CN1158385C - Clone of Japanese schistosome fatty acid-binding protein gene and its expression in Bombyx mori system - Google Patents
Clone of Japanese schistosome fatty acid-binding protein gene and its expression in Bombyx mori system Download PDFInfo
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Abstract
本发明旨在生产抗日本血吸虫病疫苗，它涉及一个编码日本血吸虫中国大陆株脂肪酸结合蛋白的基因，一个含有该基因的重组家蚕核型多角体病毒及在家蚕系统中的表达和该蛋白质的制备方法。本发明的表达产物为分子大小18kD的融合蛋白，它具有较好的免疫原性，能诱导较强的抗攻击感染作用。The present invention aims at producing anti-schistosomiasis vaccine, which relates to a gene encoding the fatty acid binding protein of Schistosoma japonicum Chinese mainland strain, a recombinant silkworm nuclear polyhedrosis virus containing the gene, its expression in the silkworm system and the preparation of the protein method. The expression product of the present invention is a fusion protein with a molecular size of 18kD, which has good immunogenicity and can induce strong anti-challenge infection effect.
Description
Technical field
The present invention relates to recombinant vaccine, be specifically related to the expression in a kind of clone of schistosoma japonicum Chinese strain fatty acid-binding protein gene and the mori system of being in.
Background technology
Schistosomicide is a kind of parasitosis of serious serious harm people, animal health, study for a long period of time and facts have proved, on the basis of using existing Prevention Technique, carry out vaccine prevention research, particularly recombinant vaccine research is that several candidate antigens of Schistosoma mansoni were preferably once recommended by the important development strategy of prevention and cure of schistosomiasis, the World Health Organization in 1994, fatty acid binding protein for example, see BergquistN K, Hall BF, James S.Schistosomiasis vaceinedeve lopment.The Immunologist.1994.2 (40): 131-134.
Summary of the invention
The objective of the invention is to schistosoma japonicum Chinese strain fatty acid binding protein (Sj14FABP) gene is cloned, and use the silkworm host and produce recombinant protein and be used to prepare vaccine.
The invention discloses a kind of clone of Schistosoma japonicum fat binding-protein gene and the expression in the mori system of being in.
Material therefor of the present invention is as follows:
The recombinant plasmid Sj14/pGEX-2T that contains the Sj14FABP gene is made up by this laboratory; The BmNPV that modifies is made up by Shanghai Inst. of Biochemistry, Chinese Academy of Sciences; The increase e. coli tg1 of recombinant plasmid and recombinant transfer plasmid of BmN cell, transferring plasmid pBacPAK Hisl (for Clontench company product) and being used to is Shanghai Inst. of Biochemistry, Chinese Academy of Sciences's preservation; Various tool enzyme, liposome,
32P mark labelling kit and low melting-point agarose are GIBCO BRL company product, nitrocellulose membrane is Chleicher ﹠amp; Schuell or Huangyan, Zhejiang product; The anti-mouse of the rabbit of horseradish peroxidase-labeled is a Sigma company product.Silkworm (75 new * 7532) is provided by Inst. of Silkworm, Chinese Academy of Agricultural Sciences.
Method of the present invention comprises the following steps:
1, the clone of Sj14FABP gene and sequential analysis
Go out the Sj14FABP gene with primer 5 '-CAGGATCCATGTCTAGTTTCTTGGGAAA-3 ' and primer 5 '-TCAGGATCCGCTTTTTTTTTTTTTTTTTT-3 ' schistosoma japonicum Chinese strain adult cDNA with PCR method quick clone, by the method in the Sambrook laboratory manual it is cloned on pSK (+) plasmid, presses USB T
7Its nucleotide sequence is measured in the operation of sequencing kit specification sheets;
2, the structure of recombinant transfer plasmid carries out genetic manipulation by the Sambrook laboratory manual, filters out the recon that contains the Sj14FABP gene, with Sal I enzyme cut and by USB sequencing kit specification sheets order-checking to determine closure and to the sign indicating number situation;
3, the virus reorganization is equipped with recombinant virus with reference to the Summers laboratory manual with liposome cotransfection legal system.The BmNPV that modifies has introduced the LacZ gene under the alfalfa californica nuclear polyhedrosis virus polyhedrin promoter regulation, introduces Bsu36 I site in the both sides of polyhedrosis gene simultaneously, cuts by enzyme and makes the genomic dna linearizing.The cotransfection mixture is made up of 2 μ g linearizing viral DNAs, 3 μ g recombinant transfer plasmid DNA and isopyknic 50 μ l liposomes.Metainfective BmN cell is got the supernatant amplification once after 27 ℃ of one weeks of cultivation;
4, the screening of recombinant virus, evaluation and purifying are got amplification cotransfection virus supernatant and are done 1: 10
-3With 1: 10
-4Dilution infects 1 * 10
5 Cell 1 hour is spread the low melting-point agarose (1%w/v) that contains 0.2mg/ml X-gal, cultivates after 4-6 days a plurality of hickies of picking in 24 well culture plates (2 * 10
4Cells/well), cultivated 3-4 days for 27 ℃, collecting cell is used
32The Sj14FABP gene probe of P mark carries out dot blot DNA hybridization analysis, selects positive colony to carry out second as stated above and takes turns the plaque analysis, gets the recombinant virus Bm-sj14FABP of purifying;
5, Sj14FABP expression of gene Bm-Sj14FABP infects 2 * 10
4Collecting cell and supernatant SDS-PAGE (15%w/v separation gel) detection respectively in 1 to 6 day behind the BmN cell.5 age silkworm at link subcutaneous injection 5 μ l Bm-sj14FABP virus supernatant or BmNPV supernatant (in contrast), begin to collect day by day hemolymph and silkworm body tissue next day.Hemolymph is through PBS 1: 3 dilution, tissue with PBS homogenate, frozen-thawed, it is ultrasonic, centrifugal that (11953g * 10min) supernatant is collected in the back, detects with SDS-PAGE (15%w/v separation gel) method;
6, the purifying expression product of expression product is a fusion rotein, contains Sj14FABP and from the part expressing protein that contains 6 Histidines of pBacPAK Hisl.Reference literature method His fusion rotein separator column NiSO
4Huge legendary turtle is closed the agarose gel column purification, and hemolymph is with containing 0.2mol Na before the upper prop
2HPO
4, the dilution of 0.2mol NaCl, PH8.0 damping fluid; Tissue with above-mentioned damping fluid homogenate, freeze thawing, it is ultrasonic, centrifugal that (11953g * 10min) prepares the upper prop sample.With 100mM EDTA wash-out, survey protein content with ultraviolet absorption method;
7, the evaluation of expression product prepares antiserum(antisera) with the sj14FABP of escherichia coli expression immunity Balb/c mouse, makes immunoblotting (Western blot) with this serum and silkworm expression product and purified product and reacts, the antigenicity of detection silkworm expression product;
The checking result of each step of the inventive method is as follows:
1, cloning one section size from schistosoma japonicum Chinese strain adult cDNA library is dna fragmentation about 600bp, and its nucleotide sequence is as follows:
1 CAGGATCCAT?GTCTTTCTTG?GGAAAGTGGA?AACTAAGCGA?ATCACACAAC?TTCGATGCTG
GTCCTAGGTA?CAGAAAGAAC?CCTTTCACCT?TTGATTCGCT?TAGTGTGTTG?AAGCTACGAC
61 TTATGTCAAA?GCTCGGTGTC?TCGTGGGCGA?CCCGACAAAT?TGGGAACACA?GTGACGCCAA
AATACAGTTT?CGAGCCACAG?AGCACCCGCT?GGGCTGTTTA?ACCCTTGTGT?CACTGCGGTT
121 CTGTCACTTT?CACAATGGAT?GGGGGATACG?ATGACCATGC?TGACAGAGTC?GACTTTCAAG
GACAGTGAAA?GTGTTACCTA?CCCCCTATGC?TACTGGTACG?ACTGTCTCAG?CTGAAAGTTC
181 AACCTCTCAG?TCACGTTCAA?ATTTGGTGAG?GAATTTGACG?AGAAAACCAG?TGATGGCAGA
TTGGAGAGTC?AGTGCAAGTT?TAAACCACTC?CTTAAACTGC?TCTTTTGGTC?ACTACCGTCT
241 AGCGTTAAGT?CAGTCGTTAC?CAAAGATTCA?GAGTCAAAGA?TAACTCAAAC?TCAAAAGGAT
TCGCAATTCA?GTCAGCAATG?GTTTCTAAGT?CTCAGTTTCT?ATTGAGTTTG?AGTTTTCCTA
301 AGTAAGAACA?CAACTGTAAT?CGTTCGTGAA?ATAGTAGGTG?ATACTATGAA?AACTACTGTA
TCATTCTTGT?GTTGACATTA?GCAAGCACTT?TATCATCCAC?TATGATACTT?TTGATGACAT
361 ACTGTCGATG?ATGTTACGGC?TATTCGGAAT?TACAAACGAT?TGTAACCCCT?GCAAACTGAT
TGACAGCTAC?TACAATGCCG?ATAAGCCTTA?ATGTTTGCTA?ACATTGGGGA?CGTTTGACTA
421 AGACTGGTCA?AATGTTTCTT?GAAAGCGCTT?GTATTTCAGT?TGACATTATT?ACGAAAATGA
TCTGACCAGT?TTACAAAGAA?CTTTCGCGAA?CATAAAGTCA?ACTGTAATAA?TGCTTTTACT
481 CCACCGGTGG?ACGTATTTGG?TTACTCGCGT?CATCCTATAT?TTAATTAATC?AGCTCCACTT
GGTGGCCACC?TGCATAAACC?AATGAGCGCA?GTAGGATATA?AATTAATTAG?TCGAGGTGAA
541 TTGGTTCAAT?AAACATTGCT?TATTATTTAA?AAAAAA
AACCAAGTTA?TTTGTAACGA?ATAATAAATT?TTTTTT
2, the structure of recombinant transfer plasmid
The building process of transferring plasmid as shown in Figure 1.Transfer vector pBacPAK Hisl is the fusion rotein transfer vector of band polyhedrosis gene promotor.Cut sj14/pGEX-2T with BamH I enzyme, separate the Sj14FABP gene fragment.This clip size is about 600bp, includes the sj14FABP gene, and this gene ORF is 399bp, 132 amino acid of encoding, and there is a Sal I site at its 163bp place.The pBacPAKHisl that this gene fragment and same enzyme are cut is connected, transforms.Recombinant plasmid is cut and is checked order through Sal I enzyme and shows direction of insertion and all correct to sign indicating number.
3, the screening of recombinant virus Bm-Sj14FABP and evaluation
23 hickies that first round plaque is chosen detect through dot-blot (DNA dot blot), and 22 hickies can quilt in the base
32The Sj14FABP gene probe identification of P mark.Select wherein 17
#The strong positive spot carries out second and takes turns the plaque analysis, and 23 plaques of picking detect with method, whole positive spots (Fig. 2).Select strong positive spot 1 wherein
#, 8
#, 17
#With 19
#Do to express and use.
4, the expression of Sj14FABP in bombyx mori cell and larva
The expression product reorganization rSj14FABP that Bm-Sj14FABP infects behind BmN cell and the larva is the fusion rotein of Sj14FABP and carrier, and molecular weight is 18KD, and wherein Sj14FABP is 14.8KD, and carrier part is 3.2KD.
2 days begin to have rSj14FABP to express behind the Bm-Sj14FABP infection BmN cell, infect the 3-6 days expression amounts in back and remain basically stable.In cells and supernatant, beginning in 3 days increases (Fig. 3) day by day behind the rSj14FABP self-infection.1 * 10
6Must measuring into about 100 μ g (the 4th day) of cell, must measure in the culture supernatant is about 33 μ g/ml.
Disease symptom occurred in 3 days behind the Bm-Sj14FABP infected silkworm larva, the state of an illness increased the weight of in 4 days, the shrinkage of silkworm body, and back silkworm body occurred dead in 4 days half.SDS-PAGE demonstration rsj14FABP is appearance in 3 days behind the equal self-infection in hemolymph and tissue, reaches peak (Fig. 4) in 4 days, and the output of rSj14FABP accounts for 33% (Fig. 5) of hemolymph total protein through GDS8000 gel scanning analysis software analysis at this moment.In hemolymph that blank silkworm and BmNPV infect and tissue, all there is not this protein band.The hemolymph in infection Bm-Sj14FABP virus-4 sky and tissue are through NiSO
4Behind-Cheling Sepherose the column purification, every milliliter of hemolymph can get the 4mg purifying protein, and every gram silkworm tissue can get the 4.6mg purifying protein, gets 0.25ml hemolymph, 0.6g silkworm tissue by every silkworm, and every silkworm can about 3.5mg expression product.Figure 6 shows that the SDS-PAGE collection of illustrative plates of purifying protein.
5, the immunological characteristic of expression product
Sj14FABP mice immunized serum with escherichia coli expression carries out western blot experiment, and collection of illustrative plates as shown in Figure 6.Infecting the hemolymph in Bmsj14FABP virus-4 sky and the rSj14FABP of purifying has obvious hybridization band (Fig. 7) at the 18kD place, illustrate that this recombinant protein has antigenicity preferably.The silkworm hemolymph that infected 4 days with BmNPV has a more weak reaction band at the 30kD place.
Japanese schistosome fatty acid-binding protein Sj14FABP (his) molecular size that obtains with the inventive method is 18kD.
Animal immune test with rSj14FABP is as follows:
Materials and methods
One, parasite: test is provided by this institute oncomelania room with schistosoma japonicum cercariae, and the schistosomicide circulation life history is kept by new zealand white rabbit and China's Mainland oncomelania hupensis.
The kunming mouse in age in two, laboratory animal: 6-8 week and Wistar rat are available from Chinese Academy of Sciences's Shanghai Experimental Animal Center; 1.0-1.5 year, male sheep all was the schistosomicide feminine gender, test first two weeks all sheep are all used Closantel (Belgian Yang Sen company) treatment, drive away other parasites that may exist in the sheep body.
The purifying of three, genetic expression, recombinant antigen and antigenic determination: the report of seeing Cai Xue loyalty etc.
Four, animal immune: successively carried out a collection of kunming mouse, Wistar big white mouse and sheep immunity test.
Five, animal is attacked and cuts open and kill: two weeks of back of immunity for the third time, mouse, big white mouse and sheep hit with 40,600 and 300 cercaria roses respectively, mouse and big white mouse hit the back in rose and cut open in 6-8 week extremely, sheep hits the back in rose and cutd open in 11 weeks extremely, collect polypide by the portal vein lavage, calculate worm reduction rate with following formula:
Six, sheep dung, organize egg count: sheep hits in rose and infects the back and the 6th to the 22nd week gathered ight soil weekly one time, cut open and gather liver, large intestine, small intestine's sample when killing, calculate every gram ight soil/organize worm's ovum number (EPG) by literature method, calculate worm reduction rate with following formula:
The result
One, rSj14FABP is mouse, rat, and inductive subtracts the worm effect in the sheep:
Mouse, rat, obtain 33%, 31.85% and 59.2% worm reduction rate in the sheep respectively.
Two, rSj14FABP inductive in sheep subtracts the ovum effect observation:
Compare with control group, rSj14FABP immunity sheep infects the 6th thoughtful the 11st week of back from attacking, and average every gram worm's ovum number (EPG) reduces 23.6%, 35.0%, 63.3%, 43.8%, 34.9% and 57.6% respectively in the ight soil, decreased average 42.97%.Hepatic tissue EPG reduces 44.9%; Small intestine EPG reduces 69.6%; But large intestine EPG does not see minimizing.
Three, again according to the literature: report such as Tendler M is used reorganization Schistosoma mansoni FABP (rSmFABP) immune mouse and rabbit, the worm reduction rate of having induced 51.4%-67.8% and 56.7%-72.1% respectively.
Above-mentioned test-results shows, Sj14FABP has better immunogenicity, and can induce stronger anti-rose to hit infection effect, it must measure very high in silkworm larva, in the silkworm hemolymph that infects BmSj14FABP virus-4 sky, the fusion rotein of Sj14FABP accounts for 33% of hemolymph total protein, and every milliliter of hemolymph can get the Sj14FABP (his) of 4mg purifying, and every Sj14FABP (his) that also can obtain the 4.6mg purifying in the silkworm body tissue that restrains, this production of vaccine to future has practical value.Silkworm is easy to raise, and production cost is low, existing at present silkworm artificial breeding technology, and method of the present invention is suitable for the scale industrial abacterial to produce vaccine.
Description of drawings
Fig. 1: the building process that shows recombinant transfer plasmid
MCS: multiple clone site; B:BamHI
Fig. 2: show that second takes turns the dot blot detected result that plaque is analyzed
Fig. 3: show behind the recombinant virus infection bombyx mori cell that the SDS-PAGE of rsj14FABP analyzes in the culture supernatant
M: lower molecular weight standard protein; 0,1,2,3,4,5,6: DAI
Fig. 4: the SDS-PAGE that infects rSj14FABP in the recombinant virus silkworm hemolymph analyzes
M: lower molecular weight standard protein; 1: normal silkworm hemolymph; 2: 4 days silkworm hemolymph behind the infection BmNPV; 3,4,5,6: 1-4 days silkworm hemolymph behind the infection recombinant virus
Fig. 5: 4 days silkworm hemolymph SDS-PAGE gel scanning analysis behind the infection recombinant virus
Fig. 6: the SDS-PAGE collection of illustrative plates of the recombinant protein rSj14FABP of purifying
M: lower molecular weight standard protein; 2: 4 days silkworm hemolymph 3 behind the infection recombinant virus: the recombinant protein rSj14FABP of purifying
Fig. 7: the immunoblotting assay M of the recombinant protein of purifying and infection recombinant virus silkworm hemolymph: lower molecular weight standard protein; 1: normal silkworm hemolymph; 2: 4 days silkworm hemolymph behind the infection BmNPV; 3: 4 days silkworm hemolymph behind the infection recombinant virus; 4: the recombinant protein of purifying
Embodiment
Embodiment one:
1, the clone of sj14FABP gene and sequential analysis,
Go out Sj14FABP gene with PCR method quick clone with primer 5 '-CAGGATCCATGTCTAGTTTCTTGGGAAA-3 ' and primer 5 '-TCAGGATCCGCTTTTTTTTTTTTTTTTTT-3 ' schistosoma japonicum Chinese strain adult cDNA in the library, by the method in the Sambrook laboratory manual it is cloned on PSK (+) plasmid, press the operation of USB T7 sequencing kit specification sheets, measure its nucleotide sequence;
2, the structure of recombinant transfer plasmid carries out genetic manipulation by the Sambrook laboratory manual, filters out the recon that contains the Sj14FABP gene, with Sal I enzyme cut and by USB sequencing kit specification sheets order-checking to determine closure and to the sign indicating number situation;
3, the virus reorganization is equipped with recombinant virus with reference to the Summers laboratory manual with Lipofectin cotransfection legal system.The BmNPV that modifies has introduced the LacZ gene under alfalfa californica nuclear polyhedrosis virus (AcNPV) the polyhedrin promoter regulation, introduces Bsu36 I site in the both sides of polyhedrosis gene (ph) simultaneously, cuts by enzyme and makes the genomic dna linearizing.The cotransfection mixture is made up of 2 μ g linearizing viral DNAs, 3 μ g recombinant transfer plasmid DNA and isopyknic 50 μ l Lipofectin.Metainfective BmN cell is got the supernatant amplification once after 27 ℃ of one weeks of cultivation;
4, the screening of recombinant virus, evaluation and purifying are got amplification cotransfection virus supernatant and are done 1: 10
-3Dilution infects 1 * 10
5 Cell 1 hour is spread the low melting-point agarose (1%) that contains 0.2mg/ml X-gal, cultivates after 4 days a plurality of hickies of picking in 24 well culture plates (2 * 10
4Cells/well), cultivated 3 days for 27 ℃, collecting cell is used
32The sj14FABP gene probe of P mark carries out dot-blot DNA hybridization analysis, selects positive colony to carry out second as stated above and takes turns the plaque analysis, gets the recombinant virus Bm-sj14FABP of purifying;
5, Sj14FABP expression of gene Bm-sj14FABP infects 2 * 10
4Collecting cell and supernatant SDS-PAGE (15% separation gel) detection respectively in 1 to 6 day behind the BmN cell.5 age silkworm at link subcutaneous injection 5 μ l Bm-sj14FABP virus supernatant or BmNPV supernatant, begin to collect day by day hemolymph and silkworm body tissue next day.Hemolymph is through PBS 1: 3 dilution, tissue with PBS homogenate, frozen-thawed, it is ultrasonic, centrifugal that (11953g * 10min) supernatant is collected in the back, detects with SDS-PAGE (15% separation gel) method;
6, the purifying expression product of expression product is a fusion rotein, contains sj14FABP and from the part expressing protein that contains 6 Histidines of pBacPAK Hisl.Reference literature method NiSO
4Hemolymph is with containing 0.2molNa before the-Cheling Sepherose column purification, upper prop
2HPO
4Dilute with 0.2mol NaCl PH8.0 damping fluid; Tissue with above-mentioned damping fluid homogenate, freeze thawing, it is ultrasonic, centrifugal that (11953g * 10min) prepares the upper prop sample.Use the 100mMEDTA wash-out, survey protein content with ultraviolet absorption method;
7, the evaluation of expression product prepares antiserum(antisera) with the Sj14FABP of escherichia coli expression immunity Balb/c mouse, makes Western blot with this serum and silkworm expression product and purified product and reacts, the antigenicity of detection silkworm expression product.
Claims (2)
1, a kind of reorganization rSj14FABP of Japanese schistosome fatty acid-binding protein Sj14FABP gene 6 * his amalgamation and expression, the product that it is characterized in that this recombinant protein gene is by the expression product behind Japanese schistosome fatty acid-binding protein gene clone, reorganization, infection BmN cell and the larva, it is the fusion rotein of Sj14FABP and carrier, molecular weight is 18KD, wherein Sj14FABP is 14.8KD, and carrier part comprises 6 Histidines.
2, the preparation method of reorganization rSj14FABP as claimed in claim 1 is characterized in that this method comprises the following steps:
(1), the clone of Sj14FABP gene and sequential analysis
Go out Sj14FABP gene with PCR method quick clone with primer 5 '-CAGGATCCATGTCTAGTTTCTTGGGAAA-3 ' and primer 5 '-TCAGGATCCGCTTTTTTTTTTTTTTTTTT-3 ' schistosoma japonicum Chinese strain adult cDNA in the library, it is cloned on the pSK+ plasmid, measures its nucleotide sequence;
(2), the structure of recombinant transfer plasmid
Filter out the recon that contains the Sj14FABP gene, cut, check order with definite closure with to the sign indicating number situation with Sal I enzyme;
(3), virus reorganization
Be equipped with recombinant virus with liposome cotransfection legal system, BmNPV has introduced the LacZ gene under the alfalfa californica nuclear polyhedrosis virus AcNPV polyhedrin promoter regulation, introduce Bsu36 I site in the both sides of polyhedrosis gene ph simultaneously, cut by enzyme and to make the genomic dna linearizing, the cotransfection mixture is made up of 2 μ g linearizing viral DNAs, 3 μ g recombinant transfer plasmid DNA and isopyknic 50 μ l liposomes, and metainfective BmN cell is got the supernatant amplification once after 27 ℃ of one weeks of cultivation;
(4), the screening of recombinant virus, evaluation and purifying
Get amplification cotransfection virus supernatant and do 1: 10
-3With 1: 10
-4Dilution infects 1 * 10
5Cell 1 hour is spread the low melting-point agarose that contains 0.2mg/ml X-gal of weightmeasurement ratio 1%, cultivates after 4-6 days a plurality of hickies of picking in 24 well culture plates, 2 * 10
4Cells/well was cultivated 3-4 days for 27 ℃, and collecting cell is used
32The Sj14FABP gene probe of P mark carries out dot blot DNA hybridization analysis, selects positive colony to carry out second as stated above and takes turns the plaque analysis, gets the recombinant virus Bm-sj14FABP of purifying;
(5), Sj14FABP expression of gene
Bm-Sj14FABP infects 2 * 10
4Respectively collecting cell and supernatant detected with the SDS-PAGE of weightmeasurement ratio 15% separation gel in 1 to 6 day behind the BmN cell, 5 age silkworm at link subcutaneous injection 5 μ l Bm-sj14FABP virus supernatant, begin to collect day by day hemolymph and silkworm body tissue next day, hemolymph is through PBS dilution in 1: 3, centrifugal supernatant is collected in the back to tissue with PBS homogenate, frozen-thawed, ultrasonic, 11953g * 10min, detects with the SDS-PAGE of weightmeasurement ratio 15% separation gel;
(6), the purifying of expression product
Expression product is reorganization rSj14FABP fusion rotein, contains sj14FABP and from the expressing protein that contains 6 Histidines of pBacPAK Hisl, with His fusion rotein separator column NiSO
4Huge legendary turtle is closed the agarose gel column purification, and hemolymph is with containing 0.2mol Na before the upper prop
2HPOX, the dilution of 0.2mol NaCl pH8.0 damping fluid; Tissue prepares the upper prop sample with above-mentioned damping fluid homogenate, freeze thawing, ultrasonic, centrifugal 11953g * 10min, with 100mM EDTA wash-out, surveys protein content with ultraviolet absorption method;
(7), the evaluation of expression product
Prepare antiserum(antisera) with the Sj14FABP of escherichia coli expression immunity Balb/c mouse, make immunoblotting reaction, the antigenicity of detection silkworm expression product with this serum and silkworm expression product and purified product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB981220436A CN1158385C (en) | 1998-11-27 | 1998-11-27 | Clone of Japanese schistosome fatty acid-binding protein gene and its expression in Bombyx mori system |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB981220436A CN1158385C (en) | 1998-11-27 | 1998-11-27 | Clone of Japanese schistosome fatty acid-binding protein gene and its expression in Bombyx mori system |
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| CN100394984C (en) * | 2005-11-15 | 2008-06-18 | 华中科技大学 | Schistosoma japonicum bivalent DNA vaccine and its preparation method |
| CN1904053B (en) * | 2006-09-30 | 2011-08-10 | 华中科技大学 | Fusion gene of Japan schistosome antigen gene and constituted DNA vaccin and preparing process |
| CN101205538B (en) * | 2006-12-15 | 2010-10-13 | 中国农业科学院上海兽医研究所 | Clone, expression and use of Schistosoma Japonicum signal transduction protein Sjwnt-4 gene |
| CN105018503B (en) * | 2015-08-11 | 2018-11-02 | 西南大学 | Rainbow trout fatty acid elongase gene, recombinant expression carrier, application |
| CN114404438B (en) * | 2019-07-18 | 2023-07-21 | 北京市农林科学院 | Use of Trifluridine in the preparation of medicines for inhibiting canine parvovirus replication |
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