CN105561301A - Application of clonorchis sinensis secreted phospholipase A2 protein in preparation of tumor therapy drug - Google Patents
Application of clonorchis sinensis secreted phospholipase A2 protein in preparation of tumor therapy drug Download PDFInfo
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- CN105561301A CN105561301A CN201610039906.3A CN201610039906A CN105561301A CN 105561301 A CN105561301 A CN 105561301A CN 201610039906 A CN201610039906 A CN 201610039906A CN 105561301 A CN105561301 A CN 105561301A
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- albumen
- csspla2
- protein
- recombinant
- sspla2
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- 239000011435 rock Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- 239000013595 supernatant sample Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 229940098465 tincture Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
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- 230000009466 transformation Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01004—Phospholipase A2 (3.1.1.4)
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Abstract
The invention belongs to the biotechnological field, and particularly relates to application of clonorchis sinensis secreted phospholipase A2 protein in preparation of a tumor therapy drug. Through wide and deep research, it is found that the clonorchis sinensis secreted phospholipase A2 protein (CssPLA2 protein) has the significant inhibition effect on growth of FRH cells of human cholangiocarcinoma (hilar cholangiocarcinoma) for the first time, and the inhibition rate reaches up to 86% or above. That is, the new application of the CssPLA2 protein in preparation of the cholangiocarcinoma therapy drug is found for the first time.
Description
Technical field
The invention belongs to biological technical field, be specifically related to clonorchis sinensis TypeⅡsecretoryphospholipaseA2 albumen and preparing the purposes in anti-tumor medicine.
Background technology
Existing for clonorchis sinensis TypeⅡsecretoryphospholipaseA2 albumen (that is, CssPLA2 albumen, GenBank accession number: ABL07371.1) expression technology can only give expression to the clonorchis sinensis TypeⅡsecretoryphospholipaseA2 albumen of inactive with inclusion bodies, due to expressed go out clonorchis sinensis TypeⅡsecretoryphospholipaseA2 albumen not there is biological activity, and the functional study carrying out this albumen can not be used for.Even if after carrying out complicated protein renaturation technical finesse, the biological activity of gained albumen is still quite low.
Such as: existing document F.Huetal./Molecular & BiochemicalParasitology167 (2009) 127 – 134 is numbered the plasmid of Cs4e05 for template with clonorchis sinensis cDNA library, the special primer of Application Design, carries out pcr amplification genes of interest.By genes of interest and prokaryotic expression plasmid pET-28a enzyme action, recovery, connection and conversion, the recombinant plasmid transformed of structure is entered BL21 (DE3) competent cell by construction recombination plasmid pET-28a-CssPLA2., after CssPLA2 recombination engineering liquid mass propgation, IPTG abduction delivering, recombiant protein is expressed with inclusion bodies, and 12%SDS-PAGE electrophoresis showed is about 34kDa place appearance one obviously band at molecular weight.Because recombiant protein is expressed with inclusion bodies, the inclusion body that broken bacterium obtains is urea-denatured through 6M, after dilution and dialysis renaturation, through metal ion affinity chromatography post, after the rinsing of different low concentration imidazole gradient, with 200mmol/L imidazoles eluting, obtain the single albumen that molecular weight is about 34kDa.Mtt assay and flow cytomery CssPLA2 albumen stimulates human liver microsome proteins LX-2 propagation, and Semiquatitative RT-PCR assay have detected CssPLA2 albumen can promote that the mrna expression of human liver microsome proteins LX-2 III Collagen Type VI raises, thus causes hepatic fibrosis.
So far, there is not yet in prior art and realize clonorchis sinensis TypeⅡsecretoryphospholipaseA2 albumen (CssPLA2 albumen) report in therapeutic field of tumor.
Summary of the invention
In order to overcome in prior art existing problem, clonorchis sinensis TypeⅡsecretoryphospholipaseA2 albumen (that is, CssPLA2 albumen) is the object of the present invention is to provide to prepare the purposes in anti-tumor medicine.
To achieve these goals and other relevant objects, the present invention adopts following technical scheme:
A first aspect of the present invention, provides CssPLA2 albumen and is preparing the purposes in anti-tumor medicine.
CssPLA2 albumen, GenBank accession number: ABL07371.1.
Preferably, CssPLA2 albumen is preparing the purposes in anti-tumor medicine as effective ingredient separately.
Preferably, described tumor is selected from cancer of biliary duct.
Further preferably, described tumor is selected from hilar cholangiocarcinoma.
Preferably, described CssPLA2 albumen is restructuring CssPLA2 albumen.
Further preferably, described recombinant C ssPLA2 albumen, containing CssPLA2 albumen and MBP label protein.
Preferably, the aminoacid sequence of described CssPLA2 albumen, as shown in SEQIDNO.1, is specially:
KPRSISRDKPHAELEWSGKLSDNQTIHIWTVASKGLFGEISKPAWIQVDIRGSNSSQDAIRLIFDQEHRLRYCVFGTDTVETSVSLDDADLLTRNPIYLEHFFFTSANEFLRACKELRRASEEPAKLVRRPRTAYRANPMIMPGTLWCGKGNAATRERTFGDEIETDMCCRTHDRCFENIQSLTSKFGYYNPSPVTISNCECDDEFLSCLENAGTEAATRVGNLYFNVFKIPCFLRRTERICTHNDESGACGQFENREDIELFRPQRFVAPYITV。
Preferably, the aminoacid sequence of described MBP label protein is the aminoacid sequence on pMAL-c2X plasmid corresponding to MBP label protein coded sequence.
Preferably, the N end of CssPLA2 albumen is held with the C of MBP label protein and is connected.
Preferably, connected by connection peptides between described CssPLA2 albumen and MBP label protein.
Further preferably, the aminoacid sequence of described connection peptides is the aminoacid sequence corresponding to nucleotide sequence pMAL-c2x plasmid arrived after MBP label protein coded sequence before Xba1 restriction enzyme site.
A second aspect of the present invention, provides a kind of anti-tumor medicine preparation, containing aforementioned CssPLA2 albumen and pharmaceutically acceptable carrier.
" pharmaceutically acceptable " composition is the material being applicable to people and/or animal and namely having rational benefit/risk ratio without excessive bad side reaction (as toxicity, stimulation and allergy)." pharmaceutically acceptable carrier " is acceptable solvent, suspending agent or excipient pharmaceutically or on food for CssPLA2 albumen of the present invention being sent to animal or human.Carrier can be liquid or solid.
Pharmaceutically acceptable carrier is various pharmaceutically conventional adjuvant and/or excipient, include, but is not limited to saccharide (as lactose, dextrose plus saccharose), starch (as corn starch and potato starch), cellulose and its derivates is (as sodium carboxymethyl cellulose, ethyl cellulose and methylcellulose), tragacanth gum powder, Fructus Hordei Germinatus, gelatin, Talcum, kollag (as stearic acid and magnesium stearate), calcium sulfate, vegetable oil, as Oleum Arachidis hypogaeae semen, Oleum Gossypii semen, Oleum sesami, olive oil, Semen Maydis oil and cupu oil, polyhydric alcohol is (as propylene glycol, glycerol, Sorbitol, mannitol and Polyethylene Glycol), alginic acid, emulsifying agent is (as Tween, polyoxyethylene castor oil), wetting agent (as sodium lauryl sulfate), coloring agent, flavoring agent, tablet agent, stabilizing agent, antioxidant, antiseptic, apirogen water, isotonic saline solution and phosphate buffer etc., this carrier can improve stability, the activity and biological effectiveness etc. of formula as required.
When pharmaceutical preparation of the present invention uses, described CssPLA2 albumen is as sole active ingredient, or described CssPLA2 albumen is as one of effective ingredient, the pharmaceutical dosage form of different way of administration can be made with one or more pharmaceutically acceptable carriers or mixed with excipients.
Preferably, the form of described pharmaceutical preparation is tablet, capsule, powder, granule, syrup, solution, oral liquid, spirit, tincture, aerosol, powder spray, injection, injectable sterile powder, or suppository.Above-mentioned preparation type can according to pharmaceutics (sixth version, People's Health Publisher, Cui Fude) in related definition understand, the preparation of above-mentioned preparation can according to pharmaceutics (sixth version, People's Health Publisher, Cui Fude) in the method preparation of related preparations.
Preferably, the dosage form of described pharmaceutical composition is tablet or oral liquid.
Preferably, pharmaceutical preparation of the present invention can be passed through oral, intravenous, intramuscular or subdermal routes of administration.
Preferably, can be degerming by membrane filtration when pharmaceutical preparation of the present invention is produced, or carry out autoclaving.
A third aspect of the present invention, provides a kind of method for the treatment of cancer of biliary duct, comprises step: the anti-tumor medicine preparation containing CssPLA2 albumen is applied to patient.
The dosage used can be determined according to the state of an illness by doctor.
Compared with prior art, the present invention has following beneficial effect:
The present invention is through extensive and deep research, and Late Cambrian clonorchis sinensis TypeⅡsecretoryphospholipaseA2 albumen (CssPLA2 albumen) has remarkable inhibitory action to the growth of cholangiocarcinoma cell, and suppression ratio is up to more than 86%.That is Late Cambrian of the present invention CssPLA2 albumen is preparing the novelty teabag in cancer of biliary duct medicine.
Accompanying drawing explanation
Fig. 1: CssPLA2 genes of interest PCR primer agarose gel electrophoresis qualification figure, wherein M:DNA standard molecular weight; 1:ddH2O negative control; 2:CssPLA2 genes of interest PCR primer.
The double digestion qualification figure of Fig. 2: recombiant plasmid pMAL-c2X/CssPLA2, M1, M2:DNA standard molecular weight; 1: empty plasmid XbaI and HindIII double digestion; 2: recombiant plasmid XbaI and HindIII double digestion; 3:CssPLA2 genes of interest PCR primer.
Fig. 3: recombiant plasmid pMAL-c2X/CssPLA2 at the 12%SDS-PAGE electrophoretic analysis figure of the expression product of Escherichia coli BL21/DE3, M: protein standard molecular weight; Before 1: recombiant plasmid pMAL-c2X/CssPLA2IPTG induction; Supernatant after 2: recombiant plasmid pMAL-c2X/CssPLA2IPTG induction; 3: recombiant plasmid pMAL-c2X/CssPLA2IPTG induces postprecipitation; 4: recombinant C ssPLA2 albumen after resin affinity purification.
Fig. 4: anion exchange chromatography purification of Recombinant CssPLA2 albumen (i.e. MBP-CssPLA2 fusion rotein).
The purity of Fig. 5: 12%SDS-PAGE analysis purification recombinant C ssPLA2 albumen.
Fig. 6: Westernblotting qualification recombinant C ssPLA2 albumen.
Fig. 7: mass spectrum (Massspectrum) identifies recombinant C ssPLA2 albumen.
Fig. 8: recombinant C ssPLA2 protease activity determination figure, sPLA2 (TypeⅡsecretoryphospholipaseA2) Enzyme activity assay test kit detects that in embodiment 1, gained recombinant C ssPLA2 albumen (MBP-CssPLA2 fusion rotein) has enzymatic activity.
Fig. 9: temperature is on the impact of restructuring CssPLA2 proteinase activity.
Figure 10: enzyme concentration is on the impact of restructuring CssPLA2 proteinase activity.
Figure 11: concentration of substrate is on the impact of restructuring CssPLA2 proteinase activity.
Figure 12: CCK8 detects MBP-CssPLA2 to the growth inhibited effect of human bile duct cancer FRH cell; ESP (clonorchis sinensis excretory-secretory protein), MBP albumen (maltose-binding protein, in contrast), recombinant C ssPLA2 albumen and PBS (negative control group) hatch cancer of biliary duct FRH cell, respectively respectively with CCK8 reagent detection cell 450nm absorbance after 24 hours (A), 48 hours (B), 72 hours (C); As seen from the figure, after 72 hours, the MBP-CssPLA2 of 25ug/ml obviously inhibits the growth of human bile duct cancer FRH cell, and suppression ratio is greatly about more than 86%.
Figure 13: wherein, Figure 13 A:25ug/mlESP (clonorchis sinensis excretory-secretory protein), Figure 13 B:25ug/mlMBP albumen (maltose-binding protein, in contrast), Figure 13 C:25ug/ml recombinant C ssPLA2 albumen, Figure 13 D: short apoptosis agent (positive control), Figure 13 E:PBS (negative control) hatch human bile duct cancer FRH cell, Apoptosis by Flow Cytometry after 72 hours respectively; Early stage and the late cell apoptosis percentage ratio of FRH cell is by 13.4% (PBS group), and 26.1% (MBP group) is elevated to 44.4% (MBP-CssPLA2 group), points out MBP-CssPLA2 to facilitate the apoptosis of human bile duct cancer FRH cell.
Detailed description of the invention
Before further describing the specific embodiment of the invention, should be understood that protection scope of the present invention is not limited to following specific specific embodiments; It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments, instead of in order to limit the scope of the invention.The test method of unreceipted actual conditions in the following example, usually conveniently condition, or according to the condition that each manufacturer advises.
When embodiment provides numerical range, should be understood that except non-invention is otherwise noted, between two end points of each numerical range and two end points, any one numerical value all can be selected.Unless otherwise defined, the same meaning that all technology used in the present invention and scientific terminology and those skilled in the art of the present technique understand usually.Except the concrete grammar used in embodiment, equipment, material, according to those skilled in the art to the grasp of prior art and record of the present invention, any method of prior art that is similar with the method described in the embodiment of the present invention, equipment, material or that be equal to, equipment and material can also be used to realize the present invention.
Unless otherwise indicated, disclosed in the present invention experimental technique, detection method, preparation method all adopt the routine techniques of the molecular biology of the art routine, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and association area.These technology are existing in existing document improves explanation, specifically can see the MOLECULARCLONING:ALABORATORYMANUAL such as Sambrook, Secondedition, ColdSpringHarborLaboratoryPress, 1989andThirdedition, 2001; Ausubel etc., CURRENTPROTOCOLSINMOLECULARBIOLOGY, JohnWiley & Sons, NewYork, 1987andperiodicupdates; TheseriesMETHODSINENZYMOLOGY, AcademicPress, SanDiego; Wolffe, CHROMATINSTRUCTUREANDFUNCTION, Thirdedition, AcademicPress, SanDiego, 1998; METHODSINENZYMOLOGY, Vol.304, Chromatin (P.M.WassarmanandA.P.Wolffe, eds.), AcademicPress, SanDiego, 1999; And METHODSINMOLECULARBIOLOGY, Vol.119, ChromatinProtocols (P.B.Becker, ed.) HumanaPress, Totowa, 1999 etc.
The clonal expression of embodiment 1 recombinant C ssPLA2 albumen (i.e. MBP-CssPLA2 fusion rotein)
1.1 with the cloned plasmids (being numbered Cs4e05) of CssPLA2 gene in clonorchis sinensis Schistosoma japonicum for template (Template), the special primer of Application Design, carry out pcr amplification genes of interest:
CssPLA2 forward primer (P1): 5 '-CTAG
tCTAGAaAACCACGGTCAATTTCA-3 ' (SEQIDNO.2) underscore is XbaI enzyme cutting site);
CssPLA2 downstream primer (P2): 5 '-GGG
aAGCTTgCTCATACAGTAATGTACG-3 ' (SEQIDNO.3) underscore is HindIII restriction enzyme site).
Pass through ExTaq
tMenzyme carries out pcr amplification, and reaction condition is as follows:
95 DEG C of denaturation 10min, reaction cycle parameter is 95 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 1min, carries out 30 circulations altogether; 72 DEG C of 10min.
PCR reaction system is as follows:
By the amplified production of gained genes of interest with 1.0% agarose gel electrophoresis identify:
(1) taking 0.6g agar Icing Sugar is dissolved in 1 × TAE60ml solution, heated and boiled 3-4min, and EB substitute (10mg/ml) to the final concentration to adding 2.5 μ l when about 50 DEG C to be cooled is 0.5 μ g/ml, encapsulating after mixing;
(2) electrophoresis: by DNA sample (that is, the amplified production of genes of interest, about 5 μ l) mix with the 6 × LoadingBuffer of 1 μ l after be added in well, 5 μ lDL2000Marker are added in well, electrophoresis is started, until sample strip is to position, glue face 2/3 under 100V voltage;
(3) take out gel, take pictures under gel imaging instrument.
As shown in Figure 1, the amplified production of gained genes of interest, molecular weight is 828bp, conforms to expection.
The double digestion of 1.2 genes of interest (CssPLA2 gene) and plasmid pMAL-c2x, recovery purification:
(1) the DH5 α of inoculation containing prokaryotic expression plasmid pMAL-c2x, extracts empty plasmid pMAL-c2x (by the operation of test kit description) with plasmid Mini Kit, with the agarose gel electrophoresis qualification of 1.0%, conforms to expection;
(2) gained genes of interest amplified production in 1.1 is carried out the agarose gel electrophoresis of 1.0%, glue is cut according to gene molecule amount size, with DNA gel reclaim test kit carry out recoverys purify (by test kit description operate), reclaim product take a morsel carry out 1.0% agarose gel electrophoresis qualification, conform to expection;
(3) plasmid pMAL-c2x and the genes of interest amplified production that reclaimed purification are carried out double digestion with XbaI and HindIII respectively simultaneously, double digestion system is as follows:
| 10×K Buffer | 2.0μl |
| PCR Produt/pMAL-c2Xvector | 8.0μl |
| XbaI | 1.0μl |
| HindIII | 1.0μl |
| dd H 2O | 8.0μl |
| Total volume | 20.0μl |
(4) mixing of above-mentioned double digestion system is placed in 37 DEG C of water-baths reacts 30min, product is carried out agarose gel electrophoresis respectively, cut glue according to respective molecular size range, reclaim test kit with DNA gel and reclaim purification double digestion product, be stored in-20 DEG C for subsequent use.
The connection of 1.3 genes of interest (CssPLA2 gene) and expression vector pMAL-c2x:
The genes of interest double digestion product of purification will be reclaimed in 1.2 and pMAL-c2x double digestion product is 4:1 mixing by volume, sets up the coupled reaction system of 20 μ l:
| T4DNA ligase Buffer | 2.0μl |
| Purified PCR Product | 4.0μl |
| Purified pMAL-c2X vector | 1.0μl |
| T4DNA ligase(5U/μl) | 1.0μl |
| dd H 2O | 12.0μl |
| Total volume | 20.0μl |
By each component crawl of above-mentioned coupled reaction system mixing (that is, adopts a little centrifuge each sample component to be mixed) room temperature reaction 1h afterwards, obtain connection product.
The conversion of 1.4 connection products
(1) gained in 10 μ l above-mentioned 1.3 is connected product to add in 200 μ lDH5 α/DE3 competent cells, softly mix, ice bath 30min;
(2) 42 DEG C of water-bath 90sec (time needs strict control and can not shake) are then placed in;
(3) taking-up is placed in 5min on ice immediately;
(4) super-clean bench is interior softly mixes in adding 800 μ lLB culture medium in EP pipe, sealing;
(5) 37 DEG C, 150rpm, shakes 1 ~ 1.5h slowly;
(6) 4 DEG C, the centrifugal 5min of 4,000rpm, abandons 800 μ l supernatants;
(7) play precipitation mixing with pipettor featheriness, then 200 μ l bacterial suspensions are coated on amicillin resistance solid agar culture plate, be placed in 37 DEG C of incubators and cultivate, be inverted dull and stereotyped after dull and stereotyped drying, continue cultivation 14 ~ 16h.
1.5 positive clone identification:
(1) monoclonal bacterium colony picking culture plate grown, access 5ml contains in the LB culture medium of 5 μ l ampicillin, and 37 DEG C, 250rpm jolting is spent the night;
(2) with bacterium liquid for template, with the described primer in above-mentioned 1.1 and reaction condition, pcr amplification is carried out to genes of interest, identifies with 1.0% agarose gel electrophoresis;
(3) positive bacteria liquid (containing genes of interest) is chosen, the recombiant plasmid of positive bacteria is extracted with plasmid Mini Kit, double digestion system enzyme action in above-mentioned 1.2, digestion products carries out 1.0% agarose gel electrophoresis to identify that whether restructuring is successful.Result as shown in Figure 2, shows that recombiant plasmid pMAL-c2X/CssPLA2 recombinates successfully.
(4) successful for qualification positive colony is carried out check order (Ai Ji Bioisystech Co., Ltd completes by Guangzhou), ensure the accuracy of Insert Fragment in positive colony recombiant plasmid.
According to sequencing result, nucleotide sequence such as the SEQIDNO.2 of genes of interest shows, is specially:
AAACCACGGTCAATTTCAAGGGACAAGCCACATGCTGAATTGGAATGGAGTGGAAAGTTGTCAGACAATCAGACAATTCATATATGGACAGTAGCATCGAAAGGACTATTTGGAGAGATCTCGAAACCGGCTTGGATCCAGGTTGATATCCGAGGCTCAAATTCCAGTCAAGACGCCATTCGTCTGATATTCGATCAAGAGCATCGACTCCGTTATTGTGTGTTCGGTACAGACACTGTCGAAACATCAGTCAGTCTGGACGACGCGGATTTACTGACCAGAAATCCCATCTATCTGGAGCACTTTTTTTTCACGTCGGCCAATGAATTCCTTCGAGCGTGTAAAGAGCTCCGGAGGGCGTCAGAAGAGCCGGCCAAACTGGTCCGTCGTCCGAGAACTGCCTACCGGGCTAACCCGATGATAATGCCTGGCACACTATGGTGTGGCAAAGGTAATGCTGCCACGCGCGAACGAACATTTGGTGACGAAATCGAGACTGACATGTGCTGTCGAACTCATGACCGATGTTTTGAAAACATCCAGAGTCTGACGAGTAAATTCGGATACTACAATCCATCGCCAGTGACGATTTCAAATTGTGAATGCGACGATGAGTTCCTCAGCTGTCTTGAAAACGCAGGAACTGAAGCAGCCACTCGAGTTGGAAACCTGTACTTCAATGTATTCAAAATTCCGTGTTTCTTGCGGCGCACCGAACGCATTTGTACCCACAATGACGAAAGCGGAGCATGTGGACAATTTGAAAATAGAGAAGATATTGAACTGTTCCGCCCGCAACGTTTTGTTGCTCCGTACATTACTGTATGA。Absolutely prove that in positive colony recombiant plasmid, Insert Fragment is correctly complete.
The abduction delivering of 1.6 recombiant plasmid in e. coli bl21/DE3:
(1) by through order-checking and correct recombiant plasmid pMAL-c2X/CssPLA2 transformation of E. coli BL21 (DE3) competent cell of comparison, coat on amicillin resistance agar plate, be placed in 37 DEG C of incubators to cultivate, after dull and stereotyped drying, flat board is inverted, continues cultivation 14 ~ 16h; Picking monoclonal, in access 5mlLB fluid medium, 37 DEG C, 250rpm jolting cultivate after through bacterium liquid PCR and double digestion qualification;
(2) recombinant correct for empirical tests and empty plasmid pMAL-c2x are accessed in the LB culture medium of 5ml amicillin resistance respectively, 37 DEG C, 250rpm jolting overnight incubation;
(3) next day according to the volume ratio (volume of bacterium liquid: the volume of culture medium) of 1:100 by fever at night bacterium switching LB culture medium, 37 DEG C, 250rpm jolting is cultured to OD600=0.5 ~ 0.6;
(4) contrast of 1ml bacterium liquid as non-abduction delivering is drawn; Add the IPTG that final concentration is 0.3mmol/L respectively, 37 DEG C, 250rpm inducing culture 4h; Draw 1ml bacterium liquid as the sample after abduction delivering;
(5) contrast and the centrifugal rear picking of sample are precipitated a little, add 5 μ lDTT and 95 μ l1 × SDS respectively, after mixing crawl, boil 5-10min;
The centrifugal 1min of (6) 13,000rpm, draws 10 μ l supernatants and carries out SDS-PAGE (12%);
(7) contaminate glue 30min under coomassie brilliant blue R250 room temperature, observe recombinant C ssPLA2 albumen after boiling decoloring and whether express.
The great expression of 1.7 recombinant C ssPLA2 albumen (i.e. MBP-CssPLA2 fusion rotein) and purification
First, the great expression of recombinant C ssPLA2 albumen is carried out:
(1) recombinant is connected in 5ml amicillin resistance LB culture medium, 37 DEG C, 250rpm shakes and spend the night;
(2) be forwarded in LB culture medium by the volume ratio of 1:100 from the bacterium that spends the night next day, 37 DEG C, 250rpm jolting is cultured to OD600=0.5 ~ 0.6; Add the IPTG that final concentration is 0.3mmol/L respectively, 37 DEG C, 250rpm abduction delivering 4h;
(3) by bacterium liquid 4 DEG C, centrifugal 15min collects thalline under 8,000rpm conditions;
(4) resuspended thalline, adds 1 × binding buffer liquid (20mMTris7.4) by the volume ratio (20mMTris7.4: culture medium) of 1:20;
(5) thalline suspension is placed in ultrasonication on ice 2 times (stopping 2sec after ultrasonic 1sec, each ultrasonic 15min);
(6) 4 DEG C, collect supernatant and precipitation respectively after 13,000rpm centrifugal 20min, draw supernatant 30 μ l, add 2 μ lDTT and 8 μ l4 × SDS sample-loading buffers; Take a small amount of precipitation, add 5 μ lDTT and 95 μ l1 × SDS sample-loading buffers, boil 5-10min after sample blending, 12%SDS-PAGE observes recombinant C ssPLA2 protein expression situation, and result as shown in Figure 3.
Next, the affinity purification of recombinant C ssPLA2 albumen is carried out:
(1) Amyloseresin (amylose resin) affinity column pretreatment: 3 times of volumes of deionized water; 3 times of volume 0.1%SDS; 1 times of volumes of deionized water; 3 times of volumes cross post buffer (20mMTris7.4,200mMNacl, 1mMEDTA);
(2) by supernatant obtained above 0.45 μm of membrane filtration twice; When buffer is down to close to resin surface, add Supernatant samples, cross post three times, retain effluent simultaneously;
1 × binding buffer liquid of (3) at least 100 times of volumes rinses to remove foreigh protein removing;
(4) 10mMmaltose (maltose) elution buffer adding 10 times of volumes carries out eluting, collects eluent;
(5) by 3 times of volumes of deionized water; 3 times of volume 0.1%SDS; 1 times of volumes of deionized water; 3 times of volumes cross post buffer; 3 times of volume 20% alcohol flushings Amyloseresin (amylose resin), and resin is stored in 20% ethanol;
(6) draw eluent 30 μ l, add 2 μ lDTT and 8 μ l4 × SDS sample-loading buffers, after boiling 5-10min, 12%SDS-PAGE analyzes the purity of purification recombinant C ssPLA2 albumen, as shown in Figure 3;
(7) according to electrophoresis result, the eluent containing purer recombinant C ssPLA2 albumen is loaded in bag filter, 4 DEG C, the middle dialysis of Tris buffer (pH8.0), change a dialysis solution every 4 hours, altogether change liquid 3 ~ 4 times;
(8) mesohigh tomographic system and anion-exchange column (HiTrapQFF) method of anion-exchange chromatography (Anionexchangechromatography) is used to be further purified recombinant C ssPLA2 albumen; First use 15ml buffer (20mMTris-HCL, PH8.0) to rinse pillar, then add recombinant C ssPLA2 protein sample, use the Nacl buffer (20mMTris-HCL of 5mM-500mM again, the NaCl of PH8.0,5mM-500mM) gradient elution, collect eluent; The visible obviously eluting peak of difference when using 100mM and 300-400mMNacl buffer solution elution, as shown in Figure 4.Draw the eluent 30 μ l of each gradient respectively, add 2 μ lDTT and 8 μ l4 × SDS sample-loading buffers, after boiling 5-10min, 12%SDS-PAGE analyzes the purity of purification recombinant C ssPLA2 albumen; Recombinant C ssPLA2 albumen (MBP-CssPLA2 fusion rotein) for the purpose of eluting peak during 300-400mMNacl buffer solution elution, 12%SDS-PAGE is as in Fig. 5, visible a part amount is about the single band of 76kDa, illustrates that the molecular size range of gained recombinant C ssPLA2 albumen (MBP-CssPLA2 fusion rotein) conforms to expection.CssPLA2 molecular weight of albumen is about 34kDa, and the molecular weight of MBP label protein subsidiary on pMAL-c2x plasmid is about 42kDa, and therefore, the molecular weight of recombinant C ssPLA2 albumen (MBP-CssPLA2 fusion rotein) is about 76kDa.
1.8Westernblot and Mass Spectrometric Identification recombinant C ssPLA2 albumen:
CssPLA2 monoclonal antibody and mass spectrometry method is used to identify recombinant C ssPLA2 albumen respectively.
First, Westernblotting is adopted to identify recombinant C ssPLA2 albumen:
(1), after recombinant C ssPLA2 albumen carries out 12%SDS-PAGE, gel is placed in electrotransfer buffer and soaks 15min;
(2) prepare pvdf membrane and several filter paper, pvdf membrane processes in the following order successively: soak 10min in 5min in 10sec → deionized water → electrotransfer buffer in 100% methanol, is directly soaked in by filter paper in electrotransfer liquid simultaneously;
(3) orifice plate erection sequence is followed successively by: black holes plate, gelatinous fibre pad, filter paper, gel, pvdf membrane, filter paper, gelatinous fibre pad, white orifice plate; Gel, pvdf membrane, filter paper will align, and the bubble of every layer all will drain, and installs and is correctly placed in electrotransfer groove;
(4) 100V constant voltage, on ice transferring film 1 ~ 1.5h;
(5) pvdf membrane is immersed in 10sec in methanol, taking-up filter paper makes its bone dry;
(6) be dipped in 5% skim milk powder solution by pvdf membrane, room temperature closes 2h;
(7) film is washed 3 times, 5min/ time with PBST;
(8) pvdf membrane and the clonorchis sinensis CssPLA2 monoclonal antibody of diluting (diluent is 1%BSA) by 1:300 are hatched, 4 DEG C are spent the night;
(9) film is washed 5 times, 5min/ time with PBST;
(10) the rabbit anti-mouse IgG (1:2,000 dilution) of pvdf membrane and HRP labelling is hatched, incubated at room 1h;
(11) film is washed 5 times, 5min/ time with PBST;
(12) with filter paper by film bone dry, immerse ECL luminescent solution colour developing (A, B liquid respectively gets 1ml), use fluorescence, chemiluminescence imaging analytical system takes pictures.Result as shown in Figure 6, illustrates that embodiment 1 gained recombinant C ssPLA2 albumen is correct.
The Mass Spectrometric Identification of recombinant C ssPLA2 albumen is identified by Zhongshan University's Zhongshan Medical College protein science center.Result as shown in Figure 7, illustrates equally, and embodiment 1 gained recombinant C ssPLA2 albumen is correct.
Concrete, recombinant C ssPLA2 albumen, containing CssPLA2 albumen and MBP label protein.The aminoacid sequence of described CssPLA2 albumen, as shown in SEQIDNO.1, is specially:
KPRSISRDKPHAELEWSGKLSDNQTIHIWTVASKGLFGEISKPAWIQVDIRGSNSSQDAIRLIFDQEHRLRYCVFGTDTVETSVSLDDADLLTRNPIYLEHFFFTSANEFLRACKELRRASEEPAKLVRRPRTAYRANPMIMPGTLWCGKGNAATRERTFGDEIETDMCCRTHDRCFENIQSLTSKFGYYNPSPVTISNCECDDEFLSCLENAGTEAATRVGNLYFNVFKIPCFLRRTERICTHNDESGACGQFENREDIELFRPQRFVAPYITV。The aminoacid sequence of MBP label protein described in CssPLA2 is the aminoacid sequence on pMAL-c2x plasmid corresponding to MBP label protein coded sequence.The N end of albumen is held with the C of MBP label protein and is connected.Connected by connection peptides between described CssPLA2 albumen and MBP label protein.The nucleotide coding sequence of described connection peptides specifically on pMAL-c2x plasmid after MBP label protein coded sequence to the nucleotide sequence before Xba1 restriction enzyme site.
Brief summary: the present invention is through extensive and deep research, once attempted multiple prokaryotic expression plasmid, as pET-30a, pET-32a, pET-26b, pGEX-4T-1, pMAL-c2x, finally find only have pMAL-c2x plasmid that solubility can be given expression to and there is bioactive CssPLA2 albumen.
The enzyme assay of embodiment 2 embodiment 1 gained recombinant C ssPLA2 albumen
Use the enzymatic activity of secreting type PLA2 (sPLA2) enzyme activity determination kit measurement embodiment 1 gained recombinant C ssPLA2 albumen, with Apis secreting type PLA2 as positive control, with MBP label protein (maltose-binding protein) as negative control.
Principle: phospholipase A2 (PhospholipaseA2), be called for short PLA2, it can be hydrolyzed the second acyl key (sn-2) of phosphoglyceride, generates lysophosphatide and fatty acid.
Operating procedure:
(1) negative control group: add people 10ulDTNB in ELISA Plate aperture, 5ul reaction buffer and 10ulMBP reference protein;
(2) positive controls: add people 10ulDTNB in ELISA Plate aperture, 5ul reaction buffer and 10ul Apis PLA2 albumen;
(3) experimental group: add people 10ulDTNB in ELISA Plate aperture, 5ul reaction buffer and 10ulMBP-CssPLA2 fusion rotein (make MBP-CssPLA2 fusion rotein concentration reach 1-4mg/ml as far as possible, be equivalent in every 10ul testing sample containing 10-40 μ g albumen);
(4) in each aperture, add 200ul phospholipid substrate (DiheptanoylThio-PC) start reaction, softly rock ELISA Plate mixing reactant liquor;
(5) multi-functional microplate reader is used to detect 414nm place absorbance.
As shown in Figure 8, sPLA2 (TypeⅡsecretoryphospholipaseA2) Enzyme activity assay test kit detects that in embodiment 1, gained recombinant C ssPLA2 albumen (MBP-CssPLA2 fusion rotein) has enzymatic activity to result.And existing expression technology, such as the CssPLA2 albumen of existing document F.Huetal./Molecular & BiochemicalParasitology167 (2009) 127 – 134 gained is inclusion body, there is no enzymatic activity, even if enzymatic activity is also lost very large after renaturing inclusion bodies, those skilled in the art repeated this experiment, after found that renaturing inclusion bodies, enzymatic activity is very faint, well below the activity of gained recombinant C ssPLA2 albumen of the present invention.
Temperature, enzyme concentration, concentration of substrate on the impact of enzymatic activity respectively as shown in Fig. 9 ~ 11.Always in Fig. 9, can find out, the embodiment of the present invention 1 gained recombinant C ssPLA2 albumen still has good enzymatic activity 50 degree time, illustrates that its stability is good especially.
Embodiment 3 removes the endotoxin of recombinant C ssPLA2 albumen
3.1 according to Detoxi-Gel
tMendotoxinRemovingGel test kit description, operates as follows:
(1) treat that resin is about 30min activity recovery in room temperature natural subsidence;
(2) Detoxi-Gel resin is regenerated: with 1% NaTDC cleaning resin of 5 times of resin volumes, the water for injection then adding 3 ~ 5 times of volumes removes cleaning agent;
(3) resin is balanced: the water for injection adding 3 ~ 5 times of volumes in pillar.
(4) application of sample: loaded by embodiment 1 gained recombinant C ssPLA2 protein solution in bag filter, 4 DEG C, the middle dialysis of PBS buffer (pH7.4), changes a dialysis solution every 4 hours, altogether changes liquid 3-4 time; The bag filter of having dialysed is put into sucrose, utilizes sucrose water absorption, protein concentrate; Add 1ml concentrate after recombinant C ssPLA2 protein solution, in order to obtain higher joint efficiency, CssPLA2 albumen to be reorganized enters in resin, incubated at room 1-1.5h; And then add water for injection, collect effluent;
(5) step (2) is repeated to remove remaining endotoxin, regenerating resin;
(6) resin is stored in 25% ethanol, 2 ~ 8 DEG C of placements;
(7) each pipe flow fluid of collecting is carried out 12%SDS-PAGE.
Whether 3.2 tachypleus amebocyte lysate detect the recombinant C ssPLA2 albumen after removing endotoxin qualified
Experimental group: 100 μ l recombinant C ssPLA2 protein sample+100 μ l detection water; Matched group: 200 μ l detection water.
After being mixed gently by solution in detector tube, sealed membrane closes the mouth of pipe, vertically puts into 37 DEG C of water-baths, insulation 60min, takes out detector tube gently after terminating, slowly reverses, if form gel in pipe, and gel is indeformable, not from tube wall slippage person be positive (namely containing endotoxin); The gel deformation not forming gel or formation is negative (namely not containing endotoxin) from tube wall slippage person.Note not rocking between soak in order to avoid affect formation gel.
After testing, the recombinant C ssPLA2 albumen removed after endotoxin is qualified, can be used for follow-up cell experiment.
Embodiment 4CCK8 method detects recombinant C ssPLA2 albumen to the growth inhibited effect of human bile duct cancer FRH cell
(1) inoculating cell is in 20ml Tissue Culture Flask, and 10%FBS-164037 DEG C, 5%CO2 cellar culture go down to posterity and be inoculated in 96 orifice plates, cell density is 1 × 10
5individual/ml, every pore volume 100 μ l, cultivate 24h for 37 DEG C;
(2), after cell attachment, cultivate 24h in serum-free medium after, culture fluid is abandoned; The clonorchis sinensis excretory-secretory protein (ESP) of 25ug/ml is prepared respectively by 2%FBS-1640 culture medium, the MBP label protein of 25ug/ml, the recombinant C ssPLA2 albumen of 25ug/ml, using not containing recombinant C ssPLA2 albumen and the 2%FBS-1640 culture fluid adding PBS as negative control; Central part cell culture well got by every block plate, divide 4 groups, wherein 4 groups of cell holes add containing 25 μ g/mlESP, 25ug/mlMBP reference proteins, 25ug/ml recombinant C ssPLA2 albumen respectively and add the 2%FBS-1640 culture fluid 100ul of PBS not containing recombiant protein, put into CO
2incubator, 37 DEG C are continued to cultivate;
(3) colour generation: respectively at cultivation 24h, 48h, 72h with after changing liquid containing 100 μ l/ hole 2%FBS-1640 culture medium often hole add 10 μ lCCK8, put into CO
2incubator, 37 DEG C are continued to cultivate 3.5h;
(4) colorimetric: select 450nm wavelength, measure each hole absorbance by full-automatic microplate reader, record result.
CCK8 detects MBP-CssPLA2 to the growth inhibited effect of human bile duct cancer FRH cell, and result as shown in figure 12.
ESP (clonorchis sinensis excretory-secretory protein), MBP albumen (maltose-binding protein, in contrast), recombinant C ssPLA2 albumen (MBP-CssPLA2) and PBS (negative control group) hatch cancer of biliary duct FRH cell, respectively respectively with CCK8 reagent detection cell 450nm absorbance after 24 hours (A), 48 hours (B), 72 hours (C); As seen from Figure 12, after 72 hours, the MBP-CssPLA2 of 25ug/ml obviously inhibits the growth of human bile duct cancer FRH cell, and suppression ratio is greatly about more than 86%.MBP albumen (maltose-binding protein) in contrast, do not suppress the effect of human bile duct cancer FRH Growth of Cells, therefore, absolutely prove, be the effect that CssPLA2 albumen serves the growth that obviously inhibit human bile duct cancer FRH cell, suppression ratio is greatly about more than 86%.
Embodiment 5 Flow cytometry recombinant C ssPLA2 albumen is to the growth inhibited effect of human bile duct cancer FRH cell
(1) by human bile duct carcinoma in 10%FBS-1640 culture medium in 37 DEG C, 5%CO
2cellar culture in incubator, is passaged in six porocyte culture plates, and 1 × 10
5individual/ml, every pore volume 2ml;
(2), after cell attachment, cultivate 24h in serum-free medium after, culture fluid is abandoned.The clonorchis sinensis excretory-secretory protein (ESP) of 25ug/ml is prepared respectively by 2%FBS-1640 culture medium, the MBP label protein of 25ug/ml, the recombinant C ssPLA2 albumen of 25ug/ml, using not containing recombiant protein and the 2%FBS-1640 culture fluid adding PBS as negative control, urge the 2%FBS-1640 culture fluid of apoptosis agent as positive control to add 1:2000; First Nature enemy (serum-free culture) is carried out to all cells, culture fluid is abandoned after cultivating 24h, add respectively and urge apoptosis agent and the 2%FBS-1640 culture fluid containing PBS containing 25 μ g/mlESP, 25ug/mlMBP reference proteins, 25ug/ml recombinant C ssPLA2 albumen, 1:2000, cellar culture 72 hours respectively;
(3) with 0.25% trypsin digestion cell in 15ml centrifuge tube; Suspension cell centrifugal (the centrifugal 5min of 2000rpm) is collected; The collected by trypsinisation (note: trypsinization time not easily long, otherwise easily cause false positive) of attached cell not containing EDTA;
(4) 1 ~ 5 × 10 are collected with PBS washed cell secondary (the centrifugal 5min of 2000rpm)
5cell;
(5) the BindingBuffer suspension cell of 500 μ L is added;
(6), after adding 5 μ LAnnexinV-FITC mixings, add 10 μ LPropidiumIodide and mix, room temperature, lucifuge, reaction 5 ~ 15min; In 1h, carry out flow cytomery;
(7) with flow cytomery, during flow cytomery, cell quantity should lower than 1 × 10
5.
Result as shown in figure 13, Figure 13: wherein, Figure 13 A:25ug/mlESP (clonorchis sinensis excretory-secretory protein), Figure 13 B:25ug/mlMBP albumen (maltose-binding protein, in contrast), Figure 13 C:25ug/ml recombinant C ssPLA2 albumen, Figure 13 D: short apoptosis agent (positive control), Figure 13 E:PBS (negative control) hatch human bile duct cancer FRH cell, Apoptosis by Flow Cytometry after 72 hours respectively; Early stage and the late cell apoptosis percentage ratio of FRH cell is by 13.4% (PBS group), 26.1% (MBP group) is elevated to 44.4% (MBP-CssPLA2 group), prompting MBP-CssPLA2 facilitates the apoptosis of human bile duct cancer FRH cell, therefore, absolutely proving, is the effect that CssPLA2 albumen serves the growth that obviously inhibit human bile duct cancer FRH cell.
Brief summary, by the experimental result of embodiment 4 and embodiment 5, absolutely prove, CssPLA2 albumen is to the growth inhibited effect of cancer of biliary duct FRH cell (hilar cholangiocarcinoma).Late Cambrian of the present invention, CssPLA2 albumen is preparing the novelty teabag in cancer of biliary duct medicine.
The above; be only preferred embodiment of the present invention; not to any formal and substantial restriction of the present invention; should be understood that; for those skilled in the art; under the prerequisite not departing from the inventive method, also can make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.All those skilled in the art, without departing from the spirit and scope of the present invention, a little change made when utilizing disclosed above technology contents, the equivalent variations of modifying and developing, be Equivalent embodiments of the present invention; Meanwhile, all according to substantial technological of the present invention to the change of any equivalent variations that above-described embodiment is done, modify and differentiation, all still belong in the scope of technical scheme of the present invention.
Claims (10)
1.CssPLA2 albumen is preparing the purposes in anti-tumor medicine.
2. purposes according to claim 1, is characterized in that, described tumor is selected from cancer of biliary duct.
3. purposes according to claim 1, is characterized in that, described CssPLA2 albumen is restructuring CssPLA2 albumen.
4. purposes according to claim 3, is characterized in that, described recombinant C ssPLA2 albumen, containing CssPLA2 albumen and MBP label protein.
5. purposes according to claim 4, is characterized in that, the aminoacid sequence of described CssPLA2 albumen is as shown in SEQIDNO.1.
6. purposes according to claim 4, is characterized in that, the aminoacid sequence of described MBP label protein is the aminoacid sequence on pMAL-c2X plasmid corresponding to MBP label protein coded sequence.
7. purposes according to claim 4, is characterized in that, the N end of CssPLA2 albumen is held with the C of MBP label protein and is connected.
8. purposes according to claim 4, is characterized in that, is connected between described CssPLA2 albumen and MBP label protein by connection peptides.
9. an anti-tumor medicine preparation, containing CssPLA2 albumen and pharmaceutically acceptable carrier.
10. treat a method for cancer of biliary duct, comprise step: the anti-tumor medicine preparation containing CssPLA2 albumen is applied to patient.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1343772A (en) * | 2000-09-18 | 2002-04-10 | 中山大学 | Phosphatidase A2 of sea serpent and gene for coding it |
| CN101102790A (en) * | 2005-01-14 | 2008-01-09 | 诺瓦提斯公司 | Identification of phospholipase A2 as target in cancer treatment, with special emphasis on colorectal cancer and its mechanism of action |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1343772A (en) * | 2000-09-18 | 2002-04-10 | 中山大学 | Phosphatidase A2 of sea serpent and gene for coding it |
| CN101102790A (en) * | 2005-01-14 | 2008-01-09 | 诺瓦提斯公司 | Identification of phospholipase A2 as target in cancer treatment, with special emphasis on colorectal cancer and its mechanism of action |
Non-Patent Citations (2)
| Title |
|---|
| ARAYA C ET AL.: "Antitumor effects of cationic synthetic peptides derived from Lys49 phospholipase A2 homologues of snake venoms", 《CELL BIOL INT》 * |
| HU F ET AL.: "Clonorchis sinensis secretory phospholipase A2 and investigation of its potential contribution to hepatic fibrosis. Mol Biochem Parasitol", 《MOL BIOCHEM PARASITOL》 * |
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