CN101613680B - Moloney murine leukemia virus reverse transcriptase mutant as well as expression method and application thereof - Google Patents
Moloney murine leukemia virus reverse transcriptase mutant as well as expression method and application thereof Download PDFInfo
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- CN101613680B CN101613680B CN2009100369299A CN200910036929A CN101613680B CN 101613680 B CN101613680 B CN 101613680B CN 2009100369299 A CN2009100369299 A CN 2009100369299A CN 200910036929 A CN200910036929 A CN 200910036929A CN 101613680 B CN101613680 B CN 101613680B
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Abstract
The invention provides a moloney murine leukemia virus reverse transcriptase mutant as well as an expression method and application thereof. The mutant is a protein formed by substituting a praline residue at the 196th site of the moloney murine leukemia virus reverse transcriptase from the N end by an alanine residue. The expression method of the moloney murine leukemia virus reverse transcriptase comprises the steps of transforming an expression vector containing the coding gene of the moloney murine leukemia virus reverse transcriptase into Escherichia coli, culturing positive clones and expressing to obtain the moloney murine leukemia virus reverse transcriptase mutant. The mutant can be applied to RNA synthesis, and the reverse transcriptase mutant obtained from reconstructing the moloney murine leukemia virus reverse transcriptase has a fidelity function.
Description
Technical field
The present invention relates to gene engineering technology field, particularly moloneys mouse leukemia virus reverse transcriptase mutant and expression method thereof and application.
Background technology
When reverse transcription is reverse transcription virus gene group rna replicon, viral RNA synthetic DNA and be incorporated into process in the host cell gene group under reversed transcriptive enzyme catalysis.Reversed transcriptive enzyme is the distinctive enzyme of this viroid.It has exercised following function: 1. dependenc RNA or DNA are the DNA polymerization of template; 2. chain transformation; 3. the RNase H function of RNA in degradation of rna-DNA heterozygote.Therefore reversed transcriptive enzyme is usually used in the structure in cDNA library.The transformation of moloneys mouse leukosis virus on the market (Moloney MurineLeukemia Virus, be called for short MMLV) reversed transcriptive enzyme mainly is to transform at the RNaseH position of enzyme, with reach reduction to DNA-RNA the function of the degraded of RNA in the assorted and body.After transforming, this kind of enzyme can go out longer single stranded DNA product by reverse transcription.Because the MMLV reversed transcriptive enzyme can mix wrong base when synthetic DNA, caused the high mutation rate in the viral genome reproduction process, therefore need obtain reversed transcriptive enzyme with fidelity function.To reversed transcriptive enzyme transform need to guarantee be: need the reactive site of its functionating to keep original activity and characteristic, need the reactive site of reduction when transforming, to guarantee not influence the implementation of other function.In theory, there is very big mutability in natural enzyme and exists improved potentiality, as: an enzyme of forming by 100 amino acid, have the not homotactic possibility of 20100 powers, obviously nature is impossible have the corresponding enzyme of all sequences.Simultaneously, correspondingly will inevitably produce different in kind in existing enzyme with the difference on the so huge sequence, also will inevitably exist the character of some aspect to be superior to the new enzyme of natural enzyme, the mutability on this sequence will inevitably be embodied on the 26S Proteasome Structure and Function.On the basis of understanding catalyst mechanism, the catalytic efficiency of artificial raising enzyme is the major objective of molecular enzymology engineering, and creates the economical and practical enzyme of high catalytic efficiency thus.In past 20 years, with the technology of rite-directed mutagenesis, the engineering enzyme has been obtained very great achievement, nearly all structure-function relationship clearly enzyme all by this technological improvement.This is because the directional property of sudden change and the alternative that replaces residue, the interference of higher structure is not influenced or influences less, the reliability of measuring means.Whether certain amino-acid residue that this method can spell out in the enzyme molecule participates in substrate combination and catalysis, and the suspicious amino-acid residue of the candidate's reactive site that draws from other method is further verified, draws believable conclusion.
Present existing MMLV reversed transcriptive enzyme crystalline structure comprises the crystalline structure of the reversed transcriptive enzyme N end that does not contain DNA under the crystalline structure of the total length reversed transcriptive enzyme that contains DNA under the on-catalytic state and the catalysis state, therefore aspect structure and machine-processed enzyme transformation certain difficulty is being arranged.The structural research of HIV-1 reversed transcriptive enzyme is more, contains the crystalline structure of template/primer, the triple complex bodys of dNTP under the existing catalysis state.
Summary of the invention
The objective of the invention is to overcome above-mentioned technological deficiency, a kind of mutant of moloneys mouse leukemia virus reverse transcriptase is provided, the preparation method and the application thereof of this mutant are provided simultaneously.
For realizing purpose of the present invention, adopt following technical scheme:
Hold the 196th proline residue to be substituted by the protein of L-Ala from N the moloneys mouse leukemia virus reverse transcriptase.
Described moloneys mouse leukemia virus reverse transcriptase mutant has the described aminoacid sequence as SEQ ID NO:1.
The encoding gene of moloneys mouse leukemia virus reverse transcriptase mutant provided by the invention has the described nucleotide sequence as SEQ ID NO:2.
The method of moloneys mouse leukemia virus reverse transcriptase mutant of the present invention is that the expression vector that will contain moloneys mouse leukemia virus reverse transcriptase mutant encoding gene is transformed in the intestinal bacteria, cultivate positive colony, express obtaining the moloneys mouse leukemia virus reverse transcriptase mutant.
The above-mentioned expression vector that contains moloneys mouse leukemia virus reverse transcriptase mutant encoding gene is the plasmid pET-28a that has as the described nucleotide sequence of SEQ ID NO:2.
Described intestinal bacteria are Escherichia coli BL21.
The present invention also protects the expression vector that contains moloneys mouse leukemia virus reverse transcriptase mutant encoding gene and the host bacterium of moloneys mouse leukemia virus reverse transcriptase mutant encoding gene.
Above-mentioned moloneys mouse leukemia virus reverse transcriptase mutant can be applicable to RNA synthetic in.
Another object of the present invention provides a kind of method of expressing the mutant of moloneys mouse leukemia virus reverse transcriptase.
The mutant that moloneys mouse leukemia virus reverse transcriptase of the present invention is transformed resulting reversed transcriptive enzyme has the fidelity function.
Description of drawings
Fig. 1 is the misincorporation experimental result picture of mononucleotide;
Fig. 2 extends experimental result picture for after the mispairing of mononucleotide.
Embodiment
For making the present invention easier to understand,, further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.
Embodiment 1: contain the structure and the structural analysis thereof of the MMLV reversed transcriptive enzyme structural models of template-primer, dNTP under the active condition
Structure contains the structural models of template/primer and dNTP: present existing MMLV crystalline structure comprises the crystalline structure of the MMLV total length that contains DNA, this DNA is away from catalytic site, and the crystalline structure that does not contain the MMLV reversed transcriptive enzyme N end of DNA under the catalysis state, therefore aspect the enzyme function modifications of structure, certain difficulty is being arranged.The structural research of HIV-1 reversed transcriptive enzyme is more, contains the crystalline structure of template/primer, the triple complex bodys of dNTP under the existing catalysis state.The structure height homology of MMLV reversed transcriptive enzyme and HIV-1 reversed transcriptive enzyme, we are in conjunction with the triple complex bodys of the HIV-1 reversed transcriptive enzyme crystalline structure of (comprising enzyme, template-primer, dNTP) (PDB (protein data bank) number: the crystalline structure (PDB (protein data bank) number: 1mml) made up the MMLV reversed transcriptive enzyme structural models that contains template-primer, dNTP under the active condition of MMLV reversed transcriptive enzyme N end 1rtd) and under the catalysis state.Concrete grammar is as follows: with the 6th, 9 and 10 βZhe Die of HIV-1 reversed transcriptive enzyme and the C of E and F bar α spiral
αThe the 7th, 10 and 11 βZhe Die of atom and MMLV reversed transcriptive enzyme and the C of H and I bar α spiral
αAtom is overlapping.Carry out energy minimization by GROMOS 96, make the C of HIV-1 reversed transcriptive enzyme
αThe rootmean-square of the catalysis carboxyl of atom and MMLV reversed transcriptive enzyme correspondence position carboxyl (root mean square, be called for short r.m.s) depart from less than
Adopt Swiss-PdbViwer and InsightII molecular simulation software to analyze at primer binding site, simulate all kinds of mutant reversed transcriptive enzymes, by AMBER 99 Force Field Analysis carry out the minimizing of iteration (iterative minimizations) up to energy variation less than 0.0001kcal/mol per
Predict that the 196th site amino acid is relevant with MMLV reversed transcriptive enzyme fidelity.
Embodiment 2: 196 proline(Pro) (P) of rite-directed mutagenesis MMLV reversed transcriptive enzyme are L-Ala (A)
196 proline(Pro) (P) of rite-directed mutagenesis MMLV reversed transcriptive enzyme (Genebank:AF033811) are L-Ala (A).Adopt the method that merges PCR to carry out.At first design the upstream and downstream primer RTUp and the RTDown of MMLV pol gene:
RTUp：5’ATGCATATGACATGGCTGTCTGATTTT 3’
RTDown：5’ATTACTCGAGTTAGAGGAGGGTAGAGGTGTCTGGAGTC 3’
And at mutational site design primer 196Up and 196Down:
196Up 5’CCA CAG GGT TTC AAA AAC AGT GCC ACC CTG TTT 3’
196Down 5’AAA CAG GGT GGC ACT GTT TTT GAA ACC CTG TGG 3’
As primer, MMLV pol gene fragment is a template amplification N end dna fragmentation with RTUp and 196Down.Be primer amplification C end dna fragmentation with RTDown and 196Up then.After the N of pcr amplification end and C end dna fragmentation reclaimed respectively jointly as template, the total length MMLV reversed transcriptive enzyme that utilizes the export-oriented primer amplification of RTup and RTDown to suddenly change simultaneously.
The expression and purification of embodiment 3:MMLV reverse transcriptase mutant
Clone: will cut rear clone in the pET-28a carrier with Nde I/Xho I enzyme behind MMLV mutant pol gene (P196A) purifying, make to add the His label behind the MMLV pol gene generation pET-28a-RTP196A expression plasmid.Recombinant expression plasmid is identified correct through order-checking.Express: recombinant expression plasmid is converted into the BL21 at E.coli.It is 0.6 o'clock that picking list bacterium colony is cultured to the OD600 value for 37 ℃, induces 3 hours with 0.2mM IPTG, and centrifugal 35 minutes of 4200rpm collects bacterium liquid ,-80 ℃ of preservations.Purifying: with damping fluid I (the 50mM NaH that contains the 10mM imidazoles
2PO
4(pH 7.8), 5% glycerine, 0.3M NaCl) suspension bacteria liquid, add N,O-Diacetylmuramidase to final concentration 1mg/ml, hatch cracking in 30 minutes on ice.Cracking bacterium liquid 35, centrifugal 40 minutes of 000rpm.Supernatant is crossed nickel post (Ni-NTA columns), washes post twice with the damping fluid I that contains the 20mM imidazoles.With the damping fluid I eluted protein that contains the 250mM imidazoles, collect albumen again, damping fluid I is replaced with damping fluid II (200mMNaCl, 50% glycerine) with filter Amicon 30 centricons.Measure protein concentration with the Bradford method.
The fidelity of embodiment 4:MMLV reverse transcriptase mutant is identified
Determine unit of enzyme activity: with poly (rA)-(dT) 18 is template-primer,
32The dTTP of P mark is that substrate reacts.The reaction system cumulative volume is 6 μ L, comprises 50mM Tris HCl (pH 8.0), 100 μ g/mL BSA, 5mM MgCl
2, 1mM DTT, 50mM KCl, 100nM template-primer, 100 μ MdTTP, 10nM MMLV mutant reversed transcriptive enzyme and 50 μ M
32The dTTP of P mark (0.4 μ Ci/nmol).Be reflected under 37 ℃ and hatch 15min, add 6 μ L sample-loading buffer termination reactions, reactant carries out 1.4% agarose gel electrophoresis 90 ℃ of heating after 5 minutes, with the exposure of X-ray sheet.
The misincorporation of mononucleotide: with the single stranded DNA of 24bp, 5 ' GCA CCG GCG CTC GAACAG GGA CTG 3 ' is template; With
32The single stranded DNA of the 21bp of P mark, 3 ' GGC CGC GAGCTT GTC CCT GAC 5 ' is primer, carries out the misincorporation experiment of mononucleotide.Reaction system is 2.5nM template/primer, 50mM Tris-HCl (pH 7.8), 1mM DTT, 0.01%BSA, 60mM KCl, 5mM MgCl
2And 500 μ M dATP, to cumulative volume 5 μ L.Be reflected at 25 ℃ carry out 30min after, add 5 μ l sample-loading buffer termination reactions.Reactant is by behind 12% the denaturing polyacrylamide gel electrophoresis, with the exposure of X-ray sheet.Fig. 1 is the experimental result picture of the misincorporation of mononucleotide, and as seen from Figure 1,196 mutant enzymes are similar with the ability that wild-type enzyme mixes wrong base.
Extend after the mispairing of 3 ' mononucleotide: with the single stranded DNA of 24bp, 5 ' GCA CCG GCG CTCGAA CAG GGA CTG 3 ' is template; With
32The single stranded DNA of the 21bp of P mark, 3 ' CGC CGCGAG CTT GTC CCT GAC 5 ' is a primer, carries out extending experiment after the mispairing of mononucleotide.Reaction system is 2.5nM template/primer, 50mM Tris-HCl (pH 7.8), 1mM DTT, 0.01%BSA, 60mM KCl, 5mM MgCl
2And 500 μ M dATP or dTTP, to cumulative volume 5 μ L.Be reflected at 25 ℃ carry out 30min after, add 5 μ l sample-loading buffer termination reactions.Reactant is by behind 12% the denaturing polyacrylamide gel electrophoresis, with the exposure of X-ray sheet.Fig. 2 is shown as the experimental result picture that extends after the mononucleotide mispairing, and among Fig. 2,1,2 is wild-type enzyme, and 3,4 is 193 mutant enzymes, and 5,6 is 196 mutant enzymes.With dATP is that substrate reacts (1,3,5), or is that substrate carries out reaction product (2,4,6) to observe dTTP.Wild-type enzyme can to a certain degree extend after can observing mispairing; 193 mutant enzymes can continue to extend, thereby can insert sudden change on template; 196 mutant enzymes almost can not extend, and difficult the insertion on template suddenlyd change.In view of this 196 mutant enzymes have higher fidelity than wild-type.
Last institute should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although the present invention has been done detailed description with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can make amendment or be equal to replacement technical scheme of the present invention, and not break away from the essence and the scope of technical solution of the present invention.
Sequence table
＜120〉moloneys mouse leukemia virus reverse transcriptase mutant and expression method thereof and application
<170>PatentIn version 3.2
<210>1
<211>672
<212>PRT
<213>Moloney Murine Leukemia Virus
<400>1
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Ala Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile
660 665 670
＜120〉moloneys mouse leukemia virus reverse transcriptase mutant and expression method thereof and application
<170>PatentIn version 3.2
<210>2
<211>2016
<212>DNA
<213>Moloney Murine Leukemia Virus
<400>2
accctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60
ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120
ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180
ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240
ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300
gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360
cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420
ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480
caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540
ggacaattga cctggaccag actcccacag ggtttcaaaa acagtgccac cctgtttgat 600
gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660
cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720
cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780
atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840
actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900
agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960
gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020
caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080
gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140
ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200
ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260
aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320
gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380
caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440
gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500
gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560
tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620
accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680
gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740
tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800
cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860
ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920
ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980
atcacagaga ctccagacac ctctaccctc ctcata 2016
Claims (7)
1. the moloneys mouse leukemia virus reverse transcriptase mutant is characterized in that, described moloneys mouse leukemia virus reverse transcriptase mutant is the aminoacid sequence shown in the SEQ ID NO:1.
2. the encoding gene of the described moloneys mouse leukemia virus reverse transcriptase mutant of claim 1.
3. encoding gene according to claim 2 is characterized in that, described encoding gene is the nucleotide sequence shown in the SEQID NO:2.
4. method of expressing the described moloneys mouse leukemia virus reverse transcriptase mutant of claim 1, it is characterized in that, the expression vector that will contain the described moloneys mouse leukemia virus reverse transcriptase mutant of claim 2 encoding gene is transformed in the intestinal bacteria, cultivate positive colony, express obtaining the moloneys mouse leukemia virus reverse transcriptase mutant.
5. method according to claim 4 is characterized in that, described intestinal bacteria are Escherichia coli BL21.
6. the expression vector that contains claim 2 or 3 described moloneys mouse leukemia virus reverse transcriptase mutant encoding genes.
7. the host bacterium that contains claim 2 or 3 described moloneys mouse leukemia virus reverse transcriptase mutant encoding genes.
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| CN110382690A (en) * | 2017-02-16 | 2019-10-25 | 生物辐射实验室股份有限公司 | Novel reverse transcriptase and application thereof |
| CN112795551B (en) * | 2019-11-13 | 2024-01-30 | 广州达安基因股份有限公司 | High Wen Ni-resistant transcriptase mutant and application thereof |
| CN112795550A (en) * | 2019-11-13 | 2021-05-14 | 中山大学达安基因股份有限公司 | High temperature resistant reverse transcriptase mutants |
| CN114480330A (en) * | 2020-11-13 | 2022-05-13 | 广州达安基因股份有限公司 | Reverse transcriptase mutant with multiple mutation sites |
| CN112458120B (en) * | 2020-11-25 | 2022-04-12 | 云舟生物科技(广州)股份有限公司 | Self-inactivation vector based on Moloney murine leukemia virus and application thereof |
| CN112695019B (en) * | 2021-03-23 | 2021-06-25 | 翌圣生物科技（上海）有限公司 | Reverse transcriptase mutant and application thereof |
| CN114317485B (en) * | 2021-12-30 | 2023-04-11 | 南京巨匠生物科技有限公司 | Recombinant murine leukemia virus reverse transcriptase mutant, preparation method and application |
| CN116622669A (en) * | 2023-06-30 | 2023-08-22 | 翌圣生物科技(上海)股份有限公司 | Murine leukemia virus reverse transcriptase mutant and application thereof |
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| 莫洛尼鼠白血病病毒逆转录酶的原核表达纯化及其生物学活性;王贤松等;《生物工程学报》;20080531;第24卷(第5期);第903-906页 * |
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