CN100999553A - IIR2 analogous protein and its coding gene and application - Google Patents
IIR2 analogous protein and its coding gene and application Download PDFInfo
- Publication number
- CN100999553A CN100999553A CN 200610144153 CN200610144153A CN100999553A CN 100999553 A CN100999553 A CN 100999553A CN 200610144153 CN200610144153 CN 200610144153 CN 200610144153 A CN200610144153 A CN 200610144153A CN 100999553 A CN100999553 A CN 100999553A
- Authority
- CN
- China
- Prior art keywords
- apmv
- simulated
- simulated albumin
- cdv
- ccv
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention discloses one kind of HR2 simulation protein, which is fusion protein obtained through connecting 2-5 HR2's of one or several of viruses APMV-1, APMV-2, CDV and CCV serially or cross serially. The APMV-1 HR2 has the amino acid residue sequence as shown in the SEQ ID No. 1 of the sequence list; the APMV-2 HR2 has the amino acid residue sequence as shown in the SEQ ID No. 2; the CDV HR2 has the amino acid residue sequence as shown in the SEQ ID No. 3; and the CCV HR2 has the amino acid residue sequence as shown in the SEQ ID No. 4. Experiment shows that the HR2 simulation protein of the present invention can inhibit the formation of one or several syncytia of APMV-1, APMV-2, CDV and CCV and has high effect of preventing and treating APMV-1, APMV-2, CDV and CCV infected viral diseases.
Description
Technical field
The present invention relates to have antiviral HR2 simulated albumin and encoding gene and the application that suppresses the son effect, particularly relate to the application in HR2 simulated albumin and encoding gene thereof and its one or more virus infection medicines in preparation control APMV-1, APMV-2, CDV and CCV.
Background technology
The paramyxovirus membrane glycoprotein mainly contains two kinds, i.e. hemagglutinin neuraminidase (HN) (or with merit albumen H, G) and fusion rotein (F), and wherein, F albumen is by its precursor forms (F
0) cracking produces the F that fusion-activity is arranged
1, F
2Albumen.Avian paramyxoviruses-2 (Avian paramyxovirus-2, APMV-2) (being also referred to as fowl parainfluenza virus-2) is the essential items for inspection of the used SPF chicken of preparation human biological products, this virus infects the SPF chicken separately and only causes slight respiratory symptom, but obviously strengthens the latter's pathogenic effects with other fowl respiratory tract disease cause of disease polyinfection meeting.(Avian paramyxovirus-1 is that (Newcastle disease virus NDV) (is also referred to as fowl parainfluenza virus-1) to Avian pneumo-encephalitis virus, is height contagiousness, the lethal virus of mainly encroaching on bird APMV-1) to avian paramyxoviruses-1.Canine distemper is that (Canine distemper virus CDV) infects the height contagious disease of a kind of dog cause, the illness complexity by canine distemper virus, the lethality rate height, be the first transmissible disease of harm dog class, it is reported that this virus also can make the national treasure giant panda cause a disease.(Canine coronavirus virus CCV) can make dog produce the gastro-enteritis that weight differs to canine coronavirus, is that current coronavirus cyst membrane surface has only spike protein (S) to supporting the bigger a kind of transmissible disease of dog industry hazardness, and it has fusion.APMV-1, APMV-2, CDV and CCV can make infected cells form polykaryocyte, i.e. synplasm.
(paramyxovirus is a F albumen for APMV-1, APMV-2, CDV and CCV fusion rotein, coronavirus is a S albumen) heptad repeat region territory (Heptad repeat, HR) HR1 and HR2 can mutually combine and form six aggressiveness coiled structure, this six aggressiveness structure distance between host cell membrane and the virus envelope that furthered, impelling virus envelope and host cell membrane to melt is a film, thereby realizes infecting host cell.The HR2 polypeptide is solvable, but can not self-polymerization, can not with other viral HR2 polypeptide polymerization.
Summary of the invention
The purpose of this invention is to provide a kind of HR2 simulated albumin (inhibitor) that one or more virus infections among APMV-1, APMV-2, CDV and the CCV is had prevention and therapeutic action.
For solving above technical problem, the present invention takes following technical scheme: a kind of HR2 simulated albumin is the fusion rotein that one or more viral 2-5 HR2 series connection among the CCV of APMV-1, APMV-2, CDV and the crown Viraceae of Paramyxoviridae or the series connection that intersects are obtained; The amino acid residue sequence of described APMV-1 HR2 is the SEQ ID NO:1 in the sequence table; The amino acid residue sequence of described APMV-2 HR2 is the SEQ ID NO:2 in the sequence table; The amino acid residue sequence of described CDV HR2 is the SEQ ID NO:3 in the sequence table; The amino acid residue sequence of described CCV HR2 is the SEQ ID NO:4 in the sequence table.
SEQ ID NO:1 in the sequence table is made up of 33 amino-acid residues, and its encoding sequence can be the SEQ ID NO:5 in the sequence table, by 99 based compositions; SEQ ID NO:2 in the sequence table is made up of 33 amino-acid residues, and its encoding sequence can be the SEQ ID NO:6 in the sequence table, by 99 based compositions; SEQ IDNO:3 in the sequence table is made up of 34 amino-acid residues, and its encoding sequence can be the SEQ ID NO:7 in the sequence table, by 102 based compositions; SEQ ID NO:4 in the sequence table is made up of 51 amino-acid residues, and its encoding sequence can be the SEQ ID NO:8 in the sequence table, by 153 based compositions.
Described with APMV-1, APMV-2, the bind mode of 2-5 HR2 in the HR2 simulated albumin that one or more viral 2-5 among the CCV of CDV and crown Viraceae HR2 series connection or the series connection that intersects obtain and be arbitrarily in proper order, it both can be the HR2 series connection of virus of the same race, it also can be the series connection (several HR2 of identical virus link) of above-mentioned 2-4 kind different virus HR2, and can also adopt the mode of intersection series connection (being linked with other viral HR2 between the HR2 of identical virus) between the HR2 of different virus, it is the aminoterminal that the HR2 of a certain virus can be in other viral HR2, can be in the carboxyl terminal of other viral HR2, the change of connection order does not have influence to activity yet.
The described HR2 simulated albumin that one or more viral 2-5 among the CCV of APMV-1, APMV-2, CDV and crown Viraceae HR2 series connection or the series connection that intersects are obtained is preferably: the fusion rotein that the HR2 of 2-5 APMV-2 is connected and obtains.
The molecular weight of albumen of the HR2 of APMV-1, APMV-2, CDV and CCV is between 3-6kDa, wherein, the molecular weight of albumen of APMV-1HR2 is 4.8kDa, and the molecular weight of albumen of APMV-2 HR2 is 4.1kDa, the molecular weight of albumen of CDV HR2 is 4.5kDa, and the molecular weight of albumen of CCV HR2 is 6.0kDa.The molecular weight of albumen of the above-mentioned HR2 simulated albumin that one or more viral 2-5 among the CCV of APMV-1, APMV-2, CDV and crown Viraceae HR2 series connection or the series connection that intersects are obtained should be controlled between the 8-30kDa, wherein, molecular weight is that the HR2 simulated albumin of 8-20kDa demonstrates stronger antiviral activity.
The gene of above-mentioned HR2 simulated albumin of encoding also belongs to protection scope of the present invention.
Expression carrier, transgenic cell line and the host bacterium that contains described HR2 simulated albumin also is that the present invention will protect.
Second purpose of the present invention provides a kind of expression method of above-mentioned HR2 simulated albumin.
The expression method of HR2 simulated albumin provided by the present invention is to make up the recombinant expression vector that contains HR2 simulated albumin gene, and the recombinant expression vector that makes up is imported host cell, cultivates host cell and makes the genetic expression of HR2 simulated albumin.
The carrier that sets out that is used to make up described recombinant expression vector can be at expression in escherichia coli expression of exogenous gene carrier, as pGEX-6p-I, pET-28a, pET-28b, pET-28c, pET-21a (+) or pET-30a etc., is preferably pGEX-6p-I.
Be the carrier that sets out with pGEX-6p-I, the recombinant expression vector that contains HR2 simulated albumin gene of structure is pGEX-(APMV2-HR2-HR2-HR2) or pGEX-(CCVHR2-APMV1HR2-APMV2HR2-CDVHR2).
Described host can be intestinal bacteria, yeast, mammalian cell, insect cell or Bacillus subtilus etc., is preferably intestinal bacteria.
Described intestinal bacteria can be E.coli BL21 (DE3), E.coli BL21 (DE3) plys, E.coli BLR (DE3) or E.coli B834 etc.
Above-mentioned recombinant expression vector and reorganization bacterium all can make up according to ordinary method.
Cultivation contains the substratum and the culture condition of the host cell of HR2 simulated albumin gene of the present invention, all can be substratum and the culture condition of cultivating the host that sets out.Wherein, need add inductor when cultivating described recombination bacillus coli host, as IPTG etc., add IPTG concentration be 0.8-1.2mmol/L, be preferably 1mmol/L, inducing temperature is 30-39 ℃, is preferably 30 ℃, induction time is 2-4 hour, is preferably 4 hours.
The present invention also provides the medicine of one or more virus infections among a kind of APMV-1 of control, APMV-2, CDV and the CCV.
The activeconstituents of described medicine is above-mentioned HR2 simulated albumin.
When needing, in said medicine, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises thinner, absorption enhancer or the tensio-active agent etc. of pharmaceutical field routine.
This medicine can be made acceptable drug form on any technology of pharmaceutics such as capsule, tablet, granule, pulvis, injection liquid.
The consumption of said medicine is generally 10-50 μ g/kg body weight/day, and be 7-20 days the course of treatment.
For improving curative effect, medicine of the present invention can also carry out combined therapy with microbiotic, immunostimulant etc.
The invention provides a kind of HR2 simulated albumin.This albumen is with one or more the viral HR2 series connection among APMV-1, APMV-2, CDV and the CCV.Experimental results show that, HR2 simulated albumin of the present invention can become six aggressiveness coiled structure in conjunction with its part with the HR1 that suppresses virus self by competition with the HR2 allosteric, then check the viromembrane fusion process, thereby can suppress one or more viral plasmodial formation among APMV-1, APMV-2, CDV and the CCV, one or more virus infections among APMV-1, APMV-2, CDV and the CCV are had prevention and therapeutic action preferably.More single HR2, the present invention increases through the molecular weight of the HR2 of series connection or modification, and the increasing of space and volume can prolong the proteic transformation period, be difficult for degraded, and artificial constructed simulated albumin also may reduce the immunogenicity and the toxic side effect of protein drug.In addition, the preparation method of HR2 simulated albumin of the present invention is simple and easy to do, has the feasibility of suitability for industrialized production.The present invention not only can be used for the usually control of several virus mixed infection diseases of APMV-1, APMV-2, CDV and CCV of appearance in aquaculture and the raising pets, development to other disease of viral infection medicine simultaneously also has certain reference, has important application prospects and innovative significance in the animal medicine field.
Below in conjunction with specific embodiment the present invention is described in further details.
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, and concrete steps can be referring to " Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, David W., Molecular Cloning:A Laboratory Manual, 3
RdEdition, 2001, NY, Cold SpringHarbor).It is synthetic that the primer and dna sequence dna are given birth to the worker by Shanghai.Described percentage concentration is mass/volume percentage concentration or volume/volume percentage concentration.Used DMEM nutritive medium all contains 2% foetal calf serum and 1% pair of anti-(penicillin and Streptomycin sulphate 1: 1 are purchased glad through company of section in Beijing) and 1% glutamine.
The acquisition of the HR2 simulated albumin of embodiment 1, APMV-2 and prevention and cure of viruses effect detection thereof
One, the acquisition of 3 placed in-line HR2 simulated albumins of APMV-2 HR2
With 3 placed in-line HR2 simulated albumins of APMV-2 HR2 of following method preparation, may further comprise the steps:
1, the clone and the construction of prokaryotic expression vector thereof of 3 the placed in-line HR2 simulated albumin of APMV-2 HR2 genes
1) design primer
With the proteic aminoacid sequence of avian paramyxoviruses-2 (APMV-2) F (GenBank Accession No.D13977) input LearnCoil-VMF software (http://nightingale.lcs.mit.edu/cgi-bin/vmf), with the proteic HR2 zone location of APMV-2F from aminoterminal 442-474 amino acids sequence, i.e. SEQ ID NO:2 in the sequence table.Gene order (the SEQ ID NO:6 in the sequence table) according to coding APMV-2 HR2 designs primer again, wherein, upstream primer contains restriction enzyme BamH I recognition site, downstream primer contains Xho I recognition site, and establish terminator codon before the Xho I recognition site of downstream primer, primer sequence is as follows:
Upstream primer the P1:5 '-gtc in amplification APMV-2 HR2 zone
GgatccTcgctggatataagcagt-3 ' (band underscore base is a restriction enzyme BamH I recognition site)
Downstream primer the P2:5 '-ta in amplification APMV-2 HR2 zone
TctcgagTcagttcacagcgttcagcca-3 ' (band underscore base is a restriction enzyme Xho I recognition site).
2) amplification of APMV-2 HR2 gene
CDNA (GenBank Accession No.D13977) with APMV-2 F protein gene is a template, pcr amplification APMV-2 HR2 gene under the guiding of primer P1 and P2, and the PCR reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 1min then, 55 ℃ of 1min, 72 ℃ of 1min, totally 28 circulations; Last 72 ℃ of 10min.After reaction finishes, pcr amplification product is carried out 10% agarose gel electrophoresis to be detected, the result has obtained size and has been the dna fragmentation of 99bp, conform to expected results, reclaim and this purpose fragment of purifying, with its with behind restriction enzyme BamH I and the XhoI double digestion be connected through the carrier pGEX-6p-I of same enzyme double digestion (available from east scientific ﹠ technical corporation), 16 ℃ of connections are spent the night after (12-24 hour), to connect product transformed into escherichia coli JM109 competent cell, random choose transformed bacteria list colony inoculation is to 5mL LB liquid nutrient medium, 37 ℃ of following overnight incubation, extract plasmid, and carry out PCR as template with primer P1 and P2 and identify, can pcr amplification going out size is candidate's positive colony for the plasmid of the dna fragmentation of 99bp, gained candidate positive plasmid is carried out dna sequencing, and the sequencing result shows and has obtained the correct APMV-2 HR2 gene of sequence.
3) it is as follows to contain 8 primer sequences of acquisition of 3 the placed in-line HR2 simulated albumin of APMV-2 HR2 Prokaryotic Expression carriers:
Pa:5 '-ga
GggatccAacctcaacatctcaactgagcttggtaatgtcaacaacagcatcagc-3 ' (band underscore base is a restriction enzyme BamH I recognition site)
Pb：5’-gcaacagcaagcttgacaaggtcaacaacctcaacatctcaactgaacttggg-3’
Pc：5’-cagcaatgctctcgataagttggaggagagcaacagcaagctggacaaggtcaac-3’
Pd：5’-tcaactgaacttgggaacgtcaacaactcgatcagcaacgctctcgataagttggaggaa-3’
Pe：5’-caagcttgctgttgctttcctcgagcttgtccagagcgttgctgatgctgttgttg-3’
Pf：5’-atcgagagcattgctgatcgagttgttgacgttcccaagttcagttgag-3’
Pg：5’-cccaagttcagttgagatgttgaggttgttgaccttgtccag-3’
Ph:5 '-cgc
CtcgagTcagttgaccttgtcgagcttgctattgctttcctccaacttatc-3 (band underscore base is a restriction enzyme Xho I recognition site).
3 placed in-line HR2 simulated albumins of APMV-2 HR2 of method pcr amplification gene with bridging PCR, first round PCR reaction conditions is: 8 primers of design are dissolved in respectively in the sterilization deionized water with the concentration of 50pmol/ μ l, respectively get 4 μ l (primer is made template mutually) during use.The PCR program is: 94 ℃ of 5min; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, 28 circulations; 72 ℃ of 10min.In first round PCR product, add the 3M NaAC of 1/10 volume, mix with the ice-cold ethanol of 3 times of cumulative volumes again ,-20 ℃ of 30min, the centrifugal 10min of 12000r/min uses 75% washing with alcohol again, is dissolved in after the seasoning in the sterilization deionized water of 10-20 μ l.Getting 1-2 μ l and take turns the PCR reaction template as second, is that the upstream and downstream primer carries out second and takes turns pcr amplification with primer Pa and primer Ph, and the PCR program is: 94 ℃ of 5min; 94 ℃ of 1min, 55 ℃ of 1.5min, 72 ℃ of 1.5min, 28 circulations; 72 ℃ of 5min.
After reaction finishes, pcr amplification product is carried out 10% agarose gel electrophoresis to be detected, reclaim the also purpose fragment of purifying 300bp, it is cloned among the carrier pGEX-6p-I, obtain containing 3 the placed in-line HR2 simulated albumin of APMV-2 HR2 Prokaryotic Expression carriers, with its called after pGEX-(APMV-2-HR2-HR2-HR2).
2, the expression and the purifying thereof of 3 placed in-line HR2 simulated albumins of APMV-2 HR2
Contain 3 the placed in-line HR2 simulated albumin of APMV-2 HR2 Prokaryotic Expression carrier pGEX-(APMV-2-HR2-HR2-HR2) transformed into escherichia coli BL21 (DE3) competent cell with what step 1 made up, the picking mono-clonal is inoculated in LB liquid nutrient medium (the Tryptones 10g that contains the 100mg/L penbritin, yeast extract 5g, sodium-chlor 10g, water 1000mL) in, 37 ℃ of following shake overnight incubation as seed liquor, again seed liquor is inoculated in the fresh LB liquid nutrient medium in 1: 100 ratio, is cultured to A at 37 ℃ of following shakes
590Be 0.8-1.0, add inductor IPTG, continue to cultivate 4h at 30 ℃ to final concentration 1mmol/L.After cultivating end, the centrifugal 15min of 5000r/min collects thalline, adds an amount of PBS damping fluid (140mmol/L NaCl, 2.7mmol/L KCl, 10mmol/L Na
2HPO
4, 1.8mmol/L KH
2PO
4PH7.3) resuspended thalline, the ultrasonic treatment thalline, the adding final concentration is 1% TritonX-100, ice bath 30min, at 4 ℃, centrifugal 15min under the 12000rpm, get supernatant, supernatant is passed through through PBS equilibrated Glutathione-Sepharose 4B affinity column (available from east scientific ﹠ technical corporation), again with at least 10 column volumes of PBS washing affinity column, then use reduced glutathion solution (the 10mmol/L reduced glutathione of at least 3 column volumes, 50mmol/L Tri sHCl, pH8.0) wash-out, collect elutriant and obtain gst fusion protein, be after the proteic ultrafiltration pipe of 5K concentrates with the molecular mass of damming then, change enzyme cutting buffering liquid (50mmol/L TrisHCl (pH7.0) into, 150mmol/L NaCl, 1mmol/L DTT, 1mmol/LEDTA, pH8.0), add 4 ℃ of enzymes of excessive GST-3C proteolytic enzyme (available from east scientific ﹠ technical corporation) and cut 16h, change the PBS damping fluid again into after concentrating, remove GST and the GST-3C that enzyme downcuts with Glutathione-Sepharose 4B affinity column, method is: add in right amount through the affine filler of PBS equilibrated Glutathione-Sepharose 4B, the light shaking mixing, middle constantly vibration is to prevent the filler precipitation, behind the about 1h of room temperature effect (or long-time slightly), the centrifugal 15min of 500rpm, the gentle aspiration supernatant, promptly obtain the target protein (3 placed in-line HR2 simulated albumins of APMV-2 HR2) of purifying, be concentrated into concentration with 3K ultrafiltration pipe and be about 3mg/mL, the target protein of purifying is carried out 15%SDS-polyacrylamide gel electrophoresis (SDS-PAGE) detect, the result has obtained the target protein that the higher molecular weight of purity is about 12KD.With target protein-80 ℃ frozen standby (can be placed on 4 ℃ of refrigerators preservations in the ice chest) if use in several days.
Two, form the prevention and cure of viruses effect that detects 3 placed in-line HR2 simulated albumins of APMV-2 HR2 with synplasm formation and inhibition experiment with the APMV-2 synplasm
1, the preparation of Hela cell
To put into 37-38 ℃ warm water fast after the Hela cell taking-up frozen in the liquid nitrogen; do not stop to rock; after treating that frozen liquid in pipe melts fully; the centrifugal 10min of 1000rpm; precipitation suspends with the DMEM nutritive medium that contains the 10-20% foetal calf serum, the piping and druming mixing, and horizontal is cultivated in 37 ℃, 5%CO2 incubator; be changed to the DMEM nutritive medium that contains 10% foetal calf serum behind the 12h again, obtain individual layer Hela cell at last.
2, the cytotoxic propagation of APMV-2 Hela
The individual layer Hela cell of cultivating behind the 24h is cleaned 2-3 time with PBS, with tiring of ratio inoculation step 2 acquisitions of 1mL APMV-2 virus liquid/10mL DMEM nutritive medium 2
4Above fresh APMV-2 chick embryo fibroblast poison, 37 ℃, CO
2Discard supernatant after cultivating 1.5h in the incubator, add the DMEM nutritive medium (promptly keeping liquid) that contains 1% foetal calf serum, at 37 ℃, 5%CO
2Cultivated 5-6 days in the incubator, receive poison behind the multigelation 3 times, and survey hemagglutinative titer, method is: get 96 hole blood-coagulation-boards, every hole adds the physiological saline of 25 μ l, and the 1st hole adds APMV-2 Hela cell toxicant, get 25 μ l after surplus the mixing 10 time and add the 2nd hole, so doubling dilution to the 11 holes are drawn 25 μ l and are abandoned behind the 11st hole mixing, and the 12nd hole is stayed and done negative control.Then, every hole adds the physiological saline of 25 μ l and the red corpuscle of 25 μ l 1% again, the vibration mixing, and room temperature leaves standstill 20-30min, observations.Complete agglutinative virus greatest dilution to occur as the cytotoxic hemagglutinative titer of APMV-2 Hela.The result tires and reaches 2
4More than.Need repeatedly to breed if the hemagglutinative titer that records is very low, so that hemagglutinative titer reaches 2
4More than.
3, the APMV-2 synplasm forms experiment
To after the Hela cell that grows into individual layer on 48 orifice plates is with PBS washing 3 times, add serial dilution (1: 10
1, 1: 10
2, 1: 10
3, 1: 10
4, 1: 10
5, 1: 10
6, 1: 10
7, 1: 1
0) APMV-2 virus liquid, inoculum size is about 50 μ l/ holes, each extent of dilution is done 3 holes, establishes the DMEM nutritive medium and is contrast.37 ℃, CO
2Incubation 1.5h inhales and abandons viral liquid, adds the DMEM nutritive medium that original volume (0.3ml) contains 1%FCS, after continuing to cultivate 48h, with 0.5% violet staining 3-5min, washing back microscopy, every orifice plate are got five visuals field and are calculated the mean value of cell confluency and calculate TCID
50Cell confluency accounts for the ratio of whole cells for the synplasm sick cell.Calculate TCID according to Karber formula in " animal virology "
50, formula is LgTCID
50=L-d (S-0.5).Wherein, (high dilution is 1: 10 to L in this experiment for the logarithm of the highest viral dilution degree
1), d is dilution poor between the logarithm, S accounts for the summation of total hole count ratio for the positive hole of each extent of dilution.The mean value of cell confluency is 24% as a result, TCID
50Be 10
5
4, the APMV-2 synplasm forms and suppresses experiment
With the APMV-2 virus stock solution used with TCID
5010 times of dilution numerical value be seeded in the Hela cell that grows into individual layer on 48 orifice plates, 50 μ l/ holes add the placed in-line HR2 simulated albumin of 3 APMV-2 HR2 (50 μ l/ hole) that the step 1 of doubling dilution obtains simultaneously respectively, 37 ℃, CO
2Incubation 1.5h inhales and abandons virus and proteic mixed solution, adds the DMEM nutritive medium that 0.3mL contains 1% foetal calf serum, behind the continuation cultivation 48h (wherein changing one time of nutrition liquid), with 0.5% violet staining 3-5min, washing back microscopy, every orifice plate is got five visuals field and is calculated IC
50(half-inhibition concentration).IC as a result
50Be 4 μ M, plasmodial formation has significant inhibitory effect to APMV-2 to show the placed in-line HR2 simulated albumin of 3 APMV-2 HR2 of the present invention, can be used for preventing and treating the disease of APMV-2 virus infection.
The acquisition of the HR2 simulated albumin of embodiment 2, APMV-1 and APMV-2 and prevention and cure of viruses effect detection thereof
One, the acquisition of 1 APMV-1 HR2 and 1 placed in-line HR2 simulated albumin of APMV-2 HR2
With following method 1 APMV-1 HR2 of preparation and 1 placed in-line HR2 simulated albumin of APMV-2 HR2, may further comprise the steps:
1, the structure that contains 1 APMV-1 HR2 and 1 the placed in-line HR2 simulated albumin of APMV-2 HR2 Prokaryotic Expression carrier
1) primer of design amplification APMV-1 HR2 gene
With the proteic aminoacid sequence of avian paramyxoviruses-1 (APMV-1) F (GenBank Accession No.AF079172) input LearnCoil-VMF software (http://nightingale.lcs.mit.edu/cgi-bin/vmf), with the proteic HR2 zone location of APMV-1 F from aminoterminal 359-391 amino acids sequence, i.e. SEQ ID NO:1 in the sequence table.Gene order (the SEQ ID NO:5 in the sequence table) according to coding APMV-1 HR2 designs primer again, wherein, upstream primer contains restriction enzyme BamH I recognition site, downstream primer contains Xho I recognition site, and establish terminator codon before the Xho I recognition site of downstream primer, primer sequence is as follows:
Upstream primer the P3:5 '-gtc in amplification APMV-1 HR2 zone
GgatccAatctcaatatctcaact-3 ' (band underscore base is a restriction enzyme BamH I recognition site)
Downstream primer the P4:5 '-cagc in amplification APMV-1 HR2 zone
CtcgagTcaattgactttgtcaagttt-3 ' (band underscore base is a restriction enzyme Xho I recognition site).
2) amplification of APMV-1 HR2 gene
CDNA (GenBank Accession No.AF079172) with APMV-1 F protein gene is a template, pcr amplification APMV-1 HR2 gene under the guiding of primer P3 and P4, and the PCR reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 1min then, 55 ℃ of 1min, 72 ℃ of 1min, totally 28 circulations; Last 72 ℃ of 10min.After reaction finishes, pcr amplification product is carried out 10% agarose gel electrophoresis to be detected, the result has obtained size and has been the dna fragmentation of 99bp, conform to expected results, reclaim and this purpose fragment of purifying, with its with behind restriction enzyme BamH I and the Xho I double digestion be connected through the carrier pGEX-6p-I of same enzyme double digestion (available from east scientific ﹠ technical corporation), 16 ℃ of connections are spent the night after (12-24 hour), to connect product transformed into escherichia coli JM109 competent cell, random choose transformed bacteria list colony inoculation is to 5mL LB liquid nutrient medium, 37 ℃ of following overnight incubation, extract plasmid, and carry out PCR as template with primer P3 and P4 and identify, can pcr amplification going out size is candidate's positive colony for the plasmid of the dna fragmentation of 99bp, gained candidate positive plasmid is carried out dna sequencing, and the sequencing result shows and has obtained the correct APMV-1 HR2 gene of sequence.
3) contain the structure of 1 APMV-1 HR2 and 1 the placed in-line HR2 simulated albumin of APMV-2 HR2 Prokaryotic Expression carrier
With APMV-1 HR2 gene and gene constructed 1 APMV-1 HR2 and 1 the placed in-line HR2 simulated albumin of the APMV-2 HR2 Prokaryotic Expression carrier of containing of APMV-2 HR2, concrete grammar is: with APMV-1 HR2 and APMV-2 HR2 gene is template, with the upstream primer P3 of amplification APMV-1 HR2 gene and the downstream primer P2 of amplification APMV-2 HR2 gene is primer, 1 APMV-1 HR2 of pcr amplification and 1 the placed in-line HR2 simulated albumin of APMV-2 HR2 gene.After reaction finishes, pcr amplification product is carried out 10% agarose gel electrophoresis to be detected, reclaim the also purpose fragment of purifying 198bp, it is cloned among the carrier pGEX-6p-I, obtain containing 1 APMV-1 HR2 and 1 the placed in-line HR2 simulated albumin of APMV-2 HR2 Prokaryotic Expression carrier, with its called after pGEX-(APMV1-HR2-APMV2-HR2).
2, the expression and the purifying thereof of 1 APMV-1 HR2 and 1 placed in-line HR2 simulated albumin of APMV-2 HR2
Contain 1 APMV-1 HR2 and 1 the placed in-line HR2 simulated albumin of APMV-2 HR2 Prokaryotic Expression carrier pGEX-(APMV1-HR2-APMV2-HR2) transformed into escherichia coli BL21 (DE3) competent cell with what step 1 made up, use then with embodiment 1 step 1 in identical method screening positive recombinant, it is carried out inducing culture with IPTG, and the purified target protein (1 APMV-1 HR2 and 1 placed in-line HR2 simulated albumin of APMV-2 HR2) that obtains purifying, be concentrated into concentration with 3K ultrafiltration pipe and be about 3mg/mL, the albumen of purifying is carried out 15%SDS-polyacrylamide gel electrophoresis (SDS-PAGE) detect, the result has obtained the target protein that the higher molecular weight of purity is about 9KD.Target protein-80 is ℃ frozen standby.
Two, form the prevention and cure of viruses effect that suppresses experiment 1 APMV-1 HR2 of detection and 1 placed in-line HR2 simulated albumin of APMV-2HR2 with APMV-1 and APMV-2 synplasm
With with embodiment 1 step 2 in identical method detect 1 APMV-1 HR2 and the inhibition effect of 1 placed in-line HR2 simulated albumin of APMV-2 HR2 of step 1 preparation to APMV-1 and the formation of APMV-2 synplasm.Concrete grammar is: with the hybrid virus stoste of APMV-1, APMV-2 or APMV-1 and APMV-2 respectively with TCID
5010 times of dilution numerical value inoculate the Hela cell that grows into individual layer on 48 orifice plates, 50 μ l/ holes add 1 APMV-1 HR2 and 1 placed in-line HR2 simulated albumin of APMV-2 HR2 (50 μ l/ hole) of doubling dilution simultaneously respectively, 37 ℃, CO
2Incubation 1h, virus and proteic mixed solution are abandoned in suction, add the DMEM nutritive medium that 0.3mL contains 2% foetal calf serum, continue to cultivate behind the 14h (or 48h) with 0.5% violet staining 3-5min, washing back microscopy, every orifice plate is got five visuals field and is observed the cytogamy situation.When the placed in-line HR2 simulated albumin of 1 the APMV-1 HR2 that does not add doubling dilution and 1 APMV-2 HR2, the TCID of the individual layer Hela cell of the infection APMV-1 of APMV-1 experimental group
50Be 10
7, the TCID of the individual layer Hela cell of the infection APMV-2 of APMV-2 experimental group
50Be 10
5After adding 1 APMV-1 HR2 and 1 placed in-line HR2 simulated albumin of APMV-2 HR2 of doubling dilution, cell confluency is obvious reduction trend, show that 1 APMV-1 HR2 of the present invention and 1 placed in-line HR2 simulated albumin of APMV-2 HR2 all have significant inhibitory effect to APMV-1 and the plasmodial formation of APMV-2, can be used for preventing and treating the disease of APMV-1 and/or APMV-2 virus infection.
Sequence table
<160>8
<210>1
<211>33
<212>PRT
＜213〉avian paramyxoviruses 1 (APMV-1)
<400>1
Asn?Leu?Asn?Ile?Ser?Thr?Glu?Leu?Gly?Asn?Val?Asn?Asn?Ser?Ile?Ser
1 5 10 15
Asn?Ala?Leu?Asp?Lys?Leu?Glu?Glu?Ser?Asn?Ser?Lys?Leu?Asp?Lys?Val
20 25 30
Asn
<210>2
<211>33
<212>PRT
＜21S〉avian paramyxoviruses-2 (APMV-2)
<400>2
Ser?Leu?Asp?Ile?Ser?Ser?Glu?Ile?Gly?Ser?Ile?Asn?Asn?Thr?Val?Asn
1 5 10 15
Arg?Val?Asp?Glu?Leu?Ile?Lys?Glu?Ser?Asn?Glu?Trp?Leu?Asn?Ala?Val
20 25 30
Asn
<210>3
<211>34
<212>PRT
＜213〉canine distemper virus (CDV)
<400>3
Leu?Asp?Val?Gly?Thr?Asn?Leu?Gly?Asn?Ala?Leu?Lys?Lys?Leu?Asp?Asp
1 5 10 15
Ala?Lys?Val?Leu?Ile?Asp?Ser?Ser?Asn?GIn?Ile?Leu?Glu?Thr?Val?Arg
20 25 30
Arg?Ser
<210>4
<211>51
<212>PRT
＜213〉canine coronavirus (CCV)
<400>4
Leu?Pro?Leu?Asp?Ile?Phe?Asn?Ala?Thr?Tyr?Leu?Asn?Leu?Thr?Gly?Glu
1 5 10 15
Ile?Asn?Asp?Leu?Glu?Phe?Arg?Ser?Glu?Lys?Leu?His?Asn?Thr?Thr?Val
20 25 30
Glu?Leu?Ala?Ile?Leu?Ile?Asp?Asn?Ile?Asn?Asn?Thr?Leu?Val?Asn?Leu
35 40 45
Glu?Trp?Leu
50
<210>5
<211>99
<212>DNA
＜213〉avian paramyxoviruses 1 (APMV-1)
<400>5
aatctcaata?tctcaactga?acttgggaat?gtcaacaact?cgataagtaa?tgctttggat 60
aagttagagg?aaagcaatag?caaacttgac?aaagtcaat 99
<210>6
<211>99
<212>DNA
＜213〉avian paramyxoviruses-2 (APMV-2)
<400>6
tcgctggata?taagcagtga?aattggtagc?attaacaaca?cagttaatcg?ggtcgacgag 60
ttaatcaagg?aatccaacga?gtggctgaac?gctgtgaac 99
<210>7
<211>102
<212>DNA
＜213〉canine distemper virus (CDV)
<400>7
ttggacgtag?gtacgaactt?aggcaacgcc?ctgaagaagc?tggacgacgc?taaggtactg 60
atcgacagca?gcaaccagat?ccttgagacg?gttcgccagt?ct 102
<210>8
<211>153
<212>DNA
＜213〉canine coronavirus (CCV)
<400>8
ctgccacttg?acattttcaa?tgcaacgtac?ctgaacctga?cgggtgaaat?caacgacctg 60
gaattccgca?gcgaaaagct?gcataacacg?acggtggagc?tggctatcct?gatcgacaac 120
atcaataaca?cgctggtcaa?ccttgagttg?ctc 153
Claims (9)
1, a kind of HR2 simulated albumin is with one or more the viral 2-5 among APMV-1, APMV-2, CDV and the CCV the HR2 series connection or the fusion rotein of connecting and obtaining that intersects; The amino acid residue sequence of described APMV-1 HR2 is the SEQ ID NO:1 in the sequence table; The amino acid residue sequence of described APMV-2 HR2 is the SEQ IDNO:2 in the sequence table: the amino acid residue sequence of described CDV HR2 is the SEQ ID NO:3 in the sequence table; The amino acid residue sequence of described CCV HR2 is the SEQ ID NO:4 in the sequence table.
2, HR2 simulated albumin according to claim 1 is characterized in that: described HR2 simulated albumin is the fusion rotein that the HR2 of 2-5 APMV-1 series connection is obtained.
3, HR2 simulated albumin according to claim 1 and 2 is characterized in that: the molecular weight of albumen of the described HR2 simulated albumin that one or more viral 2-5 among the CCV of APMV-1, APMV-2, CDV and crown Viraceae HR2 series connection or the series connection that intersects are obtained is 8-30kDa.
4, the gene of coding claim 1 or 2 or 3 described HR2 simulated albumins.
5, contain the described HR2 simulated albumin of claim 4 expression carrier, transgenic cell line and host bacterium.
6, a kind of expression method of HR2 simulated albumin, be to make up the recombinant expression vector that contains the described HR2 simulated albumin of claim 4 gene, the recombinant expression vector that makes up is imported host cell, cultivate host cell and make the genetic expression of HR2 simulated albumin, obtain the HR2 simulated albumin.
7, expression method according to claim 6 is characterized in that: the carrier that sets out that is used to make up described recombinant expression vector is pGEX-6p-I, pET-28a, pET-28b, pET-28c, pET-21a (+) or pET-30a; With pGEX-6p-I is that the set out recombinant expression vector of containing of vector construction described HR2 simulated albumin gene is pGEX-(APMV-2-HR2-HR2-HR2) or pGEX-(CCVHR2-APMV1HR2-APMV2HR2-CDVHR2); Described host is E.coli BL21 (DE3), E.coli BL21 (DE3) plys, E.coli BLR (DE3) or E.coli B834.
8, according to claim 6 or 7 described expression methods, it is characterized in that: need add the IPTG inductor when cultivating described recombination bacillus coli host, add IPTG concentration be 0.8-1.2mmol/L, inducing temperature is 30-39 ℃, induction time is 2-4 hour.
9, the application in claim 1 or 2 or 3 described HR2 simulated albumins one or more virus infection medicines in preparation control APMV-1, APMV-2, CDV and CCV.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2006101441539A CN100999553B (en) | 2006-11-28 | 2006-11-28 | HR2 analogous protein and its coding gene and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2006101441539A CN100999553B (en) | 2006-11-28 | 2006-11-28 | HR2 analogous protein and its coding gene and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN100999553A true CN100999553A (en) | 2007-07-18 |
| CN100999553B CN100999553B (en) | 2010-11-03 |
Family
ID=38258386
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2006101441539A Expired - Fee Related CN100999553B (en) | 2006-11-28 | 2006-11-28 | HR2 analogous protein and its coding gene and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100999553B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013075660A1 (en) * | 2011-11-25 | 2013-05-30 | 畿晋庆三联（北京）生物技术有限公司 | Method for highly expressing recombinant protein of engineering bacteria and use thereof |
-
2006
- 2006-11-28 CN CN2006101441539A patent/CN100999553B/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013075660A1 (en) * | 2011-11-25 | 2013-05-30 | 畿晋庆三联（北京）生物技术有限公司 | Method for highly expressing recombinant protein of engineering bacteria and use thereof |
| CN103131724A (en) * | 2011-11-25 | 2013-06-05 | 畿晋庆三联（北京）生物技术有限公司 | Efficient engineering bacteria recombinant protein expression method and applications thereof |
| CN103131724B (en) * | 2011-11-25 | 2015-02-25 | 畿晋庆三联（北京）生物技术有限公司 | Efficient engineering bacteria recombinant protein expression method and applications thereof |
| EA025689B1 (en) * | 2011-11-25 | 2017-01-30 | Протеин Дизайн Лаб, Лтд. | Method for highly expressing recombinant protein of engineering bacteria and use thereof |
| US9611299B2 (en) | 2011-11-25 | 2017-04-04 | Protein Design Lab, Ltd. | Method for highly expressing recombinant protein of engineering bacteria and use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100999553B (en) | 2010-11-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2223934B1 (en) | Polypeptides for inhibiting influenza virus infection | |
| TWI380993B (en) | Uses of recombinant super-compound interferons | |
| CN102883736B (en) | Peptides for promoting angiogenesis and a use thereof | |
| RU2004112768A (en) | VII HUMAN COAGULATION FACTOR POLYPEPTIDES | |
| CN101948845B (en) | Optimization gene for coding chicken interferon alpha and application thereof in preparing chicken interferon alpha | |
| CN102355906A (en) | Method for preventing and treating hyperpermeability | |
| CN101186637A (en) | Method for inhibiting influenza virus infection and medicament thereof | |
| CN107893087A (en) | A kind of preparation method and applications of the interferon of Recombinant Swine λ 3 | |
| CN100400549C (en) | Nerve growth factor combined with collagen specificity and its coading gene and application | |
| CN102397559B (en) | Broad spectrum type influenza vaccine and preparation method thereof | |
| CN101879312B (en) | Broad spectrum type influenza vaccine and preparation method thereof | |
| CN100484959C (en) | Modified recombinant porcine alpha interferon protein and coding gene and uses thereof | |
| CN100999553B (en) | HR2 analogous protein and its coding gene and application | |
| WO2005080419A1 (en) | Polypeptide agent for inhibiting sars coronavirus and its derivatives and use thereof | |
| CN102884073B (en) | Peptides for promoting angiogenesis and a use thereof | |
| Foroozanfar et al. | ACE2 as a potential target for management of novel coronavirus (nCoV-2019) | |
| CN101897953B (en) | Non-invasive high-penetrability epidermal growth factor and application thereof | |
| IE852103L (en) | Preparing a vaccinal influenza virus polypeptide | |
| Storey et al. | Nucleotide Sequence of the Coding and Flanking Regions of the Human Parainfluenza Virus 3 Hemagglutinin–Neuraminidase Gene: Comparison with Other Paramyxoviruses | |
| WO2021164576A1 (en) | Anti-coronavirus infection medicine and use thereof | |
| CN106832004A (en) | A kind of oral fowl interferon fusion protein and its application as immunopotentiator | |
| CN110343164A (en) | A kind of pig interferon alpha-mutant and its preparation method and application of 7 site mutation of high activity | |
| CN104151420A (en) | Long-acting interferon, preparation method therefor and applications thereof | |
| CN101899116B (en) | Non-invasive high-penetrability epidermal growth factor and application thereof | |
| CN100540047C (en) | The application of TRAP albumen in the medicine of preparation treatment infection of staphylococcus aureus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20101103 Termination date: 20131128 |