CN104761628A - Babesia mocroti Bm7 recombinant antigenic protein, preparation method and purpose thereof - Google Patents
Babesia mocroti Bm7 recombinant antigenic protein, preparation method and purpose thereof Download PDFInfo
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Abstract
The invention discloses a babesia mocroti Bm7 recombinant antigenic protein, which comprises protein with an amino acid sequence shown in a SEQ ID NO.2, or a protein with the same functions formed by replacing and deleting or inserting the protein. In addition, the invention also discloses a preparation method of the babesia mocroti Bm7 recombinant antigenic protein, which comprises the steps of babesia mocroti Bm7 gene sequence amplification, babesia mocroti Bm7 recombinant plasmid construction and identification, and inducible expression and purifying of the recombinant protein. In addition, the invention also discloses an application of babesia mocroti Bm7 recombinant antigenic protein in preparation of a reagent or a kit for detecting babesia mocroti disease patients (animal or human) serum antibody. Through verification, the babesia mocroti Bm7 recombinant antigenic protein used for babesia mocroti diagnosis has high sensitivity and singularity, is latent diagnosis candidate target, and can be taken as a target antigen for babesia mocroti disease diagnosis.
Description
Technical field
The present invention relates to molecule, RESEARCH ON CELL-BIOLOGY field, be specifically related to recombinant antigen protein of the unnamed albumen of vole babesia (Bm7) and preparation method thereof.In addition, the invention still further relates to the application of this vole babesia Bm7 recombinant antigen protein in the reagent or test kit of preparation detection vole babesiosis Serum Antibodies.
Background technology
Babesia (Babesia) parasitizes an important tick matchmaker protozoon in people or other mammalian erythropoietin, can cause Zoonosis babesiosis (zoonotics babesiosis), all have distribution in Asia, Europe, the United States, Africa etc.Babesia tool opportunistic pathogenesis, to the pathogenic enhancing of immunologic hypofunction person, very can threat to life to immunodeficiency person.Babesiosis is main in animal and veterinary field in China's research as a Zoonosis tick matchmaker parasitosis, zoogenetic infection rate is higher and study less at human infection babesia, correlative study weak foundation, and in recent years along with pace of population aging's quickening, HIV (human immunodeficiency virus, full name is human immunodeficiency virus) infect etc. cause immunologic hypofunction crowd to increase, babesia waits cases of infection in immunodeficiency person to appear in the newspapers repeatly as significant opportunity protozoon of causing a disease patient HIV, and serious threat patients ' lives; The expansion of another people's scope of activity, enters Zoonosis pathogenic agent natural focus etc., and Parasitic Infections In People spectrum constantly changes, Shi You babesiosis grave infection Case report.In China, babesiosis belongs to new and sends out parasitosis, and have human infection Case report during recent years, current babesiosis is popular exists following characteristics with preventing and controlling:
(1) high-risk areas is widely distributed: the Asia comprising China mainland and Taiwan in recent years has human infection babesia Case report successively, main based on vole BABEI worm sample (B.microti-like) babesia, Saito-Ito etc. are once to China Zhejiang, the ground such as Fujian, Sun etc. carry out tick matchmaker and/or the investigation of host B.microti infection conditions to Chinese Heilongjiangdistrict and Zamoto etc. to the Eurasia the Northeast comprising autonomous region of Xinjiang, China Uygur and show that above area all has and comprise the tick infecting babesia and pass pathogenic agent and Natur al foca host thereof and exist.Though babesia natural focus extensively distribute in the whole nation and time have Case report, these high-risk areas babesiosis population infections and carrier's situation be it be unclear that.
(2) babesia infects generally invisible: babesia tool opportunistic pathogenesis, population infection babesia can be carried asymptomatic to babesia infection immunity functional defect as hiv infected patient or low occur obvious clinical symptom by health, even dead, it is by donate blood or pathogen propagation is caused babesiosis to the blood of immunologic hypofunction or organ recipient by organ that many asymptomatic carriers detect, therefore babesia infection tool in healthy population is invisible, threatens the safety of blood product.And at present the blood donors of China to inapparent infection babesia there is no examination standard and instrument, in the urgent need to the higher screening method of sensitivity and specificity to guarantee to transfuse blood and the safety of relevant blood product.
(3) babesia Morphologic Diagnosis difficulty: babesia merozoite is B.microti and the parasitic red blood cell phase plesiomorphism of plasmodium falciparum (Plasmodiumfalciparum) ring bodies especially.At part malarious area, malaria patients' concurrent infection babesia and babesiosis patient can be misdiagnosed as malaria or to fail to pinpoint a disease in diagnosis and out in the cold.In the ground tick matchmakers such as Yunnan Province of China and reservoir host field rodent, B.microti investigates and finds that its natural focus is also malaria prevalence area simultaneously, this area has in malaria sample fever patient crowd and has detected babesia cases of infection, and these cases may concurrent infection plasmodiums and out in the cold.Plasmodium and babesia are difficult to distinguish on morphology, and diagnosis of molecular biology is subject to the restriction of work on the spot, needs badly and carries out dependent diagnostic and differential diagnosis technical study.
Immunologic diagnostic method have easy and simple to handle, susceptibility is higher, specificity is better and the compliance of the Endemic Area masses serves very important effect compared with advantages of higher in the evaluation etc. of the extensive examination of relative disease, seroepidemiological survey and prevention effect.The analysis of domestic and international babesiosis present Research shows, existing babesiosis diagnostic techniques quite backwardness, can be less for serology quick diagnosis antigen, and the antibody horizontal that human body produces lags behind parasitemia, causes different diagnostic method susceptibilitys, specificity all can not practical requirement.
At present, the most frequently used antigen of babesiosis immunodiagnosis is polypide crude antigen, but somatic antigen complicated component, and preparation process is loaded down with trivial details, limited source and not easily Quality Control, is difficult to the demand adapting to extensive on-the-spot general investigation of desease.The recombinant antigen molecule relying on gene engineering method to produce can be the antigen-like material that field popularization application provides a large amount of, and definite ingredients, specificity are good.
Vole babesia Bm7 mono-unknown function albumen is the protein sequence that babesia is new.The present invention goes out vole babesia Bm7 full genome by pcr amplification, and in intestinal bacteria this gene recombinant expressed, obtain the recombinant protein that size is about 24kDa, confirm that gained recombinant protein is used for the piroplasmotic diagnosis of vole and has higher Sensitivity and Specificity through ELISA test, be potential diagnostic antigen candidate targets, thus complete the present invention.
Before making the present invention, also do not occur relating to vole babesia Bm7 recombinant antigen protein of the present invention and the open report for babesiosis diagnosis thereof.
Summary of the invention
One of the technical problem to be solved in the present invention is to provide a kind of vole babesia Bm7 recombinant antigen protein.
Two of the technical problem to be solved in the present invention is to provide a pair Auele Specific Primer for the amplification of vole babesia Bm7 gene PCR.
Three of the technical problem to be solved in the present invention is to provide the preparation method of this vole babesia Bm7 recombinant antigen protein.
Four of the technical problem to be solved in the present invention is to provide the application of this vole babesia Bm7 recombinant antigen protein in the reagent or test kit of preparation detection babesiosis patients serum antibody.
The present invention obtains the coding gene sequence (as shown in SEQ IDNO.1 sequence, Genbank CCF73510.1) of vole babesia Bm7 by bioinformatics technique analysis.Protocols in Molecular Biology is utilized to carry out pcr amplification to vole babesia Bm7 gene, purifying amplified production, products therefrom, plasmid vector pET-28a (+) are carried out enzyme respectively cut, reclaim, connect recombinant plasmid pET-28a (+)-Bm7 being built into Bm7 gene prokaryotic, pass through transformation and selection, extracting recombinant plasmid, cuts after qualification confirmation through PCR, order-checking and enzyme, is converted in host cell colibacillus and expresses; Get the clone that expression amount is higher, fermentation, for thalline, identifies the solvability of recombinant protein; Because the recombinant protein of expressing is for forgiving and being insoluble in denaturing agent, therefore direct whole cell is carried out SDS-PAGE, object band is cut after the dyeing of Camas light blue, reclaim test kit with PAGE glue albumen trace and reclaim purifying target protein, finally obtain the recombinant protein Bm7 of purifying, complete the body outer clone Expression and purification of Bm7 gene.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
The present invention first aspect is to provide a kind of vole babesia Bm7 recombinant antigen protein, its albumen with aminoacid sequence shown in SEQ ID NO.2 or the albumen with identical function by this albumen, one or more amino acid whose displacement, disappearance or insertion occurring and formed; This vole babesia Bm7 recombinant antigen protein is adopted and is prepared with the following method:
1) amplification of vole babesia Bm7 gene order: the nucleotide sequence shown in design SEQ ID NO:3 or the nucleotide sequence shown in its complementary strand and SEQ ID NO:4 or its complementary strand are primer, with containing vole babesia cDNA for template carries out pcr amplification; Gained PCR primer through agarose gel electrophoresis, and reclaims purifying;
2) structure of vole babesia Bm7 recombinant plasmid and qualification;
3) by this recombinant plasmid transformed in host cell, and to express in host cell, obtain the recombinant protein of expressing;
4) PAGE glue albumen trace reclaims the recombinant protein that test kit reclaims purifying expression.
The present invention second aspect there is provided a pair Auele Specific Primer for the amplification of vole babesia Bm7 gene PCR, and its sequence is:
Upstream: 5'-CCGGAATTC ATGCATATCAACTACAAATTAATTATA-3'(is as shown in SEQ ID NO:3) or its complementary strand;
Downstream: 5'-CCGCTCGAG TTAAGCAGCATTAGGTGTG-3'(is as shown in SEQ ID NO:4) or its complementary strand.
Third aspect of the present invention provides a kind of preparation method of vole babesia Bm7 recombinant antigen protein, comprises the steps:
1) amplification (obtaining the DNA fragmentation of Bm7 with round pcr from vole babesia cDNA) of vole babesia Bm7 gene order;
2) structure of vole babesia Bm7 recombinant plasmid and qualification: with pET-28a (+) vector construction vole babesia Bm7 recombinant expression plasmid pET-28a (+)-Bm7;
3) by pET-28a (+)-Bm7 recombinant plasmid transformed in host cell, and to express in host cell, obtain the recombinant protein Bm7 expressed;
4) PAGE glue albumen trace reclaims the vole babesia Bm7 recombinant protein that test kit reclaims purifying expression.
Step 1) amplification of vole babesia Bm7 gene order, be specially:
With the cDNA clone containing vole babesia Bm7 gene order for template, SEQ ID NO.3 (CCGGAATTCATGCATATCAACTACAAATTAATTATA) or its complementary strand are 5 ' primer, SEQ ID NO.4 (CCGCTCGAG TTAAGCAGCATTAGGTGTG) or its complementary strand are 3 ' primer, carry out pcr amplification, gained PCR primer, through agarose gel electrophoresis, is used
glue recovery test kit (
gel Extraction Kit) reclaim purifying.The reaction conditions of described pcr amplification is 95 DEG C of denaturation 5min; 94 DEG C of sex change 1min, 55 DEG C of annealing 1min, 72 DEG C extend 1min, and 72 DEG C extend 10min, 30 circulations; Finally be stored in 4 DEG C.
Step 2) build recombinant plasmid pET-28a (+)-Bm7 that can express vole babesia Bm7 encoding gene in host cell, be specially:
The vole babesia Bm7 gene PCR product fragment of purifying and plasmid vector pET-28a (+) are carried out enzyme with restriction enzyme EcoR I and Xho I respectively cut, use
glue recovery test kit (
gel Extraction Kit) reclaim purifying.Goal gene fragment after purifying is connected with the ratio of carrier endonuclease bamhi according to 3:1 (mol ratio), is built into recombinant plasmid pET-28a (+)-Bm7 of Bm7 encoding gene prokaryotic expression.This plasmid is passed through CaCl
2method is transformed into bacillus coli DH 5 alpha competent cell, coat on the LB agar plate containing kantlex after cultivation, bacterium colony on the above-mentioned flat board of random picking, thalline is collected after cultivation, with recombinant plasmid contained in AxyPrep Plasmid Miniprep Kit extracting thalline, carry out PCR qualification, order-checking qualification and enzyme and cut qualification.
Step 3) expression of recombinant plasmid pET-28a (+)-Bm7 in colibacillus (host cell), be specially:
Transform above-mentioned extracting gained pET-28a (+)-Bm7 recombinant plasmid transformed to enter in e. coli bl21 (DE3) by calcium, coat after cultivation on the LB agar plate containing kantlex.Bacterium colony on the above-mentioned flat board of random picking, cultivates bacterium liquid to logarithmic phase, adds inductor (IPTG) and continues to cultivate.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) identifies abduction delivering result, directly with whole cell electrophoresis showed.
Step 4) purifying vole babesia Bm7 recombinant protein:
Getting the frozen bacterial classification of expressing vole babesia Bm7 recombinant protein amount higher lines on the LB agar plate containing kantlex, after overnight incubation, colony inoculation on the above-mentioned flat board of random picking is to the same liquid nutrient medium containing kantlex, cultivate bacterium liquid to logarithmic phase, add inductor (IPTG) to continue to cultivate, finally collect thalline; In above-mentioned thalline, add Protein Extraction agent cracking bacterium, centrifugal rear reservation supernatant liquor and precipitation, by SDS-PAGE electroresis appraisal result.According to the above results, by denaturing agent cracking gained precipitation, precipitation is insoluble in denaturing agent.The whole cell of expressing recombinant protein is carried out SDS-PAGE, and gel, after coomassie brilliant blue staining, cuts object band, and carry out recovery with PAGE glue albumen trace recovery test kit and purify, SDS-PAGE checks purification result.
The present invention the 4th aspect provides the application of a kind of vole babesia Bm7 recombinant antigen protein in the reagent or test kit of preparation detection vole babesiosis patients serum antibody.The present invention is with the vole babesia Bm7 recombinant antigen protein coated elisa plate of purifying, and 4 DEG C are spent the night.Add confining liquid, 100 μ l/ holes, the nonspecific binding site on sealase target.Add the vole babesiosis human serum of 1:100 dilution, malaria disease human serum and normal human serum respectively, 100 μ l/ holes, every increment product all did multiple hole, in 37 DEG C of reactions 2 hours.The HRP that every hole adds 100 μ l marks the full molecule of goat anti-human igg (Sigma-Aldrich, suitable extent of dilution), and 37 DEG C are reacted 1 hour.Add the tmb substrate solution in 100 μ l/ holes, after color development at room temperature, add the 2M H in 100 μ l/ holes
2sO
4termination reaction.OD450 is read by microplate reader
nmnumerical value.PBST (phosphoric acid salt Tween buffer) is used to wash above between each step respectively.
Through the checking that the indirect ELISA of above-mentioned vole babesia Bm7 recombinant protein is tested, vole babesia Bm7 recombinant antigen protein of the present invention is used for the piroplasmotic diagnosis of vole and has higher Sensitivity and Specificity, potential diagnostic antigen candidate targets, can as the object antigen of vole babesiosis immunodiagnosis.
Accompanying drawing explanation
Fig. 1 is the amplification schematic diagram of vole babesia Bm7 gene order in embodiment 1.In Fig. 1, M:DNA molecular weight standard; 1:Bm7;
Fig. 2 is vole babesia Bm7 gene recombination plasmid bacterium colony PCR qualification result schematic diagram in embodiment 3.In Fig. 2, M:DNA molecular weight standard; 1-4:Bm7; Through the BL21 E. coli clones pcr amplification transformed, 1-4 represents 4 bacterium colonies of random selecting, all successfully connects conversion.
Fig. 3 is the SDS-PAGE analytical results schematic diagram of vole babesia Bm7 in embodiment 4.In Fig. 3, M: Protein Marker; 1: do not induce contrast; 2: 4h (1mM/L IPTG) after induction; 3: supernatant after cellular lysate; 4: cellular lysate postprecipitation.
Fig. 4 is the SDS-PAGE analytical results schematic diagram of vole babesia Bm7 recombinant protein purification effect in embodiment 5.In Fig. 4, M: Protein Marker; 1: do not induce contrast; 2: 4h (1mM/L IPTG) after induction; 3: Bm7 recombinant protein after purifying.
Fig. 5 be in embodiment 6 vole babesia Bm7 recombinant protein for the Sensitivity and Specificity test-results schematic diagram of Serum Antibody Detection.
Embodiment
Following examples are only not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in embodiment, usually conveniently condition, or according to the condition that manufacturer advises.
Main agents is as shown in table 1:
Table 1
Key instrument equipment is as shown in table 2:
Table 2
| PCR instrument | U.S. Bio-Rad |
| Protein electrophoresis instrument | U.S. Bio-Rad |
| Table-type high-speed refrigerated centrifuge | Germany eppendorf |
| High speed freezing centrifuge (CR22F) | FDAC |
| Milli-Q pure water system | U.S. Millipore |
Test materials:
Vole babesia (Babesia micoti type strain) cDNA, host strain E. coli DH5 α, BL21 (DE3), plasmid pET-28a (+), provide by Prevention & Control Station of Parasitic Disease, China Diseases Prevention & C.
The clone of embodiment 1 vole babesia Bm7 gene
The amplification of 1.1 Bm7 gene fragments and purifying:
1.1.1 according to the sequence (as shown in SEQ ID NO.1 sequence) of Bm7 gene (Genbank CCF73510.1), PrimerPremier5.0 software design a pair Auele Specific Primer is utilized, as follows:
PF:5 '-CCG GAATTC ATGCATATCAACTACAAATTAATTATA-3 ' (as shown in SEQ ID NO.3);
PR:5 '-CCG CTCGAG TTAAGCAGCATTAGGTGTG-3 ' (as shown in SEQ ID NO.4);
The protectiveness base of what italicized item represented is upstream and downstream primer, bolded section is the EcoR I restriction enzyme site of upstream primer and the Xho I restriction enzyme site of downstream primer.Auele Specific Primer is synthesized by prompt base (Shanghai) trade Co., Ltd in the English Weihe River.
1.1.2 with vole babesia cDNA for template, carry out PCR reaction, amplification Bm7 gene, reaction system is as follows:
Reaction conditions:
With 1.2% sepharose (GoldView
tMnucleic acid dye) electrophoresis detection PCR primer, observe whether there is object band, result as shown in Figure 1, M:DNA molecular weight standard, 1:Bm7.The result display of PCR reaction, there is a band clearly at about 654bp place, conforms to, show successfully from cDNA, to amplify Bm7 gene with the size of expection fragment.
1.1.3 the purifying of PCR primer reclaims (QIAGEN company
glue reclaims test kit)
1), after electrophoresis terminates, with clean, sharp blade, DNA object fragment is cut down from sepharose.
2) gel piece cut is smashed to pieces be placed in centrifuge tube and weigh, add 3 times of dissolving binding buffer liquid (IodineSodium Solution) to gel volume (volume of 100 μ l is about converted into by the gel of 100mg).IodineSodium Solution is dissolving and binding buffer liquid (with pH indicator), for DNA recovery experiment.
3) centrifuge tube hatches 10min in 50 DEG C of water-baths, for accelerating gel dissolves, every 2-3min, centrifuge tube is taken out mixing of turning upside down.
4) after gel dissolves completely, observe liquid color in centrifuge tube whether with the non-colloidal sol of sodium iodide damping fluid before color basically identical; If liquid in pipe becomes orange or purple, sodium-acetate (pH5.0) adjust ph of 10 μ l 3M need be added.
5) in centrifuge tube, 1 times is added to the Virahol of gel volume, mixing.
6) the QIAquick spin column (rubber tapping column spinner) in test kit is placed in 2ml collection tube; Added by liquid in centrifuge tube in QIAquick spin column, 13000rpm, centrifugal 1min, abandons filtrate.
7) put back in former centrifuge tube by QIAquick spin column, add 500 μ l Buffer QG, 13000rpm, centrifugal 1min, abandons filtrate.
8) in QIAquick spin column, add 750 μ l and wash post damping fluid, leave standstill 2-5min, 13000rpm, centrifugal 1min, abandons filtrate.
9) in 13000rpm, recentrifuge 2min, to remove residual Buffer PE.
10) QIAquick spin column is placed in new 1.5ml centrifuge tube; Add 30 μ l Buffer EB in the film central authorities of QIAquick spincolumn, leave standstill 4min, 13000rpm, centrifugal 1min, collect elutriant, be stored in-20 DEG C.
11) detect organic efficiency with agarose gel electrophoresis, and estimate the concentration of DNA fragmentation.
The structure of embodiment 2 Bm7 gene recombination plasmid pET-28a (+)-Bm7
2.1 PCR primer double digestion and recovery
The PCR object fragment of recovery spent the night in 37 DEG C of water-bath double digestions, endonuclease reaction system is as follows:
Digestion products carries out 1.2% sepharose (GoldView
tMnucleic acid dye) electrophoresis, cut object band, again reclaim DNA molecular, way of recycling, with 1.1.3 in embodiment 1, reclaims product and is stored in-20 DEG C.
The preparation (AxyPrep Plasmid Miniprep Kit) of 2.2 pET-28a (+) empty plasmid
Single bacterium colony of pET-28a (+) plasmid is contained in 5mlLB substratum (kantlex containing 50 μ g/ml), 37 DEG C of overnight incubation at the upper picking of LB flat board (kantlex containing 50 μ g/ml).Next day, get 1ml nutrient solution and proceed in 1.5ml centrifuge tube, be stored in 4 DEG C as bacterial classification.By remaining nutrient solution in 5000rpm, centrifugal 10 minutes, abandon supernatant.The resuspended bacterial precipitation of Buffer S1 (suspend and need evenly, should not leave little bacterium block, otherwise the cracking of thalline can be affected) of RNaseA1 has been added with 250 μ l.Add 250 μ l Buffer S2, gentle but spin upside down mixing fully 6 times, until form bright solution.Add 400 μ l Buffer S3, gentleness also spins upside down mixing 10 times fully, and room temperature leaves standstill 2 minutes, centrifugal 10 minutes of 14000rpm (if still have suspended substance, can put upside down the rear recentrifuge of mixing gently 3 minutes).Being transferred to by supernatant liquor after centrifugal prepares in pipe, is placed in 2ml centrifuge tube, centrifugal 1 minute of 1400rpm.Filtrate is transferred to again preparation pipe to repeat in the same way to combine once.Abandon filtrate, put get back to preparing pipe in former centrifuge tube, add 500 μ l Buffer W1 and wash, centrifugal 1 minute of 14000rpm.Abandon filtrate, put get back to preparing pipe in former centrifuge tube, add 700 μ l Buffer W2 (having added the dehydrated alcohol of proper volume), centrifugal 1 minute of 14000rpm, abandons filtrate; Wash once with 700 μ l Buffer W2 more in the same way, abandon filtrate.Put preparing pipe and get back in 2ml centrifuge tube, centrifugal 1 minute of 14000rpm.Moving into new 1.5ml centrifuge tube by preparing pipe, adding 20 μ l elutriants (elutriant is heated to 65 DEG C in advance, can improve elution efficiency) in the film central authorities of preparing pipe, room temperature leaves standstill 1 minute; 14000rpm carries out wash-out in centrifugal 1 minute.Rejoined by elutriant and prepare periosteum central authorities, room temperature leaves standstill 1 minute, centrifugal 1 minute of 14000rpm eluted dna again.Elutriant is placed in-20 DEG C of preservations, detects the plasmid DNA extracted with agarose gel electrophoresis.(with reference to AxyPrep Plasmid Miniprep Kit operational manual)
The double digestion of 2.3 pET-28a empty plasmid
Add digestion with restriction enzyme pET-28a (+) empty plasmid, 37 DEG C of water-baths spend the night, and reaction system is as follows:
Digestion products carries out 1% sepharose (GoldView
tMnucleic acid dye) electrophoresis, cut object fragment and carry out recovery purifying, way of recycling is with 1.1.3 in embodiment 1, and sample retention is in-20 DEG C.
The structure of 2.4 recombinant plasmids
Be connected with expression vector fragment by exogenous genetic fragment after recovery according to the ratio of 3:1 (mol ratio), linked system is as follows:
Reaction system connects in 16 DEG C of water-baths spends the night, and builds pET-28a (+)-Bm7 recombinant plasmid.
The qualification of embodiment 3 vole babesia Bm7 expression vector
3.1 connect product conversion E.coli DH5 α competent cell
Connection product in 2.4 of embodiment 2 is mixed gently with E.coli DH5 α competent cell, ice bath 30 minutes, in 42 DEG C of water-bath heat shocks 1.5 minutes, ice bath 5 minutes again.In culture tube, the SOC substratum of 900 μ l is added, 37 DEG C, 200rpm, shaking culture 1 hour under aseptic condition.By cultured bacterium liquid in 3500rpm, centrifugal 3 minutes, discard most of supernatant, stay the resuspended thalline of about 100 μ l substratum.Re-suspension liquid is spread evenly across on LB flat board (kantlex containing 50 μ g/ml), is inverted overnight incubation for 37 DEG C, observes colony growth situation.
The PCR qualification of 3.2 recombinant plasmids
Single bacterium colony on random picking flat board carries out bacterium colony PCR qualification, PCR primer detects (result as shown in Figure 2 through agarose gel electrophoresis, in Fig. 2,1-4 represents 4 bacterium colonies of random selecting, all successfully connect conversion), there is object band in the molecular size range place being presented at expectation through PCR qualification, shows the insertion having exogenous genetic fragment.
The order-checking qualification of 3.3 recombinant plasmids
Whether single bacterium colony Song Yingweijie base trade Co., Ltd that selection can amplify object band checks order, to check the sequence of Insert Fragment correct.Sequencing analysis result shows that the exogenous genetic fragment sequence inserted is correct, construction of recombinant plasmid success.
The expression in E.coli of the recombinant plasmid of embodiment 4 vole babesia Bm7 and qualification
The abduction delivering of 4.1 pET-28a (+)-Bm7 recombinant plasmids
1) get pET-28a (+)-Bm7 recombinant plasmid transformed E.coli BL21 (DE3) competent cell that 1 μ l checks order correct, method for transformation is with 3.1 in embodiment 3.
2) next day, respectively at the LB substratum (kantlex containing containing 50 μ g/mls) of each random picking 6 single colony inoculations on each flat board in 5ml, 37 DEG C, 200rpm, shaking culture is to OD
600=0.6.
3) take out 1ml bacterium liquid as bacterial classification, then take out 2ml bacterium liquid and do not add IPTG in contrast, it is that the IPTG of 1mM induces that remaining 2ml bacterium liquid adds final concentration, and 37 DEG C, 250rpm, continues cultivation 4 hours.
4) by cultured bacterium liquid in 4 DEG C, centrifugal 10 minutes of 5000rpm collects thalline, supernatant discarded.
4.2 SDS-PAGE identify induced product
The resuspended bacterial sediment of 200 μ l 1 × PBS is added respectively in induction pipe and control tube.From induction pipe and control tube, take out the re-suspension liquid of 5 μ l respectively, add 2 × SDS-PAGE sample-loading buffer 5 μ l, after mixing, boil sex change in 5 minutes in 100 DEG C.Add the sample of 8 μ l in each loading hole respectively, carry out SDS-PAGE analysis, separation gel is 12%, and concentrated glue is 5%.Gel formula is as shown in table 3 below:
Table 3
The voltage arranging concentrated glue is 80V, and the voltage of separation gel is that 100V carries out electrophoresis.Powered-down when bromophenol blue indicator effusion sheet glass lower rim.Take off gel staining fluid to dye 2 hours, then with destainer decolouring, until protein band is high-visible.
As shown in Figure 3, by pET-28a (+)-Bm7 recombinant plasmid transformed E.coli BL21 (DE3) competent cell, thalline before comparatively inducing after IPTG abduction delivering has occurred that molecular weight is about the obvious band of expression of 24kDa, be the product of goal gene and Histag amalgamation and expression, size conforms to theoretical Mr.
4.3 recombinant protein solvability qualifications
To determine that E.coli BL21 (DE3) strain inoculation can expressing recombinant protein is in 100ml LB substratum (kantlex containing 50 μ g/ml), 37 DEG C, 200rpm is cultured to OD
600=0.6.The bacterium liquid cultivated is taken out 1ml as bacterial classification, separately gets 2ml in contrast.In remaining bacterium liquid, add IPTG to final concentration is 1mM, 37 DEG C, and 200rpm continues cultivation 4 hours.
Bacterium liquid after induction through 5000rpm, 4 DEG C, centrifugal 10 minutes, abandon most supernatant, in bacterial sediment, add the BugBuster Protein Extraction agent (Novagen) of 5ml, after the precipitation that fully suspends, suspension is proceeded in centrifuge tube, be placed on shaking table, lysis at room temperature thalline 1 hour.By bacterial lysate in 4 DEG C, centrifugal 10 minutes of 10000rpm, gets supernatant and is stored in another clean centrifuge tube, and picking precipitates with the PBS of proper volume resuspended on a small quantity.The upper cleer and peaceful precipitation re-suspension liquid of taking out 5 μ l respectively does SDS-PAGE analysis, the solvability of qualification recombinant protein.Electrophoresis result (Fig. 3) shows recombinant protein and is mainly arranged in precipitation, forms inclusion body.
The purifying of embodiment 5 vole babesia Bm7 recombinant protein
The purifying of 5.1 recombinant proteins
1) will determine that E.coli BL21 (DE3) mono-clonal can expressing recombinant protein is inoculated into (kantlex containing 50ug/ml) in 5ml LB substratum in proxima luce (prox. luc), 37 DEG C, 200rpm overnight incubation.
2) next day, be transferred to by the bacterium liquid of overnight incubation in LB (kantlex containing the 50 μ g/ml) substratum of 500ml, 37 DEG C, 200rpm, is cultured to OD
600be about 0.6.
3) get 1ml bacterium liquid as not inducing contrast, adding IPTG to final concentration in residue bacterium liquid is 1mM, 37 DEG C, and 200rpm, continues inducing culture 4h.
4) the bacterium liquid 10000rpm after induction, 4 DEG C, centrifugal 15min, abandons most supernatant, with the PBS gravity treatment bacterial sediment of proper volume, adds isopyknic 2 × SDS-PAGE sample-loading buffer, boils sex change in 5 minutes after mixing in 100 DEG C.
5) according in embodiment 4 4.2 method carry out SDS-PAGE, protein isolate band; Contain the part of object band after cutting decolouring with clean knife blade in blob of viscose, put into the centrifuge tube of 1.5ml, wash 2 times with distilled water, each 5min.
6) with grinding rod, the blob of viscose in centrifuge tube is ground to form tiny fragment, dry powder and DTT (dithiothreitol (DTT)) are dissolved in solution A completely, solution A (solution A, dry powder and the DTT of 400 μ l is added again in centrifuge tube, all reclaim test kit from PAGE glue albumen trace, Sangon Biotech (Shanghai) Co., Ltd., production code member: BSP062), at ambient temperature, on shaking table, (16-18h) is spent the night in concussion, period takes out, and shakes once in a while several times.
7) the centrifugal 15min of room temperature 12000rpm, transfer supernatant, in the centrifuge tube of 2ml, adds the solution B of 2ml precooling, mixing, 4 DEG C of standing 30min.
8) the centrifugal 15min of room temperature 12000rpm, removes supernatant, and the solution B again adding 0.5ml (reclaims test kit from PAGE glue albumen trace, Sangon Biotech (Shanghai) Co., Ltd., production code member: BSP062), concussion washing precipitation, 4 DEG C of standing 15min.
9) the centrifugal 15min of room temperature 12000rpm, removes supernatant, centrifuge tube is placed in stink cupboard, and evaporating of making to remain is clean.
10) add PBS (phosphate buffered saline buffer) dissolution precipitation of proper volume, SDS-PAGE detects the effect of recombinant protein purification.Electrophoresis result as shown in Figure 4, shows that rubber tapping obtains the higher recombinant protein of purity after reclaiming.
The mensuration of 5.2 recombinant protein concentration
Utilize Bradford quantification of protein test kit (sky root) to measure the concentration of recombinant protein, operate to specifications.Key step comprises:
Coomassie Brillant Blue solution first balances to room temperature before use and gentleness puts upside down mixing, preheating spectrophotometer.0,5,10,15,20,25,30 μ l bovine serum albumin (BSA) standardized solution (1mg/ml) are joined in centrifuge tube respectively, adds PBS and complement to 75 μ l.The recombinant protein of proper volume purifying is joined in centrifuge tube, and supplies 75 μ l with PBS.In each centrifuge tube, add 1425 μ l Coomassie Brillant Blue solution, mixing, room temperature leaves standstill 10 minutes.With the light absorption value at spectrophotometric determination 595nm place, and record reading.Not contain the absorbance value of BSA sample as blank, drawing standard curve, calculate the protein concentration of testing sample.If the protein concentration obtained exceeds the scope of typical curve, according to circumstances redeterminate by diluted sample or after concentrating.After calculating purifying according to the extent of dilution of trade union four and recombinant protein, the concentration of Bm7 recombinant protein is 0.815mg/ml.
Each pipe solution reaction system for drawing standard curve is as shown in table 4 below:
Table 4
The ELISA reaction of embodiment 6 vole babesia Bm7 recombinant protein
6.1 indirect ELISA methods detect the sensitivity test of serum antibody
1) recombinant protein is with the best bag by concentration coated elisa plate, and 4 DEG C are spent the night.
2) PBST washes plate 3 times, each standing 3 minutes.Then add confining liquid, 100 μ l/ holes, close in 4 DEG C and spend the night.
3) PBST washes plate 3 times, each standing 3 minutes.Add patients serum (Anhui Province) and the normal human serum (Ningxia Province) of 1:100 dilution respectively, 100 μ l/ holes, every increment product all did multiple hole, in 37 DEG C of reactions 2 hours.Every plate establishes positive reference (patient's pooled serum), negative reference (normal people's pooled serum) and blank with reference to (PBS) simultaneously.
4) plate is washed 6 times with PBST, each standing 3 minutes.The HRP that every hole adds 100 μ l marks the full molecule of goat anti-human igg (Sigma-Aldrich, 1:20000 dilute), and 37 DEG C are reacted 1 hour.
5) PBST washes plate 6 times, each standing 3 minutes.Add the tmb substrate solution in 100 μ l/ holes, after color development at room temperature, add the 2M H in 100 μ l/ holes
2sO
4termination reaction.
6) OD450 is read by microplate reader
nmnumerical value.
6.2 indirect ELISA methods detect the specific test of serum antibody
Basic skills is with in embodiment 6 6.1, and the first antibody reacted with recombinant protein is respectively Plasmodium vivax patients serum (P.v patients serum), plasmodium falciparum patients serum (P.f patients serum) and infection by Babesia microti mice serum (CDC prevention of parasitic diseases provided) and normal human serum (Jiangsu Province).Using 2.1 times of the OD average of normal human serum reaction as the threshold value (CUTOFF) judging yin and yang attribute, OD average=0.131, CUTOFF=0.275, as shown in table 5, Fig. 5, the susceptibility that Bm7 recombinant protein detects infection by Babesia microti mice serum is 100% (20/20), detect non-Endemic Area normal human serum non-false positive reaction (0/30), with falciparum infection patients serum no cross reaction (0/15), be respectively 3.33% (1/15) with the cross reacting rate of plasmodium vivax infection patient.Test-results shows that vole babesia Bm7 recombinant protein has higher Sensitivity and Specificity for the piroplasmotic diagnosis of vole, is potential diagnostic antigen.
Table 5 vole babesia Bm7 recombinant protein detects the Sensitivity and Specificity of serum antibody
Claims (8)
1. a vole babesia Bm7 recombinant antigen protein, is characterized in that, the albumen with aminoacid sequence shown in SEQ ID NO.2 or the albumen with identical function by this albumen, one or more amino acid whose displacement, disappearance or insertion occurring and formed; This vole babesia Bm7 recombinant antigen protein is adopted and is prepared with the following method:
1) amplification of vole babesia Bm7 gene order: the nucleotide sequence shown in design SEQ ID NO:3 or the nucleotide sequence shown in its complementary strand and SEQ ID NO:4 or its complementary strand are primer, with containing vole babesia cDNA for template carries out pcr amplification; Gained PCR primer through agarose gel electrophoresis, and reclaims purifying;
2) structure of vole babesia Bm7 recombinant plasmid and qualification;
3) by this recombinant plasmid transformed in host cell, and to express in host cell, obtain the recombinant protein of expressing;
4) PAGE glue albumen trace reclaims the recombinant protein that test kit reclaims purifying expression.
2. a pair Auele Specific Primer for the amplification of vole babesia Bm7 gene PCR, it is characterized in that, its sequence is:
Upstream: 5'-CCG GAATTC ATGCATATCAACTACAAATTAATTATA-3'(is as shown in SEQ ID NO:3) or its complementary strand;
Downstream: 5'-CCG CTCGAG TTAAGCAGCATTAGGTGTG-3'(is as shown in SEQ ID NO:4) or its complementary strand.
3. a preparation method for vole babesia Bm7 recombinant antigen protein as claimed in claim 1, is characterized in that, comprise the steps:
1) amplification of vole babesia Bm7 gene order: the nucleotide sequence shown in design SEQ ID NO:3 or the nucleotide sequence shown in its complementary strand and SEQ ID NO:4 or its complementary strand are primer, with containing vole babesia cDNA for template carries out pcr amplification; Gained PCR primer through agarose gel electrophoresis, and reclaims purifying;
2) structure of vole babesia Bm7 recombinant plasmid and qualification;
3) by this recombinant plasmid transformed in host cell, and to express in host cell, obtain the recombinant protein of expressing;
4) PAGE glue albumen trace reclaims the recombinant protein that test kit reclaims purifying expression.
4. the preparation method of vole babesia Bm7 recombinant antigen protein as claimed in claim 3, it is characterized in that, the reaction conditions of described pcr amplification is 95 DEG C of denaturation 5min; 94 DEG C of sex change 1min, 55 DEG C of annealing 1min, 72 DEG C extend 1min, and 72 DEG C extend 10min, 30 circulations; Finally be stored in 4 DEG C.
5. the preparation method of vole babesia Bm7 recombinant antigen protein as claimed in claim 3, it is characterized in that, step 2) be specially: by step 1) the PCR primer fragment of gained and plasmid vector pET-28a (+) carry out double digestion with restriction enzyme EcoR I and Xho I respectively, and reclaim purifying; Be connected with the concentration of carrier endonuclease bamhi according to the goal gene fragment after purifying, be built into recombinant plasmid pET-28a (+)-Bm7 of Bm7 encoding gene prokaryotic expression; This recombinant plasmid is passed through CaCl
2method is transformed into bacillus coli DH 5 alpha competent cell, and coat after cultivation containing on kantlex LB agar plate, the bacterium colony on the above-mentioned flat board of random picking, collects thalline after cultivation, contained recombinant plasmid in extracting thalline, carries out PCR qualification, order-checking qualification.
6. the preparation method of vole babesia Bm7 recombinant antigen protein as claimed in claim 3, it is characterized in that, step 3) be specially: transformed step 2 by calcium) check order and identify that errorless pET-28a (+)-Bm7 recombinant plasmid transformed enters in e. coli bl21 (DE3), coat on the LB agar plate containing kantlex after cultivation; Bacterium colony on the above-mentioned flat board of random picking, cultivates intestinal bacteria to logarithmic phase, adds inductor and continues to cultivate; SDS-PAGE electroresis appraisal abduction delivering result, directly with whole cell electrophoresis showed.
7. the preparation method of vole babesia Bm7 recombinant antigen protein as claimed in claim 3, it is characterized in that, step 4) be specially: get the higher frozen bacterial classification of expression recombinant protein Bm7 amount and line on the LB agar plate containing kantlex, after overnight incubation, colony inoculation on the above-mentioned flat board of random picking is to the same liquid nutrient medium containing kantlex, cultivate bacterium liquid to logarithmic phase, add inductor and continue to cultivate, finally collect thalline; In above-mentioned thalline, add Protein Extraction agent cracking bacterium, centrifugal rear reservation supernatant liquor and precipitation, by SDS-PAGE electroresis appraisal result; According to the above results, by denaturing agent cracking gained precipitation, precipitation is insoluble in denaturing agent; The whole cell of expressing recombinant protein is carried out SDS-PAGE, and gel, after coomassie brilliant blue staining, cuts object band, and carry out recovery with PAGE glue albumen trace recovery test kit and purify, SDS-PAGE checks purification result.
8. the application of recombinant antigen protein as claimed in claim 1 in the reagent or test kit of preparation detection vole babesiosis patients serum antibody.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108893476A (en) * | 2018-06-28 | 2018-11-27 | 中国疾病预防控制中心寄生虫病预防控制所 | Babesiamicrofti 2D97 antigen protein and its application |
| CN108893477A (en) * | 2018-06-28 | 2018-11-27 | 中国疾病预防控制中心寄生虫病预防控制所 | Babesiamicrofti 2D41 antigen protein and its application |
| US20180372742A1 (en) * | 2015-12-10 | 2018-12-27 | Immport Therapeutics, Inc. | Babesia Biomarkers for Diagnostic and Screening In Vitro Diagnostic Test |
| CN109371043A (en) * | 2018-06-28 | 2019-02-22 | 复旦大学 | Babesiamicrofti 2D33,2D36 antigen protein and its application |
| CN111926083A (en) * | 2020-08-09 | 2020-11-13 | 现代牧业(合肥)有限公司 | High-yield cow cluster gene breeding method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103102404A (en) * | 2011-11-14 | 2013-05-15 | 中国疾病预防控制中心寄生虫病预防控制所 | Schistosoma japonicum katsurada recombinant antigen protein containing SjSap (sphingolipid activator protein) and preparation method and application thereof |
| CN103374567A (en) * | 2012-04-17 | 2013-10-30 | 中国疾病预防控制中心寄生虫病预防控制所 | Schistosoma japonicum calcium-binding EF-hand domain containing protein (SjEFCAB) recombinant antigen protein and preparation method and application thereof |
-
2015
- 2015-03-20 CN CN201510124956.7A patent/CN104761628A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103102404A (en) * | 2011-11-14 | 2013-05-15 | 中国疾病预防控制中心寄生虫病预防控制所 | Schistosoma japonicum katsurada recombinant antigen protein containing SjSap (sphingolipid activator protein) and preparation method and application thereof |
| CN103374567A (en) * | 2012-04-17 | 2013-10-30 | 中国疾病预防控制中心寄生虫病预防控制所 | Schistosoma japonicum calcium-binding EF-hand domain containing protein (SjEFCAB) recombinant antigen protein and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| EBI: "UniProtKB - I7IQ54", 《EMBL-EBI》 * |
| 孙嘉慧: "田鼠巴贝虫感染诊断抗原的筛选及相关基因的克隆表达", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180372742A1 (en) * | 2015-12-10 | 2018-12-27 | Immport Therapeutics, Inc. | Babesia Biomarkers for Diagnostic and Screening In Vitro Diagnostic Test |
| CN108893476A (en) * | 2018-06-28 | 2018-11-27 | 中国疾病预防控制中心寄生虫病预防控制所 | Babesiamicrofti 2D97 antigen protein and its application |
| CN108893477A (en) * | 2018-06-28 | 2018-11-27 | 中国疾病预防控制中心寄生虫病预防控制所 | Babesiamicrofti 2D41 antigen protein and its application |
| CN109371043A (en) * | 2018-06-28 | 2019-02-22 | 复旦大学 | Babesiamicrofti 2D33,2D36 antigen protein and its application |
| CN108893476B (en) * | 2018-06-28 | 2022-07-05 | 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) | Babesia microti 2D97 antigen protein and application thereof |
| CN108893477B (en) * | 2018-06-28 | 2022-07-05 | 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) | Babesia microti 2D41 antigen protein and application thereof |
| CN109371043B (en) * | 2018-06-28 | 2022-07-05 | 复旦大学 | Babesia microti 2D33 and 2D36 antigen proteins and application thereof |
| CN111926083A (en) * | 2020-08-09 | 2020-11-13 | 现代牧业(合肥)有限公司 | High-yield cow cluster gene breeding method |
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