CN1706496A - Genetically engineered vaccine resisting vibriosis of marine cultured animal and its prepn and use - Google Patents
Genetically engineered vaccine resisting vibriosis of marine cultured animal and its prepn and use Download PDFInfo
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- CN1706496A CN1706496A CN 200510034662 CN200510034662A CN1706496A CN 1706496 A CN1706496 A CN 1706496A CN 200510034662 CN200510034662 CN 200510034662 CN 200510034662 A CN200510034662 A CN 200510034662A CN 1706496 A CN1706496 A CN 1706496A
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- extracellular toxin
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Abstract
The present invention relates to genetically engineered vaccine resisting vibriosis of marine cultured animal and its preparation and use. The genetically engineered vaccine is the product of expressing minimal vibrio ectotoxin gene and is inactivated with formalin. It produces high immunological protection in the immunological protection rate as high as 84 %. It may be used in immunizing several kinds of marine cultured fish to raise the yield of marine culture.
Description
Technical field:
The present invention relates to recombinant vaccine of a kind of anti-vibrisis of seawater and preparation method thereof.
Background technology:
Vibriosis is an epidemic diseases common in the marine cultured animal, in existing technology, has only the production and the application of Vibrio anguillarum thalline vaccine at present, does not see the production and the application of vibrio recombinant vaccine as yet.Vibrio anguillarum thalline vaccine has more single-minded anti-vibrio specificity in aquaculture production, the disease that anti-vibrio anguillarum is caused has higher resistance, but it is single-minded just because of its effect, defense reaction to the pathogenic microbial disease of other vibrio is relatively poor, has very significantly limitation in production application.In addition, the deactivation of link adding in the end medicine residues in the vaccine on the production process of traditional bacterial vaccine, causes existing side effect.
Summary of the invention:
At the deficiencies in the prior art, the object of the present invention is to provide a kind of recombinant vaccine of anti-vibrisis of seawater, this vaccine is fit to do the immunizing agent that multiple marine cultured animal improves the anti-vibriosis of cultured output, extracellular toxin expression of gene product is made after the formalin deactivation, the generation strain is a vibrio mimicus, and its characteristic can be entirely the effect animal and accept.The preparation of the recombinant vaccine of anti-vibrisis of seawater of the present invention realizes by following steps:
1, the design of the mensuration of extracellular toxin aminoacid sequence and genes of interest probe
The extracellular toxin of having purified is carried out amino acid sequencing, according to the anti-possible nucleotide sequence of extracellular toxin gene coding region of releasing of aminoacid sequence, use the external synthetic oligonucleotide fragment of dna synthesizer in view of the above, with making probe behind the DIG labelling, in order to screening and detection extracellular toxin gene.
2, the clone of extracellular toxin gene and expression
Utilize method separation target DNA fragments such as gel electrophoresis separation.Target DNA fragment is connected back transformed into escherichia coli competent cell with cloning vector plasmids, with the alkaline lysis method of extracting plasmid, detect with vibrio mimicus extracellular toxin gene probe, confirm that inserting fragment in the recombiant plasmid is the target DNA fragment that contains vibrio mimicus extracellular toxin gene.
Target DNA and expression vector plasmid reorganization back transformed into escherichia coli, yeast culture thing ultrasonic disruption, lysate is made SDS-PAGE, and makes Western blotting with vibrio mimicus extracellular toxin monoclonal antibody, to detect expressed extracellular toxin.
3, the preparation of recombinant vaccine
Extracellular toxin gene and expression vector plasmid reorganization back transformed into escherichia coli adopt biochemical methods such as grade ammonium sulfate salting-out, DEAE-cellulose chromatography and Sephadex-G100 chromatography the culture supernatant to be carried out the purification of extracellular toxin gene expression product.Be that 0.2% formalin carries out inactivation treatment to expression product with concentration, the inactivated vaccine that obtains is a recombinant vaccine.
The production technology of vibrio recombinant vaccine provided by the invention, specific solution is achieved in that
1, after the common sea water Nutrient medium of vibrio mimicus activation back inoculation, 30 ℃, 200rpm shaking table were cultivated 18-20 hour, and culture centrifugal 30 minutes through 10000rpm is got supernatant.
2, ectotoxic purification: adopt biochemical methods such as ammonium sulfate precipitation, DEAE-cellulose chromatography and Sephadex-G100 chromatography to purify.
3, the extracellular toxin of having purified is carried out amino acid sequencing,, use the external synthetic oligonucleotide fragment of dna synthesizer in view of the above, with making probe behind the DIG labelling according to the anti-possible nucleotide sequence of extracellular toxin gene coding region of releasing of aminoacid sequence.
4, use phenol-chloroform method and extract the vibrio mimicus chromosomal DNA.Select suitable restricted enzyme to utilize local digestion method to handle genomic DNA, gel electrophoresis separates, and makes Southern blotting, and the DNA that is transferred on the NC film detects with vibrio mimicus extracellular toxin gene probe.Cut and the corresponding gel strips in positive reaction band position, reclaim test kit with DNA and reclaim target DNA fragment.
Behind the cloning vector plasmids enzyme action, be connected with the target DNA fragment that reclaims, connect product transformed into escherichia coli competent cell, after cultivating 16-18 hour, with the alkaline lysis method of extracting plasmid, detect with vibrio mimicus extracellular toxin gene probe, confirm that inserting fragment in the recombiant plasmid is the target DNA fragment that contains vibrio mimicus extracellular toxin gene.
5, extracellular toxin gene and expression vector plasmid reorganization back transformed into escherichia coli carries out the purification of extracellular toxin gene expression product to the culture supernatant.Be that 0.2% formalin carries out inactivation treatment to expression product with concentration, the inactivated vaccine that obtains is the recombinant vaccine Seedling.
The effect of vaccine of the present invention in production application:
The purposes of anti-vibrisis of seawater recombinant vaccine provided by the present invention is the immunity that can be applicable to vibriosis of marine cultured animal, and this vaccine can produce immune protective preferably, and its immune protective rate on average can reach 84%.This vaccine is applicable to immune multiple marine fish, and has the effect that improves cultured output.The vaccine that utilizes above-mentioned preparation is through the immersion immunity cultivated animals, and its protection effect sees the following form:
The trial effect of table 1 vaccine in the cage culture snapper
| The place | Group | Experimental period (moon) | Experiment fish (tail) | Dead fish (tail) | Mortality rate (%) | Survival rate (%) |
| Shenzhen | The immune group matched group | ??6 ??6 | ??400 ??400 | ??65 ??180 | ??16.3 ??45 | ??83.7 ??55 |
| Zhanjiang | The immune group matched group | ??6 ??6 | ??400 ??400 | ??55 ??155 | ??13.8 ??38.8 | ??86.2 ??61.2 |
The trial effect of table 2 vaccine in the cage culture cabrilla
| The place | Group | Experimental period (moon) | Experiment fish (tail) | Dead fish (tail) | Mortality rate (%) | Survival rate (%) |
| Shenzhen | The immune group matched group | ??6 ??6 | ??400 ??400 | ??96 ??211 | ??24.0 ??52.8 | ??76.0 ??47.2 |
| Zhanjiang | The immune group matched group | ??6 ??6 | ??400 ??400 | ??85 ??199 | ??21.3 ??49.8 | ??78.7 ??50.2 |
The specific embodiment:
Example 1:
1, the activatory vibrio mimicus 10ml that learns from else's experience is inoculated in the filtered common sea water Nutrient medium of 1000ml, and 30 ℃, 200rpm shaking table were cultivated 18-20 hour, and culture centrifugal 30 minutes through 10000rpm is got supernatant.
2, ectotoxic purification: adopt biochemical methods such as ammonium sulfate precipitation, DEAE-cellulose chromatography and Sephadex-G100 chromatography to purify.
Grade ammonium sulfate salting-out: the saturation that adds solid ammonium sulfate to 20% in the centrifugal supernatant, place 4 ℃ to leave standstill 4 hours, centrifugal 30 minutes of 10000rpm abandons precipitation, adds the saturation of solid ammonium sulfate to 60% again in supernatant, place 4 ℃ to leave standstill 4 hours, centrifugal 30 minutes of 10000rpm removes supernatant, and precipitation is dissolved in the Tris-Hcl that concentration is 50mmol/L (pH7.8) buffer, to the same buffer dialysed overnight, collect to be and slightly carry extracellular toxin 1.
DEAE-cellulose ion-exchange chromatography: slightly carry extracellular toxin 1 and add on the Balanced DEAE-cellulose column (Phamacia product, 2.6cm * 30cm).With concentration is the NaCl of 0 ~ 1mol/L and Tris-Hcl (pH7.8) buffer solution for gradient elution that concentration is 50mmol/L, and flow velocity is 5ml/cm
2/ hour, every 4ml collects a pipe, measures the haemolysis valency of every pipe simultaneously.Merging the protein peak eluent, is Tris-Hcl (pH7.8) the buffer dialysed overnight of 50mmol/L with concentration.Concentrate glue with Polyethylene Glycol PEG100 and concentrate, and then be Tris-Hcl (pH7.8) the buffer dialysed overnight of 50mmol/L, be and slightly carry extracellular toxin 2 concentration.
Sephadex-G100 chromatography: slightly carry extracellular toxin 2 and add in the Balanced Sephadex-G100 chromatographic column that (flow velocity is 6ml/cm for Phamacia product, 1.6cm * 100cm), be Tris-Hcl (pH7.8) buffer solution elution of 50mmol/L with concentration
2/ hour, every 4ml collects a pipe, measures the haemolysis valency of every pipe simultaneously.Merge protein peak, concentrate glue with PEG100 and concentrate, and then be Tris-Hcl (pH7.8) the buffer dialysed overnight of 50mmol/L, be the purification extracellular toxin concentration.
3, the extracellular toxin of having purified is carried out amino acid sequencing, according to the anti-possible nucleotide sequence of extracellular toxin gene coding region of releasing of aminoacid sequence, use the external synthetic oligonucleotide fragment of dna synthesizer in view of the above, with making probe behind the DIG labelling.
4, use phenol-chloroform method and extract the vibrio mimicus chromosomal DNA.Select suitable restricted enzyme to utilize local digestion method to handle genomic DNA, gel electrophoresis separates, and makes Southernblotting, and the DNA that is transferred on the NC film detects with vibrio mimicus extracellular toxin gene probe.Cut and the corresponding gel strips in positive reaction band position, reclaim test kit with DNA and reclaim target DNA fragment.
Behind the cloning vector plasmids enzyme action, be connected with the target DNA fragment that reclaims, connect product transformed into escherichia coli competent cell, after cultivating 16-18 hour, with the alkaline lysis method of extracting plasmid, detect with vibrio mimicus extracellular toxin gene probe, confirm that inserting fragment in the recombiant plasmid is the target DNA fragment that contains vibrio mimicus extracellular toxin gene.
5, extracellular toxin gene and expression vector plasmid reorganization back transformed into escherichia coli adopt biochemical methods such as ammonium sulfate precipitation, DEAE-cellulose chromatography and Sephadex-G100 chromatography the culture supernatant to be carried out the purification of extracellular toxin gene expression product.Be that 0.2% formalin carries out inactivation treatment to expression product with concentration, the inactivated vaccine that obtains is a recombinant vaccine.
Example 2:
1, the activatory vibrio mimicus 10ml that learns from else's experience is inoculated in the filtered common sea water Nutrient medium of 1000ml, and 27 ℃, 200rpm shaking table were cultivated 20-24 hour, and culture centrifugal 30 minutes through 10000rpm is got supernatant.
2, ectotoxic purification: adopt biochemical methods such as ammonium sulfate precipitation, DEAE-cellulose chromatography and Sephadex-G100 chromatography to purify.
Grade ammonium sulfate salting-out: the saturation that adds solid ammonium sulfate to 20% in the centrifugal supernatant, place 4 ℃ to leave standstill 4 hours, centrifugal 30 minutes of 10000rpm abandons precipitation, adds the saturation of solid ammonium sulfate to 60% again in supernatant, place 4 ℃ to leave standstill 4 hours, centrifugal 30 minutes of 10000rpm removes supernatant, and precipitation is dissolved in the Tris-Hcl that concentration is 50mmol/L (pH7.8) buffer, to the same buffer dialysed overnight, collect to be and slightly carry extracellular toxin 1.
DEAE-cellulose ion-exchange chromatography: slightly carry extracellular toxin 1 and add on the Balanced DEAE-cellulose column (Phamacia product, 2.6cm * 30cm).With concentration is the NaCl of 0 ~ 1mol/L and Tris-Hcl (pH7.8) buffer solution for gradient elution that concentration is 50mmol/L, and flow velocity is 5ml/cm
2/ hour, every 4ml collects a pipe, measures the haemolysis valency of every pipe simultaneously.Merging the protein peak eluent, is Tris-Hcl (pH7.8) the buffer dialysed overnight of 50mmol/L with concentration.Concentrate glue with Polyethylene Glycol PEG100 and concentrate, and then be Tris-Hcl (pH7.8) the buffer dialysed overnight of 50mmol/L, be and slightly carry extracellular toxin 2 concentration.
Sephadex-G100 chromatography: slightly carry extracellular toxin 2 and add in the Balanced Sephadex-G100 chromatographic column that (flow velocity is 6ml/cm for Phamacia product, 1.6cm * 100cm), be Tris-Hcl (pH7.8) buffer solution elution of 50mmol/L with concentration
2/ hour, every 4ml collects a pipe, measures the haemolysis valency of every pipe simultaneously.Merge protein peak, concentrate glue with PEG100 and concentrate, and then be Tris-Hcl (pH7.8) the buffer dialysed overnight of 50mmol/L, be the purification extracellular toxin concentration.
3, the extracellular toxin of having purified is carried out amino acid sequencing, according to the anti-possible nucleotide sequence of extracellular toxin gene coding region of releasing of aminoacid sequence, use the external synthetic oligonucleotide fragment of dna synthesizer in view of the above, with making probe behind the DIG labelling.
4, use phenol-chloroform method and extract the vibrio mimicus chromosomal DNA.Select suitable restricted enzyme to utilize local digestion method to handle genomic DNA, gel electrophoresis separates, and makes Southernblotting, and the DNA that is transferred on the NC film detects with vibrio mimicus extracellular toxin gene probe.Cut and the corresponding gel strips in positive reaction band position, reclaim test kit with DNA and reclaim target DNA fragment.
Behind the cloning vector plasmids enzyme action, be connected with the target DNA fragment that reclaims, connect product transformed into escherichia coli competent cell, after cultivating 16-18 hour, with the alkaline lysis method of extracting plasmid, detect with vibrio mimicus extracellular toxin gene probe, confirm that inserting fragment in the recombiant plasmid is the target DNA fragment that contains vibrio mimicus extracellular toxin gene.
5, extracellular toxin gene and expression vector plasmid reorganization back transformed into escherichia coli adopt biochemical methods such as ammonium sulfate precipitation, DEAE-cellulose chromatography and Sephadex-G100 chromatography the culture supernatant to be carried out the purification of extracellular toxin gene expression product.Be that 0.2% formalin carries out inactivation treatment to expression product with concentration, the inactivated vaccine that obtains is a recombinant vaccine.
Example 3:
1, the activatory vibrio mimicus 10ml that learns from else's experience is inoculated in the filtered common sea water Nutrient medium of 1000ml, and 37 ℃, 200rpm shaking table were cultivated 18-20 hour, and culture centrifugal 30 minutes through 10000rpm is got supernatant.
2, ectotoxic purification: adopt biochemical methods such as ammonium sulfate precipitation, DEAE-cellulose chromatography and Sephadex-G100 chromatography to purify.Grade ammonium sulfate salting-out: the saturation that adds solid ammonium sulfate to 20% in the centrifugal supernatant, place 4 ℃ to leave standstill 4 hours, centrifugal 30 minutes of 10000rpm abandons precipitation, adds the saturation of solid ammonium sulfate to 60% again in supernatant, place 4 ℃ to leave standstill 4 hours, centrifugal 30 minutes of 10000rpm removes supernatant, and precipitation is dissolved in the Tris-Hcl that concentration is 50mmol/L (pH7.8) buffer, to the same buffer dialysed overnight, collect to be and slightly carry extracellular toxin 1.
DEAE-cellulose ion-exchange chromatography: slightly carry extracellular toxin 1 and add on the Balanced DEAE-cellulose column (Phamacia product, 2.6cm * 30cm).With concentration is the NaCl of 0 ~ 1mol/L and Tris-Hcl (pH7.8) buffer solution for gradient elution that concentration is 50mmol/L, and flow velocity is 5ml/cm
2/ hour, every 4ml collects a pipe, measures the haemolysis valency of every pipe simultaneously.Merging the protein peak eluent, is Tris-Hcl (pH7.8) the buffer dialysed overnight of 50mmol/L with concentration.Concentrate glue with Polyethylene Glycol PEG100 and concentrate, and then be Tris-Hcl (pH7.8) the buffer dialysed overnight of 50mmol/L, be and slightly carry extracellular toxin 2 concentration.
Sephadex-G100 chromatography: slightly carry extracellular toxin 2 and add in the Balanced Sephadex-G100 chromatographic column that (flow velocity is 6ml/cm for Phamacia product, 1.6cm * 100cm), be Tris-Hcl (pH7.8) buffer solution elution of 50mmol/L with concentration
2/ hour, every 4ml collects a pipe, measures the haemolysis valency of every pipe simultaneously.Merge protein peak, concentrate glue with PEG100 and concentrate, and then be Tris-Hcl (pH7.8) the buffer dialysed overnight of 50mmol/L, be the purification extracellular toxin concentration.
3, the extracellular toxin of having purified is carried out amino acid sequencing, according to the anti-possible nucleotide sequence of extracellular toxin gene coding region of releasing of aminoacid sequence, use the external synthetic oligonucleotide fragment of dna synthesizer in view of the above, with making probe behind the DIG labelling.
4, use phenol-chloroform method and extract the vibrio mimicus chromosomal DNA.Select suitable restricted enzyme to utilize local digestion method to handle genomic DNA, gel electrophoresis separates, and makes Southernblotting, and the DNA that is transferred on the NC film detects with vibrio mimicus extracellular toxin gene probe.Cut and the corresponding gel strips in positive reaction band position, reclaim test kit with DNA and reclaim target DNA fragment.
Behind the cloning vector plasmids enzyme action, be connected with the target DNA fragment that reclaims, connect product transformed into escherichia coli competent cell, after cultivating 16-18 hour, with the alkaline lysis method of extracting plasmid, detect with vibrio mimicus extracellular toxin gene probe, confirm that inserting fragment in the recombiant plasmid is the target DNA fragment that contains vibrio mimicus extracellular toxin gene.
5, extracellular toxin gene and expression vector plasmid reorganization back transformed into escherichia coli adopt biochemical methods such as ammonium sulfate precipitation, DEAE-cellulose chromatography and Sephadex-G100 chromatography the culture supernatant to be carried out the purification of extracellular toxin gene expression product.Be that 0.2% formalin carries out inactivation treatment to expression product with concentration, the inactivated vaccine that obtains is a recombinant vaccine.
Claims (3)
1, a kind of recombinant vaccine of anti-vibrisis of seawater is characterized in that this vaccine by preparing for vibrio mimicus extracellular toxin expression of gene product described in the claim 2 after the formalin deactivation, the generation strain is a vibrio mimicus.
2, the described anti-vibrisis of seawater recombinant vaccine of a kind of preparation claim 1 method is characterized in that this method comprises the steps:
(1), after the common sea water Nutrient medium of vibrio mimicus activation back inoculation, 30 ℃, 200rpm shaking table were cultivated 18-20 hour;
(2), adopt ammonium sulfate precipitation, DEAE-cellulose chromatography and Sephadex-G100 chromatography method to culture supernatant extracellular toxin separate, purification;
(3), the extracellular toxin of having purified is carried out amino acid sequencing, according to the anti-possible nucleotide sequence of extracellular toxin gene coding region of releasing of aminoacid sequence, use the external synthetic oligonucleotide fragment of dna synthesizer in view of the above, with making probe behind the DIG labelling, in order to screening and detection extracellular toxin gene;
(4), extracellular toxin gene and expression vector plasmid reorganization back transformed into escherichia coli, the culture supernatant is carried out the purification of extracellular toxin gene expression product, be that 0.2% formalin carries out inactivation treatment to expression product with concentration, the inactivated vaccine that obtains is the anti-vibrisis of seawater recombinant vaccine.
3, the purposes of the recombinant vaccine of anti-vibrisis of seawater described in a kind of claim 1 is characterized in that this vaccine is applicable to immune multiple marine fish, and has the effect that improves cultured output.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101926986A (en) * | 2010-08-20 | 2010-12-29 | 中国科学院南海海洋研究所 | Vibriosis-resisting attenuated live vaccine and preparation method and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101926986A (en) * | 2010-08-20 | 2010-12-29 | 中国科学院南海海洋研究所 | Vibriosis-resisting attenuated live vaccine and preparation method and application thereof |
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