CN1064405C - Systematic production of recombined Nippon blood-flukes glutathione transferase by using domestic silk-worm - Google Patents
Systematic production of recombined Nippon blood-flukes glutathione transferase by using domestic silk-worm Download PDFInfo
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Abstract
The present invention relates to preparation technology of a polypeptide medicine. The present invention provides preparation technology of recombined Japanese blood fluke glutathione-transferase. The technology uses a bombyx mori nuclear polyhydrosis virus as a carrier, and bombyx mori (or pupa) is used as an expression system of a biological reactor. The present invention has the characteristics of high expression efficiency, good product activity, low preparation cost, etc. The bombyx mori resources are abundant, and the present invention for expressing an antigenic gene of a blood fluke is a new technical path with wide prospects and benefit. The present invention is suitable for mass industrial preparation.
Description
The present invention relates to the production technique of polypeptide drugs, be specifically related to the production technique of recombination Japanese blood-flukes glutathione-transferring enzyme.
Glutathione-transferase (GST) is one of first-selected vaccine of anti-schistosomiasis, at home and abroad broad research.France Pasteur's Institute has reported the clone of coding Schistosoma mansoni 28kDaGST (Sm28) and the expression in intestinal bacteria in the Capron laboratory the earliest; recombinant antigen obtains higher protection and subtracts the worm's ovum effect in the animal immune test; and prove that reorganization Sm28 has the GTS vigor, sees Nature (1987) 326:149, ParasiteImmunol (1991) 13:473 and EMBOJ (1988) 7:465 report.Nucleic acid and the aminoacid sequence of Sm28 and Schistosoma japonicum 28kDaGST (Sj28) have 77% homology, but that both contain T cell antigen determinant functional zone is different.Australia has carried out clone, reorganization and the expression work of Schistosoma japonicum Philippines strain 26 kDa-GST (SjP26) in the Mitchell laboratory, see ProNatl Acad Sci USA (1986) 83:8703 and MOL BiochemParasitol (1988) 27:249 report, but its immune effect instability, and Sj28 and Sj26 are completely different, amino acid sequence homology is seen Trans RSOC Trop MedHyg (1988) 82:885 and Mol Biothem Parasitol (1990) 40:23 report less than 20%.
The objective of the invention is to overcome above-mentioned weak point, research and development are the host with the silkworm, substitute other expression systems such as intestinal bacteria, efficiently express the SjC28kDaGST with enzymic activity in silkworm larva (or pupa).
The invention provides a kind of new production technique of recombination Japanese blood-flukes glutathione-transferring enzyme.
Technology of the present invention is to utilize tame mori system to produce recombination Japanese blood-flukes glutathione S-transferring enzyme.
Production technique of the present invention specifically comprises the following steps: (see figure 1)
The structure of Fig. 1 transferring plasmid SjC28-pBacPAK8
The SjC28 gene; Fusion gene
AcNPV flanking sequence MCS, multiple clone site; Tac, the Tac promotor; Ph polyhedron promotor.
(1) contains the structure of the recombinant bombyx mori nuclear polyhedrosis virus Bm-Sj28 of SjC28kDaGST gene; The SjC28kDaGST gene coding region is 636bp, 211 amino acid of encoding, gene 5 ' end Bam HI recognition sequence ATG 2bp of being separated by, owing to have only Hind III site in the flanking sequence in pBacPAK8 multiple clone site downstream, and also there is a Hind III site at the 21bp place of SjC28 gene, so work as the SjC28 gene with Bam HI, the KpnI orientation is packed into behind the pBacPAK8, recombinant plasmid is through Hind III restriction enzyme digestion and electrophoresis, demonstrate two kinds of fragments of 5Kb and 1.1Kb, 5.1Kb fragment then only appears in non-recombinant plasmid, the silkworm BmN cell of the recombinant transfer plasmid DNA after the amplification purification and the BmNPV DNA of linearization for enzyme restriction cotransfection monolayer culture under liposome (Lipofectin) effect, carry out dna homology reorganization in the cell, after 27 ℃ of 1 weeks of cultivation, collect supernatant liquor, do 10 respectively
-3, 5 * 10
-4With 10
-4Dilution infects 1 * 10
5 Cell 1 hour, spread 27 ℃ of the low melting-point agaroses after 4 days that contain 0.2mg/lX-gal, blue, white plaque appears, the a plurality of hickies of picking are in 24 well culture plates (1 * 104 cells/well), cultivated 72 hours in 27 ℃, collecting cell carries out SDS-PAGE (12% separation gel), and 350mA, 1 hour electrotransfer filter out positive recombinant virus plaque with the SjC28kDaGST of escherichia coli expression immunity sheep serum (r-SjC28ISS) to nitrocellulose filter (NC).Carry out second then and take turns the plaque analysis, obtain the recombinant virus Bm-Sj28 of purifying, titre is 7 * 10
6PFU/ml; SjC28 KDaGST gene is the site except that a Hind III, at 90bp, 111bp an Eco RI site is arranged respectively, and 503bp has a Bcl I site, Bm-Sj28 after these three kinds of enzyme enzymes are cut, all can in [α-
32P]-the Sjc28 probe hybridization of dCTP mark, prove that further the SjC gene is inserted into the tram (see figure 2) among the BmNPV DNA.
Fig. 2: the Southern hybridization of recombinant virus Bm-Sj28
(2) SjC28kDaGST gene efficiently expressing in silkworm larva (or pupa);
The Bm-Sj28 of purifying infects * 10 with 200ul (Mol=1.4PFU/ of infestation index cell)
6The BmN cell is cultivated after 3 days for 27 ℃, collects supernatant liquor as seed culture of viruses, and the silkworm that mulberry leaf are raised plays silkworm (or pupa) 5 ages, injects 10ul (7 * 10 under intersegmental membrane
4The PFU/ silkworm) recombinant virus is in infecting the silkworm body tissue that the back was collected hemolymph respectively on the 4th and removed midgut; See Fig. 3, Fig. 3 is that the SDS-PAGE collection of illustrative plates and the protein immunoblotting (Western Blotting) of expressing SjC28 in the silkworm larva hemolymph are analyzed.
1-4 wherein: infect the silkworm hemolymph (infecting the back the 2nd to the 5th) of Bm-Sj28,
5-6: the silkworm hemolymph (infecting the back the 2nd, the 4th) that infects BmNPV
7-8: healthy silkworm hemolymph (the 2nd, the 5th day)
(3) separation and purification SjC28kDaGST from silkworm hemolymph and tissue;
Silkworm hemolymph and tissue that above-mentioned steps is obtained add in advance with PBST (PH7.2PBS that contains 1% Triton X-100) equilibrated glutathione agarose affinity column, slowly circulate 3-5 time, fully wash post with PBST and PBS successively then, use PH9.1 at last, 50mmol/L Tris-7mmol GSH wash-out is collected each component.See Fig. 4, Fig. 4 is a SDS-PAGE purifying collection of illustrative plates.
H wherein: the purified product of silkworm larva hemolymph,
T: the purified product in the silkworm larva tissue,
A: silkworm larva hemolymph or tissue homogenate supernatant,
B: the effluent liquid behind the mistake post,
1-10 or 1-7; Each wash-out component.
The plasmid pTrcHiSB that contains the SjC28 gene in the technology of the present invention is that Huggins doctor M.C is so kind as to give by London university health and tropical medicine institute medical science parasite, and transferring plasmid pBacPAK8 (clontech company product) is the non-fusion rotein transfer vector of band polyhedrosis gene promotor.Infect the SjC28kDaGST energy that purifying obtains in the silkworm hemolymph of Sj28KDaGST and the tissue and play significant immune association reaction, prove that the SjC28kDaGST that expresses in the silkworm has stronger Schistosoma japonicum 28kD GST antigenicity with different dilution r-SJC28ISS.Preliminary mouse immune protectiveness test-results shows that the SjC28kDaGST of silkworm expression has induced 17.26% worm reduction rate in BALB/c mouse, and the 6th all ight soil egg reduction rates reach 85.38%.
Identify by Southern hybridization with explained hereafter recombination Japanese blood-flukes glutathione S-transferring enzyme of the present invention.
Characteristic measurement result with explained hereafter recombination Japanese blood-flukes glutathione S-transferring enzyme of the present invention is as follows: (Nethodes in Enzymology (1985) 113:499) carries out by literature method, total reaction volume is 1ml, PH6.0 wherein, 0.1mol/L sodium phosphate buffer 880ul, substrate GSH and 2,4-dinitrochlorobenzene (CDNB) final concentration are 1mmol/l, respectively getting the 20ul sample measures, 25 ℃ were reacted 1 minute, make blank with the substrate buffer solution that does not add sample, in 751 spectrophotometric determination OD
340, be converted into then and compare vigor.
(2) antigenic determination: carry out (Chinese animal doctor's science and technology (1991) 21:42) by the method that this laboratory is set up, purifying SjC28 with 5ug/ml concentration bag by 96 hole enzyme plates, 4 ℃ are spent the night, after PBST (PH7.2PBS that the contains 0.05%Tween-20) washing, 37 ℃ of sealings of 2% bovine serum albumin 1 hour, after the PBST washing, adding is since 1: 50 two-fold dilution's r-SjC28ISS, make 12 extent of dilution altogether, repeat 2 holes, 37 ℃ act on 2 hours, PBST washing back adds the anti-sheep IgG of horseradish peroxidase-labeled of dilution in 1: 12000,37 ℃ were reacted 1 hour, carried out the colour developing of 0.1% O-Phenylene Diamine substrate solution after the PBST washing, measured OD with the automatic microplate reader of BIO-TEK312 at last
490
(3) mouse immune protectiveness test: 6 ages in week, female BALB/c mouse was available from Shanghai cell research institute of Chinese Academy of Sciences experimental animal room.Three immune mouses of purifying SjC28kDaGST average mark, subcutaneous in conjunction with Freund's complete adjuvant (CFA) injection mouse foot pad and back for the first time, subcutaneous after 2 weeks in conjunction with Freund's incomplete adjuvant (IFA) injection mouse back, 2 weeks back intramuscular injection booster immunization, in 1 week of back of immunity for the third time, every mouse infects 40 of schistosoma japonicum cercariaes (this group oncomelania room provides) through skin of abdomen.With the freund's adjuvant group is control group, infects the 6th week of back and collects stool in mice, calculates the ight soil egg reduction rate; Infect the 7th week of back and dissect mouse, collect imago of blood fluke, calculate worm reduction rate with the portal vein perfusion art.
The above results shows: the recombinant bombyx mori nuclear polyhedrosis virus Bm-Sj28 that the present invention makes up, polyhedron gene is substituted by SjC28kDaGST structure gene, be positioned at the powerful polyhedrosis gene promotor control of BmNPV down, in silkworm larva, along with the infection of recombinant virus with duplicate, SjC28kDaGST is efficiently expressed, expression product obtains SDS-PAGE electrophoresis pure products by easy single stage method purifying, its purity can satisfy the requirement of domestic animal prevention and cure of schistosomiasis vaccine, and this is a simple and feasible production technique.Compare with the SjC28kDaGST that utilizes the intestinal bacteria system expression: the purifying SjC28kDaGST output of a silkworm is equivalent to the above culture of Escherichia coli of 50ml; GST is than vigor suitable (e. coli expression product 3.94umol/min.mg); High nearly 1 times of antigenicity (e. coli expression product is 1.5 to the highest OD value of r-SjC28ISS reaction); To the protection of mouse, then with the purify protection close (Chinese animal doctor's parasitosis (1996) 4:5) of natural GST of the SjC28kDaGST (25-31%) of escherichia coli expression and polypide, the ight soil egg reduction rate is then up to 85.38%.Once there was animal experiment to show, though to subtract the worm effect undesirable for reorganization or natural GST, higher (ParasiteImmunol (1991) 13:473 of egg reduction rate; (1993) 15:383).Because worm's ovum is the important step that schistosomicide is propagated, so the worm's ovum the number of discharge that reduces significantly in host's ight soil has very important practical significance to the control schistosomicide.In addition, be fusion rotein with the product of intestinal bacteria system expression, and with the BmNPV system expression be non-fusion rotein, therefore more approach the space conformation of natural GST.Silkworm is easy to raise, and production cost is low, and the silkworm artificial breeding technology of Jian Liing for the heavy industrialization sterile production provides condition, utilizes BmNPV-family's mori system production schistosomicide vaccine to have bright prospects and economic benefit undoubtedly in recent years.
Example 1, transferring plasmid SjC28-pBacPAK8 make up
With BumHI, the KpnI orientation pBacPAK8 that packs into, recombinant plasmid demonstrates two kinds of fragments of 5Kb and 1.1Kb through Hind III restriction enzyme digestion and electrophoresis with the SjC28kDaGST gene, and non-recombinant plasmid has only 5.1Kb fragment.The BmNPV DNA of recombinant transfer plasmid DNA and linearization for enzyme restriction is cotransfection monolayer culture silkworm BmN cell under the Lipofetin effect, carries out homologous recombination in the cell.After 27 ℃ of 1 weeks of cultivation, collect supernatant and do 10 respectively
-3, 5 * 10
-4With 10
-4Dilution infects 1 * 10
6 BmN cell 1 hour is spread the low melting point gelose that contains 0.2mg/ml X-gal, and 27 ℃ of 20 hickies of picking after 4 days are in 24 well culture plates (1 * 10
4Cells/well), cultivated 72 hours for 27 ℃, collecting cell carries out SDS-PAGE (12% separation gel) and Western Blotting, use r-SjC28ISS and screen 19 positive recombinant virus plaques, carry out second again and take turns the plaque analysis, obtain the recombinant virus Bm-Sj28 of purifying, titre is 7 * 10
6PFU/ml.
Example 2, SjC28kDaGST gene efficiently expressing and purifying in silkworm larva (or pupa).
5 silkworm larvas in age (silkworm industry institute of the Chinese Academy of Agricultural Sciences provides), product are 57A.57B * 24.46, with 7 * 10
4The dosage of PFU/ silkworm infects recombinant virus, cut off abdominal foot on the 4th day, collect the silkworm tissue of hemolymph and removal midgut, use glutathione agarose affinity chromatography column purification respectively, measure calculating through Folin-phenol method (JBiol Chem (1951) 193:265), get the 0.35ml hemolymph in every silkworm, get purifying SjC28kDaGST 1.66mg in every silkworm hemolymph, GST is 3.98umol/min.mg than vigor; The whole piece silkworm is organized on average in 0.8g (weight in wet base), can get purifying SjC28kDaGST 4.03mg, and GST is 3.74umol/min.mg than vigor.
Claims (1)
1, a kind of production technique of utilizing tame mori system to produce recombination Japanese blood-flukes glutathione-transferring enzyme is characterized in that this production technique comprises the following steps:
(1) contains the structure of the recombinant bombyx mori nuclear polyhedrosis virus Bm-Sj28 of SjC28kDaGST gene; The SjC28kDaGST gene coding region is 636bp, 211 amino acid of encoding, gene 5 ' end Bam HI recognition sequence ATG 2bp of being separated by, owing to have only Hind III site in the flanking sequence in pBacPAK8 multiple clone site downstream, and also there is a Hind III site at the 21bp place of SjC28 gene, so work as the SjC28 gene with Bam HI, the KpnI orientation is packed into behind the pBacPAK8, recombinant plasmid is through Hind III restriction enzyme digestion and electrophoresis, demonstrate two kinds of fragments of 5Kb and 1.1Kb, 5.1Kb fragment then only appears in non-recombinant plasmid, the silkworm BmN cell of the recombinant transfer plasmid DNA after the amplification purification and the BmNPV DNA of linearization for enzyme restriction cotransfection monolayer culture under the liposome effect, carry out dna homology reorganization in the cell, after 27 ℃ of 1 weeks of cultivation, collect supernatant liquor, do 10 respectively
-3, 5 * 10
-4With 10
-4Dilution infects 1 * 10
5Cell 1 hour, spread 27 ℃ of the low melting-point agaroses after 4 days that contain 0.2mg/lX-gal, blue, white plaque occurs, a plurality of hickies of picking were cultivated 72 hours in 27 ℃ in 24 well culture plates, collecting cell carries out SDS-PAGE, 350mA, 1 hour electrotransfer filter out positive recombinant virus plaque with the immune sheep serum of the SjC28kDaGST of escherichia coli expression to nitrocellulose filter, carry out second then and take turns the plaque analysis, obtain the recombinant virus Bm-Sj28 of purifying, titre is 7 * 10
6PFU/ml; SjC28 KDaGST gene is the site except that a Hind III, at 90bp, 111bp an Eco RI site is arranged respectively, and 503bp has a Bcl I site, Bm-Sj28 after these three kinds of enzyme enzymes are cut, all can with [α-
32P]-the Sjc28 probe hybridization of dCTP mark, prove that further the SjC gene is inserted into the tram among the BmNPVDNA;
(2) SjC28kDaGST gene efficiently expressing in silkworm larva;
The Bm-Sj28 of purifying infects 1 * 10 with 200ul (Mol=1.4PFU/ of infestation index cell)
6The BmN cell is cultivated after 3 days for 27 ℃, collects supernatant liquor as seed culture of viruses, and the silkworm silkworm in 5 length of time that mulberry leaf are raised is injected 10ul (7 * 10 under intersegmental membrane
4The PFU/ silkworm) recombinant virus is in infecting the silkworm body tissue that the back was collected hemolymph respectively on the 4th and removed midgut;
(3) separation and purification SjC28kDaGST from silkworm hemolymph and tissue;
Silkworm hemolymph and tissue that above-mentioned steps is obtained add in advance with PBST (PH7.2PBS that contains 1% Triton X-100) equilibrated glutathione agarose affinity column, slowly circulate 3-5 time, fully wash post with PBST and PBS successively then, use PH9.1 at last, 50mmol/L Tris-7mmol GSH wash-out is collected each component.
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| CN113045671A (en) * | 2021-01-29 | 2021-06-29 | 长春万成生物电子工程有限公司 | Recombinant antigen for detecting schistosoma japonicum, preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN85104701A (en) * | 1984-06-21 | 1986-12-17 | 第一制药株式会社 | Produce the method for useful matter |
| CN86107784A (en) * | 1985-11-14 | 1987-06-03 | 第一制药株式会社 | method for producing peptide |
| CN87104489A (en) * | 1986-05-19 | 1988-05-04 | 希巴-盖吉股份公司 | Herbicide tolerant plants containing glutathione S-transferase gene |
| CN1096541A (en) * | 1994-01-24 | 1994-12-21 | 南京大学 | Recombination of human macrophage colony stimulating factor by domestic silkworm gene engineering |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN85104701A (en) * | 1984-06-21 | 1986-12-17 | 第一制药株式会社 | Produce the method for useful matter |
| CN86107784A (en) * | 1985-11-14 | 1987-06-03 | 第一制药株式会社 | method for producing peptide |
| CN87104489A (en) * | 1986-05-19 | 1988-05-04 | 希巴-盖吉股份公司 | Herbicide tolerant plants containing glutathione S-transferase gene |
| CN1096541A (en) * | 1994-01-24 | 1994-12-21 | 南京大学 | Recombination of human macrophage colony stimulating factor by domestic silkworm gene engineering |
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