CN109082419A - A kind of 3alpha-Hydroxysteroid dehydrogenase mutant, coding nucleotide sequence and kit - Google Patents
A kind of 3alpha-Hydroxysteroid dehydrogenase mutant, coding nucleotide sequence and kit Download PDFInfo
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- CN109082419A CN109082419A CN201811048063.9A CN201811048063A CN109082419A CN 109082419 A CN109082419 A CN 109082419A CN 201811048063 A CN201811048063 A CN 201811048063A CN 109082419 A CN109082419 A CN 109082419A
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- C12Y101/01213—3Alpha-hydroxysteroid 3-dehydrogenase (A-specific) (1.1.1.213)
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Abstract
3alpha-Hydroxysteroid dehydrogenase mutant provided by the invention, 3 α-HSD mutant amino acid sequence as shown in SEQ ID NO:2 is through obtained by point mutation, the point mutation is the amino acid substitution at least one site in the 25th~37, the 100th~120 and the 145th~166.By point mutation, obtaining, there is the 3alpha-Hydroxysteroid dehydrogenase mutant protein enzyme of high catalytic activity can be obviously improved 60% or more enzymatic activity relative to 3 α-HSD, reach as high as to 2 times of original enzymatic activity.Kit total cholic acid content enzyme process detection efficiency is greatly improved using it as toolenzyme, is reduced production cost, is improved the market competitiveness of corresponding product.In addition, the present invention also provides a kind of nucleotide sequences and kit for encoding 3alpha-Hydroxysteroid dehydrogenase mutant.
Description
Technical field
The present invention relates to molecular biology and field of biotechnology more particularly to 3alpha-Hydroxysteroid dehydrogenase mutant,
Coding nucleotide sequence and kit.
Background technique
Bile acid plays an important role in metabolism as the important component of bile, and hepatic disease causes Bile Acids In Serum to contain
The increase of amount, thus Determination of Bile Acids becomes the common counter of clinical examination liver function, 3 α of 3alpha-Hydroxysteroid dehydrogenase-
HSD is one kind of a variety of steroid dehydrogenases of Comamonas testosteroni secretion, may act on a variety of steroids matrix, clinically uses
3 α-HSD measure total bile acid (TBA) concentration in human serum as toolenzyme, are the cores in Enzymatic cycling TBA kit
Ingredient, reagent kit product quality depend on the quality of 3 α-HSD enzymes.It is false single that current existing 3 α-HSD derives from testosterone
Born of the same parents bacterium is extracted or gene engineering expression, but extracted purifying or the 3 α-HSD enzymatic activitys by obtaining after gene engineering expression are not
Height so that catalytic efficiency is lower in enzyme process detection process, thus has to be hoisted.
Summary of the invention
In order to overcome in the prior art 3 α-HSD enzymatic activities it is low, the low deficiency of corresponding enzyme process detection efficiency, this hair
It is bright to be designed to provide a kind of higher 3alpha-Hydroxysteroid dehydrogenase mutant of catalytic activity, encoding gene and its application,
To promote its efficiency as toolenzyme in enzyme process detection.
In order to achieve the object of the present invention, present invention firstly provides a kind of 3alpha-Hydroxysteroid dehydrogenase mutant, by
Amino acid sequence shown in SEQ ID NO:2 is through obtained by point mutation, the point mutation includes being selected from the 25th~37, the 100th~120
The amino acid substitution at least one site in position and the 145th~166.
Optionally, the point mutation includes the ammonia at least one site in the 32nd, the 114th and the 155th
The replacement of base acid.
Optionally, the 3alpha-Hydroxysteroid dehydrogenase mutant further includes amino acid sequence shown in SEQ ID NO:2
In other sites in addition to the 25th~37, the 100th~120 and the 145th~166 conservative substitution form, increase or lack
Lose one or several amino acid forms, aminoterminal clipped form, amino shown in c-terminus clipped form and SEQ ID NO:2
The form of tandem repeats of some or all of acid sequence.
Optionally, the point mutation specifically: the 32nd aspartic acid in amino acid sequence shown in SEQ ID NO:2
(D) glutamic acid (E) is sported.
Optionally, the point mutation specifically: the 114th serine in amino acid sequence shown in SEQ ID NO:2
(S) glycine (G) is sported.
Optionally, the point mutation specifically: the 155th tyrosine in amino acid sequence shown in SEQ ID NO:2
(Y) histidine (H) is sported.
Further, the 3alpha-Hydroxysteroid dehydrogenase mutant has any one of SEQ ID NOs:3-5 freely
Shown in amino acid sequence.
In order to achieve the object of the present invention, another aspect of the present invention provides a kind of nucleotide sequence, encodes noted earlier
3alpha-Hydroxysteroid dehydrogenase mutant.Specifically, the nucleotide sequence can be DNA sequence dna, RNA sequence or mRNA
Sequence.
In order to achieve the object of the present invention, another aspect of the present invention provides a kind of reagent for total bile acid content detection
Box, the kit include mentioned-above 3alpha-Hydroxysteroid dehydrogenase mutant.
Beneficial effects of the present invention
In conclusion 3alpha-Hydroxysteroid dehydrogenase mutant, encoding gene and its application provided by the invention, pass through
Rite-directed mutagenesis is carried out to the amino acid in specific region site in 3alpha-Hydroxysteroid dehydrogenase gene, final expression is had
The 3alpha-Hydroxysteroid dehydrogenase mutant protein enzyme of high catalytic activity can be obviously improved enzymatic activity relative to 3 α-HSD
60% or more, it reaches as high as to 2 times of original enzymatic activity.Kit total cholic acid is greatly improved as toolenzyme using it
Content enzyme process detection efficiency, reduces production cost, improves the market competitiveness of corresponding product.
Specific embodiment
The present invention provides a kind of 3alpha-Hydroxysteroid dehydrogenase mutant, encoding gene and its application, of the invention to make
Purpose, technical solution and effect are clearer, clear, and the present invention is described in more detail below.It should be appreciated that this place is retouched
The specific embodiment stated is only used to explain the present invention, is not intended to limit the present invention.
The present invention will be further described below in conjunction with the embodiments
Embodiment 1
The building of 3 α-HSD-pET22b expression vectors
According to gene pool (GenBank ID:11140882) gene order design primer HSD-F and HSD-R.Primer is as follows:
HSD-F:CGCaagcttATGTCCATCATCGTGATAAGC
HSD-R:ATActcgagtcaatgatgatgatgatgatggaactgtgtcgggc
With primer HSD-F and HSD-R from Comamonas testosteroni Comamonas testosteroni genome PCR
Amplification obtains 3 α-HSD full-length genes, and nucleotide sequence is as shown in SEQ ID NO:1, corresponding amino acid sequence such as SEQ ID
Shown in NO:2.Wherein, PCR amplification system totally 50 μ l, specifically includes: Comamonas testosteroni genome (2 μ l), HSD-F (1 μ
L), HSD-R (1 μ l), 2 × Phata master mix (25 μ l), ddH2O (21 μ l)；The reaction condition of PCR amplification are as follows: 95 DEG C
Initial denaturation 3min, 95 DEG C of denaturation 30s, 60 DEG C of annealing 15s, 72 DEG C of extensions 30s, 72 DEG C of overall elongation 3min expand 30 circulations.
After PCR amplification, take wherein 5 μ L by agarose gel electrophoresis confirmation stripe size it is consistent it is errorless after, it is remaining
Pcr amplification reaction liquid is recycled using PCR purification and recovery kit.Product after the recovery uses restriction enzyme
The pET22b carrier of HindIII and XhoI digestion, digestion with restriction enzyme identical as process carries out enzyme company, and enzyme connects at 16 DEG C
After 2 hours, converts into E.coli DH5a competent cell, sequencing is sent after cultivating and extracting plasmid, correct matter will be sequenced
Grain conversion obtains 3 α-HSD-pET22b of plasmid to Rosetta (DE3).
Embodiment 2
The rite-directed mutagenesis of 32 amino acids
Mutant D32E is obtained in order to which the Asp (D) in the 32nd site in parent amino acid sequence is sported Glu (E), with reality
3 α-the HSD-pET22b of plasmid applied in example 1 is template, and design primer D32EF and D32ER is as follows:
D32EF:gtaggcatcgaaatacgcgatgc
D32ER:gcatcgcgtatTtcgatgcctac
Full plasmid PCR amplification is carried out with the primer, wherein PCR amplification system totally 50 μ l is specifically included: 3 α-HSD-
PET22b (2 μ l), D32EF (1 μ l), D32ER (1 μ l), 2 × Phata master mix (25 μ l), ddH2O (21 μ l)；PCR expands
The reaction condition of increasing are as follows: 95 DEG C of initial denaturation 3min, 95 DEG C of denaturation 15s, 60 DEG C of annealing 15s, 72 DEG C of extension 6min, 72 DEG C of overall elongations
5min, coamplification 20 circulations.
After PCR amplification, wherein 5 μ l electrophoresis detection is taken, remaining PCR product carries out 37 DEG C of DpnI enzyme and digests 0.5 hour,
Digestion reaction system is DpnI enzyme (1 μ l), 10 × DpnI buffer (2 μ l), PCR product (10 μ l), ddH2O (7 μ l).Digestion
After carry out conversion DH5 α, choose and carry out whether sequence verification mutation succeeds after Colony Culture.Successful D32E bacterium will be mutated
Strain converts after extracting plasmid to Rosetta (DE3), and carries out guarantor bacterium.
Embodiment 3
The rite-directed mutagenesis of 114 amino acids
Mutant S114G is obtained in order to which the Ser (S) in the 114th site in parent amino acid sequence is sported Gly (G), with
3 α-HSD-pET22b of plasmid in embodiment 1 is template, and design primer S114GF and S114GR is as follows:
S114GF:agccgtcgtcatctcgGGcgtggcttccgcgcatctg
S114GR:cagatgcgcggaagccacgCCCgagatgacgacggct
Full plasmid PCR amplification is carried out with the primer, wherein PCR amplification system totally 50 μ l is specifically included: 3 α-HSD-
PET22b (2 μ l), S114GF (1 μ l), S114GR (1 μ l), 2 × Phata master mix (25 μ l), ddH2O (21 μ l)；PCR
The reaction condition of amplification are as follows: 95 DEG C of initial denaturation 3min, 95 DEG C of denaturation 15s, 60 DEG C of annealing 15s, 72 DEG C of extension 6min, 72 DEG C are always prolonged
Stretch 5min, coamplification 20 circulations.
After PCR amplification, wherein 5 μ l electrophoresis detection is taken, remaining PCR product carries out 37 DEG C of DpnI enzyme and digests 0.5 hour,
Digestion reaction system is DpnI enzyme (1 μ l), 10 × DpnI buffer (2 μ l), PCR product (10 μ l), ddH2O (7 μ l).Digestion
After carry out conversion DH5 α, choose and carry out whether sequence verification mutation succeeds after Colony Culture.Successful S114G bacterium will be mutated
Strain converts after extracting plasmid to Rosetta (DE3), and carries out guarantor bacterium.
Embodiment 4
The rite-directed mutagenesis of 155 amino acids
Mutant Y155H is obtained in order to which the Tyr (Y) in the 155th site in parent amino acid sequence is sported His (H), with
3 α-HSD-pET22b of plasmid in embodiment 1 is template, and design primer Y155HF and Y155HR is as follows:
Y155HF:gaaatctggccCatgcgggcagc
Y155HR:gctgcccgcatGggccagatttc
Full plasmid PCR amplification is carried out with the primer, wherein PCR amplification system totally 50 μ l is specifically included: 3 α-HSD-
PET22b (2 μ l), Y155HF (1 μ l), Y155HR (1 μ l), 2 × Phata master mix (25 μ l), ddH2O (21 μ l)；PCR
The reaction condition of amplification are as follows: 95 DEG C of initial denaturation 3min, 95 DEG C of denaturation 15s, 60 DEG C of annealing 15s, 72 DEG C of extension 6min, 72 DEG C are always prolonged
Stretch 5min, coamplification 20 circulations.
After PCR amplification, wherein 5 μ l electrophoresis detection is taken, remaining PCR product carries out 37 DEG C of DpnI enzyme and digests 0.5 hour,
Digestion reaction system is DpnI enzyme (1 μ l), 10 × DpnI buffer (2 μ l), PCR product (10 μ l), ddH2O (7 μ l).Digestion
After carry out conversion DH5 α, choose and carry out whether sequence verification mutation succeeds after Colony Culture.Successful Y155H bacterium will be mutated
Strain converts after extracting plasmid to Rosetta (DE3), and carries out guarantor bacterium.
Embodiment 5
The extraction and purifying of 3 α-HSD
According to embodiment 1-4,4 groups of bacterial strain D32E/Rosetta (DE3), S114G/Rosetta of bacterium will be protected respectively
(DE3), (benzyl of ammonia containing 100mg/L is anti-in LB plate by Y155H/Rosetta (DE3) and 3 α-HSD-pET22b/Rosetta (DE3)
Raw element) it is 37 DEG C of cultures 12 hours upper.Inoculation is individually cloned in 5mL LB liquid medium (benzyl of ammonia containing 100mg/L mycin), and 37
DEG C culture OD600 to 0.6~0.8, be forwarded in 1L LB liquid medium (benzyl of ammonia containing 100mg/L mycin), 37 DEG C cultivate
OD600 to 0.6~0.8, adds IPTG to final concentration of 0.2Mm, 30 DEG C Fiber differentiation 12 hours, thalline were collected by centrifugation, and suspends
In 10mL 50mM PBS buffer solution (pH 7.5).Then ultrasonic treatment bacterial cell is used.(4 DEG C, 15000g, 10 points of centrifugation
Clock) collect supernatant, as thick leach protein.The supernatant was subjected to ni-sepharose purification and obtains pure protein liquid.
3 α-HSD and its mutein concentration mensuration
Using the concentration of Bradford method measurement protein, step are as follows:
Preparing one group of concentration is respectively 0.10mg/ml, 0.08mg/ml, 0.06mg/ml, 0.04mg/ml, 0.02mg/ml,
Bovine serum albumin (BSA) solution of 0mg/ml pipettes one group of BSA solution that 50ul is prepared respectively with liquid-transfering gun, is added drop-wise to orifice plate
In, then it is separately added into the Coomassie brilliant blue (CBB) of 200ul.After standing 10min, the extinction of this group of BSA solution is measured with microplate reader
Degree.Protein concentration is obtained to a standard curve of absorbance.
Protein solution (totally 4 groups) each 100ul of 3 α-HSD and its mutant D32E, S114G, Y155H are drawn respectively, then
It is separately added into the Coomassie brilliant blue (CBB) of 900ul.After standing 10min, the absorbance of 4 kinds of protein solutions is measured with microplate reader,
Corresponding standard curve calculates the concentration of protein, and four kinds of protein solutions are uniformly adjusted to 100 μ g/Ml.
α-HSD and mutant enzyme determination of activity
Four groups of reaction systems are configured, it is 30 DEG C, total reaction volume 0.5ml that reaction temperature, which is respectively set, in reaction system
Contain: 100mmol/L sodium pyrophosphate buffer solution (pH 11), 0.5mmol/L NAD+, 012mmol/L androsterone, 0.5g/L BSA；
It is separately added into the above-mentioned α-HSD for having been adjusted to 100 μ g/Ml and 100 μ L of mutant enzyme solution starting to every group to react, 15 points
The absorbance change at 340nm is read after clock, and is to calculate referring to the relative activity for calculating different mutants enzyme with α-HSD
It as a result is respectively D32E (175.8%), S114G (205.1%), Y155H (93.2%).
Above embodiments absolutely prove that the enzymatic activity of mutant D32E, S114G, Y155H are compared to α-HSD before mutation
Enzymatic activity has been obviously improved 75.8%, 105.1%, 93.2% respectively.
It is noted that in an embodiment of the present invention, it is above-mentioned to α-HSD enzyme D32E, S114G, Y155H amino acids
Mutation be only to technical solution of the present invention specifically enumerating, in the present invention, the above-mentioned ammonia to site specific in α-HSD enzyme
Base acid mutation is not limited to the acid and alkali such as sport particular kind of amino acid, but can be mutated into Arg, Lys, Thr
Property and polar amino acid, in addition, it is above-mentioned to α-HSD enzyme amino acid mutation further include to its 25~37,100~120,145~166
The combination of the mutation of an optional site amino acids or the mutation of optionally multiple site amino acids in position, and can obtain and this
Inventive embodiments are essentially identical or the enzymatic activity of approximate mutant.Specifically, to the prominent of multiple site amino acids of α-HSD enzyme
Become can be equal replace in its 25~37,100~120,145~166 any 2, it is 3 any, 4 any
Or the mutation of the amino acid in any 5 sites.
Certainly, when being mutated to 3 α-HSD, keep or it is basic keep such as mutant primary activity in the present embodiment or
Obtain it is more preferable it is active on the basis of, can also with to removed in amino acid sequence shown in sequence 2 the 25th~37, the 100th~
Conservative substitution form, increase or one or several amino acid forms of missing in other sites outside 120 and the 145th~166,
The tandem sequence repeats shape of some or all of amino acid sequence shown in aminoterminal clipped form, c-terminus clipped form and sequence 2
Formula.
In conclusion 3alpha-Hydroxysteroid dehydrogenase mutant, encoding gene and its application provided by the invention, pass through
Rite-directed mutagenesis is carried out to the amino acid in specific region site in 3alpha-Hydroxysteroid dehydrogenase gene, final expression is had
The 3alpha-Hydroxysteroid dehydrogenase mutant protein enzyme of high catalytic activity can be obviously improved enzymatic activity relative to 3 α-HSD
60% or more, it reaches as high as to 2 times of original enzymatic activity.Kit total cholic acid is greatly improved as toolenzyme using it
Content enzyme process detection efficiency, reduces production cost, improves the market competitiveness of corresponding product.
Sequence table
<120>a kind of 3alpha-Hydroxysteroid dehydrogenase mutant, coding nucleotide sequence and kit
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1073
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
catggctggt ttcctcacac gcaaacgttg cgccagtgta ggaagggcca cggtaaattt 60
tccctagggg aattacatcg tccgtttgca tgtagcctgg ttgttcatga tgggctttga 120
tgtgcgccat caccacccag acaaaaggag acaagacatg tccatcatcg tgataagcgg 180
ctgcgccacc ggcattggtg cggctacgcg caaggtcctg gaggcggccg gtcaccagat 240
cgtaggcatc gatatacgcg atgcggaagt gattgccgat ctctcgacgg ccgaaggtcg 300
aaagcaggcg attgccgatg tactggcgaa gtgcagcaag ggcatggacg gcctggtgct 360
gtgcgccggc ctgggaccgc agaccaaggt gcttggcaat gtggtttcgg tcaattattt 420
tggcgcgacc gagctgatgg atgccttttt gccagcgctg aaaaaaggcc atcagcccgc 480
agccgtcgtc atctcgtccg tggcttccgc gcatctggct tttgacaaga acccactggc 540
gctggcactg gaagccggcg aggaagccaa ggcccgcgcc attgtcgaac atgcgggaga 600
gcagggcgga aatctggcct atgcgggcag caagaatgct ttgacggtgg ctgtgcgcaa 660
acgcgccgcc gcctggggcg aggctggcgt gcgcctgaac accatcgccc ccggtgcaac 720
cgagactccc ttgctgcagg cgggcctgca ggacccgcgc tatggcgaat ccattgccaa 780
gttcgttcct cccatgggcc gccgtgccga gccgtccgag atggcgtcgg tcatcgcctt 840
tttgatgagc ccggccgcaa gctatgtgca tggcgcgcag atcgtcattg atggcggcat 900
tgatgcggtg atgcgcccga cacagttctg acctctcatg tggcgctttg ccagagggcc 960
tgcgccttcc ccccctcgct gtgcgggagg gggaaggcgg cctcgctgca aggctttttt 1020
ttgttcaccg cccccgtttg ggcattgccg gttttcaaac ggcgcctgct aga 1107
<210> 2
<211> 257
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Met Ser Ile Ile Val Ile Ser Gly Cys Ala Thr Gly Ile Gly Ala Ala
1 5 10 15
Thr Arg Lys Val Leu Glu Ala Ala Gly His Gln Ile Val Gly Ile Asp
20 25 30
Ile Arg Asp Ala Glu Val Ile Ala Asp Leu Ser Thr Ala Glu Gly Arg
35 40 45
Lys Gln Ala Ile Ala Asp Val Leu Ala Lys Cys Ser Lys Gly Met Asp
50 55 60
Gly Leu Val Leu Cys Ala Gly Leu Gly Pro Gln Thr Lys Val Leu Gly
65 70 75 80
Asn Val Val Ser Val Asn Tyr Phe Gly Ala Thr Glu Leu Met Asp Ala
85 90 95
Phe Leu Pro Ala Leu Lys Lys Gly His Gln Pro Ala Ala Val Val Ile
100 105 110
Ser Ser Val Ala Ser Ala His Leu Ala Phe Asp Lys Asn Pro Leu Ala
115 120 125
Leu Ala Leu Glu Ala Gly Glu Glu Ala Lys Ala Arg Ala Ile Val Glu
130 135 140
His Ala Gly Glu Gln Gly Gly Asn Leu Ala Tyr Ala Gly Ser Lys Asn
145 150 155 160
Ala Leu Thr Val Ala Val Arg Lys Arg Ala Ala Ala Trp Gly Glu Ala
165 170 175
Gly Val Arg Leu Asn Thr Ile Ala Pro Gly Ala Thr Glu Thr Pro Leu
180 185 190
Leu Gln Ala Gly Leu Gln Asp Pro Arg Tyr Gly Glu Ser Ile Ala Lys
195 200 205
Phe Val Pro Pro Met Gly Arg Arg Ala Glu Pro Ser Glu Met Ala Ser
210 215 220
Val Ile Ala Phe Leu Met Ser Pro Ala Ala Ser Tyr Val His Gly Ala
225 230 235 240
Gln Ile Val Ile Asp Gly Gly Ile Asp Ala Val Met Arg Pro Thr Gln
245 250 255
Phe
<210> 3
<211> 257
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Met Ser Ile Ile Val Ile Ser Gly Cys Ala Thr Gly Ile Gly Ala Ala
1 5 10 15
Thr Arg Lys Val Leu Glu Ala Ala Gly His Gln Ile Val Gly Ile Glu
20 25 30
Ile Arg Asp Ala Glu Val Ile Ala Asp Leu Ser Thr Ala Glu Gly Arg
35 40 45
Lys Gln Ala Ile Ala Asp Val Leu Ala Lys Cys Ser Lys Gly Met Asp
50 55 60
Gly Leu Val Leu Cys Ala Gly Leu Gly Pro Gln Thr Lys Val Leu Gly
65 70 75 80
Asn Val Val Ser Val Asn Tyr Phe Gly Ala Thr Glu Leu Met Asp Ala
85 90 95
Phe Leu Pro Ala Leu Lys Lys Gly His Gln Pro Ala Ala Val Val Ile
100 105 110
Ser Ser Val Ala Ser Ala His Leu Ala Phe Asp Lys Asn Pro Leu Ala
115 120 125
Leu Ala Leu Glu Ala Gly Glu Glu Ala Lys Ala Arg Ala Ile Val Glu
130 135 140
His Ala Gly Glu Gln Gly Gly Asn Leu Ala Tyr Ala Gly Ser Lys Asn
145 150 155 160
Ala Leu Thr Val Ala Val Arg Lys Arg Ala Ala Ala Trp Gly Glu Ala
165 170 175
Gly Val Arg Leu Asn Thr Ile Ala Pro Gly Ala Thr Glu Thr Pro Leu
180 185 190
Leu Gln Ala Gly Leu Gln Asp Pro Arg Tyr Gly Glu Ser Ile Ala Lys
195 200 205
Phe Val Pro Pro Met Gly Arg Arg Ala Glu Pro Ser Glu Met Ala Ser
210 215 220
Val Ile Ala Phe Leu Met Ser Pro Ala Ala Ser Tyr Val His Gly Ala
225 230 235 240
Gln Ile Val Ile Asp Gly Gly Ile Asp Ala Val Met Arg Pro Thr Gln
245 250 255
Phe
<210> 4
<211> 257
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Met Ser Ile Ile Val Ile Ser Gly Cys Ala Thr Gly Ile Gly Ala Ala
1 5 10 15
Thr Arg Lys Val Leu Glu Ala Ala Gly His Gln Ile Val Gly Ile Asp
20 25 30
Ile Arg Asp Ala Glu Val Ile Ala Asp Leu Ser Thr Ala Glu Gly Arg
35 40 45
Lys Gln Ala Ile Ala Asp Val Leu Ala Lys Cys Ser Lys Gly Met Asp
50 55 60
Gly Leu Val Leu Cys Ala Gly Leu Gly Pro Gln Thr Lys Val Leu Gly
65 70 75 80
Asn Val Val Ser Val Asn Tyr Phe Gly Ala Thr Glu Leu Met Asp Ala
85 90 95
Phe Leu Pro Ala Leu Lys Lys Gly His Gln Pro Ala Ala Val Val Ile
100 105 110
Ser Gly Val Ala Ser Ala His Leu Ala Phe Asp Lys Asn Pro Leu Ala
115 120 125
Leu Ala Leu Glu Ala Gly Glu Glu Ala Lys Ala Arg Ala Ile Val Glu
130 135 140
His Ala Gly Glu Gln Gly Gly Asn Leu Ala Tyr Ala Gly Ser Lys Asn
145 150 155 160
Ala Leu Thr Val Ala Val Arg Lys Arg Ala Ala Ala Trp Gly Glu Ala
165 170 175
Gly Val Arg Leu Asn Thr Ile Ala Pro Gly Ala Thr Glu Thr Pro Leu
180 185 190
Leu Gln Ala Gly Leu Gln Asp Pro Arg Tyr Gly Glu Ser Ile Ala Lys
195 200 205
Phe Val Pro Pro Met Gly Arg Arg Ala Glu Pro Ser Glu Met Ala Ser
210 215 220
Val Ile Ala Phe Leu Met Ser Pro Ala Ala Ser Tyr Val His Gly Ala
225 230 235 240
Gln Ile Val Ile Asp Gly Gly Ile Asp Ala Val Met Arg Pro Thr Gln
245 250 255
Phe
<210> 5
<211> 257
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 5
Met Ser Ile Ile Val Ile Ser Gly Cys Ala Thr Gly Ile Gly Ala Ala
1 5 10 15
Thr Arg Lys Val Leu Glu Ala Ala Gly His Gln Ile Val Gly Ile Asp
20 25 30
Ile Arg Asp Ala Glu Val Ile Ala Asp Leu Ser Thr Ala Glu Gly Arg
35 40 45
Lys Gln Ala Ile Ala Asp Val Leu Ala Lys Cys Ser Lys Gly Met Asp
50 55 60
Gly Leu Val Leu Cys Ala Gly Leu Gly Pro Gln Thr Lys Val Leu Gly
65 70 75 80
Asn Val Val Ser Val Asn Tyr Phe Gly Ala Thr Glu Leu Met Asp Ala
85 90 95
Phe Leu Pro Ala Leu Lys Lys Gly His Gln Pro Ala Ala Val Val Ile
100 105 110
Ser Ser Val Ala Ser Ala His Leu Ala Phe Asp Lys Asn Pro Leu Ala
115 120 125
Leu Ala Leu Glu Ala Gly Glu Glu Ala Lys Ala Arg Ala Ile Val Glu
130 135 140
His Ala Gly Glu Gln Gly Gly Asn Leu Ala His Ala Gly Ser Lys Asn
145 150 155 160
Ala Leu Thr Val Ala Val Arg Lys Arg Ala Ala Ala Trp Gly Glu Ala
165 170 175
Gly Val Arg Leu Asn Thr Ile Ala Pro Gly Ala Thr Glu Thr Pro Leu
180 185 190
Leu Gln Ala Gly Leu Gln Asp Pro Arg Tyr Gly Glu Ser Ile Ala Lys
195 200 205
Phe Val Pro Pro Met Gly Arg Arg Ala Glu Pro Ser Glu Met Ala Ser
210 215 220
Val Ile Ala Phe Leu Met Ser Pro Ala Ala Ser Tyr Val His Gly Ala
225 230 235 240
Gln Ile Val Ile Asp Gly Gly Ile Asp Ala Val Met Arg Pro Thr Gln
245 250 255
Phe
Claims (9)
1. a kind of 3 α-HSD mutant of 3alpha-Hydroxysteroid dehydrogenase, which is characterized in that the 3 α-HSD mutant is by SEQ ID
Amino acid sequence shown in NO:2 is through obtained by point mutation, the point mutation is selected from the 25th~37, the 100th~120 and the 145th
The amino acid substitution at least one site in~166.
2. 3alpha-Hydroxysteroid dehydrogenase mutant according to claim 1, which is characterized in that the 3 α-HSD mutation
Body, including in amino acid sequence shown in SEQ ID NO:2 in addition to the 25th~37, the 100th~120 and the 145th~166
The conservative substitution form in other sites, increase or one or several amino acid forms of missing, aminoterminal clipped form, c-terminus are cut
The form of tandem repeats of some or all of amino acid sequence shown in disconnected form and SEQ ID NO:2.
3. 3alpha-Hydroxysteroid dehydrogenase mutant according to claim 1, which is characterized in that the point mutation is choosing
Amino acid substitution from least one site in the 32nd, the 114th and the 155th.
4. 3alpha-Hydroxysteroid dehydrogenase mutant according to claim 3, which is characterized in that the point mutation is SEQ
The 32nd aspartic acid (D) in amino acid sequence shown in ID NO:2 sports glutamic acid (E).
5. 3alpha-Hydroxysteroid dehydrogenase mutant according to claim 3, which is characterized in that the point mutation is SEQ
The 114th serine (S) in amino acid sequence shown in ID NO:2 sports glycine (G).
6. 3alpha-Hydroxysteroid dehydrogenase mutant according to claim 3, which is characterized in that the point mutation is SEQ
The 155th tyrosine (Y) in amino acid sequence shown in ID NO:2 sports histidine (H).
7. 3alpha-Hydroxysteroid dehydrogenase mutant according to claim 1, which is characterized in that the 3 Alpha-hydroxy class is solid
Alcohol dehydrogenase mutant, which has, is selected from the amino acid sequence as shown in any one of SEQ ID NOs:3-5.
8. a kind of nucleotide sequence, which is characterized in that described nucleotide sequence coded such as claimed in any one of claims 1 to 63
Alpha-hydroxysteroid dehydrogenase mutant.
9. a kind of kit for total bile acid content detection, which is characterized in that the kit includes such as claim 1-5
Described in any item 3alpha-Hydroxysteroid dehydrogenase mutant.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111235275A (en) * | 2020-03-13 | 2020-06-05 | 青岛市中心医院 | Gene marker of lung cancer and application thereof |
| CN111235122A (en) * | 2019-01-29 | 2020-06-05 | 武汉生之源生物科技股份有限公司 | 3 α hydroxysteroid dehydrogenase mutant and application thereof in total bile acid detection |
| CN113151207A (en) * | 2021-04-21 | 2021-07-23 | 重庆第二师范学院 | NAD (H) -dependent 3 alpha-hydroxysteroid dehydrogenase and gene encoding same |
| CN114026245A (en) * | 2019-07-08 | 2022-02-08 | 西姆莱斯有限公司 | Biotechnological production of diols |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090246783A1 (en) * | 2008-02-28 | 2009-10-01 | Asahi Kasei Pharma Corporation | Method of measuring pyrophosphate |
| CN101698834A (en) * | 2009-09-08 | 2010-04-28 | 北京利德曼生化股份有限公司 | 3 alpha-hydroxysteroid dehydrogenase, nucleotide sequence thereof, recombinant vector thereof, recombinant host cells thereof and kit |
| CN102768190A (en) * | 2012-07-04 | 2012-11-07 | 中国科学院过程工程研究所 | Determining reagent of serum total bile acid and detecting method |
-
2018
- 2018-09-07 CN CN201811048063.9A patent/CN109082419B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090246783A1 (en) * | 2008-02-28 | 2009-10-01 | Asahi Kasei Pharma Corporation | Method of measuring pyrophosphate |
| CN101698834A (en) * | 2009-09-08 | 2010-04-28 | 北京利德曼生化股份有限公司 | 3 alpha-hydroxysteroid dehydrogenase, nucleotide sequence thereof, recombinant vector thereof, recombinant host cells thereof and kit |
| CN102768190A (en) * | 2012-07-04 | 2012-11-07 | 中国科学院过程工程研究所 | Determining reagent of serum total bile acid and detecting method |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111235122A (en) * | 2019-01-29 | 2020-06-05 | 武汉生之源生物科技股份有限公司 | 3 α hydroxysteroid dehydrogenase mutant and application thereof in total bile acid detection |
| CN111235122B (en) * | 2019-01-29 | 2020-10-30 | 武汉生之源生物科技股份有限公司 | 3 alpha hydroxysteroid dehydrogenase mutant and application thereof in total bile acid detection |
| CN114026245A (en) * | 2019-07-08 | 2022-02-08 | 西姆莱斯有限公司 | Biotechnological production of diols |
| CN111235275A (en) * | 2020-03-13 | 2020-06-05 | 青岛市中心医院 | Gene marker of lung cancer and application thereof |
| CN111235275B (en) * | 2020-03-13 | 2020-11-06 | 青岛市中心医院 | Gene marker of lung cancer and application thereof |
| CN113151207A (en) * | 2021-04-21 | 2021-07-23 | 重庆第二师范学院 | NAD (H) -dependent 3 alpha-hydroxysteroid dehydrogenase and gene encoding same |
| CN113151207B (en) * | 2021-04-21 | 2023-03-31 | 重庆第二师范学院 | NAD (H) -dependent 3 alpha-hydroxysteroid dehydrogenase and gene encoding same |
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| CN109082419B (en) | 2022-03-01 |
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