任何类型的大肠杆菌进行蓝白斑筛选阳性克隆；
(2)本申请对野生型LacZα基因和LacZω基因进行了密码子优化，构建的pWizard质粒在宿主菌中的LacZα基因和LacZω基因拷贝数远高于基因组拷贝数，显著提高了宿主细胞对α肽和ω肽的表达量，通过α-互补作用提高了具有β-半乳糖苷酶活性的酶的表达量，增强了显色效果和显色速度，缩短了显色时间。
附图说明
图1为pWizard质粒图谱；
图2为pUC57质粒转化DH5α菌株、涂布含IPTG和X-gal平板；
图3为pUC57质粒转化Top10F’菌株、涂布含IPTG和X-gal平板；
图4为pUC57质粒转化STBL3菌株、涂布含IPTG和X-gal平板；
图5为pWizard质粒转化DH5α菌株、涂布含X-gal平板；
图6为pWizard质粒转化Top10F’菌株、涂布含X-gal平板；
图7为pWizard质粒转化STBL3菌株、涂布含X-gal平板；
图8为pET-30a质粒转化STBL3菌株、涂布含IPTG和X-gal平板；
图9为pWizard质粒平末端克隆外源DNA转化DH5α菌株、涂布含X-gal平板；
图10为pWizard质粒平末端克隆外源DNA转化STBL3菌株、涂布含X-gal平板；
图11为平末端克隆外源DNA菌检结果，其中，泳道1-8为DH5α菌检结果，泳道9-16为STBL3菌检结果，泳道M为DNA ladder，条带大小分别为100bp、250bp、500bp、750bp、1kb、1.5kb、2kb、3kb和5kb；
图12为pWizard质粒粘性末端克隆外源DNA转化DH5α菌株、涂布含X-gal平板；
图13为pWizard质粒粘性末端克隆外源DNA转化STBL3菌株、涂布含X-gal平板；
图14为粘性末端克隆外源DNA菌检结果，其中，泳道1-8为DH5α菌检结果，泳道9-16为STBL3菌检结果，泳道M为DNA ladder，条带大小分别为100bp、250bp、500bp、750bp、1kb、1.5kb、2kb、3kb和5kb。
具体实施方式
为进一步阐述本申请所采取的技术手段及其效果，以下结合实施例和附图对本申请作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本申请，而非对本申请的限定。
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：广泛应用于基因表达调控研究的一种基因，编码的β-半乳糖苷酶(简称β-gal)是由4个亚基组成的四聚体，可以催化乳糖水解，β-gal稳定性好，用X-Gal作底物进行染色时，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β-半乳糖苷酶活性的酶，将无色化合物X-gal(5-溴-4-氯-3-吲哚-β-D-半乳糖苷)切割为半乳糖和深蓝色物质5-溴-4-靛蓝；
核酸内切酶：水解核酸分子链内部磷酸二酯键生成寡核苷酸的酶；
PCR技术：即聚合酶链式反应，DNA在体外95℃高温时变性为单链，低温(60℃左右)时引物与单链按照碱基互补配对原则进行结合，再将温度调节至DNA聚合酶的最适反应温度(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仪是一个温控设备，能够在变性温度、复性温度和延伸温度之间很好地控制温度的变化。
材料：
DH5α感受态细胞赛默飞世尔科技公司
Top10F’感受态细胞赛默飞世尔科技公司
STBL3感受态细胞赛默飞世尔科技公司
EcoRV限制性内切酶NEB公司
KpnI限制性内切酶NEB公司
HindIII限制性内切酶NEB公司
T4DNA连接酶NEB公司
λDNA NEB公司
Gibson
Figure PCTCN2020125162-appb-000011
Master Mix试剂盒NEB公司
引物合成苏州金唯智生物科技有限公司
实施例1 pWizard质粒的构建
(1)设计并合成SEQ ID NO:3所示的基因表达组件bla-α-RBS-ω，序列全长3700bp，其中，自5’端起，第146～3127bp的反向互补序列为LacZω基因，编码ω肽，第3128～3176bp的反向互补序列为RBS序列，起始LacZω基因的转录翻译，第3177～3542bp的反向互补序列为LacZα基因，编码α肽，第3445～3533bp为多克隆位点(MCS)序列，第3543～3692bp的反向互补序列为bla启动子；
(2)设计并合成SEQ ID NO:4所示的pBR322复制子，序列全长1030bp，自5’端起，第265～947bp为pBR322基因序列；
(3)设计并合成SEQ ID NO:5所示的Kan基因，序列全长1055bp，自5’端起，第42～857bp为Kan基因序列；
(4)上述三个片段之间具有30bp的同源序列，采用Gibson
Figure PCTCN2020125162-appb-000012
Master Mix(NEB)试剂盒进行连接，反应体系为SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:5三个基因片段各2μL，灭菌去离子水4μL，Gibson
Figure PCTCN2020125162-appb-000013
Master Mix 10μL(含有T5核酸外切酶、Phusion DNA聚合酶和Taq DNA连接酶)，反应条件为50℃反应1h，组装形成环状克隆载体，命名为pWizard质粒；
(5)反应产物转化DH5α感受态细胞，涂布Kan抗性平板；
(6)次日挑取数个单克隆，培养后抽提质粒，进行Sanger测序，测序正确的质粒即为pWizard质粒，质粒长度为5695bp。
如图1所示，pWizard质粒中主要元件的位置分别为：自5’端起，第146～3127bp的反向互补序列为LacZω基因，编码ω肽，第3128～3176bp的反向互补序列为RBS序列，起始LacZω基因的转录翻译，第3177～3542bp的反向互补序列为LacZα基因，编码α肽，第3445～3533bp为多克隆位点(MCS)序列，第3543～3692bp的反向互补序列为bla启动子，第3935～4617bp为pBR322复制子序列，第4712～5527bp的反向互补序列为Kan基因序列。
实施例2 pWizard质粒转化大肠杆菌在非IPTG诱导条件下形成蓝斑
(1)分别吸取约50ng pWizard质粒加入到Top10F’、DH5α、STBL3感受态细胞中，分别吸取约50ng pUC57质粒(金唯智保存)加入到Top10F’、DH5α、STBL3感受态细胞中，同时，吸取约50ng pET-30a质粒(金唯智保存)加入到STBL3感受态细胞中；
(2)冰浴30min；
(3)42℃热激90s，冰浴3min；
(4)向感受态细胞中分别加入700μL预热的LB培养基，并于37℃、220rpm的条件下复苏1小时；
(5)从转化pWizard质粒的感受态细胞中吸取适量菌液均匀涂布于含X-gal和Kan抗性的LB平板上，同时，从转化pUC57和pET-30a质粒的感受态细胞中吸取适量菌液均匀涂布于含IPTG、X-gal和Kan抗性的LB平板上；
(6)将平板置于37℃条件下过夜培养。
次日观察菌落表型，结果见图2～图8，其中，图2为pUC57质粒转化DH5α菌株、涂布含IPTG和X-gal平板，图3为pUC57质粒转化Top10F’菌株、涂布含IPTG和X-gal平板，图4为pUC57质粒转化STBL3菌株、涂布含IPTG和X-gal平板，图5为pWizard质粒转化DH5α菌株、涂布含X-gal平板，图6为pWizard质粒转化Top10F’菌株、涂布含X-gal平板，图7为pWizard质粒转化STBL3菌株、涂布含X-gal平板，图8为pET-30a质粒转化STBL3菌株、涂布含IPTG和X-gal平板。
可以看出，所有平板的克隆均显蓝色，表明pWizard质粒不需要IPTG诱导便可以表达具有β-半乳糖苷酶活性的物质，STBL3感受态细胞不适用于普通的需要IPTG诱导的蓝白斑筛选载体；此外，与转化pUC57质粒的感受态细胞相比，转化pWizard质粒的感受态细胞形成的菌落蓝色更深，说明在相同的培养时间内，转化pWizard质粒的感受态细胞能够表达更多的活性β-半乳糖苷酶，使蓝白斑更易区分，更易于挑取阳性克隆。
实施例3 使用pWizard质粒平末端克隆外源DNA片段
(1)以λDNA为模板，F-λDNA-Blunt和R-λDNA-Blunt为引物进行PCR扩增，PCR扩增体系和扩增程序如表1和表2所示；
F-λDNA-Blunt(SEQ ID NO:7)：ACGTTTGAGCAGAATAACCATGTGG；
R-λDNA-Blunt(SEQ ID NO:8)：AAATAACGTTCTCCACCGACCTCTG；
表1
试剂 用量
ddH 2O 75μL
5×Buffer 20μL
dNTP 1μL
F-λDNA-Blunt 1μL
R-λDNA-Blunt 1μL
λDNA 1μL
pfu 1μL
总体积 100μL
表2
Figure PCTCN2020125162-appb-000014
(2)PCR反应结束后使用1％琼脂糖凝胶进行电泳检测，目的片段大小600bp，对目的片段进行切胶，采用Axygen公司的凝胶回收试剂盒进行回收纯化，采用Nanodrop2000测定纯化的DNA浓度约120ng/μL；
(3)使用EcoRV限制性内切酶酶切pWizard质粒，酶切反应体系如表3所示，37℃酶切1小时；
表3
10×buffer(μL) pWizard(μL) EcoRV(μL) 无菌ddH 2O(μL)
6 10(～3μg) 3 41
(4)使用Axygen公司的凝胶回收试剂盒对酶切产物进行回收纯化，Nanodrop2000测定纯化的酶切产物的浓度约20ng/μL；
(5)按照表4配制连接反应体系，将纯化的PCR片段与EcoRV酶切的pWizard质粒进行连接反应，22℃连接1小时；
表4
10×buffer(μL) 线性pWizard质粒(μL) λDNA扩增片段(μL) 无菌ddH 2O(μL)
1 1 2 6
(6)分别取5μL连接产物转化DH5α和STBL3感受态细胞，菌液涂布含X-gal和Kan抗性的LB平板；
(7)置于37℃下过夜培养。
次日观察菌落表型，结果见图9～图10，其中，图9为pWizard质粒平末端克隆外源DNA转化DH5α菌株、涂布含X-gal平板，图10为pWizard质粒平末端克隆外源DNA转化STBL3菌株、涂布含X-gal平板。
可以看出，pWizard质粒作为克隆载体不需要IPTG诱导便可以形成明显的蓝白斑，表明pWizard质粒适用于STBL3等LacZ基因未突变的菌株，可以通过蓝白斑筛选阳性克隆。
从平板中随机挑取8个单克隆进行菌落PCR，使用JJ-F-pWizard和JJ-R-pWizard作为引物，电泳结果如图11所示，其中，泳道1-8为DH5α菌检结果，泳道9-16为STBL3菌检结果，可以看出菌检产物大小约900bp；从每个平板中随机挑取两个阳性克隆进行Sanger测序，测序结果显示序列正确；
JJ-F-pWizard(SEQ ID NO:9)：CTTCTTCGCTGTTGCGCCAGCTCGC；
JJ-R-pWizard(SEQ ID NO:10)：GTATCCGCTCATGAGACAATAACCC。
实施例4 使用pWizard质粒粘性末端克隆外源DNA片段
(1)以λDNA为模板，F-λDNA-Sticky和R-λDNA-Sticky为引物进行PCR扩增，PCR扩增体系和扩增程序如表1和表2所示；
F-λDNA-Sticky(SEQ ID NO:11)：
CTGACCGC GGTACCCGTGGAACCCACCGAGTGAAAGTGT(下划线为KpnI酶切位点)；
R-λDNA-Sticky(SEQ ID NO:12)：
CAATGTTG AAGCTTCTGCGTATCCAGCTCACTCTCAATG(下划线为HindⅢ酶切位点)；
(2)PCR反应结束后使用1％琼脂糖凝胶进行电泳检测，目的片段大小约758bp，对目的片段进行切胶，采用Axygen公司的凝胶回收试剂盒进行回收纯化，采用Nanodrop2000测定纯化的DNA浓度约160ng/μL；
(3)使用KpnI和HindⅢ限制性内切酶酶切回收的PCR产物，酶切反应体系如表5所示，37℃酶切1小时；
表5
10×buffer(μL) 纯化的PCR产物(μL) KpnI(μL) HindⅢ(μL) 无菌ddH 2O(μL)
4 13 2 2 19
(4)使用Axygen公司的凝胶回收试剂盒对酶切产物进行回收纯化，Nanodrop2000测定纯化的酶切产物的浓度约20ng/μL；
(5)使用KpnI和HindⅢ限制性内切酶酶切pWizard质粒，酶切反应体系如表6所示，37℃酶切1小时；
表6
10×buffer(μL) pWizard(μL) KpnI(μL) HindⅢ(μL) 无菌ddH 2O(μL)
6 10(～3μg) 3 3 38
(6)使用Axygen公司的凝胶回收试剂盒对酶切产物进行回收纯化，Nanodrop2000测定纯化的酶切产物的浓度约15ng/μL；
(7)按照表4配制连接反应体系，将纯化的PCR片段与KpnI和HindⅢ酶切的pWizard质粒进行连接反应，22℃连接1小时；
(8)分别取5μL连接产物转化DH5α和STBL3感受态细胞，菌液涂布含X-gal和Kan抗性的LB平板；
(9)置于37℃下过夜培养。
次日观察菌落表型，结果见图12～图13，其中，图12为pWizard质粒粘性末端克隆外源DNA转化DH5α菌株、涂布含X-gal平板，图13为pWizard质粒粘性末端克隆外源DNA转化STBL3菌株、涂布含X-gal平板。
可以看出，形成的菌落几乎全是白斑，仅有极少数蓝斑出现，极少数蓝斑很可能是由没有被酶切的残留质粒转化形成。
从平板中随机挑取8个单克隆进行菌落PCR，使用JJ-F-pWizard和JJ-R-pWizard作为引物，电泳结果如图14所示，其中，泳道1-8为DH5α菌检结果，泳道9-16为STBL3菌检结果，可以看出菌检产物大小约1kb；从每个平板中随机挑取两个阳性克隆进行Sanger测序，测序结果显示序列正确。
综上所述，本申请采用组成型启动子调控LacZα基因和LacZω基因的表达，使得LacZα基因和LacZω基因可以在宿主细胞中持续稳定地表达，构建的克隆载体具有蓝白斑筛选功能，几乎适用于所有类型的大肠杆菌，蓝斑显色快，不需要IPTG诱导就能够大量表达具有β-半乳糖苷酶活性的蛋白或多肽，具有重要意义和广泛的应用前景。
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. 一种基因表达组件，其编码β-半乳糖苷酶α肽和ω肽；
    其中所述基因表达组件包括组成型启动子，和所述组成型启动子启动表达的LacZα基因和LacZω基因。
  2. 根据权利要求1所述的基因表达组件，其中，所述LacZα基因中含有多克隆位点；并且
    所述LacZα基因和LacZω基因之间具有核糖体结合位点。
  3. 根据权利要求1或2所述的基因表达组件，其中，所述组成型启动子包括抗性基因启动子，优选为bla启动子。
  4. 根据权利要求1至3中任一项所述的基因表达组件，其中，所述LacZα基因包括SEQ ID NO:1所示的核酸序列；
    优选地，所述LacZω基因包括SEQ ID NO:2所示的核酸序列；
    优选地，所述基因表达组件包括SEQ ID NO:3所示的核酸序列。
  5. 一种克隆载体，其包括权利要求1至4中任一项所述的基因表达组件。
  6. 根据权利要求5所述的克隆载体，其中，所述克隆载体还包括大肠杆菌复制子，优选为pBR322复制子；
    优选地，所述pBR322复制子包括SEQ ID NO:4所示的核酸序列。
  7. 根据权利要求5或6所述的克隆载体，其中，所述克隆载体还包括抗生素抗性基因；
    优选地，所述抗生素抗性基因包括卡那霉素抗性基因；
    优选地，所述卡那霉素抗性基因包括SEQ ID NO:5所示的核酸序列；
    优选地，所述克隆载体包括SEQ ID NO:6所示的核酸序列。
  8. 一种权利要求5至7中任一项所述的克隆载体的构建方法，包括：
    将基因表达组件、pBR322复制子和卡那霉素抗性基因的核酸分子混合，加入核酸外切酶、DNA聚合酶和DNA连接酶，40～60℃反应0.5～1h，得到闭合的环状双链DNA；
    将所述闭合的环状双链DNA转化感受态细胞，挑取单克隆培养后抽提质粒进行测序鉴定，得到所述克隆载体。
  9. 根据权利要求8所述的构建方法，其中，所述基因表达组件、pBR322复制子和卡那霉素抗性基因的核酸分子的摩尔比为1:1:1。
  10. 一种重组载体，其中，所述重组载体为插入有外源基因的权利要求5至7中任一项所述的克隆载体；
    优选地，所述外源基因插入权利要求5至7中任一项所述的克隆载体的多克隆位点。
  11. 一种宿主细胞，其中，所述宿主细胞的基因组中整合有权利要求1至4中任一项所述的基因表达组件，和/或所述宿主细胞包括权利要求5至7中任一项所述的克隆载体和/或权利要求10所述的重组载体；
    优选地，所述宿主细胞包括β-半乳糖苷酶野生型基因工程菌和/或β-半乳糖苷酶缺陷型基因工程菌；
    优选地，所述宿主细胞包括Top10、DH5α、STBL2或STBL3。
  12. 一种试剂盒，其中，所述试剂盒包括权利要求1至4中任一项所述的基因表达组件、权利要求5至7中任一项所述的克隆载体、权利要求10所述的重组载体或权利要求11所述 的宿主细胞中的任意一种或至少两种的组合。
  13. 一种外源基因克隆方法，其中，所述方法包括：
    将外源基因插入权利要求5至7中任一项所述的克隆载体的多克隆位点，导入宿主细胞，培养后根据蓝白斑筛选获得表达外源基因的阳性克隆。
  14. 根据权利要求13所述的外源基因克隆方法，其中，所述方法还包括从阳性克隆培养上清或阳性克隆菌体中分离纯化得到蛋白质的步骤。
  15. 一种权利要求1至4中任一项所述的基因表达组件、权利要求5至7中任一项所述的克隆载体、权利要求10所述的重组载体、权利要求11所述的宿主细胞或权利要求12所述的试剂盒在基因克隆、蛋白质制备或测序领域中的应用。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113980995A (zh) * 2021-10-12 2022-01-28 中国热带农业科学院热带作物品种资源研究所 一种植物基因表达载体的构建方法和应用

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111378679B (zh) * 2020-03-20 2023-10-03 苏州金唯智生物科技有限公司 一种基因表达组件及其构建的克隆载体和应用
CN113549652A (zh) * 2021-07-21 2021-10-26 华农（肇庆）生物产业技术研究院有限公司 SFV-helper质粒、pSFVCs-LacZ病毒样颗粒及其制备方法和应用
CN114164225B (zh) * 2021-12-28 2023-10-03 苏州金唯智生物科技有限公司 一种使大肠杆菌获得有效nhej系统的高通量筛选工具及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106086060A (zh) * 2016-06-13 2016-11-09 福建师范大学 一种筛选毕赤酵母启动子的系统和方法
CN106350534A (zh) * 2016-08-25 2017-01-25 深圳市职业病防治院 以lacZα短肽编码基因为第二基因编码框的双顺反子特异DNA及其应用
CN111378679A (zh) * 2020-03-20 2020-07-07 苏州金唯智生物科技有限公司 一种基因表达组件及其构建的克隆载体和应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103320433A (zh) * 2013-06-06 2013-09-25 中国科学院广州生物医药与健康研究院 一种用于高效构建无抗性标记重组分枝杆菌的抗性表达盒
CN104480130B (zh) * 2014-11-20 2017-11-14 苏州金唯智生物科技有限公司 一种pTerm‑SC质粒及其构建方法和应用
CN104388454B (zh) * 2014-11-20 2018-03-23 苏州金唯智生物科技有限公司 一种高拷贝pTerm质粒及其构建方法和应用
CN104404069B (zh) * 2014-11-20 2018-02-16 苏州金唯智生物科技有限公司 一种低拷贝pTerm质粒及其构建方法和应用
CN108118059B (zh) * 2017-12-30 2021-03-19 苏州金唯智生物科技有限公司 一种改进的启动子及其组成的载体和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106086060A (zh) * 2016-06-13 2016-11-09 福建师范大学 一种筛选毕赤酵母启动子的系统和方法
CN106350534A (zh) * 2016-08-25 2017-01-25 深圳市职业病防治院 以lacZα短肽编码基因为第二基因编码框的双顺反子特异DNA及其应用
CN111378679A (zh) * 2020-03-20 2020-07-07 苏州金唯智生物科技有限公司 一种基因表达组件及其构建的克隆载体和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"Bacterial Protein Secretion Systems : Methods and Protocols; IN: Methods in Molecular Biology,", vol. 1615, 31 December 2017, SPRINGER NEW YORK, New York, NY, ISBN: 978-1-4939-7033-9, ISSN: 1064-3745, article KARIMOVA GOUZEL, LADANT DANIEL: "Defining Membrane Protein Topology Using pho-lac Reporter Fusions", pages: 129 - 142, XP009530923, DOI: 10.1007/978-1-4939-7033-9_10 *
HUANG YIDE, ZHANG YAFEI, LI SUHUAN, LIN TING, WU JINGWEN, LIN YAO: "Screening for functional IRESes using α-complementation system of β-galactosidase in Pichia pastoris", BIOTECHNOLOGY FOR BIOFUELS, vol. 12, no. 1, 27 December 2019 (2019-12-27), XP055851724, DOI: 10.1186/s13068-019-1640-3 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113980995A (zh) * 2021-10-12 2022-01-28 中国热带农业科学院热带作物品种资源研究所 一种植物基因表达载体的构建方法和应用

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