WO2005071072A1 - Transcriptase inverse du virus de la leucemie murine recombinee, gene codant pour ladite transcriptase inverse et methode permettant d'exprimer ladite transcriptase inverse - Google Patents

Transcriptase inverse du virus de la leucemie murine recombinee, gene codant pour ladite transcriptase inverse et methode permettant d'exprimer ladite transcriptase inverse Download PDF

Info

Publication number
WO2005071072A1
WO2005071072A1 PCT/CN2004/000039 CN2004000039W WO2005071072A1 WO 2005071072 A1 WO2005071072 A1 WO 2005071072A1 CN 2004000039 W CN2004000039 W CN 2004000039W WO 2005071072 A1 WO2005071072 A1 WO 2005071072A1
Authority
WO
WIPO (PCT)
Prior art keywords
reverse transcriptase
leukemia virus
virus reverse
recombinant mouse
mouse leukemia
Prior art date
Application number
PCT/CN2004/000039
Other languages
English (en)
French (fr)
Inventor
Guangxia Gao
Shufeng Liu
Original Assignee
The Institute Of Biophysics Chinese Academy Of Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Institute Of Biophysics Chinese Academy Of Sciences filed Critical The Institute Of Biophysics Chinese Academy Of Sciences
Priority to EP04701574A priority Critical patent/EP1712617A4/en
Priority to CNB2004800371754A priority patent/CN100537756C/zh
Priority to PCT/CN2004/000039 priority patent/WO2005071072A1/zh
Priority to US10/585,863 priority patent/US7794996B2/en
Publication of WO2005071072A1 publication Critical patent/WO2005071072A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase

Definitions

  • the invention relates to a recombinant reverse transcriptase, a coding gene and an expression method thereof in the field of biotechnology, and particularly to a recombinant mouse leukemia virus reverse transcriptase, a coding gene and an expression method thereof.
  • Reverse Transcriptase is a DNA polymerase encoded by a retrovirus. It can synthesize DNA using RNA or DNA as a template. Reverse transcriptase is a commonly used tool for molecular biology research. It can reverse transcribe RNA into cDNA. It is mainly used for the construction of cDNA library or combined with polymerase chain reaction (PCR) (RT-PCR) to detect RNA in biological samples. content.
  • PCR polymerase chain reaction
  • RT-PCR polymerase chain reaction
  • MLV-RT consists of two functional domains and contains two kinds of enzyme activities, namely DNA polymerase (Pol) at the N-terminus and RNase H at the C-terminus. The two functions can be expressed separately without affecting the activity of any enzyme.
  • the first-generation recombinant MLV-RT used for cDNA synthesis contained only the DNA polymerase portion and not the RNaseH portion. Although the enzyme activity was similar to the full-length MLV-RT, its ability to continuously synthesize was weak and the synthesized cDNA was shorter .
  • RNaseH part does not affect the DNA polymerase activity of MLV-RT, it does affect its ability to continuously synthesize, because the RNaseH domain can bind the template-primer complex and increase the affinity between MLV-RT and template-primer.
  • site-directed mutagenesis was used to mutate aspartic acid (Asp) at position 524 in the active center of RNaseH to asparagine (Asn) to become MLV-RT-D524N, the enzyme did not have lyaseH activity, but retained its DNA polymerase activity and high affinity for template-substrate, because Ife has higher sustained synthesis ability.
  • the mutant enzyme is a patented product of American Invitrogen Company under the trade name Superscript, which is currently the most widely used reverse transcriptase.
  • Superscript is not perfect, and its ability to continuously synthesize is not ideal. Most of the synthesized cDNA is still incomplete.
  • the object of the present invention is to provide a recombinant mouse leukemia virus reverse transcriptase.
  • MLV-RT-Q84X The recombinant mouse leukemia virus reverse transcriptase provided by the present invention, named MLV-RT-Q84X, is a protein in which MLV-RT has a glutamine residue at the 84th position of the N-terminus as an amino acid residue X, where X Amino acids whose side chains are shorter than the side chains of glutamine residues.
  • murine leukemia virus The aspartic acid residue at position 524 of the terminal was replaced with an asparagine residue.
  • the name of the obtained recombinant mouse leukemia virus reverse transcriptase was MLV-RT-Q84X-D524N.
  • X is preferably alanine, serine, aspartic acid or asparagine, and particularly preferably alanine.
  • the gene encoding the above-mentioned recombinant mouse leukemia virus reverse transcriptase also belongs to the protection scope of the present invention.
  • Another object of the present invention is to provide a method for expressing a recombinant mouse leukemia virus reverse transcriptase.
  • a method for expressing a recombinant mouse leukemia virus reverse transcriptase is to transform an expression vector containing a gene encoding a recombinant mouse leukemia virus reverse transcriptase into E. coli, cultivate a positive clone, and express the recombinant mouse leukemia virus reverse transcriptase;
  • the recombinant mouse leukemia virus reverse transcriptase is a protein in which the glutamine residue at position 84 from the N-terminus of MLV-RT is replaced with an amino acid residue X, wherein X is a side chain shorter than the glutamine side Chain of amino acids.
  • the recombinant mouse leukemia virus reverse transcriptase preferably replaces MLV-RT from the N-terminal glutamine residue at the 84th position with amino acid residue X, and from the N-terminal asparagine residue at the 524th position.
  • the expression vector containing the recombinant mouse leukemia virus reverse transcriptase-encoding gene may be a plasmid pTacRT-Q84A-D524N having the nucleotide sequence of sequence 1 in the sequence list or a plasmid having the nucleotide sequence of sequence 3 in the sequence list pTacRT- Q84N- D524N, the Escherichia coli is Escherichia coli BL21, the amino acid sequence of the recombinant mouse leukemia virus reverse transcriptase expressed by the plasmid pTacRT- Q84A- D524N and the plasmid pTacRT- Q84N- D524N is shown in sequence 2 in the sequence listing And shown in sequence 4.
  • sequence 1 and sequence 3 in the sequence list are composed of 7488 bases, and their open reading frames are from the 1515th base to the 3527th base at the 5 ′ end, and the sequences 2 and 4 in the sequence list are both 671 Amino acid residues.
  • Figure 1 shows the purified MLV RT- Q84A- D524N SDS-PAGE electrophoresis
  • Figure 2 shows the kinetic analysis of MLV-RT-Q84A D524N and MLV-RT-D524N enzymes.
  • Figure 3 is a comparison of cDNA synthesized using MLV-RT-Q84A-D524N and MLV-RT-D524N.
  • Figure 4 is a purified MLV-RT-Q84N-D524N SDS-PAGE electrophoresis detection chart.
  • Figure 5 is an enzyme activity measurement chart of MLV- RT- Q84N- D524N
  • Primer Q84A-AP (5 'CGGAATTCCCGCGTCCAACAGTCTCTGTA) also contains an EcoRI restriction site due to synonymous mutations and a mutant alanine codon (in bold, which is amplified by PCR with the upstream primer (5, GTGGA TGTGAGCCGA)
  • a 300 bp Aflll-EcoRI fragment was obtained.
  • Afl II-EcoRI fragment (ntl467-1770) and EcoRI / Mfel double-cut EcoRI-Mfel (ntl770-2058) fragment treated with Af II / EcoRI and The 6. 9 kb fragment double-cut by the Aflll-Mfel vector P TacRT-D524N was ligated to transform E.coli Topo10.
  • the resulting recombinant plasmid pTacRT-Q84A-D524N was identified as a positive clone by digestion, and dideoxy sequencing showed that it has the sequence listing The nucleotide sequence shown in Sequence 1.
  • PTacRT-Q84A-D524N was transformed into Escherichia coli BL21, and a single colony was picked and inoculated in LB liquid medium containing 100 ⁇ g / ml ampicillin, and cultured at 37 C to 0D 6 . . 0.5 pm, add IPTG to a final concentration of 0.5 mM, continue to culture at 37 ° C for 2-3 hours, collect the bacterial cells, wash the cells with pre-chilled 50 mM Tris-HCl (pH 7. 5) and reserve for later use .
  • the collected cells were suspended in buffer A (20mM PBS, pH 7. 4, 0.5M NaCl), and after thawing, lysozyme was added to a final concentration of 0.5mg / ml, digested at 4 ° C for 30min, and sonicated (power : 190W) 3 times, 20 seconds each time, centrifuge, collect the supernatant. After being separated and purified by a metal chelate chromatography column HiTrap chelating HP column (purchased from Pharmacia), the purity can reach more than 80%. The molecular weight of SDS-PAGE is used to determine and collect the active protein peaks.
  • the "radiation intensity retained in DE81 filter paper / total radiation intensity of the same sample” indicates the amount of dTTP incorporated into the product.
  • Kinetic analysis showed that when dTTP was used as the substrate, MLV-RT-Q84A-D524N and RT-D524N had similar Km (11.04 ⁇ M and 12.94 ⁇ M, respectively), while
  • the maximum reaction rate Vmax of MLV-RT-Q84A-D524N is 3.2 times that of MLV-RT-D524N (0.41 ⁇ 1 ⁇ ⁇ 1 ⁇ - l.mg-1 and 0.13 ⁇ 1. ⁇ - l.mg-1) ( (See Table 1).
  • Table 1 Comparison of MLV-RT-Q84A-D524 and MLV-RT- D524N enzyme kinetic parameters
  • This step compares the MLV-RT-Q84A-D524N and MLV-RT-D524N in their ability to synthesize cDNA.
  • RNA of 293T cells was used as a template, and oligo (dT) 18 was used as a primer to synthesize the first-strand cDNA.
  • the product contained 32 P as a marker.
  • 20 ⁇ 1 reaction system includes total RNA of lyg293T cells, 50 raM Tris. Hcl (pH 8.3), 75raMKCl, 3 raM MgCl 2 , 10 raM DTT, 500 ⁇ M dTTP, 50 ⁇ g / ml oligo (dT) 18 , 2 ⁇ Ci 32 PdTTP, 1 ⁇ g RT pure enzyme sample. Reaction at 42 ° C for 1 hour.
  • Fig. 3 shows that the cDNA fragments synthesized by MLV-RT-Q84A-D524N are not only longer than those synthesized by RT-D524N, but also the amount of synthesized cDNA is larger than that of RT-D524N.
  • the glutamine at position 84 in MLV-RT-D524N is mutated to asparagine to make it MLV-RT-Q84N-D524N.
  • Primer Q8 N-AP (5 'CGGGATCCCGnGTCCAACAGTCTCTGTA) also contains a BamHI restriction site due to synonymous mutations and a mutated asparagine codon (indicated in bold), which is similar to the upstream primer
  • the obtained recombinant plasmid pTacRT-Q84N-D524N was identified as a positive clone by enzyme digestion, and dideoxy sequencing showed that it had the nucleotide sequence shown in Sequence 3 in the Sequence Listing.
  • the pTacRT-Q84N-D524N transformed Escherichia coli BL21 Individual colonies were picked inoculated in liquid LB medium containing 100 yg / m l ampicillin, 37 ° C to culture 0D 6Q. 0.5 pm, add IPTG to a final concentration of 0.5 mM, continue incubation at 37 ° C for 2-3 hours, collect the bacterial cells, wash the cells with pre-cooled 50 mM Tris-HCl ( ⁇ ⁇ ⁇ 5) once and reserve .
  • the collected cells were suspended in buffer A (20mM PBS, pH 7. 4, 0.5M NaCl), and after thawing, lysozyme was added to a final concentration of 0.5mg / ml, digested at 4 ° C for 30min, and ultrasound (power : 190W) Crush 3 times for 20 seconds each time, centrifuge and collect the supernatant. After separation and purification with a metal chelate chromatography column HiTrap chelating HP column (purchased from Pharmacia), the purity can reach more than 80%. The molecular weight of the protein is determined according to SDS-PAGE, and the active protein peak is determined and collected.
  • FIG. 4 shows that the protein is a uniform band with a molecular weight of about 76KD, and no impurities are seen.
  • M is a molecular weight standard; 1 means that the sample contains 1 microgram of protein; 2 means that the sample contains 2 microgram of protein; 3 means that the sample contains 5 microgram of protein. 4. Determination of enzyme activity and kinetic analysis
  • MLV-RT-Q84A-D524N has comparable vitality.
  • the glutamine (Q84) at the 84th position of the enzyme is located near the active center of the enzyme and participates in regulating the catalytic ability of the enzyme, and its longer side chain hinders the extension of the synthetic product Affects the activity and sustained synthesis of DNA polymerase.
  • the MLV-RT gene is mutated, so that the Q84 residue is mutated into a residue with a shorter side chain, and a new mutant enzyme MLV-RT-Q84X is generated, where X is a side chain shorter than the glutamine side chain.
  • Amino acids such as alanine (Ala), serine (Ser), asparagine (Asn), or aspartic acid (Asp).
  • the present invention introduces a Q84X mutation into MLV-RT-D524N to make it MLV-RT-Q84X-D524N.
  • the recombinant mouse leukemia virus reverse transcriptase of the present invention has higher enzyme activity and continuous synthesis ability than Superscript, and will play an important role in the field of biotechnology, and can be widely used for cDNA synthesis.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Description

重組小鼠白血病病毒逆转录酶及其编码基因与表达方法 技术领域
本发明涉及生物技术领域中一种重组逆转录酶及其编码基因与表达方 法， 特别涉及一种重组小鼠白血病病毒逆转录酶及其编码基因与表达方法。 背景技术
逆转录酶（Reverse Transcriptase, 简称 RT) 是由逆转录病毒编码的 DNA聚合酶， 能以 RNA或 DNA作为模板合成 DNA。逆转录酶是分子生物学研究 的常用工具酶，可以将 RNA逆转录成 cDNA，主要用于 cDNA文库的构建或与聚 合酶链式反应（PCR)合用 （RT- PCR)检测生物样品中 RNA的含量。 目前市场 上占主导地位的逆转录酶是小鼠白血病病毒 （Murine Leukemia Virus, 简称 MLV) 的逆转录酶（MLV- RT) 。
MLV-RT由两个功能域组成， 同时含有两种酶活性， 即位于 N端的 DNA聚 合酶（Pol )和位于 C端的 RNase H。 两个功旨 域可分别表达， 而不影响任何 —种酶的活性。 用于 cDNA合成的第一代重组 MLV-RT只含有 DNA聚合酶部分 而不含有 RNaseH部分， 虽然酶的活性与全长的 MLV-RT相似， 但其持续合成 能力较弱，合成的 cDNA较短。后来人们发现， RNaseH部分虽然不影响 MLV-RT 的 DNA聚合酶活性， 却影响其持续合成能力， 原因是 RNaseH结构域能结合模 板一引物复合体， 增加 MLV-RT与模板一引物的亲和力。 当利用定点突变方法 将位于 RNaseH活性中心的第 524位的天门冬氨酸（Asp) 突变为天门冬酰胺 (Asn) ， 成为 MLV-RT- D524N, 该酶不具有丽 aseH活性， 但保留了其 DNA聚 合酶的活性和对模板一底物的高亲和力， 因 Ife具有更高的持续合成能力。 该 突变酶为美国 Invitrogen公司专利产品， 商品名为 Superscript, 是目前使 用最为广泛的逆转录酶。但是， Superscript也并非完美无缺，其持续合成能 力尚不理想， 合成的 cDNA中绝大部分还是不完整的。
发明公开
本发明的目的是提供一种重组小鼠白血病病毒逆转录酶。
本发明所提供的重组小鼠白血病病毒逆转录酶， 名称为 MLV-RT-Q84X, 是将 MLV- RT的自 N端第 84位谷氨酰胺残基¾代为氨基酸残基 X的蛋白质， 其中 X为侧链短于谷氨酰胺残基侧链的氨基酸。
为了进一步提高其活性， 再将上述重组 4、鼠白血病病毒逆转录酶的自 N 端第 524位天门冬氨酸残基取代为天门冬酰胺残基， 所得到的重组小鼠白血 病病毒逆转录酶的名称为 MLV-RT- Q84X- D524N。
其中， 所述 X优选为丙氨酸、 丝氨酸、 天门冬氨酸或天门冬酰胺， 尤其 优选为丙氨酸。
上述重组小鼠白血病病毒逆转录酶的编码基因也属于本发明的保护范 围。
本发明的另一个目的是提供一种表达重组小鼠白血病病毒逆转录酶的方 法。
一种表达重组小鼠白血病病毒逆转录酶的方法， 是将含有重组小鼠白血 病病毒逆转录酶编码基因的表达载体转化到大肠杆菌中， 培养阳性克隆， 表 达重组小鼠白血病病毒逆转录酶； 所述重组小鼠白血病病毒逆转录酶， 是将 MLV-RT的自 N端第 84位谷氨酰胺残基取代为氨基酸残基 X的蛋白质，其中 X 为侧链短于所述谷氨酰胺侧链的氨基酸。
其中， 所述重组小鼠白血病病毒逆转录酶优选为将 MLV- RT 的自 N端第 84位谷氨酰胺残基取代为氨基酸残基 X， 自 N端第 524位天门冬氨酸残基取 代为天门冬酰胺残基的蛋白质， 其中 X为侧链短于所述谷氨酰胺侧链的氨基 酸；尤其优选为将 MLV-RT的自 N端第 84位谷氨酰胺残基取代为丙氨酸残基， 自 N端第 524位天门冬氨酸残基取代为天门冬酰胺残基的蛋白质。
所述含有重组小鼠白血病病毒逆转录酶编码基因的表达载体可以是具有 序列表中序列 1的核苷酸序列的质粒 pTacRT- Q84A-D524N或具有序列表中序 列 3的核苷酸序列的质粒 pTacRT- Q84N- D524N,所述大肠杆菌为 Escherichia coli BL21， 质粒 pTacRT- Q84A- D524N和质粒 pTacRT- Q84N- D524N所表达的重 组小鼠白血病病毒逆转录酶的氨基酸序列分别如序列表中的序列 2和序列 4 所示。 其中， 序列表中的序列 1和序列 3均由 7488个碱基组成， 它们的开放 阅读框架均自 5 ' 端第 1515位碱基 -3527位碱基， 序列表中序列 2和 4均由 671个氨基酸残基组成。
附图说明
图 1为纯化后的 MLV RT- Q84A- D524N SDS- PAGE电泳检测图
图 2为 MLV-RT- Q84A D524N与 MLV- RT- D524N酶动力学分析图 .
图 3为利用 MLV-RT- Q84A-D524N和 MLV- RT-D524N合成的 cDNA的比较图 图 4为纯化后的 MLV- RT- Q84N-D524N SDS-PAGE电泳检测图 图 5为 MLV- RT- Q84N- D524N的酶活测定图
实施发明的最佳方式
实施例 1、 MLV- RT- Q84A- D524N的制备
1、 质粒 pTacRT- Q84A- D524N的构建
本步骤中将 MLV- RT- D524N中的第 84位的谷氨酰胺突变为丙氨酸， 使之 成为 MLV- RT- Q84A- D524N0
MLV-RTH38 A-D524N的定点突变 将两段 PCR产物 Af ΠΙ EcoRI和 EcoRI- Mf el 撤掉 pTacRT-D524 iBlain, S. W. &Goff> S. P. (1995) J. Virol. 69， 4440-4452。 ) 的 Aflll- Mfel片段（nt 1467- 2058)。引物 Q84A- SP (5， CGGAATTCTGGTACCCTGCCAGTC) 包含有一个由于同义突变产生的 EcoRI酶切位点 （用下划线表示） ， 它和下游 引物（5， TGGGAGTCTGGTCCAGG) PCR扩增得到一个 300bp的 EcoRI- Mfel片段。 引物 Q84A - AP (5 ' CGGAATTCCCGCGTCCAACAGTCTCTGTA)同样包含有一个由于同 义突 ^生的 EcoRI酶切位点以及突变的丙氨酸密码子（黑体 ， 它和上游引物 (5， GTGGA TGTGAGCCGA)经 PCR扩增得到一个 300bp的 Aflll- EcoRI片段。 将 分别用 Af ΠΙ/EcoRI双酶切处理过的 Aflll- EcoRI片段（ntl467- 1770)以及 EcoRI/Mfel双切处理过的 EcoRI- Mfel (ntl770- 2058)片段与经 Aflll- Mfel双 切过的载体 PTacRT-D524N 6. 9kb片段连接，转化大肠杆菌 Τορ10。所得重组质 粒 pTacRT- Q84A-D524N通过酶切鉴定为阳性克隆， 双脱氧法测序表明其具有 序列表中序列 1所示的核苷酸序列。
2、 重组逆转录酶在大肠杆菌中的诱导和表达
将 pTacRT- Q84A-D524N转化 Escherichia coli BL21,挑取单菌落接种于 含 100 y g/ml氨苄青霉素的 LB液体培养基中， 37 C培养至 0D6。。 0. 5时， 加 入 IPTG至终浓度 0. 5mM, 37°C继续培养 2-3小时，收集菌体，将菌体用预冷的 50 mM Tris-HCl (pH 7. 5)洗涤一次后备用。
3、 重组逆转录酶的纯化
将收集的菌体悬浮于缓冲液 A (20mM PBS, pH 7. 4, 0. 5M NaCl) ,冻融后， 加入溶菌酶至终浓度 0. 5mg/ml， 4°C消化 30min,超声 （功率： 190W) 破碎 3 次， 每次 20秒， 离心， 收集上清。 经过金属螯合层析柱 HiTrap chelating HP column (购自 Pharmacia)分离纯化后， 纯度即可达 80%以上， 根据 SDS-PAGE 显示分子量大小， 确定并收集活性蛋白峰。 经过离子交换层析柱 MonoS (购自 Pharmacia)进一步分离纯化后， 电泳检测考马斯亮蓝染色， 结果如图 1所示， 表明该蛋白为一分子量约为 76KD的均一条带， 未见杂质。 图 1中， M为分子 量标准； 1表示样品中含 5微克蛋白； 2表示样品中含 2微克蛋白。
4、 酶活性测定及动力学分析
50μ1反应体系中包括 10ng RT纯酶样品， 60 mM Tris. Hcl (pH 8.0)， 75 ηιΜ NaCl, 0.7 raM MnCl2, 5 rnM DTT, 12 μ g/ml Poly (rA)模板， 6 μ g/ral oligo(dT) 18 引物， 10 μ Ci/ml (lCi=37 GBq)32P标记的 dTTP以及 12 μ M未标记的 dTTP，反 应于 37DC进行。 分别于反应开始后不同时间点取样 4μ 1点于 DE81滤纸， 终 止反应。 然后将 DE81滤纸用 2 XSSC洗涤三次， 95%乙醇漂洗两次， 将滤纸 晾干后， 放射自显影。 定量分析采用液闪计数仪测定。
动力学分析釆用公知的双倒数作图法进行， 如图 2所示。 用每个样品中
"保留在 DE81滤纸的放射强度 /同一样品总的放射强度"来表示掺入到产物 中的 dTTP的量。 动力学分析显示， 在以 dTTP为底物时， MLV- RT- Q84A- D524N 和 RT- D524N具有相似的 Km (分别为 11.04 μ M和 12.94 μ Μ) ，而
MLV-RT-Q84A-D524N的最大反应速率 Vmax值则是 MLV- RT- D524N的 3.2倍（分 别为 0·41μηιο1·πΰη- l.mg- 1和 0.13μηιο1.πάη- l.mg- 1) (如表 1) 。 表 1. MLV-RT-Q84A-D524 与 MLV- RT- D524N酶动力学参数比较
Figure imgf000005_0001
5. cDNA合成
本步骤比较了 MLV- RT-Q84A- D524N与 MLV- RT- D524N在合成 cDNA能力方 面的差异。
cDNA合成采用 293T细胞的总 RNA为模板， oligo(dT) 18为引物合成第一 链 cDNA, 产物中惨有 32P作为标记。 20μ 1反应体系中包括 lyg293T细胞总 RNA, 50 raM Tris. Hcl (pH 8.3), 75raMKCl， 3 raM MgCl2, 10 raM DTT, 500 μ M dTTP, 50 μ g/ml oligo (dT) 18， 2 μ Ci 32PdTTP， 1 μ g RT纯酶样品。 42°C反应 1小时。 碱变性电泳检测实验结果。 结果如图 3所示， 表明 MLV- RT- Q84A-D524N合成 的 cDNA片段不但较 RT- D524N合成的长， 而且合成的 cDNA量也比 RT- D524N 多。
实施例 2、 MLV-RT-Q84N-D524 的制备 1、 质.粒 pTacRT- Q84N- D524N的构建
本步骤中将 MLV- RT- D524N中的第 84位的谷氨酰胺突变为天门冬酰胺， 使之成为 MLV- RT- Q84N- D524N。
MLV-RTH384N-D524N的定点突^ 将两段 PCR产物 Α ΙΙ- BamHI和 BamHI- Mfel ^ pTacRT- D52 ain, S. W. &Goff, S. P. (1995) J. Virol. 69, 4440-4452。 ) 的 Af III- Mfel片段（nt 1467- 2058) 0引物 Q84N- SP (5， CGGGATCCTGGTACCCTGCCAGTC) 包含有一个由于同义突变产生的 BamHI酶切位点 （用下划线表示）， 它和下游引物 (5， TGGGAGTCTGGTCCAGG)PCR扩增得到一个 300bp的 BamHI- Mfel片段。引物 Q8 N-AP (5' CGGGATCCCGnGTCCAACAGTCTCTGTA)同样包含有一个由于同义突 ^生的 BamHI 酶切位点以及突变的天门冬酰胺密码子 （黑体表示） ， 它和上游引物
(5' GTGGAATTGTGAGCCGA)经 PCR扩增得到一个 300bp的 Afl II- BamHI片段。 将分别用 Af ΙΠ/BamHI双酶切处理过的 Af III- BamHI片段 (ntl467- 1770)以及 BamHI/Mfel双切处理过的 BamHI- Mfel (ntl770- 2058)片段与经 Af III- Mfel双 切过的载体 pTacRT- D524N 6. 9kb片段连接,转化大肠杆菌 Τορ10。所得重组质 粒 pTacRT- Q84N- D524N通过酶切鉴定为阳性克隆， 双脱氧法测序表明其具有 序列表中序列 3所示的核苷酸序列。
2、 重组逆转录酶在大肠杆菌中的诱导和表达
将 pTacRT-Q84N-D524N转化 Escherichia coli BL21，挑取单菌落接种于 含 100 y g/ml氨苄青霉素的 LB液体培养基中， 37°C培养至 0D6Q。 0. 5时， 加 入 IPTG至终浓度 0. 5mM， 37°C继续培养 2-3小时，收集菌体，将菌体用预冷的 50 mM Tris-HCl (ρΗ Ί· 5)洗涤一次后备用。
3、 重组逆转录酶的纯化
将收集的菌体悬浮于缓冲液 A (20mM PBS, pH 7. 4, 0. 5M NaCl)，冻融后， 加入溶菌酶至终浓度 0. 5mg/ml， 4°C消化 30min，超声 (功率: 190W) 破碎 3 次， 每次 20秒， 离心， 收集上清。 经过金属螯合层析柱 HiTrap chelating HP column (购自 Pharmacia)分离纯化后， 纯度即可达 80%以上， 根据 SDS- PAGE 显示分子量大小， 确定并收集活性蛋白峰。 经过离子交换层析柱 MonoS (购自 Pharmacia)进一步分离纯化后， 电泳检测考马斯亮蓝染色， 结果如图 4所示， 表明该蛋白为一分子量约为 76KD的均一条带， 未见杂质。 图 4中， M为分子 量标准； 1表示样品中含 1微克蛋白； 2表示样品中含 2微克蛋白； 3表示样 品中含 5微克蛋白。 4、 酶活性测定及动力学分析
50μ1反应体系中包括 10ng RT纯酶样品， 60 mM Tris. Hcl (pH 8. 0) , 75 mM NaCl, 0. 7 mM MnCl2， 5 mM DTT, 12 μ g/ml Poly (rA)模板， 6 μ g/ml oligo (dT) 18 引物， 10 μ Ci/ml (lCi=37 GBq) 32P标记的 dTTP以及 12 μ M未标记的 dTTP,反 应于 37°C进行。 分别于反应开始后不同时间点取样 4 μ 1点于 DE81滤纸， 终 止反应。 然后将 DE81滤纸用 2 X SSC洗涤三次， 95%乙醇漂洗两次， 将滤纸 晾干后， 放射自显影。 结果如图 5所示， 图中黑点颜色的深浅代表酶活力的 高低。 结果显示 MLV- RT- Q84N- D524N的酶活力较 MLV- RT- D524N高， 跟
MLV-RT-Q84A-D524N的活力相当。
工业应用
本发明通过分析 MLV- RT的晶体结构模型， 发现该酶第 84位的谷氨酰胺 (Q84)位于酶的活性中心附近， 参与调控酶的催化能力， 其较长的侧链妨碍 合成产物的延伸， 影响 DNA聚合酶的活性和持续合成能力。 本发明将 MLV- RT 基因进行突变， 使 Q84残基突变成侧链较短的残基， 产生了新的突变酶 MLV-RT-Q84X, 其中 X为侧链短于谷氨酰胺侧链的氨基酸， 如丙氨酸（Ala)、 丝氨酸（Ser) 、 天门冬酰胺 （Asn) 、 或天门冬氨酸 （Asp) 等。 同时， 本发 明将 Q84X突变引入 MLV- RT- D524N中，使之成为 MLV-RT- Q84X- D524N。本发明 的重组小鼠白血病病毒逆转录酶具有比 Superscript更高酶活性和持续合成 能力， 将在生物技术领域中发挥重要作用， 可以广泛用于 cDNA的合成。

Claims

权利要求书
1、 一种重组小鼠白血病病毒逆转录酶， 是将 MLV- RT的自 N端第 84位谷 氨酰胺残基取代为氨基酸残基 X的蛋白质， 其中 X为侧链短于所述谷氨酰胺 残基侧链的氨基酸。
2、 根据权利要求 1所述的重组小鼠白血病病毒逆转录酶， 其特征在于： 所述重组小鼠白血病病毒逆转录酶的自 N端第 524位天门冬氨酸残基取代为 天门冬酰胺残基。
3、根据权利要求 1或 2所述的重组小鼠白血病病毒逆转录酶，其特征在 于： 所述 X为丙氨酸、 丝氨酸、 天门冬氨酸或天门冬酰胺。
4、 根据权利要求 3所述的重组小鼠白血病病毒逆转录酶， 其特征在于： 所述 X为丙氨酸。
5、 编码权利要求 1所述重组小鼠白血病病毒逆转录酶的基因。
6、一种表达重组小鼠白血病病毒逆转录酶的方法， 是将含有重组小鼠白 血病病毒逆转录酶编码基因的表达载体转化到大肠杆菌中， 培养阳性克隆， 表达重组小鼠白血病病毒逆转录酶； 所述重组小鼠白血病病毒逆转录酶， 是 将 MLV- RT的自 N端第 84位谷氨酰胺残基取代为氨基酸残基 X的蛋白质， 其 中， X为侧链短于所述谷氨酰胺残基侧链的氨基酸。
7、 根据权利要求 6所述的方法， 其特征在于： 所述重组小鼠白血病病毒 逆转录酶的自 N端第 524位天门冬氨酸残基取代为天门冬酰胺残基。
8、 根据权利要求 7所述的方法， 其特征在于： 所述 X为丙氨酸。
9、 根据权利要求 8所述的方法， 其特征在于： 所述含有重组小鼠白血病 病毒逆转录酶编码基因的表达载体是具有序列表中序列 1 的核苷酸序列的质 粒 pTacRT- Q84A- D524N。
10、 根据权利要求 6、 7、 8或 9所述的方法， 其特征在于： 所述大肠杆 菌为 Escherichia coli BL21。
PCT/CN2004/000039 2004-01-13 2004-01-13 Transcriptase inverse du virus de la leucemie murine recombinee, gene codant pour ladite transcriptase inverse et methode permettant d'exprimer ladite transcriptase inverse WO2005071072A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP04701574A EP1712617A4 (en) 2004-01-13 2004-01-13 REVERSE TRANSCRIPTASE OF RECOMBINANT MURINE LEUKEMIA VIRUS, GENE ENCODING THE INVERSE TRANSCRIPTASE, AND METHOD FOR EXPRESSING THE INVERSE TRANSCRIPTASE TRANSCRIPTASE
CNB2004800371754A CN100537756C (zh) 2004-01-13 2004-01-13 重组小鼠白血病病毒逆转录酶及其编码基因与表达方法
PCT/CN2004/000039 WO2005071072A1 (fr) 2004-01-13 2004-01-13 Transcriptase inverse du virus de la leucemie murine recombinee, gene codant pour ladite transcriptase inverse et methode permettant d'exprimer ladite transcriptase inverse
US10/585,863 US7794996B2 (en) 2004-01-13 2004-01-13 Recombinant murine leukemia virus reverse transcriptases, the genes encoding and the method for expressing it

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2004/000039 WO2005071072A1 (fr) 2004-01-13 2004-01-13 Transcriptase inverse du virus de la leucemie murine recombinee, gene codant pour ladite transcriptase inverse et methode permettant d'exprimer ladite transcriptase inverse

Publications (1)

Publication Number Publication Date
WO2005071072A1 true WO2005071072A1 (fr) 2005-08-04

Family

ID=34800356

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2004/000039 WO2005071072A1 (fr) 2004-01-13 2004-01-13 Transcriptase inverse du virus de la leucemie murine recombinee, gene codant pour ladite transcriptase inverse et methode permettant d'exprimer ladite transcriptase inverse

Country Status (4)

Country Link
US (1) US7794996B2 (zh)
EP (1) EP1712617A4 (zh)
CN (1) CN100537756C (zh)
WO (1) WO2005071072A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102505018B (zh) * 2007-06-21 2014-06-04 日立造船株式会社 编码杜仲的甲羟戊酸途径的酶的基因

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2617790A1 (en) * 2005-08-10 2007-02-22 Stratagene California Mutant reverse transcriptase and methods of use
US9783791B2 (en) 2005-08-10 2017-10-10 Agilent Technologies, Inc. Mutant reverse transcriptase and methods of use
GB0806562D0 (en) 2008-04-10 2008-05-14 Fermentas Uab Production of nucleic acid
US9314751B2 (en) 2011-01-07 2016-04-19 Life Technologies Corporation Methods and apparatus for mixing and shipping fluids
CN116042569B (zh) * 2023-02-01 2023-08-15 珠海宝锐生物科技有限公司 Mmlv逆转录酶突变体及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6136582A (en) * 1998-01-20 2000-10-24 The Trustees Of Columbia University In The City Reverse transcriptase of moloney murine leukemia virus with RNA polymerase activity
US20030021799A1 (en) * 2001-07-11 2003-01-30 Wilkinson Jonathan Frome Reverse transcriptase formulations

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE445630T1 (de) * 1997-04-22 2009-10-15 Life Technologies Corp Verfahren zur herstellung von aslv reverser transkriptase zusammengestellt aus mehreren untereinheiten

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6136582A (en) * 1998-01-20 2000-10-24 The Trustees Of Columbia University In The City Reverse transcriptase of moloney murine leukemia virus with RNA polymerase activity
US20030021799A1 (en) * 2001-07-11 2003-01-30 Wilkinson Jonathan Frome Reverse transcriptase formulations

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, vol. 41, no. 50, 2002, pages 14831 - 14832
J. MOL. BIOL., vol. 305, no. 2, 2001, pages 341 - 359
J. VIROLOGY, vol. 69, no. 7, 1995, pages 4440 - 4452
PNAS, vol. 94, no. 2, 1997, pages 407 - 411
See also references of EP1712617A4

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102505018B (zh) * 2007-06-21 2014-06-04 日立造船株式会社 编码杜仲的甲羟戊酸途径的酶的基因
CN102505019B (zh) * 2007-06-21 2015-03-11 日立造船株式会社 编码杜仲的甲羟戊酸途径的酶的基因

Also Published As

Publication number Publication date
CN100537756C (zh) 2009-09-09
EP1712617A1 (en) 2006-10-18
US7794996B2 (en) 2010-09-14
CN1894403A (zh) 2007-01-10
US20080131950A1 (en) 2008-06-05
EP1712617A4 (en) 2007-07-18

Similar Documents

Publication Publication Date Title
EP1918370B1 (en) Mutant pcna
US20110020897A1 (en) Rna-dependent dna polymerase from geobacillus stearothermophilus
WO2010016621A1 (ja) 機能改善したｒｎａポリメラーゼ変異体
JP2015193666A (ja) 安定化された逆転写酵素融合タンパク質
CA2460546A1 (en) Dna polymerases and mutants thereof
JP2000508538A (ja) バシラス ステアロテルモフィルスｄｎａポリメラーゼの生物学的に活性な断片
WO2005071072A1 (fr) Transcriptase inverse du virus de la leucemie murine recombinee, gene codant pour ladite transcriptase inverse et methode permettant d'exprimer ladite transcriptase inverse
JP2018161129A (ja) 核酸増幅方法
JP3891330B2 (ja) 改変された耐熱性ｄｎａポリメラーゼ
JP6741061B2 (ja) 核酸増幅法
JP4917542B2 (ja) 新規ｄｎａポリメラーゼ
CA3220481A1 (en) Variants of terminal deoxynucleotidyl transferase and uses thereof
JP2010505410A (ja) 変異体ｄｎａポリメラーゼ及びそれらの遺伝子
JPWO2016084879A1 (ja) 核酸増幅試薬
JP7107345B2 (ja) Ｐｃｒ方法
WO2023143123A1 (zh) 可控合成单链dna的末端转移酶变体及应用
JPWO2004020621A1 (ja) 耐熱性リボヌクレアーゼｈ
JP4217922B2 (ja) サーモコッカス・ペプトノフィラス由来の熱安定性ｄｎａポリメラーゼ、該酵素をコードする遺伝子およびその用途
CN114958797A (zh) 突变型dna聚合酶、编码基因、重组表达载体、重组菌及其应用
CN117925558A (zh) 突变型逆转录酶及其应用
JP3624237B2 (ja) エンドヌクレアーゼ
JP2012075418A (ja) 改良されたｒｎａポリメラーゼ変異体
JP2010183842A (ja) 新規耐熱性ｄｎａポリメラーゼ
JPH03232487A (ja) Ｓｐ６ｄｎａポリメラーゼ及びその製法
JPH11206375A (ja) 熱安定性ＭｕｔＭ蛋白質

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200480037175.4

Country of ref document: CN

AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2004701574

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2004701574

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 10585863

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 10585863

Country of ref document: US