WO2020124319A1 - 融合蛋白及其应用 - Google Patents

融合蛋白及其应用 Download PDF

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Publication number
WO2020124319A1
WO2020124319A1 PCT/CN2018/121545 CN2018121545W WO2020124319A1 WO 2020124319 A1 WO2020124319 A1 WO 2020124319A1 CN 2018121545 W CN2018121545 W CN 2018121545W WO 2020124319 A1 WO2020124319 A1 WO 2020124319A1
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Prior art keywords
fusion protein
fragment
construct
present
nucleic acid
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French (fr)
Inventor
王�琦
郭斐
郭苗苗
李敬
郑越
董宇亮
章文蔚
徐崇钧
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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Priority to PCT/CN2018/121545 priority Critical patent/WO2020124319A1/zh
Priority to CN201880099747.3A priority patent/CN113166270A/zh
Publication of WO2020124319A1 publication Critical patent/WO2020124319A1/zh
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    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes

Definitions

  • the invention relates to the field of biology.
  • the invention relates to fusion proteins and their applications. More specifically, the present invention relates to fusion proteins and methods and applications for obtaining them, isolated nucleic acid molecules, constructs, and recombinant cells.
  • DNA ligase is a kind of important molecular biology tool enzyme that can catalyze the formation of phosphodiester bond between two DNA or RNA fragments.
  • the ligation reaction includes gap ligation, cohesive end ligation, TA ligation, blunt end ligation, and branch ligation. Branch), where the TA connection and the flat end connection are widely used in the library building connection, but the efficiency is low.
  • the sources of DNA ligase are diverse and cover almost all species.
  • T4 DNA ligase from T4 bacteriophage has become a unique and significant blunt end or TA end ligation activity for molecular cloning, nucleic acid modification, sequencing and library construction. There is no substitute for an enzyme.
  • junction connection based on blunt ends or TA ends is an indispensable key step in NGS database construction, and its efficiency directly affects the coverage and efficiency of subsequent sequencing results.
  • the reaction system of the linker connection reaction also has a greater impact on the upstream and downstream operations, and it can be determined whether additional nucleic acid purification steps need to be added.
  • the present invention aims to solve at least one of the problems in the prior art at least to a certain extent.
  • the present invention proposes a fusion protein, which can effectively improve the efficiency of linker connection, and is particularly suitable for the linker connection of blunt ends/TA ends, which is convenient for subsequent library construction and sequencing.
  • it simplifies the experimental process and avoids the losses caused by excessive purification.
  • the density and viscosity of the sequencing library construction system are smaller, which is more suitable for the automation platform.
  • T4 DNA ligase alone is based on blunt-end/TA-end linker connection efficiency is low, usually need to add high-density and high-viscosity auxiliary additives (such as 10-30% polyethylene glycol) to achieve Increased connection efficiency.
  • high-viscosity and high-density auxiliary additives will affect the accuracy of subsequent library construction and sequencing, and are not suitable for automated platforms.
  • the inventors found that by combining T4 DNA ligase with a protein having a DNA binding domain (eg Sso 7d binding protein, NF ⁇ B p50 protein), the protein having a DNA binding domain can fix the linker in order to T4 DNA ligase is fully functional and can significantly improve the efficiency of linker ligation in the construction of sequencing libraries. It is especially suitable for ligation of blunt-end/TA-end linkers, which facilitates subsequent sequencing, simplifies the experimental process, and avoids losses caused by excessive purification.
  • a protein having a DNA binding domain eg Sso 7d binding protein, NF ⁇ B p50 protein
  • the density and viscosity of the sequencing library construction system are small, which improves the accuracy and specificity of subsequent library construction and sequencing, and is more suitable for automated platforms.
  • the invention proposes a fusion protein.
  • the fusion protein includes: a first fragment having T4 DNA ligase activity; and a second fragment having one end of the first fragment connected to one end of the second fragment , The second fragment has a DNA binding domain.
  • the protein with a DNA binding domain can fix the linker, so that T4 DNA ligase can fully play its role
  • the efficiency of linker connection can be significantly improved, especially suitable for the connection of blunt end/TA end connection, which facilitates subsequent sequencing, simplifies the experimental process, and avoids the loss caused by excessive purification.
  • the density and viscosity of the sequencing library construction system are small, which improves the accuracy and specificity of subsequent library construction and sequencing, and is more suitable for automated platforms.
  • the fusion protein may also have the following additional technical features:
  • the second fragment has Sso 7d DNA binding protein activity or NF ⁇ B p50 protein activity.
  • the first fragment has the amino acid sequence shown in SEQ ID NO: 1.
  • the second fragment has the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 3.
  • the fusion protein further includes purified fragments and/or screening fragments.
  • the purified fragment is selected from at least one of His and Flag tags.
  • the screening fragment has kanamycin resistance and/or ampicillin resistance.
  • the fusion protein has the amino acid sequence shown in SEQ ID NO: 4 or 5.
  • the invention proposes an isolated nucleic acid molecule.
  • the isolated nucleic acid molecule encodes the aforementioned fusion protein.
  • the fusion protein encoded by the isolated nucleic acid molecule can significantly improve the efficiency of linker connection in the construction of a sequencing library, and is particularly suitable for the linker connection of blunt ends/TA ends, without the need to add high viscosity and high density auxiliary additives such as PEG, which is convenient for subsequent sequencing , Simplify the experimental process and avoid the loss caused by excessive purification.
  • the density and viscosity of the sequencing library construction system are small, which improves the accuracy and specificity of subsequent library construction and sequencing, and is more suitable for automated platforms.
  • the isolated nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 6 or 7.
  • the invention proposes a construct.
  • the construct contains the aforementioned isolated nucleic acid molecule.
  • the fusion protein expressed by the construct of the present invention can significantly improve the efficiency of linker connection in the construction of a sequencing library, and is particularly suitable for linker connection of blunt ends/TA ends. There is no need to add high viscosity and high density auxiliary additives such as PEG, which is convenient for subsequent sequencing. Simplify the experimental process and avoid the loss caused by excessive purification.
  • the density and viscosity of the sequencing library construction system are small, which improves the accuracy and specificity of subsequent library construction and sequencing, and is more suitable for automated platforms.
  • the construct is selected from at least one of plasmids, bacteriophages, artificial chromosomes, cosmids, and viruses.
  • the construct is selected from plasmids.
  • the construct is selected from pET28a plasmid.
  • the construct further includes a promoter operably linked to the nucleotide sequence shown in SEQ ID NO: 6 or 7.
  • the invention provides a recombinant cell.
  • the recombinant cell contains the aforementioned isolated nucleic acid molecule or the aforementioned construct.
  • the fusion protein expressed by the recombinant cell of the present invention can significantly improve the efficiency of linker connection in the construction of a sequencing library, and is particularly suitable for the linker connection of blunt ends/TA ends, without the need to add high viscosity and high density auxiliary additives such as PEG, which is convenient for subsequent sequencing. Simplify the experimental process and avoid the loss caused by excessive purification.
  • the density and viscosity of the sequencing library construction system are small, which improves the accuracy and specificity of subsequent library construction and sequencing, and is more suitable for automated platforms.
  • the present invention provides a method for obtaining the aforementioned fusion protein.
  • the recombinant cells described above are cultured under conditions suitable for the expression of the fusion protein, so as to obtain the fusion protein.
  • the fusion protein obtained by the method of the present invention can significantly improve the efficiency of linker connection in constructing a sequencing library, and is particularly suitable for linker connection of blunt end/TA end connection, without the need to add high viscosity and high density auxiliary additives such as PEG, which is convenient for subsequent sequencing , Simplify the experimental process and avoid the loss caused by excessive purification.
  • the density and viscosity of the sequencing library construction system are small, which improves the accuracy and specificity of subsequent library construction and sequencing, and is more suitable for automated platforms.
  • the present invention proposes the application of the aforementioned fusion protein in constructing a sequencing library.
  • the fusion protein of the present invention can significantly improve the efficiency of linker connection in the construction of a sequencing library, and is particularly suitable for linker connection of blunt ends/TA ends.
  • high viscosity and high density auxiliary additives such as PEG, which facilitates subsequent sequencing and simplifies the experimental process. Avoid losses caused by excessive purification.
  • PEG and other high-viscosity and high-density additives the density and viscosity of the sequencing library construction system are small, which improves the accuracy and specificity of subsequent library construction and sequencing, and is more suitable for automated platforms.
  • FIG. 1 shows an electrophoresis diagram of purification results of recombinant T4 DNA ligase fusion protein G1 according to an embodiment of the present invention
  • FIG. 2 shows an electrophoresis diagram of the purification result of recombinant T4 DNA ligase fusion protein G2 according to an embodiment of the present invention
  • FIG. 3 shows an electrophoresis diagram of the test result of the ligation efficiency of the blunt end of the recombinant T4 DNA ligase fusion protein G1 according to an embodiment of the present invention
  • FIG. 4 shows an electrophoresis diagram of the test result of the ligation efficiency of the blunt end of the recombinant T4 DNA ligase fusion protein G2 according to an embodiment of the present invention.
  • FIG. 5 shows an analysis diagram of the connection efficiency of the NGS library construction connector according to an embodiment of the present invention.
  • the present invention proposes fusion proteins, isolated nucleic acid molecules, constructs, recombinant cells, methods for obtaining fusion proteins, and applications of fusion proteins in constructing sequencing libraries, which will be described in detail below.
  • the invention proposes a fusion protein.
  • the fusion protein includes: a first fragment having T4 DNA ligase activity; and a second fragment, one end of the first fragment is connected to one end of the second fragment, the second fragment With DNA binding domain.
  • the protein with a DNA binding domain can fix the linker, so that T4 DNA ligase can fully play its role
  • the efficiency of linker connection can be significantly improved, especially suitable for the connection of blunt end/TA end connection, which facilitates subsequent sequencing, simplifies the experimental process, and avoids the loss caused by excessive purification.
  • the density and viscosity of the sequencing library construction system are small, which improves the accuracy and specificity of subsequent library construction and sequencing, and is more suitable for automated platforms.
  • amino acid fragments are generally divided into N-terminal and C-terminal.
  • the connection method of the first fragment and the second fragment is not strictly limited in the present invention, as long as it is the N-terminal of one fragment and the C of another fragment The end can be connected.
  • the second fragment has Sso 7d DNA binding protein activity or NF ⁇ B p50 protein activity.
  • the Sso 7d binding protein is derived from the hyperthermophilic archaebacterium Sulfolobus solfataricus, with a molecular weight of approximately 7.0 kDa, and a denaturation temperature near neutral pH close to 100°C. It has non-sequence-specific DNA binding activity and binds to the minor groove of double-stranded DNA.
  • NF ⁇ B p50 is a eukaryotic transcription factor derived from the eukaryotic Homo sapiens, with a molecular weight of approximately 36.5 kDa and a DNA binding domain, which can enhance the expression of specific anti-apoptotic genes.
  • the density and viscosity of the sequencing library construction system are small, which improves the accuracy and specificity of subsequent library construction and sequencing, and is more suitable for automated platforms.
  • the first fragment has the amino acid sequence shown in SEQ ID NO:1.
  • T4 DNA ligase is a commonly used DNA ligase in library construction, especially suitable for blunt end or TA end ligation.
  • the second fragment has the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 3.
  • the fusion protein formed by Sso 7d binding protein (SEQ ID NO: 2) or NF ⁇ B p50 protein (SEQ ID NO: 3) and T4 DNA ligase can significantly improve the efficiency of linker connection, especially suitable for blunt end/TA end connection
  • the connection facilitates subsequent sequencing, simplifies the experimental process, and avoids the loss caused by excessive purification.
  • high viscosity and high density additives such as PEG, the density and viscosity of the sequencing library construction system are small, which improves the accuracy and specificity of subsequent library construction and sequencing, and is more suitable for automated platforms.
  • the fusion protein further includes purified fragments and/or screening fragments. In order to achieve the purpose of screening and separation.
  • the purified fragment is selected from at least one of His and Flag tags. This makes it easy to screen for positive transformants.
  • the screening fragment has kanamycin resistance and/or ampicillin resistance.
  • the efficiency of screening recombinant cells that receive foreign constructs can be further improved.
  • the fusion protein has the amino acid sequence shown in SEQ ID NO: 4 or 5.
  • the fusion protein formed by T4 DNA ligase and Sso 7d DNA binding protein (SEQ ID NO: 4) or the fusion protein formed by T4 DNA ligase and NF ⁇ B p50 protein (SEQ ID NO: 5) can significantly improve the joint connection efficiency It is especially suitable for the connection of blunt end/TA end connection, which facilitates subsequent sequencing, simplifies the experimental process, and avoids the loss caused by excessive purification.
  • the density and viscosity of the sequencing library construction system are small, which improves the accuracy and specificity of subsequent library construction and sequencing, and is more suitable for automated platforms.
  • the invention proposes an isolated nucleic acid molecule.
  • the isolated nucleic acid molecule encodes the aforementioned fusion protein.
  • the fusion protein encoded by the isolated nucleic acid molecule can significantly improve the efficiency of linker connection in the construction of a sequencing library, and is particularly suitable for the linker connection of blunt ends/TA ends, without the need to add high viscosity and high density auxiliary additives such as PEG, which is convenient for subsequent sequencing , Simplify the experimental process and avoid the loss caused by excessive purification.
  • the density and viscosity of the sequencing library construction system are small, which improves the accuracy and specificity of subsequent library construction and sequencing, and is more suitable for automated platforms.
  • the isolated nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 6 or 7.
  • the nucleotide sequence shown in SEQ ID NO: 6 encodes the amino acid sequence shown in SEQ ID NO: 4
  • the nucleotide sequence shown in SEQ ID NO: 7 encodes the amino acid sequence shown in SEQ ID NO: 5.
  • the invention proposes a construct.
  • the construct contains the aforementioned isolated nucleic acid molecule.
  • the fusion protein expressed by the construct of the present invention can significantly improve the efficiency of linker connection in the construction of a sequencing library, and is particularly suitable for linker connection of blunt ends/TA ends. There is no need to add high viscosity and high density auxiliary additives such as PEG, which is convenient for subsequent sequencing. Simplify the experimental process and avoid the loss caused by excessive purification.
  • the density and viscosity of the sequencing library construction system are small, which improves the accuracy and specificity of subsequent library construction and sequencing, and is more suitable for automated platforms.
  • the construct is selected from at least one of plasmids, bacteriophages, artificial chromosomes, cosmids and viruses, preferably plasmids, more preferably pET28a plasmids. This can effectively improve the efficiency of genetic transformation using the construct of the present invention.
  • the construct of the present invention may also include other elements, so as to impart additional beneficial effects to the construct.
  • the construct further includes a promoter operably linked to the nucleotide sequence shown in SEQ ID NO: 6 or 7.
  • promoter used in the present invention refers to a nucleic acid sequence capable of directing the transcription of a nucleic acid molecule operably linked thereto.
  • operably used in the present invention refers to a functional connection between a nucleic acid expression control sequence such as a promoter, a signal sequence, an enhancer, etc. and a target nucleic acid sequence, wherein when a suitable molecule such as a transcription activation molecule is When the expression control sequence is combined, the expression control sequence affects the transcription and/or translation of the nucleic acid corresponding to the target nucleic acid sequence.
  • a specific promoter can be directly introduced into the host cell through the construct, and the promoter can be used to initiate the transcription and expression of the nucleotide sequence shown in SEQ ID NO: 6 or 7, which can improve The expression efficiency of T4 DNA ligase gene, Ssco7d binding protein gene/NF ⁇ B p50 protein in the obtained recombinant cells.
  • the type of the promoter is not particularly limited, as long as the gene carried by the construct can be efficiently expressed in the host animal cell.
  • the invention provides a recombinant cell.
  • the recombinant cell contains the aforementioned isolated nucleic acid molecule or the aforementioned construct.
  • the fusion protein expressed by the recombinant cell of the present invention can significantly improve the efficiency of linker connection in the construction of a sequencing library, and is particularly suitable for the linker connection of blunt ends/TA ends, without the need to add high viscosity and high density auxiliary additives such as PEG, which is convenient for subsequent sequencing. Simplify the experimental process and avoid the loss caused by excessive purification.
  • the density and viscosity of the sequencing library construction system are small, which improves the accuracy and specificity of subsequent library construction and sequencing, and is more suitable for automated platforms.
  • the present invention provides a method for obtaining the aforementioned fusion protein.
  • the recombinant cells described above are cultured under conditions suitable for the expression of the fusion protein, so as to obtain the fusion protein.
  • the fusion protein obtained by the method of the present invention can significantly improve the efficiency of linker connection in constructing a sequencing library, and is particularly suitable for linker connection of blunt end/TA end connection, without the need to add high viscosity and high density auxiliary additives such as PEG, which is convenient for subsequent sequencing , Simplify the experimental process and avoid the loss caused by excessive purification.
  • the density and viscosity of the sequencing library construction system are small, which improves the accuracy and specificity of subsequent library construction and sequencing, and is more suitable for automated platforms.
  • the present invention proposes the application of the aforementioned fusion protein in constructing a sequencing library.
  • the fusion protein of the present invention can significantly improve the efficiency of linker connection in the construction of a sequencing library, and is particularly suitable for linker connection of blunt ends/TA ends.
  • high viscosity and high density auxiliary additives such as PEG, which facilitates subsequent sequencing and simplifies the experimental process. Avoid losses caused by excessive purification.
  • high viscosity and high density additives such as PEG, the density and viscosity of the sequencing library construction system are small, which improves the accuracy and specificity of subsequent library construction and sequencing, and is more suitable for automated platforms.
  • T4 DNA ligase fusion protein G1 is composed of T4 DNA ligase and Sso 7d binding protein derived from the superthermophilic archaea Sulfolobus solfataricus.
  • the pET 28a vector was digested with NcoI and HindIII, and the gene sequence encoded by T4 DNA ligase and the gene sequence of Sso 7d were inserted into the cloning area of the vector pET 28a.
  • the 6 Hiss at the C-terminus of the amino acid sequence of the recombinant T4 DNA ligase fusion protein were used as purification tags.
  • the screening tag was kanamycin, and the constructed vector was named pET28a-G1.
  • T4 DNA ligase fusion protein G2 is composed of T4 DNA ligase and eukaryotic transcription factor NF ⁇ B p50 derived from the eukaryote Homo sapiens.
  • NF ⁇ B p50 eukaryotic transcription factor NF ⁇ B p50 derived from the eukaryote Homo sapiens.
  • NdeI and SacI the gene sequence encoded by T4 DNA ligase and the gene sequence of p50 were inserted into the cloning area of the vector pET28a.
  • the 6 Hiss at the N-terminus of the amino acid sequence of the recombinant T4 DNA ligase fusion protein were used as purification tags.
  • the selection tag was kanamycin, and the constructed vector was named pET28a-G2.
  • LB liquid medium tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L.
  • the recombinant expression vector pET28a-G1 or pET28a-G2 was transformed into E. coli expression strain Ecoli.BL21 (DE3), and the bacterial solution was evenly spread on a 50 ⁇ g/mL kanamycin plate, and cultured at 37°C overnight. Single colonies were picked and cultured in 5ml LB medium (containing 50 ⁇ g/mL kanamycin), 37°C, 200rpm, and cultured overnight. The bacterial solution obtained above was inoculated at 1:100 in 50 mL LB (containing 50 ⁇ g/mL kanamycin) for cultivation, 37° C., 200 rpm, 4 h.
  • Buffer A equilibration buffer 50mM Tris-HCl + 500mM NaCl + 10mM imidazole, pH 7.0.
  • Buffer B elution buffer 50mM Tris-HCl + 500mM NaCl + 500mM imidazole, pH 7.0.
  • Buffer Balance buffer 50mM Tris-HCl + 100mM NaCl, pH 7.0.
  • Buffer D elution buffer 50mM Tris-HCl+1M NaCl, pH 7.0.
  • Buffer E dilution 50mM Tris-HCl, pH 7.0.
  • Buffer F 2 ⁇ Storage solution 20mM Tris-HCl+100mM KCl+0.2mM EDTA, pH 7.0.
  • the blunt-end ligation efficiency of the fusion protein was mainly detected, and the 570bp blunt-end fragment was used as the reaction substrate.
  • the substrate was obtained by PCR, and the upstream primer was designed with a phosphate group at the 5'end, and the downstream primer was a common primer. After the PCR, the PCR product was recovered using Cycle Pure Kit (D6493, Omega).
  • the reaction system for the ligation efficiency test was 10 ⁇ L, containing substrate 120 ng, fusion protein G1 or G2 2 ⁇ M, 1 ⁇ BGI T4 DNA Ligase reaction buffer (50 mM Tris-HCl, 10 mM MgCl 2 , 5 mM DTT, 1 mM ATP, pH 7.5 25 °C ).
  • the above prepared reaction mixture was mixed well, placed on a PCR instrument for reaction, incubated at 16°C for 20 min, and heat-inactivated at 65°C for 15 min. After the reaction was completed, 1 ⁇ L of Proteinase K and 1 ⁇ L of 1% SDS were added thereto, and after thorough mixing, the agarose gel test was performed. The experimental results are shown in Figures 3 and 4. Compared with T4 DNA ligase, the fusion proteins G1 and G2 consume more substrate, higher product volume, and better ligation efficiency.
  • Example 5 The fusion protein is used to construct a linker in the NGS library to improve the conversion rate of the library
  • the conversion rate of the library that is, the efficiency of joining the DNA fragments at both ends of the linker. Only DNA that has been successfully ligated at both ends can be effectively amplified and eventually become a sequencing template.
  • a single-band PCR product (220bp) is used in a tube reaction to sequentially perform end repair, A addition, and joint addition reactions.
  • the reaction product is purified by magnetic beads and then detected by a 2100 bioanalyzer to analyze the reaction substrate. 1.
  • the peak concentration of the single-ended connector product and the double-ended connector product and then calculate the double-ended connector connection efficiency.
  • NGS terminal repair reaction the system is as follows:
  • NGS connection joint reaction the system is as follows:
  • Step 1 reaction product 50 ⁇ L Library adapter (25 ⁇ M) 2 ⁇ L 10X T4 PNK Buffer 3 ⁇ L ATP (100mM) 0.8 ⁇ L T4DNALigase(600U/ ⁇ L) 1.6 ⁇ L Nuclease Free Water up to 80 ⁇ L Total 80 ⁇ L
  • NGS terminal repair reaction the system is as follows:
  • E.coli gDNA interrupts double-selection products ⁇ 50ng 10X PNK reaction Buffer 5 ⁇ L 5:1 dATP: dNTP 0.6 ⁇ L T4PNK(10U/ ⁇ L) 0.6 ⁇ L Klenowfragment(5U/ ⁇ L) 0.1 ⁇ L rTaq(5U/ ⁇ L) 0.2 ⁇ L T4 DNA polymerase (3U/ ⁇ L) 2 ⁇ L Nuclease Free Water up to 50 ⁇ L Total 50 ⁇ L
  • the experimental groups are set as follows:
  • NGS library amplification the system is as follows:
  • the product was purified with 1 volume of Ampure XP beads, and the purified product was quantified by Qubit dsDNA HS Assay.

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Abstract

提出了融合蛋白及其获得方法和应用、分离的核酸分子、构建体以及重组细胞，所述融合蛋白包括：第一片段，所述第一片段具有T4 DNA连接酶活性；以及第二片段，所述第一片段的一端与所述第二片段的一端相连，所述第二片段具有DNA结合结构域。

Description

融合蛋白及其应用 技术领域
本发明涉及生物领域。具体地，本发明涉及融合蛋白及其应用。更具体地，本发明涉及融合蛋白及其获得方法和应用、分离的核酸分子、构建体以及重组细胞。
背景技术
DNA连接酶是一类可以催化两个DNA或者RNA片段之间形成磷酸二酯键的重要分子生物学工具酶。根据连接酶催化的核酸底物构象的不同，连接反应包括缺口连接(Nick ligation)、粘性末端连接(Cohesive end ligation)、TA连接(TA ligation)、平末端连接(Blunt end ligation)及分支连接(Branch ligation)，其中TA连接和平末端连接在建库接头连接中应用较广，但是效率较低。DNA连接酶的来源多种多样，几乎覆盖了所有物种，其中来源于T4噬菌体的T4 DNA连接酶以其独特而显著的平末端或者TA末端连接活性成为了分子克隆、核酸修饰、测序建库中无可替代的一种酶。
基于平末端或者TA末端的接头连接是NGS建库中不可缺少的关键步骤，其效率直接影响了后续测序结果的覆盖度和效率。同时，接头连接反应的反应体系也对上下游的操作有较大影响，可以决定是否需要添加额外的核酸纯化步骤。
然而，目前的DNA连接酶仍有待改进。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的问题的至少之一。为此，本发明提出了一种融合蛋白，其可以有效地提高接头连接效率，尤其适用于平末端/TA末端连接的接头连接，便于后续建库及测序。同时，简化了实验流程，避免过度纯化造成的损失。此外，测序文库构建体系的密度及粘度较小，更适合于自动化平台。
需要说明的是，本发明是基于发明人的下列发现而完成的：
在NGS建库中，单独采用T4 DNA连接酶基于平末端/TA末端连接的接头连接效率低，通常需要通过添加高密度高粘度的辅助添加剂(例如10～30％的聚乙二醇)来实现连接效率的提高。但是，这种高粘度高密度的辅助添加剂会影响后续建库及测序的准确性，也不适合自动化平台。
有鉴于此，发明人发现，通过将T4 DNA连接酶与具有DNA结合结构域的蛋白(例如Sso 7d结合蛋白、NFκB p50蛋白)形成融合蛋白，其中具有DNA结合结构域的蛋白可以 固定接头，以便于T4 DNA连接酶充分发挥作用，在构建测序文库中能够显著提高接头连接效率，尤其适用于平末端/TA末端连接的接头连接，便于后续测序，简化实验流程，避免过度纯化造成的损失。此外，由于无需添加PEG等高粘度高密度添加剂，因此测序文库构建体系的密度及粘度较小，提高了后续建库及测序的准确性和特异性，更适合于自动化平台。
为此，在本发明的一个方面，本发明提出了一种融合蛋白。根据本发明的实施例，所述融合蛋白包括：第一片段，所述第一片段具有T4 DNA连接酶活性；以及第二片段，所述第一片段的一端与所述第二片段的一端相连，所述第二片段具有DNA结合结构域。通过将T4 DNA连接酶与具有DNA结合结构域的蛋白(例如Sso 7d结合蛋白、NFκB p50蛋白)形成融合蛋白，其中具有DNA结合结构域的蛋白可以固定接头，以便于T4 DNA连接酶充分发挥作用，在构建测序文库中能够显著提高接头连接效率，尤其适用于平末端/TA末端连接的接头连接，便于后续测序，简化实验流程，避免过度纯化造成的损失。此外，由于无需添加PEG等高粘度高密度添加剂，因此测序文库构建体系的密度及粘度较小，提高了后续建库及测序的准确性和特异性，更适合于自动化平台。
根据本发明的实施例，所述融合蛋白还可以具有下列附加技术特征：
根据本发明的实施例，所述第二片段具有Sso 7d DNA结合蛋白活性或者NFκB p50蛋白活性。
根据本发明的实施例，所述第一片段具有SEQ ID NO：1所示的氨基酸序列。
根据本发明的实施例，所述第二片段具有SEQ ID NO：2所示的氨基酸序列或者SEQ ID NO：3所示的氨基酸序列。
根据本发明的实施例，所述融合蛋白进一步包括纯化片段和/或筛选片段。
根据本发明的实施例，所述纯化片段选自His和Flag标签的至少一种。
根据本发明的实施例，所述筛选片段具有卡那霉素抗性和/或氨苄青霉素抗性。
根据本发明的实施例，所述融合蛋白具有SEQ ID NO：4或5所示的氨基酸序列。
在本发明的又一方面，本发明提出了一种分离的核酸分子。根据本发明的实施例，所述分离的核酸分子编码前面所述的融合蛋白。利用该分离的核酸分子编码的融合蛋白在构建测序文库中能够显著提高接头连接效率，尤其适用于平末端/TA末端连接的接头连接，无需添加PEG等高粘度高密度的辅助添加剂，便于后续测序，简化实验流程，避免过度纯化造成的损失。此外，由于无需添加PEG等高粘度高密度添加剂，因此测序文库构建体系的密度及粘度较小，提高了后续建库及测序的准确性和特异性，更适合于自动化平台。
根据本发明的实施例，所述分离的核酸分子具有SEQ ID NO：6或7所示的核苷酸序 列。
在本发明的又一方面，本发明提出了一种构建体。根据本发明的实施例，所述构建体含有前面所述的分离的核酸分子。本发明的构建体表达的融合蛋白在构建测序文库中能够显著提高接头连接效率，尤其适用于平末端/TA末端连接的接头连接，无需添加PEG等高粘度高密度的辅助添加剂，便于后续测序，简化实验流程，避免过度纯化造成的损失。此外，由于无需添加PEG等高粘度高密度添加剂，因此测序文库构建体系的密度及粘度较小，提高了后续建库及测序的准确性和特异性，更适合于自动化平台。
根据本发明的实施例，所述构建体选自质粒、噬菌体、人工染色体、粘粒以及病毒的至少一种。
根据本发明的实施例，所述构建体选自质粒。
根据本发明的实施例，所述构建体选自pET 28a质粒。
根据本发明的实施例，所述构建体进一步包括启动子，所述启动子与SEQ ID NO：6或7所示的核苷酸序列可操纵地连接。
在本发明的又一方面，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞含有前面所述的分离的核酸分子或者前面所述的构建体。本发明的重组细胞表达的融合蛋白在构建测序文库中能够显著提高接头连接效率，尤其适用于平末端/TA末端连接的接头连接，无需添加PEG等高粘度高密度的辅助添加剂，便于后续测序，简化实验流程，避免过度纯化造成的损失。此外，由于无需添加PEG等高粘度高密度添加剂，因此测序文库构建体系的密度及粘度较小，提高了后续建库及测序的准确性和特异性，更适合于自动化平台。
在本发明的又一方面，本发明提出了一种获得前面所述融合蛋白的方法。根据本发明的实施例，在适于所述融合蛋白表达的条件下，培养前面所述的重组细胞，以便获得所述融合蛋白。利用本发明的方法所获得的融合蛋白在构建测序文库中能够显著提高接头连接效率，尤其适用于平末端/TA末端连接的接头连接，无需添加PEG等高粘度高密度的辅助添加剂，便于后续测序，简化实验流程，避免过度纯化造成的损失。此外，由于无需添加PEG等高粘度高密度添加剂，因此测序文库构建体系的密度及粘度较小，提高了后续建库及测序的准确性和特异性，更适合于自动化平台。
在本发明的又一方面，本发明提出了前面所述融合蛋白在构建测序文库中的应用。本发明的融合蛋白在构建测序文库中能够显著提高接头连接效率，尤其适用于平末端/TA末端连接的接头连接，无需添加PEG等高粘度高密度的辅助添加剂，便于后续测序，简化实验流程，避免过度纯化造成的损失。此外，由于无需添加PEG等高粘度高密度添加剂，因 此测序文库构建体系的密度及粘度较小，提高了后续建库及测序的准确性和特异性，更适合于自动化平台。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了根据本发明一个实施例的重组型T4 DNA连接酶融合蛋白G1纯化结果电泳图；
图2显示了根据本发明一个实施例的重组型T4 DNA连接酶融合蛋白G2纯化结果电泳图；
图3显示了根据本发明一个实施例的重组型T4 DNA连接酶融合蛋白G1平末端连接效率测试结果电泳图；
图4显示了根据本发明一个实施例的重组型T4 DNA连接酶融合蛋白G2平末端连接效率测试结果电泳图；以及
图5显示了根据本发明一个实施例的NGS文库构建接头连接效率分析图。
具体实施方式
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
本发明提出了融合蛋白、分离的核酸分子、构建体、重组细胞、获得融合蛋白的方法和融合蛋白在构建测序文库中的应用，下面将分别对其进行详细描述。
融合蛋白
在本发明的一个方面，本发明提出了一种融合蛋白。根据本发明的实施例，融合蛋白包括：第一片段，该第一片段具有T4 DNA连接酶活性；以及第二片段，该第一片段的一端与所述第二片段的一端相连，第二片段具有DNA结合结构域。通过将T4 DNA连接酶与具有DNA结合结构域的蛋白(例如Sso 7d结合蛋白、NFκB p50蛋白)形成融合蛋白，其中具有DNA结合结构域的蛋白可以固定接头，以便于T4 DNA连接酶充分发挥作用，在构建测序文库中能够显著提高接头连接效率，尤其适用于平末端/TA末端连接的接头连接， 便于后续测序，简化实验流程，避免过度纯化造成的损失。此外，由于无需添加PEG等高粘度高密度添加剂，因此测序文库构建体系的密度及粘度较小，提高了后续建库及测序的准确性和特异性，更适合于自动化平台。
本领域技术人员能够理解的是，氨基酸片段通常分为N端和C端，本发明对于第一片段和第二片段的连接方式不作严格限定，只要是一个片段的N端与另一个片段的C端相连即可。
根据本发明的实施例，所述第二片段具有Sso 7d DNA结合蛋白活性或者NFκB p50蛋白活性。
Sso 7d结合蛋白是来自超嗜热古细菌Sulfolobus solfataricus，分子量约7.0kDa，在中性pH附近的变性温度接近100℃，具有非序列特异性的DNA结合活性，与双链DNA的小沟结合。
NFκB p50是来源于真核生物Homo sapiens的真核转录因子，分子量约为36.5kDa，具有DNA结合结构域，能够增强特异性抗凋亡基因的表达。
发明人发现，通过将T4 DNA连接酶与Sso 7d结合蛋白或者NFκB p50蛋白形成融合蛋白，其中Sso 7d结合蛋白或者NFκB p50蛋白可以固定接头，以便于T4 DNA连接酶充分发挥作用，在构建测序文库中能够显著提高接头连接效率，尤其适用于平末端/TA末端连接的接头连接，便于后续测序，简化实验流程，避免过度纯化造成的损失。此外，由于无需添加PEG等高粘度高密度添加剂，因此测序文库构建体系的密度及粘度较小，提高了后续建库及测序的准确性和特异性，更适合于自动化平台。
根据本发明的实施例，第一片段具有SEQ ID NO：1所示的氨基酸序列。T4 DNA连接酶是建库中常用的DNA连接酶，尤其适用于平末端或者TA末端连接。
Figure PCTCN2018121545-appb-000001
根据本发明的实施例，第二片段具有SEQ ID NO：2所示的氨基酸序列或者SEQ ID NO： 3所示的氨基酸序列。Sso 7d结合蛋白(SEQ ID NO：2)或者NFκB p50蛋白(SEQ ID NO：3)与T4 DNA连接酶形成的融合蛋白均能够显著提高接头连接效率，尤其适用于平末端/TA末端连接的接头连接，便于后续测序，简化实验流程，避免过度纯化造成的损失。此外，由于无需添加PEG等高粘度高密度添加剂，因此测序文库构建体系的密度及粘度较小，提高了后续建库及测序的准确性和特异性，更适合于自动化平台。
Figure PCTCN2018121545-appb-000002
根据本发明的实施例，融合蛋白进一步包括纯化片段和/或筛选片段。由此，以便达到筛选和分离目的。
根据本发明的实施例，纯化片段选自His和Flag标签的至少一种。由此，能够方便地筛选阳性转化子。
根据本发明的实施例，筛选片段具有卡那霉素抗性和/或氨苄青霉素抗性。由此，能够进一步提高筛选接受外源构建体的重组细胞的效率。
根据本发明的实施例，融合蛋白具有SEQ ID NO：4或5所示的氨基酸序列。由T4 DNA连接酶和Sso 7d DNA结合蛋白形成的融合蛋白(SEQ ID NO：4)或者由T4 DNA连接酶和NFκB p50蛋白形成的融合蛋白(SEQ ID NO：5)均可以显著提高接头连接效率，尤其适用于平末端/TA末端连接的接头连接，便于后续测序，简化实验流程，避免过度纯化造成的损失。此外，由于无需添加PEG等高粘度高密度添加剂，因此测序文库构建体系的密度及粘度较小，提高了后续建库及测序的准确性和特异性，更适合于自动化平台。
Figure PCTCN2018121545-appb-000003
Figure PCTCN2018121545-appb-000004
分离的核酸分子
在本发明的又一方面，本发明提出了一种分离的核酸分子。根据本发明的实施例，该分离的核酸分子编码前面所述的融合蛋白。利用该分离的核酸分子编码的融合蛋白在构建测序文库中能够显著提高接头连接效率，尤其适用于平末端/TA末端连接的接头连接，无需添加PEG等高粘度高密度的辅助添加剂，便于后续测序，简化实验流程，避免过度纯化造成的损失。此外，由于无需添加PEG等高粘度高密度添加剂，因此测序文库构建体系的密度及粘度较小，提高了后续建库及测序的准确性和特异性，更适合于自动化平台。
根据本发明的实施例，分离的核酸分子具有SEQ ID NO：6或7所示的核苷酸序列。其中，SEQ ID NO：6所示的核苷酸序列编码SEQ ID NO：4所示的氨基酸序列，SEQ ID NO：7所示的核苷酸序列编码SEQ ID NO：5所示的氨基酸序列。
Figure PCTCN2018121545-appb-000005
Figure PCTCN2018121545-appb-000006
Figure PCTCN2018121545-appb-000007
Figure PCTCN2018121545-appb-000008
T4 DNA连接酶基因序列：
Figure PCTCN2018121545-appb-000009
Figure PCTCN2018121545-appb-000010
Sso 7d蛋白基因序列：
Figure PCTCN2018121545-appb-000011
NFκB p50蛋白基因序列：
Figure PCTCN2018121545-appb-000012
构建体
在本发明的又一方面，本发明提出了一种构建体。根据本发明的实施例，所述构建体含有前面所述的分离的核酸分子。本发明的构建体表达的融合蛋白在构建测序文库中能够显著提高接头连接效率，尤其适用于平末端/TA末端连接的接头连接，无需添加PEG等高粘度高密度的辅助添加剂，便于后续测序，简化实验流程，避免过度纯化造成的损失。此外，由于无需添加PEG等高粘度高密度添加剂，因此测序文库构建体系的密度及粘度较小，提高了后续建库及测序的准确性和特异性，更适合于自动化平台。
根据本发明的实施例，所述构建体选自质粒、噬菌体、人工染色体、粘粒以及病毒的至少一种，优选质粒，更优选pET 28a质粒。由此，能够有效提高利用本发明的构建体进行遗传转化的效率。
需要说明的是，本发明的构建体中还可以包含其他的元件，从而赋予构建体额外的有益效果。根据本发明的实施例，所述构建体进一步包括启动子，该启动子与SEQ ID NO：6或7所示的核苷酸序列可操纵地连接。
在本发明中所使用的术语“启动子”指的是这样一种核酸序列，其能够指导与其可操纵地连接的核酸分子的转录。在本发明中所使用的术语“可操纵地”指的是核酸表达控制序列例如启动子、信号序列、增强子等与目标核酸序列之间的功能连接，其中当合适的分子例如转录活化分子与表达控制序列结合时，表达控制序列影响着对应于目标核酸序列的核酸的转录和/或翻译。由此，可以直接通过构建体在宿主细胞中引入特定的启动子，并且该启动子可以用于启动SEQ ID NO：6或7所示的核苷酸序列的转录和表达，由此，可以提高所获得的重组细胞中T4 DNA连接酶基因、Ssco7d结合蛋白基因/NFκB p50蛋白的表达效率。根据本发明的实施例，在本发明的构建体中，所述启动子的种类不受特别限制，只要能够使构建体所携带的基因能够在宿主动物细胞中高效表达即可。
重组细胞
在本发明的又一方面，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞含有前面所述的分离的核酸分子或者前面所述的构建体。本发明的重组细胞表达的融合蛋白在构建测序文库中能够显著提高接头连接效率，尤其适用于平末端/TA末端连接的接头连接，无需添加PEG等高粘度高密度的辅助添加剂，便于后续测序，简化实验流程，避免过度纯化造成的损失。此外，由于无需添加PEG等高粘度高密度添加剂，因此测序文库构建体系的密度及粘度较小，提高了后续建库及测序的准确性和特异性，更适合于自动化平台。
获得融合蛋白的方法
在本发明的又一方面，本发明提出了一种获得前面所述融合蛋白的方法。根据本发明的实施例，在适于所述融合蛋白表达的条件下，培养前面所述的重组细胞，以便获得所述融合蛋白。利用本发明的方法所获得的融合蛋白在构建测序文库中能够显著提高接头连接效率，尤其适用于平末端/TA末端连接的接头连接，无需添加PEG等高粘度高密度的辅助添加剂，便于后续测序，简化实验流程，避免过度纯化造成的损失。此外，由于无需添加PEG等高粘度高密度添加剂，因此测序文库构建体系的密度及粘度较小，提高了后续建库及测序的准确性和特异性，更适合于自动化平台。
融合蛋白在构建测序文库中的应用
在本发明的又一方面，本发明提出了前面所述融合蛋白在构建测序文库中的应用。本发明的融合蛋白在构建测序文库中能够显著提高接头连接效率，尤其适用于平末端/TA末端连接的接头连接，无需添加PEG等高粘度高密度的辅助添加剂，便于后续测序，简化实验流程，避免过度纯化造成的损失。此外，由于无需添加PEG等高粘度高密度添加剂，因此测序文库构建体系的密度及粘度较小，提高了后续建库及测序的准确性和特异性，更适合于自动化平台。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，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，均为可以通过市购获得的常规产品。
实施例1 重组型T4 DNA连接酶融合蛋白表达载体的构建
T4 DNA连接酶融合蛋白G1是由T4 DNA连接酶和来源于超嗜热古细菌Sulfolobus solfataricus的Sso 7d结合蛋白组成。通过In-fusion的方法，采用NcoI和HindIII酶切pET 28a载体后，将T4 DNA连接酶编码的基因序列与Sso 7d的基因序列共同插入到载体pET 28a的克隆区。将重组型T4 DNA连接酶融合蛋白氨基酸序列的C端6个His作为纯化标签，其中筛选标签为卡那霉素，将构建好的载体命名为pET 28a-G1。
T4 DNA连接酶融合蛋白G2是由T4 DNA连接酶和来源于真核生物Homo sapiens的真核转录因子NFκB p50组成。通过In-fusion的方法，采用NdeI和SacI酶切pET 28a载体后，将T4 DNA连接酶编码的基因序列与p50的基因序列共同插入到载体pET 28a的克隆区。将 重组型T4 DNA连接酶融合蛋白氨基酸序列的N端6个His作为纯化标签，其中筛选标签为卡那霉素，将构建好的载体命名为pET 28a-G2。
实施例2 重组型T4 DNA连接酶融合蛋白菌株的培养和诱导
LB液体培养基：胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L。
将重组表达载体pET 28a-G1或pET 28a-G2转化到大肠杆菌表达菌株Ecoli.BL21(DE3)中，将菌液均匀涂抹在50μg/mL卡那霉素的平板上，37℃过夜培养。挑取单菌落，于5ml LB培养基(含有50μg/mL卡那霉素)培养，37℃，200rpm，过夜培养。将上述所得菌液，按1:100接种于50mL LB(含有50μg/mL卡那霉素)中培养，37℃，200rpm，4h。将扩大培养的菌液，按1:100接种于2L LB(含有50μg/mL卡那霉素)中培养，37℃，200rpm，待OD600值达0.6-0.8左右，加入IPTG至终浓度为0.4mM，25℃，200rpm，培养过夜，约16-18h。将长好的菌体于8000rpm离心收集，菌体冻存于-20℃待用。
实施例3 重组型T4 DNA连接酶融合蛋白提取及纯化
纯化Buffer配制：
1、Ni柱亲和层析
Buffer A平衡缓冲液：50mM Tris-HCl+500mM NaCl+10mM咪唑，pH 7.0。
Buffer B洗脱缓冲液：50mM Tris-HCl+500mM NaCl+500mM咪唑，pH 7.0。
2、离子交换层析
Buffer C平衡缓冲液：50mM Tris-HCl+100mM NaCl，pH 7.0。
Buffer D洗脱缓冲液：50mM Tris-HCl+1M NaCl，pH 7.0。
3、蛋白样品稀释液
Buffer E稀释液：50mM Tris-HCl，pH 7.0。
4、蛋白样品2×储存液
Buffer F 2×储存液20mM Tris-HCl+100mM KCl+0.2mM EDTA，pH 7.0。
按1g菌体加15mL亲和A液的比例重悬菌体，超声破碎细胞，直至菌体溶液至澄清。将破碎后的菌体12000rpm，4℃离心30min，取上清，0.22μm滤膜过滤后于4℃储存。
将Ni柱亲合层析柱水洗5CV，Buffer B清洗5CV，Buffer A进行平衡10CV后，进行上样。上样完成后，平衡15CV，使用Buffer B进行线性洗脱(0-100％Buffer B，10CV)，当UV值大于100mAU以上收集蛋白。
将Ni柱收集到的蛋白用Buffer E稀释5倍，将Q阴离子交换柱水洗5CV，Buffer C平衡5CV，蛋白样品上样，当UV值上升开始收样。将SP阳离子交换柱使用Buffer C平衡5CV， 将上步得到的蛋白样品上样，上样完成后，用Buffer C平衡15CV后，用洗脱缓冲液Buffer D线性洗脱(0-50％Buffer D，20CV)，收集蛋白。收集蛋白进行过夜透析，透析液为2×的储存Buffer。蛋白终浓度为2mg/mL，甘油浓度为50％。如图1和图2所示，SDS-PAGE结果显示融合蛋白的纯化效果很好，纯度合格。
实施例4 重组型T4 DNA连接酶融合蛋白连接效率测试
本实验主要检测融合蛋白的平末端连接效率，以570bp平末端片段为反应底物。底物由PCR得来，并且设计上游引物5’端带有磷酸基团，下游引物为普通引物。PCR结束后，使用Cycle Pure Kit(D6493,Omega)回收PCR产物。连接效率测试的反应体系为10μL，包含底物120ng，融合蛋白G1或G2 2μM，1×BGI T4 DNA Ligase反应缓冲液(50mM Tris-HCl，10mM MgCl 2，5mM DTT，1mM ATP，pH 7.5 25℃)。将上述配制完成的反应混合液混匀，置于PCR仪上进行反应，16℃孵育20min，65℃热失活15min。反应完成后，向其中加入1μL Proteinase K和1μL 1％SDS，充分混匀后进行琼脂糖凝胶检测。实验结果如图3和4所示，相比于T4 DNA连接酶，融合蛋白G1和G2的底物消耗更多，产物量更高，连接效率更好。
实施例5 融合蛋白用于NGS文库构建连接接头,提高文库转化率
文库转化率，即DNA片段双端接头连接效率。只有双端连接成功的DNA，才能够被有效扩增最终成为测序模板。本实施例采用单一条带PCR产物(220bp)在一管反应中顺次进行末端修复、加A、加接头等反应，反应产物经磁珠纯化后用2100生物分析仪进行检测，分析反应底物、单端接头产物、双端接头产物峰的浓度，进而计算双端接头连接效率。
1、NGS末端修复反应，体系如下：
组分 体积
220bp DNA片段 ～50ng
10X PNK reaction Buffer 5μL
5:1 dATP：dNTP 0.6μL
T4 PNK(10U/μL) 0.6μL
Klenow fragment(5U/μL) 0.1μL
rTaq(5U/μL) 0.2μL
T4 DNA polymerase(3U/μL) 2μL
Nuclease Free Water up to 50μL
Total 50μL
于冰上配制上述反应体系，Vortex混匀离心后，于PCR仪上执行以下程序：
温度 时间
热盖 on
37℃ 30min
65℃ 15min
4℃ Hold
2、NGS连接接头反应，体系如下：
组分 体积
Step 1反应产物 50μL
文库接头(25μM) 2μL
10X T4 PNK Buffer 3μL
ATP(100mM) 0.8μL
T4 DNA Ligase(600U/μL) 1.6μL
Nuclease Free Water up to 80μL
Total 80μL
于冰上配制上述反应体系，23℃反应1h，每管加入2μl Proteinase K(20mg/mL)终止反应，用AMPure XP beads纯化后取1μL 2100检测，结果见附图5，其中(A)为电泳图；(B)为连接效率图；(C)为2100检测峰图，其中0：底物峰，1：单端连接峰，2：双端连接峰。
各实验组设置及连接效率测定：
Figure PCTCN2018121545-appb-000013
Figure PCTCN2018121545-appb-000014
实施例6融合蛋白用于NGS文库构建
50ng经超声打断双选后的E.coli gDNA用于NGS文库构建，测试融合蛋白G1用于NGS文库构建的产量。
1、NGS末端修复反应，体系如下：
组分 体积
E.coli gDNA打断双选产物 ～50ng
10X PNK reaction Buffer 5μL
5:1 dATP：dNTP 0.6μL
T4 PNK(10U/μL) 0.6μL
Klenow fragment(5U/μL) 0.1μL
rTaq(5U/μL) 0.2μL
T4 DNA polymerase(3U/μL) 2μL
Nuclease Free Water up to 50μL
Total 50μL
于冰上配制上述反应体系，Vortex混匀离心后，于PCR仪上执行以下程序：
温度 时间
热盖 on
37℃ 30min
65℃ 15min
4℃ Hold
2、NGS连接接头反应体系：
Figure PCTCN2018121545-appb-000015
Figure PCTCN2018121545-appb-000016
于冰上配制上述反应体系，23℃反应1h，每管加入2ul Proteinase K(20mg/mL)终止反应，用AMPure XP beads纯化后，洗脱溶于32μL TE Buffer中。
各实验组设置如下：
Figure PCTCN2018121545-appb-000017
3、NGS文库扩增，体系如下：
组分 体积
Step 2纯化产物 15μL
2X KAPA HiFi PCR Mix 25μL
Primer F(20uM) 1.5μL
Primer R(20uM) 1.5μL
Nuclease Free Water up to 50μL
Total 50μL
于冰上配制以上反应体系，Vortex混匀离心后，于PCR仪上执行以下程序：
Figure PCTCN2018121545-appb-000018
Figure PCTCN2018121545-appb-000019
反应结束后用1倍体积Ampure XP beads纯化，纯化后产物用Qubit dsDNA HS Assay定量。
各实验组设置及文库产量对比：
Figure PCTCN2018121545-appb-000020
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (18)

  1. 一种融合蛋白，其特征在于，包括：
    第一片段，所述第一片段具有T4DNA连接酶活性；以及
    第二片段，所述第一片段的一端与所述第二片段的一端相连，所述第二片段具有DNA结合结构域。
  2. 根据权利要求1所述的融合蛋白，其特征在于，所述第二片段具有Sso 7d DNA结合蛋白活性或者NFκB p50蛋白活性。
  3. 根据权利要求1所述的融合蛋白，其特征在于，所述第一片段具有SEQ ID NO：1所示的氨基酸序列。
  4. 根据权利要求1所述的融合蛋白，其特征在于，所述第二片段具有SEQ ID NO：2所示的氨基酸序列或者SEQ ID NO：3所示的氨基酸序列。
  5. 根据权利要求1所述的融合蛋白，其特征在于，进一步包括纯化片段和/或筛选片段。
  6. 根据权利要求5所述的融合蛋白，其特征在于，所述纯化片段选自His和Flag标签的至少一种。
  7. 根据权利要求5所述的融合蛋白，其特征在于，所述筛选片段具有卡那霉素抗性和/或氨苄青霉素抗性。
  8. 根据权利要求1所述的融合蛋白，其特征在于，具有SEQ ID NO：4或5所示的氨基酸序列。
  9. 一种分离的核酸分子，其特征在于，所述分离的核酸分子编码权利要求1～8任一项所述的融合蛋白。
  10. 根据权利要求9所述的分离的核酸分子，其特征在于，所述分离的核酸分子具有SEQ ID NO：6或7所示的核苷酸序列。
  11. 一种构建体，其特征在于，含有权利要求9或10所述的分离的核酸分子。
  12. 根据权利要求11所述的构建体，其特征在于，所述构建体选自质粒、噬菌体、人工染色体、粘粒以及病毒的至少一种。
  13. 根据权利要求11所述的构建体，其特征在于，所述构建体选自质粒。
  14. 根据权利要求11所述的构建体，其特征在于，所述构建体选自pET 28a质粒。
  15. 根据权利要求11所述的构建体，其特征在于，所述构建体进一步包括启动子，所述启动子与SEQ ID NO：6或7所示的核苷酸序列可操纵地连接。
  16. 一种重组细胞，其特征在于，含有权利要求9或10所述的分离的核酸分子或者权 利要求11～15任一项所述的构建体。
  17. 一种获得权利要求1～8任一项所述融合蛋白的方法，其特征在于，在适于所述融合蛋白表达的条件下，培养权利要求16所述的重组细胞，以便获得所述融合蛋白。
  18. 权利要求1～8任一项所述融合蛋白在构建测序文库中的应用。
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