CN102925447B - peptidoglycan binding protein BjApextrin1 and BjApextrin2, and genes, production method and application thereof - Google Patents
peptidoglycan binding protein BjApextrin1 and BjApextrin2, and genes, production method and application thereof Download PDFInfo
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Abstract
The invention relates to two peptidoglycan binding proteins, encoding gene BjApextrin1 and BjApextrin2 thereof and application of the proteins in preparing medicaments for treating infectious diseases. According to the existing expressed sequence tag (EST) sequence, by means of 5'RACE and 3'RACE, BjApextrin1 and BjApextrin2 genes of branchiostoma are obtained by separating the alimentary canal of the branchiostoma in China, and deoxyribonucleic acid (DNA) sequences of the BjApextrin1 and BjApextrin2 genes are indicated by a sequence 1 and a sequence 2 in a sequence table. Amino acid sequences of the proteins (BjApextrin1 and BjApextrin2) encoded by the genes are indicated by a sequence 3 and a sequence 4 in the sequence table. The protein BjApextrin1 and BjApextrin2 encoded by the genes can indentify and agglutinate gram-positive bacteria, and can be specifically bonded with biological macromolecular peptidoglycan on the surfaces of the bacteria to be used for preparing the medicaments for treating infectious diseases.
Description
Technical field
The present invention relates to a kind of peptidoglycan binding-protein gene of novelty and protein B jApextrin1 and the BjApextrin2 of coding thereof, and the application of this albumen in preparation treatment infectious disease medicament.
Background technology
Branchiostoma is in Chordata (Chordata), Cephalochordata (Cephalochordata), being that questions of invertebrate evolution arrives vertebrate transitional type, is the similar model of vertebrates original ancestry before 500,000,000 years, is the good material that research vertebrates grows all the time.In recent years, the immune research of lancelet has been caused widely and paid attention to, found therein many significant immunogenes.
Peptidoglycan (Peptidoglycan, PGN) be one of the main component of the cell walls of nearly all bacterium, especially be rich in gram-positive microorganism, also be a kind of important pathogeny associated molecular pattern (Pathogen-associated molecular patterns, PAMPs) of the invasion of host's perception pathogenic bacteria, activate immunity reaction.Peptidoglycan can be identified molecular recognition by various modes, as peptidoglycan recognition protein (Peptidoglycan recognition proteins, PGRPs), and Toll sample acceptor 2(Toll-like receptor2, TLR2) etc.
The poor aobvious technology of the utilizations such as Haag contrasts the different sea urchin of fetal development pattern, gene expression abundance is apparently higher than the tubercle sea urchin of indirect development in the embryo of the Red sea of direct development pattern courage (Heliocidaris erythrogramma) to find Apextrin, and embryo's in situ hybridization shows that Apextrin specifically expressing is in Anthocidaris crassispina embryo's ectoderm.Immunohistochemical experiment proof Apextrin albumen is stored in ovum the inside with the form of secretory vacuole, after completing fertilization, ovum starts gradually secretion, when embryo's Apextrin albumen after polarization blastula stage is just positioned embryo top extracellular matrix, therefore be called top extracellular matrix protein (Apical extracellular matrix protein, Apextrin).People, again respectively hydra, find Apextrin gene in coral subsequently, and embryo's in situ hybridization shows that Apextrin also may participate in the embryo development procedure of hydra and coral.
The expression amount of lancelet Apextrin gene has raised thousands of times after bacterium stimulates 12h; The expression amount of Red sea courage Apextrin albumen after being subject to vibrio infection 20h also significantly increases.We find that the Apextrin of lancelet can identify and aggegation gram-positive microorganism, and the biomacromolecule peptidoglycan of specific combination bacterium surface, can be used for the medicine as preparation treatment infectious diseases, in fields such as aquaculture, medical and health, there is wide DEVELOPMENT PROSPECT.
Summary of the invention
The object of the present invention is to provide a kind of new peptidoglycan in conjunction with the gene of protein B jApextrin1 and BjApextrin2 and this albumen of encoding.
Another object of the present invention is to provide the production method of this albumen.
The 3rd object of the present invention is to provide the application of this albumen in preparation treatment infectious disease medicament.
The present invention according to existing est sequence by 5 ' and 3 ' RACE, from the digestive tube of Chinese Amphioxus, separation obtains lancelet BjApextrin1 and BjApextrin2 gene, its DNA sequence dna is as shown in sequence in sequence table 1 and sequence 2 sequences.
The protein B jApextrin1 of genes encoding of the present invention and BjApextrin2, its aminoacid sequence is as shown in sequence in sequence table 3 and sequence 4 sequences; This albumen iso-electric point is respectively 6.05 and 5.03, and molecular weight is respectively 56,671 and 52,516 dalton.
It is upper that BjApextrin1 and BjApextrin2 are cloned into coli expression carrier pET32a, obtains recombinant expression vector pET32a-BjApextrin1 and pET32a-BjApextrin2.
This albumen can be expressed with insoluble inclusion body form intestinal bacteria by expression vector pET32a-BjApextrin1 and pET32a-BjApextrin2.
Described expression vector pET32a-BjApextrin1 and pET32a-BjApextrin2 be take Trx as the amalgamation and expression companion body, and this system makes BjApextrin1 and the BjApextrin2 inclusion body of expressing by becoming renaturation, correctly be folded into soluble albumen under the help of Trx.
The selected Qingdao branchiostoma of the present invention (Branchiostoma japonicum) is collected in Shandong Province's sand Estuary Waters.
The present invention according to existing est sequence by 5 ' and 3 ' RACE, from separation the digestive tube of Chinese Amphioxus, obtain lancelet BjApextrin1 and BjApextrin2 gene (its DNA sequence dna is as shown in sequence in sequence table 1 and sequence 2 sequences).504 and 462 amino acid (its aminoacid sequence is as shown in sequence in sequence table 3 and sequence 4 sequences) of encoding respectively, iso-electric point is respectively 6.05 and 5.03, and molecular weight is respectively 56,671 and 52,516 dalton.
The present invention, by having designed two pairs of primers, is cloned into coli expression carrier pET32a(Novagen company by gene BjApextrin1 and BjApextrin2), construction expression plasmid is also transformed e. coli bl21 (DE3).Expression vector pET32a-BjApextrin1 and pET32a-BjApextrin2 be take Trx as the amalgamation and expression companion body, and this system makes BjApextrin1 and the BjApextrin2 inclusion body of expressing by becoming renaturation, correctly be folded into soluble albumen under the help of Trx.This recombinant protein 670 and 628 amino acid of encoding respectively, iso-electric point is respectively 5.80 and 5.14, and molecular weight is respectively 74,374 and 70,219 dalton.By to incubation time, induction time, the groping and optimize of the conditions such as temperature, the expression amount of BjApextrin1 and BjApextrin2 fusion rotein is higher, and the overwhelming majority exists with soluble inclusion body form.
The present invention also gropes and has optimized the purification condition of BjApextrin1 and BjApextrin2 recombinant protein, and thalline carries out after ultrasonication, and centrifuging and taking precipitation becomes renaturation and can obtain purer albumen.
Expression plasmid clone method of the present invention: with reference to Sambrook(Sambrook, et al.1989, Molecular cloing.Cold Spring Harbor Labroratory Press.USA) method, use CaCl
2method by plasmid Transformed E .coli.DH5 α or BL21(DE3) bacterial strain, with transforming bacterial strain containing the LB culture medium culturing of penbritin (100 μ g/mL), with Omega company test kit, extract plasmid.
The present invention by bacterium in conjunction with evidence this recombinant protein there is the activity in conjunction with gram positive bacterium.Bacterial agglutination experiment shows that this recombinant protein can make middenstead coccus and staphylococcus aureus generation aggegation.Enzyme-linked immunosorbent assay (Enzyme-linked-immunosorbent assay, ELISA) show that this recombinant protein can be specifically in peptidoglycan combination, fusion rotein and peptidoglycan are done drop-down experiment (Pulldown assay) and are shown that the increase with peptidoglycan concentration strengthens this recombinant protein to the combination activity of peptidoglycan.
By building deletion mutant, we find that the pattern recognition function of BjApextrin1 is that one section of conserved sequence being held by C determines.
Accompanying drawing explanation
Fig. 1 a and Fig. 1 b are respectively BjApextrin1 and the BjApextrin2 gene object fragment that 5 ' RACE and 3 ' RACE amplification obtains.1.5 ' RACE amplified fragments; 2.3 ' RACE amplified fragments; 3.DNA molecular weight standard (DL2000).
Fig. 2 a and Fig. 2 b are respectively BjApextrin1 and the BjApextrin2 full length gene fragment that amplification obtains.
Fig. 3 is recombinant expression plasmid pET32a-BjApextrin1 and pET32a-BjApextrin2 design of graphics.
Fig. 4 a and Fig. 4 b are respectively restructuring BjApextrin1 and the protein induced expression of BjApextrin2, become Purification electrophorogram.M, protein marker; 1, the ultrasonic supernatant of not inducing; 2, the ultrasound precipitation of not inducing; 3, the ultrasonic supernatant of induction; 4, the ultrasound precipitation of induction; 5, the TRX-Apextrin1 after purifying and TRX-Apextrin2.
Fig. 5 be the bacterium of recombinant protein BjApextrin1 and BjApextrin2 in conjunction with test-results: by approximately 2 * 10
7individual various bacterium TBS(pH7.5) after washing balance, with 1 μ g target protein at 1ml TBS(pH7.5) 4 ℃ of oscillation incubations spend the night in damping fluid; 4 ℃, the centrifugal 1min of 12000 * g, precipitation is washed 5 times with 1ml TBS damping fluid; With the resuspended thalline of 100 μ l TBS, then add 20 μ l6 * sample-loading buffers, after mixing, boil and boil 10min; Then carry out Western blot analysis, by His monoclonal antibody, carry out testing goal albumen, Sepharose4B bead carries out parallel laboratory test as negative control.
Fig. 6 is the bacterial agglutination experimental result of recombinant protein BjApextrin1 and BjApextrin2: the enterococcus faecalis that FITC mark is crossed and streptococcus aureus and recombinant protein are hatched altogether, in the aggegation situation of fluorescent microscope norma basilaris micrometer biology.
Fig. 7 a and Fig. 7 b are respectively the ELISA experimental result of recombinant protein BjApextrin1 and BjApextrin2 and bacteria cell wall pure component: recombinant protein respectively with LPS, LTA, PGN, Mannan and Zymosan carry out ELISA experiment.
Fig. 8 is recombinant protein BjApextrin1 and BjApextrin2 and peptidoglycan Pulldown experimental result: 1 μ g target protein is carried out to Pulldown experiment with the PGN of 5,10,20,50 μ g respectively.
Fig. 9 is recombinant protein BjApextrin1 deletion mutant abduction delivering, change Purification electrophorogram.M, protein marker; The 1st swimming lane, the not ultrasonic supernatant of TRX-△ C of induction; The 2nd swimming lane, not the TRX-△ C ultrasound precipitation of induction; The 3rd swimming lane, the ultrasonic supernatant of TRX-△ C of induction; The 4th swimming lane, the TRX-△ C ultrasound precipitation of induction; The 5th swimming lane, the TRX-△ C after purifying; The 6th swimming lane, the not ultrasonic supernatant of TRX-△ N of induction; The 7th swimming lane, not the TRX-△ N ultrasound precipitation of induction; The 8th swimming lane, the ultrasonic supernatant of TRX-△ N of induction; The 9th swimming lane, the TRX-△ N ultrasound precipitation of induction; The 10th swimming lane, the TRX-△ N after purifying.
Figure 10 a and 10b are respectively the peptidoglycan PGN(S.a in recombinant protein BjApextrin1 deletion mutant and streptococcus aureus source place) and the peptidoglycan PGN(B.s in subtilis source) ELISA experimental result.
Embodiment
Below implement to contribute to those of ordinary skill in the art further to understand the present invention, but do not limit in any form the present invention.
Embodiment 1: the extraction of the total RNA of Chinese Amphioxus digestive tube and cDNA's is synthetic
The extraction of total RNA and RACE cDNA are synthetic: collect Chinese Amphioxus digestive tube, adopt Trizol reagent method to extract the total RNA of digestive tube.Get the total RNA of 1 μ g, according to the cDNA of TOYOBO company mono-chain synthetic agent box (ReverTra Ace-α-
tM, code No.FSK-100) illustrate and synthesize cDNA mono-chain.Adopt the GeneRacer of Invitrogen
tMkit is according to synthetic RACE cDNA mono-chain of explanation.
Embodiment 2:RACE amplification BjApextrin1 and BjApextrin2 gene 5 ' and 3 ' end.
According to the gene-specific primer of 5 ' RACE amplification of known est sequence design BjApextrin1, be 5 '-GSP:5 '-CGTCATCTTCATCGTCCCA ACGGAA-3 '; The gene-specific primer of 3 ' RACE amplification of BjApextrin1 is 3 '-GSP:5 '-TTCCGCTTGGGACGATGAAGATG-3 '.The gene-specific primer of 5 ' RACE amplification of BjApextrin2 is 5 '-GSP:5 '-CGTCTTCATCGTCCCACCGGAA-3 '; The gene-specific primer of 3 ' RACE amplification of BjApextrin2 is 3 '-GSP:5 '-TTCCGGTGGGACGATGAAGAC-3 '.Adopt the LA Taq polysaccharase of Takara company according to GeneRacer
tMthe reaction system of Kit is carried out pcr amplification.The current fragment that amplification obtains is as Fig. 1.Object fragment is connected to pGEX Teasy vector(promega) the rear bacillus coli DH 5 alpha that transforms, select recombinant clone order-checking.After being spliced, the sequencing result of 5 ' RACE and 3 ' RACE obtained the full length sequence of BjApextrin1 and BjApextrin2 gene.The full length sequence of BjApextrin1 contains the open reading frame that a length is 1515bp (open reading frame, ORF), 504 the amino acid whose albumen of encoding; The full length sequence of BjApextrin2 contains the open reading frame that a length is 1389bp (open reading frame, ORF), 462 the amino acid whose albumen of encoding.
Amplification and the analysis of embodiment 3:BjApextrin1 and BjApextrin2 full length gene sequence.
According to PCR primer 5 '-AACGGCGGTTCTTCTCTGA-3 ' and the 5 '-TGTAGTCGCATCAAGCTCTTTATT-3 ' of the sequences Design BjApextrin1 full length gene sequence that splicing obtains above; The PCR primer 5 of BjApextrin2 full length gene sequence '-CAAAGGAACGTCAGAACATTC-3 ' and 5 '-TTTAGAGTGAGCCAGTTTGT-3 ', adopt the full gene of the LA Taq polymeric enzymatic amplification BjApextrin1 of Takara, the 1578bp that amplification obtains respectively and 1433bp fragment, electrophoretogram is as Fig. 2.The object fragment that amplification is obtained transforms bacillus coli DH 5 alpha after being connected to pGEX Teasy vector, selects recombinant clone order-checking.Sequencing result analysis is shown, obtained BjApextrin1 and BjApextrin2 full length gene sequence.
Embodiment 4: the structure of restructuring pET32a-BjApextrin1 and pET32a-BjApextrin2 expression plasmid
According to the synthetic pair of primers of the sequence of BjApextrin1 gene, upstream primer is containing BamH I cleavage site, and downstream primer is containing Xho I cleavage site.
Upstream primer (FL-F1):
5'-CGC
GGATCCGCACCGACCGAAGTCGTACA-3'
BamH?I
Downstream primer (FL-R1):
5'-CCG
CTCGAGCGAATAGTAACAGTAGTG?GAGTCT-3'
Xho?I
According to the synthetic pair of primers of the sequence of BjApextrin2 gene, upstream primer is containing BamHI cleavage site, and downstream primer is containing Xho I cleavage site.
Upstream primer (FL-F2):
5'-GGA
AGATCTATGAAATTCGCGCTGCTCG-3'
Bgl?II
Downstream primer (FL-R2):
5'-CCG
CTCGAGAGAGTAGTAGCAGTAGTGAATCCG-3'
Xho?I
The pGEX Teasy plasmid that contains BjApextrin1 and BjApextrin2 gene of take is respectively template, and FL-F1/FL-R1 and FL-F2/FL-R2 are primer PCR amplification, obtains the single band of specific amplified, and product size is in 1500 left and right.Pcr amplification product is cloned into prokaryotic fusion expression vector pET32a above, obtains its building process of recombinant expression vector pET32a-BjApextrin1 and pET32a-BjApextrin2(as shown in Figure 3).Exogenous gene sequence in expression vector is identified correct through order-checking.
Embodiment 4: expression and the purifying of Chinese Amphioxus BjApextrin1 and BjApextrin2 fusion rotein
By pET32a-BjApextrin1 and pET32a-BjApextrin2 plasmid transformation escherichia coli BL21(DE3).Genetic engineering bacterium ultrasonic degradation supernatant liquor and precipitation show through SDS-PAGE electrophoretic analysis, and bacterial strain has obvious specifically expressing product band after induction in ultrasonic degradation precipitation, molecular weight conform to the theoretical value of prediction (Fig. 4).
To the conditions such as incubation time, induced concentration, temperature grope draw.The culture condition of genetic engineering bacterium is: order bacterium colony is in 50ml ammonia benzyl resistance 2 * YT liquid nutrient medium, 37 ℃, 250rpm overnight incubation, get in ammonia benzyl resistance 2 * YT substratum that overnight culture 5ml is inoculated in 500ml, 37 ℃, 250rpm is cultured to the about 4h of OD600=0.6(enlarged culturing), add 100mM IPTG to be respectively 0.1mM to final concentration, 18 ℃, centrifugal collection thalline after 250rpm inducing culture 24h.Through SDS-PAGE electrophoretic analysis, show, the expression amount of BjApextrin1 and BjApextrin2 fusion rotein accounts for the more than 20% of bacterial protein with this understanding, in insoluble inclusion body state.By the ultrasonic degradation of collecting TBS damping fluid for precipitation (50mM TrisCl, 150mM NaCl, pH7.5) resuspended after, the power with ultrasonic cell disruptor with 200W, the on ice ultrasonic 4s of ultrasonic 30min(, intermittently 4s), 4 ℃, 12, the centrifugal 30min of 000rpm.Repeat twice.To be precipitated according to 8M urea-TBS for 1g:50ml ratio and dissolve, by adding magnetic stirring apparatus to accelerate it, dissolve room temperature 12, the centrifugal lysate 10min of 000rpm.Pack the inclusion body supernatant liquor of dissolving into processed dialysis tubing, dialysis tubing is transferred to buffer A(50mM TrisCl, pH6.0, 150mM NaCl, 2mM reduced glutathione (GSH), 0.2mM oxidized form Triptide (GSSG), 10% glycerine, 4M urea), 4 ℃ of dialysis 12h, then dialysis tubing is transferred to buffer B(50mM TrisCl, pH6.0, 150mM NaCl, 2mM GSH, 0.2mM GSSG, 10% glycerine, 2M urea), 4 ℃ of dialysis 12h, again dialysis tubing is transferred to the TrisCl of buffer C(50mM, pH6.0, 150mM NaCl, 2mM GSH, 0.2mM GSSG, 10% glycerine, 1M urea), 4 ℃ of dialysis 12h, finally dialysis tubing is transferred to buffer D(50mM TrisCl, pH6.0, 150mM NaCl, 10% glycerine), 4 ℃ of dialysis 12h, change fresh dialysis buffer D, repeat previous step.Recombinant protein is at 4 ℃, and the centrifugal 15min of 12,000rpm, gets supernatant and carry out SDS-PAGE electrophoresis detection (Fig. 4).
Embodiment 5: the bacterium of Chinese Amphioxus BjApextrin1 and BjApextrin2 fusion rotein is in conjunction with activation analysis
The bacterium of using comprises: gram negative bacterium e. coli k12 (Escherichia coli K12), Vibrio anguillarum (Vibro anguillarum), Vibrio parahaemolyticus (Vibro paraheamolyticus) and Acinetobacter calcoaceticus (Acinetobacter caloacetius); Gram positive bacterium streptococcus aureus (Staphylococcus aureus), staphylococcus haemolyticus (Staphylococcus haemolyticus), middenstead coccus (Enterococcus faecium), subtilis (Bacillus subtilis) and micrococcus flavus (Micrococcus luteus).By approximately 2 * 10
7individual various bacterium TBS(pH7.5) after washing balance, with the BjApextrin1 of 1 μ g and BjApextrin2 fusion rotein at 1ml TBS(pH7.5) 4 ℃ of oscillation incubations spend the night in damping fluid; 4 ℃, the centrifugal 1min of 12000 * g, precipitation is washed 5 times with 1ml TBS damping fluid; With the resuspended thalline of 100 μ lTBS, then add 20 μ l6 * sample-loading buffers, after mixing, boil and boil 10min; Then carry out Western blot analysis, by His monoclonal antibody, carry out testing goal albumen, Sepharose4B bead carries out parallel laboratory test as negative control.BjApextrin1 and BjApextrin2 recombinant protein can be in conjunction with gram positive bacteriums as shown in Figure 5, and not in conjunction with gram negative bacterium.
Embodiment 6: the Bacterial agglutinative activity analysis of Chinese Amphioxus BjApextrin1 and BjApextrin2 recombinant protein
After enterococcus faecalis and streptococcus aureus shaking culture are spent the night, the centrifugal collection 2 * 10 of 6,000rpm
9various bacteriums after, with TBS(pH7.5) clean 2 times.Add after 50 μ l FITC solution (be dissolved in DMSO, final concentration is 10mg/ml), with TBS, fill to cumulative volume 1ml, lucifuge is rocked 1h.With TBS, clean 7-8 all over colourless to solution, be finally suspended in the CaCl that 1ml contains 10mM
2tBS solution in standby.On 96 hole flat boards, carry out, every hole adds the various microbial suspensions (final concentration about 1 * 10 of 10 μ l
7the bacterium of CFU/ml or 1 * 10
6cFU/ml yeast), the target protein of 10 μ g, fills to 100 μ l with TBS, detects agglutination activity after mixing the standing 1～2h of rear lucifuge under fluorescent microscope.BjApextrin1 and BjApextrin2 recombinant protein can be in conjunction with enterococcus faecalis and streptococcus aureus generation aggegations as shown in Figure 6.
Embodiment 7: the bacteria cell wall pure component of Chinese Amphioxus BjApextrin1 and BjApextrin2 recombinant protein is in conjunction with activation analysis
By ELISA, test to detect recombinant protein active to the combination of bacteria cell wall pure component.Six kinds of bacteria cell wall pure components that use are respectively: derive from streptococcus aureus peptidoglycan (PGN (S.a)), derive from peptidoglycan (PGN (B.s)), zymosan (Zymosan), teichoic acid (the Lipoteichoic acid of Bacillus subtillis, LTA), lipopolysaccharides (Lipopolysaccharides, LPS) and mannosans (Mannan).Because PGN, Mannan and Zymosan are as insolubles, use ultrasonic wave hydrotropy after these 3 kinds of components being resuspended in carbonic acid buffer before experiment, centrifuging and taking supernatant carries out experiment below.
1) coated: with carbonic acid buffer (0.1M NaHCO3,2.5mM Na2CO3, pH9.6), to dissolve various cell wall polysaccharides component.Then in the reacting hole of each polystyrene microtiter plates, add 20 each components of μ g, supplement coated damping fluid to 100 μ l, hatch 3h(blank hole for 37 ℃ and do not add sugar component).
2) washing: discard solution in hole, with 200 μ l TBST(TBS, 0.05%Tween-20) wash 3 times each 3min.
3) sealing: add 4 ℃ of overnight incubation of 200 μ l sealing damping fluids (TBST, 5%BSA).Inferior daily TBST washing.
4) application of sample: add the recombinant protein of different concns in reacting hole, be placed in wet box and hatch 2h in 37 ℃.Then with TBST washing, with Trx (thioredoxin, TRX), do negative control.
5) enzyme-added mark primary antibodie: in each reacting hole, add the mouse His monoclonal antibody (1:5,000) with the dilution of sealing damping fluid that 100 μ l are fresh, be placed in wet box and hatch 1h at 37 ℃, washing.
6) add ELIAS secondary antibody: in each reacting hole, add the goat anti-mouse igg two anti-(1:20,000) of the HRP mark of sealing damping fluid dilution for 100 μ l, be placed in wet box and hatch 1h, washing in 37 ℃.
7) add substrate buffer solution (100 μ g/ml tetramethyl benzidines, 0.24%H
2o
2, 0.2M Na
2hPO
4, 0.1M citric acid, pH5.5) colour developing: in each reacting hole, add 50 μ l TMB nitrite ions, from adding first hole to start to clock.All add 37 ℃ of rear lucifuges and hatch 10min.
8) termination reaction: since 10min after the first hole timing, every hole adds 50 μ l stop buffer (2M H according to the order that adds substrate buffer solution
2sO
4).
9) result is judged: in microplate reader, in 450nm place, survey the light absorption value in each hole with blank hole after returning to zero.
As shown in Figure 7, recombinant protein TRX-BjApextrin1 and TRX-BjApextrin2 have significant combination active to the PGN in two kinds of sources, but not in conjunction with other cell-wall component.
Embodiment 8: the insoluble peptidoglycan of bacterium of Chinese Amphioxus BjApextrin1 and TRX-BjApextrin2 fusion rotein is in conjunction with activation analysis
By Pulldown, test to detect recombinant protein active to the combination of insoluble peptidoglycan (PGN).By 1 μ g target protein respectively with the PGN of two kinds of bacterial origins of 5,10,20,50 μ g at 1ml TBS(pH7.5) 4 ℃ of oscillation incubations spend the night in damping fluid; 4 ℃, the centrifugal 1min of 12000 * g, precipitation is washed 5 times with 1ml TBS damping fluid; By the resuspended precipitation of 100 μ l TBS, then add 20 μ l6 * sample-loading buffers, after mixing, boil and boil 10min; Then carry out Western blot analysis, by His monoclonal antibody, carry out testing goal albumen.As shown in Figure 8, recombinant protein TRX-BjApextrin1 and RX-BjApextrin2 have significant combination active to two kinds of PGN, and strengthen in conjunction with the active increase with peptidoglycan concentration.
Embodiment 9: expression and the purifying of Chinese Amphioxus BjApextrin1 deletion mutant fusion rotein
According to the consequence devised of the sequence of BjApextrin1 gene and homology comparison, build the primer of deletion mutant, as follows:
△C-R:
5'-CCG
CTCGAGCTGTTTGGT?AAACCGTTGGAGG-3'
BamH?I
△N-F：
5'-CGC
GGATCCCAAGGTGAAGT?TGCGGCTGT-3'
Xho?I
The pGEX Teasy plasmid that contains BjApextrin1 gene of take is template, with FL-F and △ C-R, is the segment △ C that primer PCR amplification obtains C end disappearance; With △ N-F and FL-R, be that primer PCR amplification obtains holding the segment △ N of N disappearance.Pcr amplification product is cloned into prokaryotic fusion expression vector pET32a above, obtains recombinant expression vector pET32a-△ C identical as shown in Figure 4 with its building process of pET32a-△ N(), recombinant expression vector is identified correct through order-checking.
By pET32a-△ C and pET32a-△ N plasmid transformation escherichia coli BL21(DE3).Genetic engineering bacterium ultrasonic degradation supernatant liquor and precipitation show through SDS-PAGE electrophoretic analysis, and bacterial strain has obvious specifically expressing product band after induction in ultrasonic degradation precipitation.The purification condition of recombinant protein and process are identical with embodiment 4, and insoluble inclusion body can obtain purer recombinant protein through becoming renaturation as shown in Figure 9.
Embodiment 10: the bacterial peptide glycan of Chinese Amphioxus BjApextrin1 deletion mutant fusion rotein is in conjunction with activation analysis
With ELISA method detect recombinant protein TRX-△ C and TRX-△ N to bacterial peptide glycan in conjunction with activity, method is identical with embodiment 7.As shown in figure 10, TRX-△ N has significant combination active to peptidoglycan, and TRX-△ C does not have.
Claims (4)
1.BjApextrin1 gene, its DNA sequence dna is as shown in sequence in sequence table 1.
2. by the BjApextrin1 albumen of BjApextrin1 genes encoding described in claim 1, its aminoacid sequence is as shown in sequence in sequence table 3.
3. the production method of BjApextrin1 albumen described in claim 2, carry out according to the following steps:
(1) BjApextrin1 gene clone claimed in claim 1 is arrived to coli expression carrier pET32a, obtain recombinant expression vector pET32a-BjApextrin1;
(2) pET32a-BjApextrin1 is transformed to coli strain BL21(DE3);
(3), to e. coli bl21 (DE3) liquid medium within 2 * YT transforming, cultivate that after 4 hours, to add final concentration be that the IPTG of 0.2mM spends the night 18 ℃ of inductions for 37 ℃;
(4) purifying of restructuring BjApextrin1 fusion rotein, specifically, by the centrifugal collection thalline of bacterium liquid, carries out ultrasonication, and centrifuging and taking precipitation becomes renaturation, obtains albumen.
4. the application of BjApextrin1 albumen claimed in claim 2 in the infectious disease medicament being caused by gram-positive microorganism as preparation treatment.
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