CN106801042A - Vole Babesia thioredoxin reductase molecule and its gene and application - Google Patents
Vole Babesia thioredoxin reductase molecule and its gene and application Download PDFInfo
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- C12Y108/01009—Thioredoxin-disulfide reductase (1.8.1.9), i.e. thioredoxin-reductase
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Abstract
The invention discloses a kind of amino acid sequence of vole Babesia thioredoxin reductase molecule.The invention also discloses vole Babesia thioredoxin reductase molecular gene, the nucleotide sequence comprising amino acid sequence shown in coding SEQ ID NO.1.Vole Babesia thioredoxin reductase molecule of the present invention, all has good reducing activity to DTNB, insulin, substrate B miTrx and ortholog protein EcoTrx, it is adaptable to the BmiTrxR inhibitor medicaments of screening treatment Babesia Gibsoni.
Description
Technical field
The present invention relates to technical field of bioengineering, more particularly to a kind of vole Babesia thioredoxin reductase molecule and its
Gene and application.
Background technology
Vole Babesia (Babesia microti) be it is a kind of it is new, through tick-borne Zoonosis protozoosis, colonize in people
In rodent host's red blood cell, cause the symptoms such as heating, anaemia, cachexia, elderly, extract spleen or immunity
The infection of low patient is the most serious, can cause death.The first people's infection field is detected from California, USA in 1969
Mouse Babesia so far, has had thousands of people's infection reports, and have elevated trend in recent years.Vole Babesia Gibsoni is main
US and European Countries are popular in, are all had been reported that elsewhere in the world, such as Japan, South Korea, South Africa, Brazil, India
Deng state and TaiWan, China；In China's Mainland, the case of people's infection vole Babesia also constantly has been reported that, nineteen eighty-two Li Jinfu etc.
Two suspected cases are reported first in Yunnan, stone jewellery in 1996 etc. reports a doubtful sense from Xilin Hot, Inner Mongolia
Catch an illness example, 2012, disease prevention and control center of Zhejiang Province, China province is observed by blood film and two methods of detection of nucleic acids make a definite diagnosis one
The middle aged female patient of name has infected vole Babesia.Vole Babesia bites propagation through the hard tick of medium, it is also possible to defeated by clinic
Blood approach is propagated, and with the development of diagnostic techniques, the case of more vole Babesia infection may be found.Treatment is
This new hair immediate problem that faces of disease, at present only a small number of medicines be used for clinic, effect is not sufficiently stable, it is necessary to drug combination,
New drug development is very urgent.
In mammal body, Babesia is bred in red blood cell, therefore it needs to resist active oxygen (ROS) or active nitrogen
(RNS) toxic action, because this two classes material can mainly cause the oxidative damage and lipid peroxidation of polypide DNA.Therefore,
In order to resist oxidative pressure, parasite maintains redox equilibrium using thioredoxin system (Trx).This system includes
The thioredoxin reductase (TrxR) that NADPH, Trx and NADPH are relied on.TrxR belongs to dimer flavine enzyme family, energy
Enough catalysis electronics are transferred to substrate disulphide by FAD domains from NADPH.The C-terminal redox center of protozoon TrxR
By four Amino acid scores every two cysteine residues constitute, this point is different from the TrxR of people, because the latter is by seleno half
What cystine replaced.All the time, the Trx systematic researches of plasmodium falciparum are relatively broad, but in vole Babesia
Research so far it is not yet reported that, therefore, in order to find drug targets, it is necessary to the characteristics of studying vole Babesia TrxR, so as to
For drug targets inhibitor reasonable in design.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of vole Babesia thioredoxin reductase molecule and its gene, should
Vole Babesia thioredoxin reductase molecule can be used to prepare the medicine for the treatment of Babesia Gibsoni.
In order to solve the above-mentioned technical problem, the present invention is achieved through the following technical solutions:
In one aspect of the invention, there is provided a kind of vole Babesia thioredoxin reductase molecule, its amino acid sequence
It is selected from：
(1) amino acid sequence shown in SEQ ID NO.1；
(2) obtained by lacking, add, insert or replacing one or more amino acid in amino acid sequence shown in SEQ ID NO.1
With thioredoxin reductase activity, and there is the amino acid sequence of more than 95% homology with SEQ ID NO.1.
Preferably, the amino acid sequence of the vole Babesia thioredoxin reductase molecule is as shown in SEQ ID NO.1.
In another aspect of this invention, there is provided a kind of vole Babesia thioredoxin reductase molecular gene, it is included：
The nucleotide sequence of amino acid sequence shown in coding SEQ ID NO.1.
Preferably, the nucleotide sequence of the vole Babesia thioredoxin reductase molecular gene such as SEQ ID NO.2 institutes
Show.
In another aspect of this invention, a kind of recombinant vector is additionally provided, comprising the reduction of above-mentioned vole Babesia thioredoxin
Enzyme molecule gene order or part thereof sequence.
The recombinant vector includes recombinant cloning vector or recombinant expression carrier.
In another aspect of this invention, a kind of host cell comprising above-mentioned recombinant vector is additionally provided.
In another aspect of this invention, a kind of system of vole Babesia thioredoxin reductase molecular recombination albumen is additionally provided
Preparation Method, comprises the following steps：
Build the recombinant expression carrier containing above-mentioned vole Babesia thioredoxin reductase molecular gene；
The recombinant expression carrier is transformed into e. coli host cell；
E. coli host cell of the culture comprising recombinant expression carrier, and under suitable conditions, induce the recombinant expression carrier table
Up to vole Babesia thioredoxin reductase molecular recombination albumen.
In another aspect of this invention, a kind of application of above-mentioned vole Babesia thioredoxin reductase molecule is additionally provided,
Medicine for screening treatment Babesia Gibsoni.
The medicine is the inhibitor of vole Babesia thioredoxin reductase.
In another aspect of this invention, a kind of application of above-mentioned vole Babesia thioredoxin reductase molecule is additionally provided,
For preparing disulfide reductase preparation.
Vole Babesia thioredoxin reductase molecule of the present invention, through recombinantly expressing the recombinant protein BmiTrxR for obtaining, by
Enzyme activity dynamic analysis experiment shows with good DTNB and insulin reducing activity, same for substrate B miTrx and direct line
Source protein E.coli (EcoTrx) also has reducing activity, is suitable to the BmiTrxR inhibitor medicaments of screening treatment Babesia Gibsoni.
Brief description of the drawings
The present invention is further detailed explanation with reference to the accompanying drawings and detailed description.
Fig. 1 is the amino acid alignment figure of the BmiTrxR genes with other species TrxR of the embodiment of the present invention 1；
Fig. 2 is the expression and purification result figure of the recombinant protein BmiTrxR of the embodiment of the present invention 2；
Fig. 3 is the Western Blotting testing result figures of the embodiment of the present invention 3；
Fig. 4 be the embodiment of the present invention 5 condition of different pH under recombinant protein BmiTrxR DTNB reductase activity result figures；
Fig. 5 be the embodiment of the present invention 5 in the experiment of DTNB reductase activities, recombinant protein BmiTrxR's under various concentrations
Enzyme activity initial velocity of reaction figure；
Fig. 6 is the DTNB reductase activities pair of the recombinant protein BmiTrxR and rat TrxR (RatTrxR) of the embodiment of the present invention 5
Than figure；
Fig. 7 is the Trx reduction test result figures of the embodiment of the present invention 5；
Fig. 8 is the Anranofin of the embodiment of the present invention 5 as the disulfide reductase inhibitor result of the test of recombinase BmiTrxR
Figure.
Specific embodiment
In the following example, the experimental technique of unreceipted actual conditions, generally routinely condition, such as《Molecular Cloning:A Laboratory guide》
(J. Pehanorm Brookers, D.W. Russells are write, Huang Peitang, and Wang Jiaxi, Zhu Houchu are waited and translated the 3rd edition, Beijing：Science
Publishing house, 2002) described in method carry out.
The present invention obtains a new thioredoxin reductase molecule from vole Babesia polypide merozoite first, referred to as
BmiTrxR, expression vector is cloned into by its gene coding region, is recombinantly expressed in Escherichia coli, by enzyme activity dynamics reality
Analysis BmiTrxR recombinant proteins are tested to DTNB and insulin reducing activity, and to substrate B miTrx and ortholog protein
The reducing activity of E.coli (EcoTrx).
The gene cloning of vole Babesia thioredoxin reductase molecule (BmiTrxR) of embodiment 1 and sequence analysis
1. materials and methods
1.1. parasite
Vole Babesia is quoted from American Type Culture Collection (ATCC), numbering ATCCRPRA-99TM, in this laboratory by elder brother
Bright system mouse Inoculation passage is preserved.
1.2. bacterium and plasmid
What plasmid construction and protein expression were used is the cells of Escherichia coli Top 10 (Invitrogen) and e. coli bl21 (DE3)
(Novagen).That cloning and sequencing carrier is used is pGEM-T Easy (Promega), and expression vector is pET28a vector
(Novagen)。
1.3. the molecular cloning of vole Babesia thioredoxin reductase (B.microti TrxR)
The RNA of polypide merozoite is extracted with TRIzol reagent (Invitrogen), then RNA is by DNase I
(TOYOBO) digestion, removes genomic DNA therein.CDNA is obtained from the total serum IgE of polypide by reverse transcription,
Whole step is operated according to reverse transcription reagent box (TaKaRa, Dalian, China) specification.BmiTrxR ORFs
Amplimer is F:5 '-ATGATTCTTCGGGCTGGATT-3 ' (SEQ ID NO.3) and R:5’-TCAACCACAC
TTGCCCCCAC-3 ' (SEQ ID NO.4), full length sequence by SMARTer RACE cDNA amplification kits (Clintech,
San Jose, CA, USA) obtain.According to acquired partial sequence, (5 ' hold rapid amplifying GSP1 to design specific primer:
5 '-GCCTCCTCCAATCACAGCGAAGT-3 ' (SEQ ID NO.5) and GSP2:5’-GCTTAGTTTCGGTG
AGTGATGCG-3(SEQ ID NO.6)；3 ' hold rapid amplifying GSP3:5’-TAGCACATAGGATGAAGACAA
GGCA-3 ' (SEQ ID NO.7) and GSP4:5’-CAGGGAATGGCATTAGCTATGAAAC-3’(SEQ ID NO.8)).
Pcr amplification product after purification is sequenced in being connected to cloning vector pGEM-T Easy (Promega).
2. result
From the mRNA of B.microti merozoites, the cDNA fragments (SEQ of the total length 1766bp of gene BmiTrxR is obtained
ID NO.2), a complete ORFs 1662bp is encoded, encode 553 amino acid (SEQ ID NO.1).Prediction
Molecular weight of albumen is 58.4kDa, and isoelectric point is 6.95, similar to HMW TrxR.Predict that its signal peptide sequence is preceding 18
Individual amino acid.Prediction albumen is carried out into BLAST non-redundant database analyses, the TrxR of prediction albumen and other species is as a result shown
With significant similarity.The amino acid sequence of BmiTrxR and ox Babesia Babesia bovis TrxR (XP_
001608869.1) homology is 58%, with plasmodium falciparum Plasmodium falciparum TrxR (XP_002808951.1)
Homology is 53%, is 54% with the homology of people Cryptosporidium Cryptosporidium hominis TrxR (XP_666572.1),
Homology with Theileria annulata Theileria annulata strain Ankara (XP_954814.1) is 55%, with Human
The homology of TrxR (3QFB_B) is 44%, as shown in Figure 1.It was found that there is the N-terminal of prediction albumen sulfydryl disulphide to aoxidize
Reducing activity center (- CVNVGC-), C-terminal includes one section of conservative sequence GCGGGKCG, this section of sequence exactly TrxR tool
There is the domain of catalysis activity.
The recombination expression of vole Babesia thioredoxin reductase molecule (BmiTrxR) of embodiment 2 and purifying
BmiTrxR full-length genes are subcloned into expression vector after Nco I and Xho, I two restriction enzyme sites carry out double digestion
In pET-28a (Novagen), carry out that the accuracy for ensuring sequence is sequenced.Full-length gene is by building His label proteins in E.coli
Expressed in BL21 (DE3).Expression bacterium by 1mM IPTG induce, 16 DEG C be incubated about 12 hours after collects thalline,
It is stored in -80 DEG C.Recombinant protein passes through Ni2+Affine resin (Novagen) is purified, the thalline combination buffer after induction
(NI-NTA Buffer Kit, Novagen) resuspended, then ultrasonication, 12,000 × g, 4 DEG C of centrifugation 10min, solvable supernatant
Albumen be loaded into Purification Resin, then washed twice with cleaning buffer solution, finally with elution buffer (NI-NTA Buffer Kit,
Novagen) destination protein is eluted, PAGE gel electrophoretic analysis is finally carried out and is quantified.
As a result：BmiTrxR recombinant protein successful expressions, molecular weight is about 58.4kDa, (see Fig. 2) in the same size with prediction,
Albumen is expressed with soluble form.In Fig. 2, swimming lane 1：The cell pyrolysis liquid not induced；Swimming lane 2：The cell pyrolysis liquid of induction；
Swimming lane 3：The recombinant protein BmiTrxR of purifying.
The sero-fast preparations of the recombinant protein BmiTrxR of embodiment 3 and Western Blotting detections
After recombinant protein after purification mixes with Freund's complete adjuvant, by intraperitoneal injection immune mouse (every about 200g recombinant protein),
Then, to these mouse every two weeks booster immunizations once, altogether twice, and the recombinant protein full adjuvant that needs to cannot be used up is premixed.It is logical
Cross eye socket blood sampling and collect mouse blood sample, with ELISA detection antibody titres.
Mouse red blood cell and the mouse red blood cell being uninfected by of vole Babesia infection respectively with 2 × SDS gel-loading of equivalent
Buffer mixes, and then boils 10 minutes by albuminous degeneration.Carry out being transferred to NC after gel electrophoresis in 12% SDS-PAGE
On film, closed overnight with 5% 4 DEG C of skim milk.The antiserum (1 of NC films and recombinant protein after closing:200 dilutions)
Incubation at room temperature 1 hour, is then washed once, 5 times totally for every 10 minutes with PBST.The sheep anti-mouse igg (1 marked with HRP:2000
Dilution) incubation at room temperature 1 hour after, equally wash film with primary antibody.Finally developed the color with substrate DAB.
As a result：Recombinant protein BmiTrxR immune mouses after purification, obtain polyclonal antibody.B.microti is identified with antiserum
Native protein BmiTrxR in lysate.Result in the mouse red blood cell lysate of infection B.microti as shown in figure 3, examine
Specific protein band is measured, molecular weight is about 58.4kDa, and is not detected by the cell for be uninfected by B.microti, shows
Recombinant protein has immunological effect, and antiserum there occurs specific immune response with native protein BmiTrxR.In Fig. 3,
Swimming lane 1：The mouse red blood cell lysate of B.microti infection；Swimming lane 2：The mouse red blood cell lysate being uninfected by.The He of swimming lane 1
2 are incubated with recombinant protein antiserum.
The recombinant protein BmiTrxR enzyme activity dynamic analyses of embodiment 4
1. DTNB reductase activities of recombinant protein BmiTrxR
Enzyme activity experiment is carried out under the conditions of upper 25 DEG C of multi-function microplate reader (SpectraMax M5, Molecular Devices).Restructuring egg
The TrxR activity of white BmTrxR is by detecting the recombinase to DTNB and the reducing power of insulin.Reduced in DTNB and tried
In testing, to find optimum response parameter, detection recombinant protein BmiTrxR under different pH to the reducing activity of DTNB, with
And the recombinant protein BmiTrxR under detection various concentrations, to the reducing activity of DTNB, 200 μ l reaction systems include 100mM
Kaliumphosphate buffer, 0.1mM NADPH, 2mM DTNB and 50nM BmiTrxR.By detecting first three minute of the reaction
The increase of light absorption value detects the activity of BmTrxR at 412nm, because the NADPH of 1mol produces the TNB of 2mol,
So with the extinction coefficient 13.6mM of product TNB-1cm-1Calculated.
As a result：As shown in figure 4, recombinant protein BmiTrxR after purification is under the conditions of 25 DEG C, pH 6.5,100mM phosphoric acid
Activity in salt buffer is maximum.Fig. 5 shows, when restructuring enzyme concentration is 100nM, enzyme activity reaction initial velocity is maximum.
With DTNB as substrate, and positive control is done with the rat TrxR (RatTrxR) of same concentration, find present invention restructuring
Enzyme BmiTrxR has disulfide reductase activity (Fig. 6) that significant NADPH is relied on
2. the insulin reducing activity of recombinant protein BmiTrxR
Reaction temperature is 25 DEG C, and the system of Trx reduction tests includes 0.16mM bovine insulins (Sigma), 10 μM of restructuring eggs
White BmiTrx or Escherichia coli Trx (EcoTrx), 100nM BmiTrxR or RatTrxR, 100 μM of NADPH delay
Fliud flushing is 50mM Tris-HCI (pH 6.5), and comprising 2mM EDTA, negative control is to substitute enzyme with the His polypeptides of same concentration
TrxR.By detecting the consumption of NADPH, light absorption value declines to detect the activity of recombinase at 340nm.NADPH
Extinction coefficient be 6.22mM-1cm-1.For the experiment of all of enzyme activity, blank control group system removes recombinase or substrate, often
Individual reaction minimum of three is repeated.
As a result：Recombinase BmiTrxR also has reducing activity for substrate B miTrx and ortholog protein E.coli (EcoTrx)
(Fig. 7).
In TrxR activity tests, NADPH concentration ranges are 5-100 μM, and the change in concentration scope of DTNB is 100-1000
μM, Trx is 5-30 μM, and the concentration of recombinase is fixed as 50nM.For each reaction, when a kind of change in concentration of substrate
When, system other compositions concentration immobilizes.Experimental data is carried out into nonlinear equation fitting and Michaelis-Menten equation, so as to calculate
Go out enzyme activity parameter.The calculating of enzyme activity parameter at least needs three sets of independent data.Table 1 is recombinant protein BmiTrxR for difference
The enzymatic activity parameter of substrate.
Enzymatic activity parameters of the recombinant protein BmiTrxR of table 1 for different substrates
3. Anranofin is tested as the disulfide reductase inhibitor of recombinase BmiTrxR
Anranofin is assessed as a kind of enzyme inhibitor by monitoring the increase of light absorption value at 412nm in DTNB reduction tests
The effect of inhibitor.The test system includes 100 μM of NADPH, 2mM DTNB, 50nM recombinase BmTrxR, suppression
The concentration of preparation is respectively 100 μM of 0,3.125,6.25,12.5,25,50and.Inhibitor needs premix 15 minutes with recombinase
Afterwards, NADPH and DTNB is added, most the remaining enzymatic activity of monitoring later.
As a result：When the concentration of Anranofin is 12.5 μM, enzymatic activity is almost suppressed (Fig. 8) completely.
Embodiment described above only expresses embodiments of the present invention, and its description is more specific and in detail, but can not therefore and
It is interpreted as the limitation to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, not taking off
On the premise of present inventive concept, various modifications and improvements can be made, these belong to protection scope of the present invention.Therefore,
The protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
1. a kind of vole Babesia thioredoxin reductase molecule, its amino acid sequence is selected from：
(1) amino acid sequence shown in SEQ ID NO.1；
(2) obtained by lacking, add, insert or replacing one or more amino acid in amino acid sequence shown in SEQ ID NO.1
With thioredoxin reductase activity, and there is the amino acid sequence of more than 95% homology with SEQ ID NO.1.
2. thioredoxin reductase molecule according to claim 1, it is characterised in that the vole Babesia sulphur oxygen
Also the amino acid sequence of reductase proteins molecule is as shown in SEQ ID NO.1.
3. a kind of vole Babesia thioredoxin reductase molecular gene, it is included：Amino shown in coding SEQ ID NO.1
The nucleotide sequence of acid sequence.
4. thioredoxin reductase molecular gene according to claim 3, it is characterised in that the vole Babesia
The nucleotide sequence of thioredoxin reductase molecular gene is as shown in SEQ ID NO.2.
5. a kind of recombinant vector, it is characterised in that include：Vole Babesia thioredoxin reduction described in claim 3
Enzyme molecule gene order or part thereof sequence.
6. a kind of host cell, it is characterised in that comprising the recombinant vector described in claim 5.
7. a kind of preparation method of vole Babesia thioredoxin reductase molecular recombination albumen, it is characterised in that including with
Lower step：
Build the recombinant expression carrier containing vole Babesia thioredoxin reductase molecular gene described in claim 3；
The recombinant expression carrier is transformed into e. coli host cell；
E. coli host cell of the culture comprising recombinant expression carrier, and under suitable conditions, induce the recombinant expression carrier table
Up to vole Babesia thioredoxin reductase molecular recombination albumen.
8. the application of vole Babesia thioredoxin reductase molecule described in claim 1, for screening treatment Babesia
The medicine of disease.
9. application according to claim 8, it is characterised in that the medicine is reduced for vole Babesia thioredoxin
The inhibitor of enzyme.
10. the application of vole Babesia thioredoxin reductase molecule described in claim 1, for preparing disulfide reduction
Enzyme preparation.
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| CN201510837025.1A CN106801042A (en) | 2015-11-26 | 2015-11-26 | Vole Babesia thioredoxin reductase molecule and its gene and application |
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Non-Patent Citations (4)
| Title |
|---|
| CORNILLOT E等: "登录号：NC_027206.1", 《GENBANK》 * |
| CORNILLOT E等: "登录号：XM_012793011.1", 《GENBANK》 * |
| REGNER EL等: "Biochemical characterization of thioredoxin reductase from Babesia bovis", 《BIOCHIMIE》 * |
| 赵少若等: "田鼠巴贝斯虫硫氧还蛋白还原酶的研究", 《中国畜牧兽医学会兽医寄生虫学分会第十三次学术研讨会论文集》 * |
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