CN1064405C - Systematic production of recombined Nippon blood-flukes glutathione transferase by using domestic silk-worm - Google Patents

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

Production of Recombinant Schistosoma japonicum Glutathione-Transferase Using Silkworm System

The invention relates to a production process of polypeptide medicine, in particular to a production process of recombinant Schistosoma japonicum glutathione-transferase.

Glutathione-transferase (GST) is one of the first-choice vaccines against schistosomiasis, and has been extensively studied at home and abroad. The Capron laboratory of the Institut Pasteur in France first reported the cloning and expression of Schistosoma mansoni 28kDaGST (Sm28) in Escherichia coli. Proof that recombinant Sm28 has GTS activity, see Nature (1987) 326:149, Parasite Immunol (1991) 13:473 and EMBOJ (1988) 7:465 reports. The nucleotide and amino acid sequences of Sm28 and Schistosoma japonicum 28kDaGST (Sj28) have 77% homology, but the two have different T cell antigenic determinant functional regions. Australian Mitchell laboratory carried out the cloning, recombination and expression work of Schistosoma japonicum Philippine strain 26 kDa-GST (SjP26), see ProNatl Acad Sci USA (1986) 83: 8703 and MOL Biochem Parasitol (1988) 27: 249 report, but its immune The effect is unstable, and Sj28 and Sj26 are completely different, and the amino acid sequence homology is less than 20%, see Trans RSOC Trop MedHyg (1988) 82:885 and Mol Biothem Parasitol (1990) 40:23 reports.

The purpose of the present invention is to overcome the above disadvantages, research and develop silkworm as a host, instead of other expression systems such as Escherichia coli, and efficiently express SjC28kDaGST with enzymatic activity in silkworm larvae (or pupae).

The invention provides a new production process of recombinant Schistosoma japonicum glutathione-transferase.

The technology of the invention is to use the silkworm system to produce recombinant schistosoma japonicum glutathione S-transferase.

Production technique of the present invention specifically comprises the following steps: (see Fig. 1)

Figure 1 Construction of transfer plasmid SjC28-pBacPAK8

SjC28 gene; fusion gene

  AcNPV flanking sequence MCS, multiple cloning site; Tac, Tac promoter; Ph polyhedron promoter.

(1) Construction of the recombinant silkworm nuclear polyhedrosis virus Bm-Sj28 containing the SjC28kDaGST gene; the SjC28kDaGST gene coding region is 636bp, encoding 211 amino acids, and the Bam HI recognition sequence ATG at the 5' end of the gene is separated by 2bp, due to the multiple cloning sites of pBacPAK8 There is only a HindⅢ site in the downstream side sequence, and there is also a HindⅢ site at 21 bp of the SjC28 gene, so when the SjC28 gene is loaded into pBacPAK8 with Bam HI and KpnI, the recombinant plasmid is subjected to HindⅢ digestion and electrophoresis. Two fragments of 5Kb and 1.1Kb were shown, and only one fragment of 5.1Kb appeared in the non-recombinant plasmid. Co-transfect monolayer cultured silkworm BmN cells, carry out intracellular DNA homologous recombination, and culture at 27°C for 1 week, collect the supernatant, make 10 ^-3 , 5×10 ^-4 and 10 ^-4 dilutions respectively, and infect 1 ×10 ⁵ cells for 1 hour, spread low-melting point agarose containing 0.2 mg/lX-gal at 27°C for 4 days, blue and white spots appeared, pick multiple white spots on 24-well culture plate (1×104 cells/ Wells), cultured at 27°C for 72 hours, collected cells for SDS-PAGE (12% gel), 350mA, 1 hour electrotransfer to nitrocellulose membrane (NC), immune sheep serum (r -SjC28ISS) to screen out positive recombinant virus plaques. Then the second round of plaque analysis was carried out to obtain the purified recombinant virus Bm-Sj28 with a titer of 7×10 ⁶ PFU/ml; the SjC28 KDaGST gene had an Eco RI site at 90bp and 111bp respectively except for a HindⅢ site. There is a BclⅠ site at 503bp, and Bm-Sj28 can be hybridized with [α- ^32P ]-dCTP-labeled Sjc28 probe after digested by these three enzymes, which further proves that the SjC gene is inserted into the correct position of BmNPV DNA ( See Figure 2).

Figure 2: Southern hybridization of recombinant virus Bm-Sj28

(2) High expression of SjC28kDaGST gene in silkworm larvae (or pupae);

Purified Bm-Sj28 was used to infect ×10 ⁶ BmN cells with 200ul (infection index Mol=1.4PFU/cell), cultured at 27°C for 3 days, and the supernatant was collected as virus seeds. Pupa), inject 10ul (7×10 ⁴ PFU/silkworm) recombinant virus under the intersegmental membrane, and collect the hemolymph and remove the silkworm body tissue in the midgut on the 4th day after infection; see Figure 3, Figure 3 is silkworm larvae SDS-PAGE pattern and Western Blotting analysis of SjC28 expressed in hemolymph.

Among them 1-4: hemolymph of silkworm infected with Bm-Sj28 (2nd to 5th day after infection),

5-6: Bombyx mori hemolymph infected with BmNPV (2nd and 4th day after infection)

7-8: Healthy silkworm hemolymph (day 2, day 5)

(3) Separation and purification of SjC28kDaGST from silkworm hemolymph and tissue;

The silkworm hemolymph and tissues obtained in the above steps were added to a glutathione agarose affinity chromatography column equilibrated with PBST (pH7.2PBS containing 1% Triton X-100) in advance, circulated slowly for 3-5 times, and then The column was thoroughly washed with PBST and PBS in turn, and finally eluted with pH 9.1, 50mmol/L Tris-7mmol GSH, and all fractions were collected. See Figure 4, Figure 4 is the SDS-PAGE purification profile.

Wherein H: the purified product of silkworm larval hemolymph,

T: Purified product from Bombyx mori larvae tissue,

A: Bombyx mori larva hemolymph or tissue homogenate supernatant,

B: The effluent after passing through the column,

1-10 or 1-7; each elution fraction.

The plasmid pTrcHiSB containing the SjC28 gene in the process of the present invention was obtained by Huggins M. Donated by Dr. C, the transfer plasmid pBacPAK8 (product of clontech company) is a non-fusion protein transfer vector with polyhedrin gene promoter. SjC28kDaGST purified from the hemolymph and tissues of silkworms infected with Sj28KDaGST can have significant immunological binding reactions with different dilutions of r-SJC28ISS, which proves that SjC28kDaGST expressed in silkworms has strong antigenicity to Schistosoma japonicum 28kD GST. The results of the preliminary immune protection test in mice showed that SjC28kDaGST expressed by silkworm induced a 17.26% worm reduction rate in BALB/c mice, and the fecal egg reduction rate reached 85.38% at the sixth week.

The recombinant Schistosoma japonicum glutathione S-transferase produced by the process of the invention is identified by Southern hybridization.

The characteristic measurement result of producing recombinant Schistosoma japonicum glutathione S-transferase with process of the present invention is as follows: Carry out by literature method (Nethodes in Enzymology (1985) 113:499), total reaction volume is 1ml, wherein PH6.0,0 ．1mol/L sodium phosphate buffer solution 880ul, the final concentration of substrate GSH and 2,4-dinitrochlorobenzene (CDNB) is 1mmol/l, take 20ul samples each for determination, react at 25°C for 1 minute, without adding samples The substrate buffer solution was used as a blank control, and the OD ₃₄₀ was measured with a 751 spectrophotometer, and then converted into specific activity.

(2) Determination of antigenicity: according to the method established by our laboratory (Chinese Veterinary Science and Technology (1991) 21:42), the purified SjC28 was coated with 96-well microtiter plate at a concentration of 5ug/ml, overnight at 4°C, PBST (containing 0 .05% Tween-20 pH7.2PBS) after washing, 2% bovine serum albumin at 37 ℃ for 1 hour, after washing with PBST, add r-SjC28ISS starting from 1:50, making 12 dilutions in total , repeat for 2 wells, act at 37°C for 2 hours, add horseradish peroxidase-labeled anti-sheep IgG diluted 1:12000 after washing with PBST, react at 37°C for 1 hour, wash with PBST and carry out 0.1% o-phenylenediamine substrate The solution was developed for color development, and finally the OD ₄₉₀ was measured with a BIO-TEK312 automatic microplate reader.

(3) Mice immune protection test: 6-week-old female BALB/c mice were purchased from the experimental animal room of Shanghai Institute of Cell Research, Chinese Academy of Sciences. The purified SjC28kDaGST was averagely divided into three times to immunize the mice. The first time combined with Freund's complete adjuvant (CFA) to inject the mice's foot pads and back subcutaneously, and after 2 weeks, combined with Freund's incomplete adjuvant (IFA) to inject the mouse back subcutaneously. One week later, intramuscular injection was given to boost the immunization. One week after the third immunization, each mouse was infected with 40 cercariae of Schistosoma japonicum (provided by the snail house of this group) through the abdominal skin. The Freund's adjuvant group was used as the control group. The feces of the mice were collected at 6 weeks after infection, and the fecal egg reduction rate was calculated; the mice were dissected at the 7th week after infection, and adult schistosomes were collected by portal vein perfusion, and the rate of worm reduction was calculated.

The above results show that: in the recombinant silkworm nuclear polyhedrosis virus Bm-Sj28 constructed by the present invention, the polyhedrin gene is replaced by the SjC28kDaGST structural gene, which is located under the control of the powerful polyhedrosis gene promoter of BmNPV. In silkworm larvae, along with the recombinant virus SjC28kDaGST was highly expressed, and the expression product was purified by a simple one-step method to obtain a pure product by SDS-PAGE electrophoresis. The purity can meet the requirements of livestock schistosomiasis vaccines. This is a simple and feasible production process. Compared with the SjC28kDaGST expressed in the E. coli system: the purified SjC28kDaGST output of a silkworm is equivalent to more than 50ml of E. coli culture; the specific activity of GST is equivalent (3.94umol/min.mg of E. coli expression product); the antigenicity is nearly 1 times higher (The highest OD value of Escherichia coli expression product to r-SjC28ISS reaction is 1.5); the protective power to mice is similar to that of SjC28kDaGST (25-31%) expressed in Escherichia coli and the natural GST purified from parasites (Chinese Veterinary Parasitic Diseases (1996) 4:5), while the egg reduction rate in feces is as high as 85.38%. Animal experiments have shown that although the effect of recombinant or natural GST is not satisfactory, the egg reduction rate is higher (ParasiteImmunol (1991) 13:473; (1993) 15:383). Because eggs are an important link in the transmission of schistosomiasis, it is of great practical significance to greatly reduce the number of eggs excreted in the host's feces to control schistosomiasis. In addition, the product expressed by the E. coli system is a fusion protein, while the product expressed by the BmNPV system is a non-fusion protein, so it is closer to the spatial conformation of the natural GST. Bombyx mori is easy to raise and the production cost is low. The artificial breeding technology of silkworm established in recent years has provided conditions for large-scale industrial aseptic production. The production of schistosomiasis vaccine using the BmNPV-bombyx mori system undoubtedly has broad prospects and economic benefits.

Example 1, construction of transfer plasmid SjC28-pBacPAK8

The SjC28kDaGST gene was oriented into pBacPAK8 with BumHI and KpnI, and the recombinant plasmid was subjected to HindⅢ digestion and electrophoresis, showing two fragments of 5Kb and 1.1Kb, and only one fragment of 5.1Kb in the non-recombinant plasmid. Recombinant transfer plasmid DNA and enzyme-digested linearized BmNPV DNA were co-transfected into monolayer cultured BmN cells of silkworm under the action of Lipofetin for intracellular homologous recombination. After culturing at 27°C for 1 week, collect the supernatant and make dilutions of 10 ^-3 , 5×10 ^-4 and 10 ^-4 respectively, infect 1×10 ⁶ BmN cells for 1 hour, and spread the low-lying medium containing 0.2 mg/ml X-gal Melting point agarose, after 4 days at 27°C, pick 20 white spots on a 24-well culture plate (1×10 ⁴ cells/well), culture at 27°C for 72 hours, collect cells for SDS-PAGE (12% separating gel) and Western Blotting , 19 positive recombinant virus plaques were screened by r-SjC28ISS, and the second round of plaque analysis was performed to obtain purified recombinant virus Bm-Sj28 with a titer of 7×10 ⁶ PFU/ml.

Example 2. High-efficiency expression and purification of SjC28kDaGST gene in silkworm larvae (or pupae).

5th instar silkworm larvae (provided by the Institute of Sericulture, Chinese Academy of Agricultural Sciences), strain 57A. 57B×24.46, infected with the recombinant virus at a dose of 7×10 ⁴ PFU/silkworm, cut off the gastropods on the 4th day, collected the hemolymph and removed the silkworm tissues from the midgut, and used glutathione agarose affinity layer respectively Purified by column analysis, measured and calculated by Folin-phenol method (JBiol Chem (1951) 193:265), based on 0.35ml hemolymph obtained from each silkworm, 1.66mg of purified SjC28kDaGST was obtained in each silkworm hemolymph, GST specific activity It is 3.98umol/min. mg; the average whole silkworm tissue is 0.8g (wet weight), and the purified SjC28kDaGST can be 4.03mg, and the specific activity of GST is 3.74umol/min. mg.

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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