WO2015117431A1 - 一种定点突变的Lambda噬菌体Int重组蛋白及其制备方法 - Google Patents

一种定点突变的Lambda噬菌体Int重组蛋白及其制备方法 Download PDF

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WO2015117431A1
WO2015117431A1 PCT/CN2014/090374 CN2014090374W WO2015117431A1 WO 2015117431 A1 WO2015117431 A1 WO 2015117431A1 CN 2014090374 W CN2014090374 W CN 2014090374W WO 2015117431 A1 WO2015117431 A1 WO 2015117431A1
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recombinant protein
lambda phage
int
recombinant
site
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张玮
温华杰
李春园
蒙伟能
王文忠
施金秀
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赛业（广州）生物科技有限公司
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  • the invention belongs to the field of biotechnology, and particularly relates to Lambda phage Int recombinant protein with active site mutation and its application in DNA recombination reaction under temperature control.
  • the Int protein is derived from Lambda phage (also known as Lambda phage), which plays an important role in the integration of Lambda phage DNA into host genomic DNA. Together with IHF and Xis, it is a key protein factor for the integration and release of Lambda phage DNA and host genomic DNA.
  • Lambda phage In its life history, Lambda phage needs to complete its life history by infecting host bacteria by integrating, replicating and releasing its own DNA. Both integration and release require the aid of a series of att nucleic acid sites and IHF, Int and Xis proteins present on genomic DNA.
  • the IHF gene was isolated from E. coli and the prokaryotic expression was carried out.
  • the Int gene was isolated from the Lambda phage genome and the prokaryotic expression was carried out.
  • In vitro BP reactions were performed by mixing and adjusting the ratio of IHF to Int protein.
  • the recombination reaction Since the recombination reaction has a specific att nucleic acid site, the recombination efficiency is high. It has been widely used in vector construction work. Because of its efficient recombination and site-specific recombination, it has been widely used in a variety of biotechnology work such as protein large sample expression and screening, genetic diversity analysis, and active protein screening.
  • the BP reaction is less affected by temperature under suitable temperature conditions, and the BP reaction can be carried out at a lower temperature. Therefore, the recombination activity of the enzyme cannot be affected by the temperature difference. It is not conducive to large sample recombination reactions and limits the construction of high-throughput vectors.
  • embodiments of the present invention provide a recombinant protein that initiates BP recombinant activity upon reaching a specific temperature condition.
  • the site-directed mutant Lambda phage Int recombinant protein provided by the present invention is obtained by site-directed mutagenesis of a recombinant protein gene fragment of Lambda phage Int.
  • the recombinant protein is obtained by mutation of amino acids 347 and 351 of the full-length gene of Lambda phage Int.
  • the recombinant protein is a recombinant protein of Lambda phage Int mutated by G347H and D351H.
  • the recombinant protein has a recombinant activity consistent with wild-type Lambda phage Int protease.
  • the recombinant protein has enzymatic thermostability at 15 to 37 °C.
  • the recombinant protein has enzymatic thermostability at 25 °C.
  • the recombinant protein has a temperature-sensitive, high biological activity at 25 °C.
  • the invention also provides a use of a recombinant protein in a nucleic acid recombination system.
  • the present invention provides the use of a recombinant protein for regulating nucleic acid recombination systems by temperature differences.
  • the invention also provides a method for preparing a site-directed mutant Lambda phage Int recombinant protein, comprising the following steps:
  • the recombinant plasmid obtained in the step 5) is transformed into Escherichia coli for prokaryotic expression, and then isolated and purified to obtain the recombinant protein.
  • Lambda phage Int By transforming Lambda phage Int into a recombinant protein that is active under temperature.
  • the recombinant protein does not initiate DNA recombination reaction under low temperature conditions, and initiates DNA recombination reaction under a certain temperature condition, thereby controlling DNA recombination reaction.
  • a large sample recombination reaction can be carried out to increase the flux of the recombinant sample.
  • Figure 1 is a schematic view showing the induced expression of the Int recombinant protein of the present invention
  • Figure 2 shows a histogram of BP reaction clone colonies under different temperature conditions
  • Figure 3 is a bar graph showing the recombinant activity of mutant Int recombinant protein and wild type Int protease.
  • Figure 1 shows the induction of expression of Int recombinant protein
  • Figure 2 shows the histogram of colony colonies of BP reaction under different temperature conditions.
  • lane "M” of Figure 1 is a protein marker
  • lane "1” is a bacterial induction result containing no expression plasmid
  • lane "2” is an induction result containing an expression plasmid but not containing an Int sequence
  • lane 3 is containing an Int Gene plasmid induction results
  • lane "4" is the affinity purification of nickel column
  • lane "5" is affinity purification of nickel column and then purified by molecular sieve.
  • the recombinant protein provided by the present invention is obtained by mutation of amino acids 347 and 351 of the full-length gene of Lambda phage Int (G347H, and D351H).
  • the recombinant protein of the present invention is a site-directed mutant Lambda phage Int recombinant protein.
  • the recombinant protein expression vector provided by the present invention is obtained by using a site-directed mutagenesis method to obtain Lambda phage Int recombinant protein genes (G347H, and D351H) of mutations 347 and 351.
  • the PCR products of the Lambda phage Int recombinant protein gene and the PET-22b backbone were digested with the restriction endonucleases Nde I and Ecor I, respectively.
  • the ligated product was ligated, transformed into TOP10 competent cells, cultured, and the plasmid was extracted and sequenced to obtain the correct PET 22b-Int plasmid.
  • the recombinant protein obtained by the invention is obtained by converting the constructed expression plasmid into Prokaryotic expression was carried out in E. coli BL21DE (3).
  • High-purity 345- and 351-mutant Lambda phage Int recombinant proteins (G347H, and D351H) were obtained by nickel column affinity chromatography and Superdex-200 molecular sieve chromatography.
  • the invention provides a use of a recombinant protein in homologous recombination.
  • the application is to control the activity of the recombinant protein by using a site-directed mutant Lambda phage Int recombinant protein to control the activity of the recombinant protein by temperature regulation.
  • the specific method comprises: adding a site-mutated Lambda phage Int recombinant protein and an IHF protein to the BP reaction system; adding a reaction substrate and a suitable buffer system.
  • Six temperature gradients of 4 ° C, 8 ° C, 15 ° C, 25 ° C, 30 ° C, 37 ° C were set to verify the effect of different temperatures on BP reaction.
  • the Lambda phage Int recombinant protein and IHF protein which were site-mutated at 4 °C to 25 °C, gradually increased their BP activity with increasing temperature.
  • the recombinant protein provided by the invention has the same recombination activity as the wild-type Lambda phage Int protease, and has thermal stability while maintaining the enzyme activity by being placed in a metal water bath at 25 ° C for 4 hours.
  • the recombinant protein provided by the invention is a method for site-directed mutagenesis to transform the active site of the Lambda phage Int protein into a protein which is active under the influence of temperature, and is suitable for temperature and does not initiate DNA recombination reaction under low temperature conditions, under certain temperature conditions. The DNA recombination reaction is initiated.
  • the recombinant protein (G347H, and D351H) provided by the invention that is, the mutant Int protein, has simple preparation method, large yield and high purity; the experiment proves that the thermal stability is good, and the same catalytic tendency as the wild type enzyme. Compared to wild-type enzymes, it has no catalytic activity at lower temperatures. It has the characteristics of catalytic activity at suitable temperature and has broad application prospects.
  • the length of the fragment was analyzed by 2.0% agarose gel electrophoresis, and the target band was cut according to the size of the fragment, and the cut product was recovered using a DNA purification kit.
  • the double-digested product of the PCR product and the vector was subjected to 1% agarose gel electrophoresis, and purified by a DNA gel recovery kit.
  • the bacterial liquid was transferred to a centrifuge tube, and the cells were collected at 4 ° C, 3700 rpm, 10 min. Discard the supernatant and pour out the residue.
  • the inverted plate is incubated at 37 ° C for 12-16 h.
  • the positive clone identified by the above method was submitted to the sequencing company for sequencing, and the nucleotide sequence inserted in the positive clone obtained was obtained.
  • the length of the fragment was analyzed by 2.0% agarose gel electrophoresis, and the target band was cut according to the size of the fragment, and the cut product was recovered using a DNA purification kit.
  • Example 4 Site-directed mutagenesis and wild-type Lambda phage Int protein expression and purification
  • Seed culture Positive clone colonies were picked by conventional methods and placed in 2 ml of LB containing Amp antibiotic, and cultured at 37 ° C, shaking at 250 rmp / min for 16 h.
  • Ultrasonic bacterial disruption ultrasound 3s, interval 6s, 300w, 60 times work.
  • Ni-NTA affinity chromatography The collected supernatant protein containing soluble protein was poured into a regenerated 1.5 ml Ni-NTA column and placed at 4 ° C for 3 h; after the supernatant was drained, the Wash buffer was used. (PBS + 80 mM Imidazole) was washed 6 column volumes, and the protein was washed; then the target protein was eluted with Elution buffer (50 mM Tris-HCl [pH 8.0], 1 mM DTT, 150 mM NaCl, 10 mM EDTA, 250 mM Imidazole). Clean pre-cooled beakers were collected; protein purification was detected using SDS-PAGE electrophoresis. The purification results are shown in Figure 2.
  • Example 5 Site-mutated Lambda phage Int recombinant protein (G347H, and D351H) enzyme activity stability test
  • Recombination rate (number of recombinant clones / total number of clones) ⁇ 100%
  • the PCR product containing the att B1 and attB2 sites was added, and the pDoner221 plasmid was added.
  • the reaction was carried out at 4 ° C, 8 ° C, 15 ° C, 25 ° C, 30 ° C, 37 ° C, respectively.
  • Five microliters of the reaction product at different temperatures were taken to transform competent cells.
  • Transformed competent cells were plated on Kana-resistant LB plates. After cultured at 37 ° C for 16 hours, the monoclonal was picked and cultured, and the bacterial liquid was identified by PCR. The calculation of the recombination efficiency is shown in the following table.
  • the negative control specifically adds the remaining reactants in addition to IHF and Int.

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Abstract

提供了一种定点突变的Lambda噬菌体Int重组蛋白及其制备方法。该重组蛋白为G347H和D351H突变的Lambda噬菌体Int重组蛋白，是一种受温度影响而发挥活性的重组蛋白，其在低温条件下不启动DNA重组反应，在一定温度条件下启动DNA重组反应，从而可以控制DNA重组反应。该重组蛋白可用于开展大样本重组反应，提高重组样本通量。

Description

一种定点突变的Lambda噬菌体Int重组蛋白及其制备方法 技术领域
本发明属于生物技术领域，具体涉及活性位点突变的Lambda噬菌体Int重组蛋白及其在温度控制下DNA重组反应中的应用。
背景技术
Int蛋白来源于Lambda噬菌体(又称Lambda噬菌体)，其在Lambda噬菌体DNA整合到宿主基因组DNA中具有重要作用。与IHF和Xis一同，作为Lambda噬菌体DNA与宿主基因组DNA整合与释放的关键蛋白因子。
Lambda噬菌体在其生活史上，需要通过感染宿主细菌，通过整合、复制和释放其自身DNA的方式完成其生活史。整合与释放都需要借助于系列存在基因组DNA上的att核酸位点和IHF、Int和Xis蛋白。根据Lambda噬菌体同源重组的生活史，从大肠杆菌中分离出IHF基因并开展蛋白原核表达，从Lambda噬菌体基因组中分别分离出Int基因并开展蛋白原核表达。通过混合和调节IHF和Int蛋白比例进行体外BP反应。因该重组反应具备特定att核酸位点，因此重组效率较高。目前已经广泛应用到载体构建工作中。因其具备的高效重组和位点特异性重组，已经广泛应用于蛋白大样本表达和筛选，基因遗传多样性分析，活性蛋白筛选等多种生物技术工作中。
目前BP反应在适宜温度条件下受温度影响小，在较低温度下BP反应也能进行。因此，不能够通过温度差异影响酶的重组活性。不利于开展大样本重组反应，限制高通量载体构建方法。
发明内容
有鉴于此，本发明的实施例提供了一种重组蛋白，在达到特定温度条件下，启动BP重组活性。
本发明提供的定点突变的Lambda噬菌体Int重组蛋白，由Lambda噬菌体Int的重组蛋白基因片段定点突变获得。
优选的，重组蛋白由Lambda噬菌体Int全长基因的347位和351位氨基酸突变获得。
优选的，重组蛋白为G347H和D351H突变的Lambda噬菌体Int重组蛋白。
优选的，重组蛋白具有与野生型Lambda噬菌体Int蛋白酶一致的重组活性。
优选的，重组蛋白在15～37℃具有酶活热稳定性。
优选的，重组蛋白在25℃具有酶活热稳定性。
优选的，重组蛋白在25℃具有温度敏感的高生物活性。
本发明同时提供了一种重组蛋白在核酸重组系统中的应用。
优选的，本发明提供了一种重组蛋白通过温度差异调控核酸重组系统中的应用。
本发明同时提供一种定点突变的Lambda噬菌体Int重组蛋白的制备方法，包括如下步骤：
1)设计含有突变位点的Int特异性引物；
2)克隆野生型Lambda噬菌体Int基因；
3)以野生型Lambda噬菌体Int基因为模板，在Int特异性引物的作用下进行PCR反应，获得定点突变的Lambda噬菌体Int重组基因；
4)采用限制性内切酶，分别酶切质粒载体和所述的Lambda噬菌 体Int重组基因；
5)采用DNA连接酶进行连接反应，构建含有所述的Lambda噬菌体Int重组基因的重组质粒；
6)对步骤5)得到的重组质粒转化大肠杆菌进行原核表达，然后通过分离、纯化，得到所述重组蛋白。
通过将Lambda噬菌体Int改造成受温度影响而发挥活性的重组蛋白。使得该重组蛋白在低温条件下不启动DNA重组反应，在一定温度条件下启动DNA重组反应，从而可以控制DNA重组反应。通过温度差异影响酶的重组活性，可以开展大样本重组反应，提高重组样本通量。
附图说明
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简要介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域的普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1示出本发明的Int重组蛋白诱导表达情况示意图；
图2示出不同温度条件下BP反应克隆菌落柱状图；
图3示出突变型Int重组蛋白和野生型Int蛋白酶重组活性对比柱状图。
具体实施方式
以下将参考附图详细说明本发明的各种示例性实施例、特征和方面。附图中相同的附图标记表示功能相同或相似的元件。尽管在附图中示出了实施例的各种方面，但是除非特别指出，不必按比例绘制附 图。
在这里专用的词“示例性”意为“用作例子、实施例或说明性”。这里作为“示例性”所说明的任何实施例不必解释为优于或好于其它实施例。
另外，为了更好的说明本发明，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有这些具体细节，本发明同样可以实施。在另外一些实例中，对于大家熟知的方法、手段和元件未作详细描述，以便于凸显本发明的主旨。
图1示出Int重组蛋白诱导表达情况；图2示出不同温度条件下BP反应克隆菌落柱状图。其中，图1的泳道“M”为蛋白marker；泳道“1”为不含有表达质粒的细菌诱导结果；泳道“2”为含有表达质粒，但不包含Int序列的诱导结果；泳道3为含有Int基因质粒诱导结果；泳道“4”为镍柱亲和纯化结果；泳道“5”为镍柱亲和纯化后再经过分子筛纯化结果。
本发明提供的重组蛋白，是由Lambda噬菌体Int全长基因的347位和351位氨基酸突变获得(G347H,和D351H)。本发明的重组蛋白，为定点突变的Lambda噬菌体Int重组蛋白。
本发明提供的重组蛋白表达载体获得方法是：利用定点突变方法获得347位和351位突变的Lambda噬菌体Int重组蛋白基因(G347H,和D351H)。选用核酸限制性内切酶Nde I和Ecor I分别酶切Lambda噬菌体Int重组蛋白基因的PCR产物和PET-22b骨架。连接酶切产物，通过转化TOP10感受态细胞，并培养，提取质粒，测序获得正确的PET 22b-Int质粒。
本发明提供的重组蛋白获得方法是：将构建好的表达质粒转化到 大肠杆菌BL21DE(3)中进行原核表达。通过镍柱亲和层析，Superdex-200分子筛层析的方法获得高纯度的347位和351位突变的Lambda噬菌体Int重组蛋白(G347H,和D351H)。
本发明提供一种重组蛋白在同源重组中的应用。其中：所述应用是利用定点突变的Lambda噬菌体Int重组蛋白通过温度调节方式控制重组蛋白活性，从而控制重组反应活性。
具体方法是：BP反应体系中加入定点突变的Lambda噬菌体Int重组蛋白和IHF蛋白；加入反应底物和适宜缓冲体系。设置4℃，8℃，15℃，25℃，30℃，37℃等6个温度梯度，验证不同温度对BP反应影响情况。在4℃～25℃条件下定点突变的Lambda噬菌体Int重组蛋白和IHF蛋白随着温度的升高其BP活性逐步上升。在4℃，8℃，15℃，25℃，30℃，37℃等6个温度梯度，验证温度达到25℃时，开启Int重组活性。从而证实了Lambda噬菌体Int重组蛋白的重组活性有无受到某一特定温度调控。
本发明提供的重组蛋白，具有与野生型Lambda噬菌体Int蛋白酶一致重组活性，且具有在25℃金属水浴中放置4小时依然保持酶活的热稳定性。
本发明提供的重组蛋白是通过定点突变的方法将Lambda噬菌体Int蛋白中活性位点改造成受温度影响而发挥活性的蛋白，是适宜温度内，低温条件下不启动DNA重组反应，一定温度条件下启动DNA重组反应。
本发明提供的重组蛋白(G347H,和D351H)，即突变型Int蛋白，制备方法简单，产量大，纯度高；实验证实热稳定性好，与野生型酶具有相同的催化趋势。相比野生型酶，其具有在较低温度下无催化活 性，在适合温度下具有催化活性的特征，具有广阔的应用前景。
实施例1：克隆野生型和突变型Int蛋白基因
1)克隆得到定点突变的Lambda噬菌体Int蛋白基因。
2)根据NCBI上公开的Lambda噬菌体Int蛋白基因序列设计引物，具体如下表所述：
Figure PCTCN2014090374-appb-000001
3)分别使用设计的Int-wt和Int-(G347H)-(D351H)的上游引物和下游引物进行PCR反应体系如下：
反应体系：
Figure PCTCN2014090374-appb-000002
Figure PCTCN2014090374-appb-000003
4)PCR反应条件如下：
Figure PCTCN2014090374-appb-000004
5)PCR产物回收
PCR结束后通过2.0％的琼脂糖凝胶电泳分析片段长短，根据片段大小切下目的条带，使用生工的DNA纯化试剂盒进行回收切胶产物。
实施例2：构建野生型和突变型表达菌株
将定点突变基因构建到表达质粒，并获得阳性表达菌株
1)构建含有酶切位点的定点突变的Lambda噬菌体Int蛋白基因，选用Int-全长上下游引物，以实施例1中得到的PCR产物为模板，质粒和PCR产物双酶切体系如下：
PCR产物及质粒载体的双酶切反应
PCR产物酶切体系：
Figure PCTCN2014090374-appb-000005
反应条件：37℃反应4小时
质粒载体酶切体系：
Figure PCTCN2014090374-appb-000006
反应条件：37℃反应4小时
PCR产物和载体双酶切后的产物经过1％琼脂糖凝胶电泳，并使用DNA凝胶回收试剂盒进行纯化回收。
连接反应体系：
Figure PCTCN2014090374-appb-000007
充分混匀后离心数秒，将管壁液滴收集到管底，16℃过夜连接。
2)重组质粒转化、鉴定及测序
A.感受态细胞制备
a.挑取BL21(DE3)单菌落接种于10ml液体LB，37℃，210rpm过夜培养。
b.取5ml菌液接种于500ml LB中，37℃，210rpm，摇70-80minOD可达0.375左右。
c.将菌液放置于冰水混合物上10min,同时预冷50ml离心管。
d.将菌液转移到离心管中，4℃,3700rpm，10min收集菌体。弃上清，将残液倒尽。
e.每个离心管中加入大约10ml冰预冷的激活Buffer(0.1M CaCl2),用5ml灭菌枪头打散沉淀，然后再向每个管子中加入大约30ml冰预冷的激活Buffer，颠倒混匀，冰上静置20min；
f.4℃,3700rpm，离心10min；弃上清，将残液倒净，按500ml菌液12ml冰浴冷储存buffer(0.1M CaCl2，15％甘油)的量将沉淀打散。
g.将感受态细胞分装到冰预冷的灭菌EP管中，每管100μL，置于冰上。
h.感受态-80℃冻存。
B.连接产物转化
a.将10μL连接产物加入100μL新鲜制备的感受态细胞中，轻轻混匀，冰浴30min。
b.42℃热击50s，然后迅速置于冰浴中冷却3min。
c.加入250μL的LB培养基，250rpm/min震荡培养60min，使细菌复苏，并表达质粒编码的抗生素抗性基因；
d.吸取上述菌液200μL，涂布于带有Amp抗性的LB固体培养基(Amp 100mg/L)。
e.待菌液被吸干后，倒置平板于37℃培养12-16h。
C.阳性克隆鉴定
a.菌落PCR鉴定
挑取单菌落，于10μL含有Amp抗性的LB中37℃震荡培养1.5h，按照20μL PCR体系，选用Int-全长引物进行鉴定。若为阳性克隆，通过琼脂糖凝胶电泳可检测到一条目的条带。
b.将用以上方法鉴定出的阳性克隆提交测序公司测序，得到的阳性克隆中所插入的核苷酸序列。
实施例3：attB位点PCR产物获得
1)构建含有attB1和attB2位点的PCR产物。
2)参考人类NFKB IA基因序列(NCBI No.NM_020529.2)设计一对含有attB1和attB2位点的引物。具体如下表所示：
Figure PCTCN2014090374-appb-000008
Figure PCTCN2014090374-appb-000009
3)使用设计的attB位点上游引物(attB1位点)和下游引物(attB2位点)进行PCR反应体系如下：
反应体系：
Figure PCTCN2014090374-appb-000010
4)PCR反应条件如下：
Figure PCTCN2014090374-appb-000011
5)PCR产物回收
PCR结束后通过2.0％的琼脂糖凝胶电泳分析片段长短，根据片段大小切下目的条带，使用生工的DNA纯化试剂盒进行回收切胶产物。
实施例4：定点突变和野生型的Lambda噬菌体Int蛋白表达及纯化
1)种子培养：以常规方法挑取阳性克隆菌落置于2ml的含有Amp抗生素的LB中，37℃，250rmp/min震荡培养16h。
2)菌体扩大培养：吸取2ml种子菌液，加入到含有Amp抗性100ml的LB培养液中，37℃，250rmp/min震荡培养2～3h测定OD600为～0.5。培养完毕后，向培养液中加入IPTG，使其终浓度为0.1mM,30℃诱导培养3h。
3)收集菌体：4,200rpm,4℃离心15min，弃上清，收集菌体；加入重悬液(10mM Tris-HCl[pH 8.0],150mM NaCl,10mM imidazole),震荡沉淀菌体细胞，加入蛋白酶抑制剂PMSF至终浓度为2mM。
4)超声波细菌破碎：超声3s,间隔6s，300w，工作60次。
5)高速离心：细胞裂解液于13,000rpm，4℃离心45min，收集上清液，进行下一步分离纯化。
6)Ni-NTA亲和层析：将收集的含有可溶性蛋白的上清液体倒入再生好的1.5ml Ni-NTA柱中，4℃下放置3h；待上清液流尽后，以Wash buffer(PBS+80mM Imidazole)冲洗6个柱体积，冲洗杂蛋白；后用Elution buffer(50mM Tris-HCl[pH 8.0],1mM DTT,150mM NaCl,10mM EDTA,250mM Imidazole)将目的蛋白洗脱下来，用干净预冷烧杯收集；使用SDS-PAGE电泳检测蛋白纯化情况。纯化结果见图2。
7)分子筛纯化：将收集到蛋白液进行超浓缩至2ml，上样到已 经使用平衡液(25mM Tris-HCl,pH8.0,100mM NaCl)平衡好的凝胶层析柱Superdex-200上，流速为0.4ml/min。收集蛋白峰并进行SDS-PAGE电泳，检测蛋白质的纯度及性状。纯化结果见图2。
实施例5：定点突变的Lambda噬菌体Int重组蛋白(G347H,和D351H)酶活稳定性测试
将酶液稀释合适的倍数后，设置25℃，保温4h后，与IHF蛋白混合进行BP重组反应。同时设定野生型Int混合物作为BP反应对照。BP反应完结后，统计克隆数目。结果见图3。定点突变的Lambda噬菌体Int重组蛋白(G347H,和D351H)与野生型相比酶活性相比，在25℃条件下，突变型Int蛋白和野生型Int蛋白酶重组活性相同。
实施例6：定点突变的Lambda噬菌体Int重组蛋白(G347H,和D351H)BP反应与野生型Int重组效率对比
重组率＝(重组克隆数/克隆总数)×100％
将酶液稀释合适的倍数后，添加入含有att B1和attB2位点的PCR产物，添加pDoner221质粒。分别于4℃，8℃，15℃，25℃，30℃，37℃中反应。分别取不同温度下的反应产物5微升，转化感受态细胞。将转化好的感受态细胞于卡纳抗性的LB平板上涂布。37℃培养16小时候，挑取单克隆，培养后做菌液PCR鉴定，计算重组效率如下表所示。
Figure PCTCN2014090374-appb-000012
Figure PCTCN2014090374-appb-000013
备注：阴性对照具体为除了IHF和Int外，添加其余反应物。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应所述以权利要求的保护范围为准。

Claims (10)

  1. 一种定点突变的Lambda噬菌体Int重组蛋白，其特征在于，所述重组蛋白由Lambda噬菌体I nt的重组蛋白基因片段定点突变获得。
  2. 根据权利要求1所述的重组蛋白，其特征在于，所述重组蛋白由Lambda噬菌体Int全长基因的347位和351位氨基酸突变获得。
  3. 根据权利要求2所述的重组蛋白，其特征在于，所述重组蛋白为G347H和D351H突变的Lambda噬菌体Int重组蛋白。
  4. 根据权利要求3所述的重组蛋白，其特征在于，所述重组蛋白具有与野生型Lambda噬菌体Int蛋白酶一致的重组活性。
  5. 根据权利要求4所述的重组蛋白，其特征在于，所述重组蛋白在15℃～37℃具有酶活热稳定性。
  6. 根据权利要求5所述的重组蛋白，其特征在于，所述重组蛋白在25℃具有酶活热稳定性。
  7. 根据权利要求5所述的重组蛋白，其特征在于，所述重组蛋白在25℃具有温度敏感的高生物活性。
  8. 一种如权利要求1至7任一项所述的重组蛋白在核酸重组系统中的应用。
  9. 一种如权利要求1至7任一项所述的重组蛋白通过温度差异调控核酸重组系统中的应用。
  10. 一种如权利要求1至7任一项所述的定点突变的Lambda噬菌体Int重组蛋白的制备方法，其特征在于，包括如下步骤：
    1)设计含有突变位点的Int特异性引物；
    2)克隆野生型Lambda噬菌体Int基因；
    3)以野生型Lambda噬菌体Int基因为模板，在Int特异性引物的 作用下进行PCR反应，获得定点突变的Lambda噬菌体Int重组基因；
    4)采用限制性内切酶，分别酶切质粒载体和所述的Lambda噬菌体I nt重组基因；
    5)采用DNA连接酶进行连接反应，构建含有所述的Lambda噬菌体Int重组基因的重组质粒；
    6)对步骤5)得到的重组质粒转化大肠杆菌进行原核表达，然后通过分离、纯化，得到所述重组蛋白。
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