CN102242147B - Method for synthesizing and secreting rabies virus nucleoprotein in middle silkworm silk-gland cells - Google Patents
Method for synthesizing and secreting rabies virus nucleoprotein in middle silkworm silk-gland cells Download PDFInfo
- Publication number
- CN102242147B CN102242147B CN2011101235867A CN201110123586A CN102242147B CN 102242147 B CN102242147 B CN 102242147B CN 2011101235867 A CN2011101235867 A CN 2011101235867A CN 201110123586 A CN201110123586 A CN 201110123586A CN 102242147 B CN102242147 B CN 102242147B
- Authority
- CN
- China
- Prior art keywords
- silkworm
- rabies virus
- plasmid
- gene
- virus nucleoprotein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 241000255789 Bombyx mori Species 0.000 title claims abstract description 108
- 101900236200 Rabies virus Nucleoprotein Proteins 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 24
- 230000003248 secreting effect Effects 0.000 title abstract 4
- 230000002194 synthesizing effect Effects 0.000 title abstract 4
- 239000013612 plasmid Substances 0.000 claims abstract description 36
- 230000014509 gene expression Effects 0.000 claims abstract description 27
- 108010048367 enhanced green fluorescent protein Proteins 0.000 claims abstract description 17
- 102000008579 Transposases Human genes 0.000 claims abstract description 13
- 108010020764 Transposases Proteins 0.000 claims abstract description 13
- 238000000520 microinjection Methods 0.000 claims abstract description 11
- 230000005058 diapause Effects 0.000 claims abstract description 8
- 108700019146 Transgenes Proteins 0.000 claims abstract description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 52
- 230000009261 transgenic effect Effects 0.000 claims description 41
- 230000028327 secretion Effects 0.000 claims description 19
- 230000015572 biosynthetic process Effects 0.000 claims description 18
- 238000003786 synthesis reaction Methods 0.000 claims description 18
- 239000003550 marker Substances 0.000 claims description 13
- 102000004169 proteins and genes Human genes 0.000 claims description 12
- 230000000384 rearing effect Effects 0.000 claims description 11
- 238000012216 screening Methods 0.000 claims description 10
- 241000238631 Hexapoda Species 0.000 claims description 8
- 230000001066 destructive effect Effects 0.000 claims description 8
- 230000008676 import Effects 0.000 claims description 7
- 230000003321 amplification Effects 0.000 claims description 2
- 238000009395 breeding Methods 0.000 claims description 2
- 230000001488 breeding effect Effects 0.000 claims description 2
- 239000003292 glue Substances 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 abstract description 4
- 108010013296 Sericins Proteins 0.000 abstract description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 abstract 1
- 230000035772 mutation Effects 0.000 abstract 1
- 230000017105 transposition Effects 0.000 abstract 1
- 239000012634 fragment Substances 0.000 description 9
- 235000013601 eggs Nutrition 0.000 description 7
- 125000006850 spacer group Chemical group 0.000 description 7
- 108010022355 Fibroins Proteins 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 238000003780 insertion Methods 0.000 description 6
- 230000037431 insertion Effects 0.000 description 6
- 108010061100 Nucleoproteins Proteins 0.000 description 5
- 102000011931 Nucleoproteins Human genes 0.000 description 5
- 206010037742 Rabies Diseases 0.000 description 5
- 241000711798 Rabies lyssavirus Species 0.000 description 5
- 241000700605 Viruses Species 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000002759 chromosomal effect Effects 0.000 description 3
- 230000014107 chromosome localization Effects 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000012256 transgenic experiment Methods 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 108091029795 Intergenic region Proteins 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 101150051089 A3 gene Proteins 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 108010009685 Cholinergic Receptors Proteins 0.000 description 1
- 206010011732 Cyst Diseases 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 241000711931 Rhabdoviridae Species 0.000 description 1
- 206010044221 Toxic encephalopathy Diseases 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 102000034337 acetylcholine receptors Human genes 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 101150099105 alien gene Proteins 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 208000031513 cyst Diseases 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 201000002491 encephalomyelitis Diseases 0.000 description 1
- 230000035931 haemagglutination Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000007135 neurotoxicity Effects 0.000 description 1
- 231100000228 neurotoxicity Toxicity 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 108091005981 phosphorylated proteins Proteins 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for synthesizing and secreting rabies virus nucleoproteins in middle silkworm silk-gland cells. The method comprises the following steps of: firstly constructing a carrier of pBSer1RVNP (Rabies Virus Nucleoprotein)-A3EGFP (Enhanced Green Fluorescent Protein) plasmid for synthesizing and secreting RVNP in silkworm; then introducing the plasmid and helper plasmids capable of providing transposase to a fertilized egg of a silkworm mutation variety of sericin silkworm by utilizing a microinjection transgene silkworm technology, wherein diapause of the fertilized egg is removed; and introducing a green fluorescent protein gene and a rabies virus nucleoprotein gene into a genome of the sericin silkworm by means of transposition characteristics of a piggyback transposon to obtain stable inheritance and expression, therefore, a transgene silkworm capable of specifically synthesizing and secreting the RVNP in the middle silkworm silk-gland cells can be created.
Description
Technical field
The present invention relates to utilize transgenic technology to make up a kind of method of silkworm middle division of silkgland cell synthesis secretion rabies virus nucleoprotein.
Background technology
Rabies are in world today's public health one of transmissible disease of serious threat to be arranged.Rabies are to be caused by rabies virus infection warm blooded animal or people; Cause animal or people's acute fatal encephalomyelitis; Clinical symptoms mainly shows as mad dry uneasiness, unusual, aggressive, the PP and final dead of behavior, is that a kind of people beast suffers from transmissible disease altogether.Be characterized in that latent period is long, lethality rate is high, almost 100% death.
Rabies virus belongs to the Rhabdoviridae Rhabdovirus, is strand amerism minus-stranded rna virus, and viral profile is the bullet shape, the pure circle of an end, and one holds level with both hands recessedly, and cyst membrane is arranged, and includes capsid symmetry in the shape of a spiral, and the surface has coating, contains single stranded RNA.
The about 12kb of genome total length of rabies virus; Genomic 3 ' end to 5 ' end is being arranged N, P, M, G, 5 structure genes of L; The length of each gene order is respectively 1424,991,805,1675 and 6475bp, their encode respectively nucleoprotein (N), phosphorprotein (P), stromatin (M), gp (G) and large proteins (the RNA transcriptase albumen that L or RNA rely on).
Rabies virus has two kinds of major antigens: a kind of is glycoprotein antigen on the outer virionic membrane, and it can combine with acetylcholine receptor, shows neurotoxicity, can make and produce neutralizing antibody and hemagglutination inhibition antibody in the body; Another kind is the nucleoprotein antigen of internal layer, and it makes and produces complement fixation antibody and precipitin in the body.
1424 Nucleotide of N protein gene total length. 450 amino acid of encoding, relative molecular mass is 50.5Da.In sophisticated virus particle, N albumen is one of staple that constitutes nucleocapsid spiral symplex structure, is albumen the most stable in the virus, can be used as detection antigen, also is the main foundation of rabies virus gene type.N albumen combines with RNA in the rabies virus genome, forms the RNP binding substances, can protect viral RNA to exempt from nucleicacidase and destroy.N albumen is a phosphorylated protein, and it is phosphorylation site that C holds the 389th Ser, can regulate transcribing and levels of replication of virus.N albumen can the inducing cell immunity.
Transgenic bombyx mori middle division of silkgland bio-reactor is the transgenic animal expression system of expression alien gene in the silkworm middle division of silkgland.The silkworm growth cycle is short, can accomplish a generation about 50 days, and each female moth can lay eggs about 400; Silkworm is through reaching artificial domestication, the seed selection in more than 4,000 year, and disposition is docile, has accomplished the forfeiture escape capability of circling in the air, and has very strong ability aspect, the secretion synthetic at silk-protein; Synthetic albumen can be secreted into external with fibroin, and fibroin mainly is made up of 3 kinds of silk fibroins and 3 kinds of sericins, and composition is simple.So transgenic bombyx mori sericterium bioreactor for constructing is easy, operating safety; It is high to express foreign protein efficient, the many biologically actives of foreign protein, and protein purification is convenient; Only need through raising transgenic bombyx mori; Just can keep the continuity of expression system easily, be a kind of very valuable expression system, has wide practical prospect.
Summary of the invention
The object of the present invention is to provide a kind of method of silkworm middle division of silkgland cell synthesis secretion rabies virus nucleoprotein; Be to utilize the transgenic bombyx mori technology that the rabies virus nucleoprotein gene is imported in the domestic silkworm gene group; And in silkworm middle division of silkgland cell specifically expressing; Develop the silkworm of synthesis secretion rabies virus nucleoprotein, lay the foundation for utilizing rabies virus nucleoprotein development human rabies new generation vaccine safer, efficient, economic and easy to use.
In order to achieve the above object, the step of the technical scheme of the present invention's employing is following:
(1) adopt Protocols in Molecular Biology to make up the carrier pBSer1RVNP-A3EGFP plasmid of silkworm synthesis secretion rabies virus nucleoprotein;
(2) adopt microinjection transgenic bombyx mori method with the pBSer1RVNP-A3EGFP plasmid and can provide the helper plasmid of transposase trans to import silkworm in the zygote of termination of diapause; Raise after the egg-incubation to adult, selfing continues the generation on behalf of G1, at the little silkworm rearing season of G1 for transgenic bombyx mori; Select to express the transgenic bombyx mori of EGFP marker gene through the fluorescence stereomicroscope observation; Raise to the adult selfing continuously on behalf of G2 generation, in G2 generation, all adopt the moth district to educate later on, and the adult selfing continues generation; Again through 1-3 for seed selection; In per generation, adopted transgenosis insect fluorescence gene expression relative quantity enabling non-destructive determination method, indicates for the screening of reserving seed for planting to express EGFP marker gene and silk gum silkworm proterties, and breeding the middle division of silkgland cell can synthesis secretion rabies virus nucleoprotein, the transgenic bombyx mori new variety of gene pure.
Described pBSer1RVNP-A3EGFP plasmid is to be the basis with piggyBac swivel base plasmid; Plasmid has amplification and the screening of Amp resistant gene to be used for plasmid; 2 the swivel base arm PBL and the PBR that comprise the piggyBac transposon; Comprise 2 functional expression frames; One is the green fluorescence protein gene expression cassette A3 Promoter+EGFP+SV40 that the A3 promotor starts; With the screening sign as the transgenic positive silkworm, another is domestic silkworm silk glue protein 1 promotor, has and be convenient to purify the His joint of foreign protein and the expression cassette Ser1 Promoter+His+hLYZ+SV40 of rabies virus nucleoprotein gene, to express rabies virus nucleoprotein; Helper plasmid comprises 1 swivel base arm PBR of Amp resistant gene, piggyBac transposon, the transposase trans gene expression frame A3 Promoter+ trans that the A3 promotor starts, so that transposase to be provided.
Employing microinjection transgenic bombyx mori method is with the pBSer1RVNP-A3EGFP plasmid and can provide the helper plasmid of transposase trans to import the silkworm zygote of termination of diapause; Its cultivated silkworm breed variety is a mutating variety silk gum silkworm; Described is the screening sign of reserving seed for planting in per generation with silk gum silkworm proterties, and be silk cocoon period the period of its screening.
The beneficial effect that the present invention has is:
The present invention is by fluorized marking genescreen transgenic bombyx mori; This transgenic bombyx mori can be on silkworm middle division of silkgland cell-specific ground synthesis secretion rabies virus nucleoprotein, for the production efficiency that improves rabies virus nucleoprotein, reduce production costs, and development human rabies new generation vaccine safer, efficient, economic and easy to use lay the foundation.
Description of drawings
Fig. 1 is the pBSer1RVNP-A3EGFP carrier structure figure of silkworm middle division of silkgland cell synthesis secretion rabies virus nucleoprotein.
Fig. 2 is the helper plasmid structure iron that transposase can be provided.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is described further.
Embodiment 1:
With pBSer1RVNP-A3EGFP plasmid (Fig. 1) and can provide the helper plasmid (Fig. 2) of transposase to press the 2:1 ratio mixed; The total concn of 2 kinds of plasmids is 0.4 μ g/ μ l; Plasmid is dissolved in 0.5mM, pH7.0 contains in the phosphoric acid buffer of 5mM Repone K; Adopt micro-injection method to import the silk gum silkworm then and lay eggs back 8 hours with in the zygote interior, termination of diapause, the importing TV is 20nl.The silkworm seed of microinjection is raised to adult selfing continuous generation (G1 generation) under 25 ℃, 85% humidity, 12h illumination condition.At little silkworm rearing season of the G1 generation of transgenic experiments; Observe transgenic bombyx mori 1 moth that obtains expression EGFP marker gene through fluorescent microscope (Olympus, SZX12, Japan); The excitation wavelength of fluorescent microscope is 460nm～490nm, and emission wavelength is 510 nm～550 nm.Silkworm raised to the adult selfing lay eggs continuous generation (G2).All adopt one batch rearing from G2 for later transgenic bombyx mori; Adopt transgenosis insect fluorescence gene expression relative quantity enabling non-destructive determination method in the young silkworm phase, select the transgenic bombyx mori of expressing the EGFP marker gene, raise to adult; Selecting the silk cocoon characteristic is the continuous generation of individual selfing of silk gum; Reserve seed for planting through selfing 3 generations selection,, adopt transgenosis insect fluorescence gene expression relative quantity enabling non-destructive determination method at the little silkworm rearing season in per generation; Observation is selected and remain and is expressed the transgenic bombyx mori of EGFP marker gene, is the individuality of silk gum in the silk cocoon silk cocoon characteristic of selecting and remain period.Through this breed the middle division of silkgland cell can synthesis secretion rabies virus nucleoprotein and rabies virus nucleoprotein content account for the transgenic bombyx mori new variety of the gene pure of silk-protein content higher proportion, name and be SN2.
Getting the SN2 variety genome DNA is template, adopts the insertion fragment of Inverse pcr amplification RVNP gene in the silkworm genome, to amplified fragments clone, order-checking and chromosomal localization analysis, the result shows below:
Identify in the insertion site of rabies virus nucleoprotein gene in the silkworm genome
| Strain | Karyomit(e) | Sequence fragment | Gene or transcribed spacer | The left side genome sequence | Carrier | The right side genome sequence |
| SN2 | Chr.17 | Bm_scaf33（2923746） | A3 gene | TAAGTTTTAATTTTAA | piggyBac | TTAAAATTTCGAAAGA |
The extraction fibroin is a material, adopts Western blot to analyze the expression of transgenic bombyx mori nucleoprotein.Through the analysis that mouse-anti rabies virus nucleoprotein MAb work one resists, the result obtains and the specific proteins band of expecting that the molecular weight size conforms to.
Result of study proof RVNP gene has been inserted in domestic silkworm gene group the 17th chromosomal A3 actin gene, and has obtained stable heredity and express, and has bred the transgenic bombyx mori new variety that the middle division of silkgland cell can synthesis secretion rabies virus nucleoprotein.
Embodiment 2:
With pBSer1RVNP-A3EGFP plasmid (Fig. 1) and can provide the helper plasmid (Fig. 2) of transposase to press the 2:1 ratio mixed; The total concn of 2 kinds of plasmids is 0.4 μ g/ μ l; Plasmid is dissolved in 0.5mM, pH7.0 contains in the phosphoric acid buffer of 5mM Repone K; Adopt micro-injection method to import the silk gum silkworm then and lay eggs back 8 hours with in the zygote interior, termination of diapause, the importing TV is 20nl.The silkworm seed of microinjection is raised to adult selfing continuous generation (G1 generation) under 25 ℃, 85% humidity, 12h illumination condition.At little silkworm rearing season of the G1 generation of transgenic experiments; Observe transgenic bombyx mori 2 moths that obtain expression EGFP marker gene through fluorescent microscope (Olympus, SZX12, Japan); The excitation wavelength of fluorescent microscope is 460nm～490nm, and emission wavelength is 510 nm～550 nm.Silkworm raised to the adult selfing lay eggs continuous generation (G2).All adopt one batch rearing from G2 for later transgenic bombyx mori; Adopt transgenosis insect fluorescence gene expression relative quantity enabling non-destructive determination method in the young silkworm phase, select the transgenic bombyx mori of expressing the EGFP marker gene, raise to adult; Selecting the silk cocoon characteristic is the continuous generation of individual selfing of silk gum; Reserve seed for planting through selfing 1 generation selection,, adopt transgenosis insect fluorescence gene expression relative quantity enabling non-destructive determination method at the little silkworm rearing season in per generation; Observation is selected and remain and is expressed the transgenic bombyx mori of EGFP marker gene, is the individuality of silk gum in the silk cocoon silk cocoon characteristic of selecting and remain period.Through this breed the middle division of silkgland cell can synthesis secretion rabies virus nucleoprotein and rabies virus nucleoprotein content account for the transgenic bombyx mori new variety of the gene pure of silk-protein content higher proportion, name and be SN3, SN4.
Get SN3, the SN4 genomic dna is a template, adopts the insertion fragment of Inverse pcr amplification RVNP gene in the silkworm genome, to amplified fragments clone, order-checking and chromosomal localization analysis, the result shows below:
Identify in the insertion site of rabies virus nucleoprotein gene in the silkworm genome
| Strain | Karyomit(e) | Sequence fragment | Gene or transcribed spacer | The left side genome sequence | Carrier | The right side genome sequence |
| SN3 | Chr.17 | Bm_scaf33（2923720） | Transcribed spacer | CTTGTCACTTATTTAA | piggyBac | TTAAAGTTTAGGTCGA |
| SN4 | Chr.8 | Bm_scaf19（7860408） | Transcribed spacer | ATGGTGCTGTTTTTAA | piggyBac | TTAAAATCTTTCCACT |
Extraction SN3, SN4 fibroin are material, adopt Western blot to analyze the expression of transgenic bombyx mori nucleoprotein.Through the analysis that mouse-anti rabies virus nucleoprotein MAb work one resists, the result obtains and the specific proteins band of expecting that the molecular weight size conforms to.
Result of study proof RVNP gene has been inserted in SN3 domestic silkworm gene group the 17th karyomit(e) and SN4 domestic silkworm gene group the 8th chromosomal intergenic region; And obtained stable heredity and express, bred the transgenic bombyx mori new variety that the middle division of silkgland cell can synthesis secretion rabies virus nucleoprotein.
Embodiment 3:
With pBSer1RVNP-A3EGFP plasmid (Fig. 1) and can provide the helper plasmid (Fig. 2) of transposase to press the 2:1 ratio mixed; The total concn of 2 kinds of plasmids is 0.4 μ g/ μ l; Plasmid is dissolved in 0.5mM, pH7.0 contains in the phosphoric acid buffer of 5mM Repone K; Adopt micro-injection method to import the silk gum silkworm then and lay eggs back 8 hours with in the zygote interior, termination of diapause, the importing TV is 20nl.The silkworm seed of microinjection is raised to adult selfing continuous generation (G1 generation) under 25 ℃, 85% humidity, 12h illumination condition.At little silkworm rearing season of the G1 generation of transgenic experiments; Observe transgenic bombyx mori 2 moths that obtain expression EGFP marker gene through fluorescent microscope (Olympus, SZX12, Japan); The excitation wavelength of fluorescent microscope is 460nm～490nm, and emission wavelength is 510 nm～550 nm.Silkworm raised to the adult selfing lay eggs continuous generation (G2).All adopt one batch rearing from G2 for later transgenic bombyx mori; Adopt transgenosis insect fluorescence gene expression relative quantity enabling non-destructive determination method in the young silkworm phase, select the transgenic bombyx mori of expressing the EGFP marker gene, raise to adult; Selecting the silk cocoon characteristic is the continuous generation of individual selfing of silk gum; Reserve seed for planting through selfing 2 generations selection,, adopt transgenosis insect fluorescence gene expression relative quantity enabling non-destructive determination method at the little silkworm rearing season in per generation; Observation is selected and remain and is expressed the transgenic bombyx mori of EGFP marker gene, is the individuality of silk gum in the silk cocoon silk cocoon characteristic of selecting and remain period.Through this breed the middle division of silkgland cell can synthesis secretion rabies virus nucleoprotein and rabies virus nucleoprotein content account for the transgenic bombyx mori new variety of the gene pure of silk-protein content higher proportion, name and be SN6, SN8.
Get SN6, the SN8 genomic dna is a template, adopts the insertion fragment of Inverse pcr amplification RVNP gene in the silkworm genome, to amplified fragments clone, order-checking and chromosomal localization analysis, the result shows below:
Identify in the insertion site of rabies virus nucleoprotein gene in the silkworm genome
| Strain | Karyomit(e) | Sequence fragment | Gene or transcribed spacer | The left side genome sequence | Carrier | The right side genome sequence |
| SN6 | Chr.2 | Bm_scaf83（3110498） | Transcribed spacer | CTTATTTTAAAATTAA | piggyBac | TTAATAAAGAAATC |
| SN8 | Chr.11 | Bm_scaf16（22908836） | Transcribed spacer | TATTGATCTTCTTTAA | piggyBac | TTAAAACAAATTCTAC |
Extraction SN3, SN4 fibroin are material, adopt Western blot to analyze the expression of transgenic bombyx mori nucleoprotein.Through the analysis that mouse-anti rabies virus nucleoprotein MAb work one resists, the result obtains and the specific proteins band of expecting that the molecular weight size conforms to.
Result of study proof RVNP gene has been inserted in SN6 domestic silkworm gene group the 2nd karyomit(e) and SN8 domestic silkworm gene group the 11st chromosomal intergenic region; And obtained stable heredity and express, bred the transgenic bombyx mori new variety that the middle division of silkgland cell can synthesis secretion rabies virus nucleoprotein.
Claims (2)
1. the method for a silkworm middle division of silkgland cell synthesis secretion rabies virus nucleoprotein is characterized in that the step of this method is following:
(1) adopt Protocols in Molecular Biology to make up the carrier pBSer1RVNP-A3EGFP plasmid of silkworm synthesis secretion rabies virus nucleoprotein;
Described pBSer1RVNP-A3EGFP plasmid is to be the basis with piggyBac swivel base plasmid; Plasmid has amplification and the screening of Amp resistant gene to be used for plasmid; 2 the swivel base arm PBL and the PBR that comprise the piggyBac transposon; Comprise 2 functional expression frames; One is the green fluorescence protein gene expression cassette A3 Promoter+EGFP+SV40 that the A3 promotor starts; With the screening sign as the transgenic positive silkworm, another is domestic silkworm silk glue protein 1 promotor, has and be convenient to purify the His joint of foreign protein and the expression cassette Ser1 Promoter+His+ RVNP+SV40 of rabies virus nucleoprotein gene, to express rabies virus nucleoprotein; Helper plasmid comprises 1 swivel base arm PBR of Amp resistant gene, piggyBac transposon, the transposase trans gene expression frame A3 Promoter+ trans that the A3 promotor starts, so that transposase to be provided;
(2) adopt microinjection transgenic bombyx mori method with the pBSer1RVNP-A3EGFP plasmid and can provide the helper plasmid of transposase trans to import silkworm in the zygote of termination of diapause; Raise after the egg-incubation to adult, selfing continues the generation on behalf of G1, at the little silkworm rearing season of G1 for transgenic bombyx mori; Select to express the transgenic bombyx mori of EGFP marker gene through the fluorescence stereomicroscope observation; Raise to the adult selfing continuously on behalf of G2 generation, in G2 generation, all adopt the moth district to educate later on, and the adult selfing continues generation; Again through 1-3 for seed selection; In per generation, adopted transgenosis insect fluorescence gene expression relative quantity enabling non-destructive determination method, indicates for the screening of reserving seed for planting to express EGFP marker gene and silk gum silkworm proterties, and breeding the middle division of silkgland cell can synthesis secretion rabies virus nucleoprotein, the transgenic bombyx mori new variety of gene pure.
2. the method for a kind of silkworm middle division of silkgland cell synthesis secretion rabies virus nucleoprotein according to claim 1; It is characterized in that: employing microinjection transgenic bombyx mori method is with the pBSer1RVNP-A3EGFP plasmid and can provide the helper plasmid of transposase trans to import the silkworm zygote of termination of diapause; Its cultivated silkworm breed variety is a mutating variety silk gum silkworm; Described is the screening sign of reserving seed for planting in per generation with silk gum silkworm proterties, and be silk cocoon period the period of its screening.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101235867A CN102242147B (en) | 2011-05-13 | 2011-05-13 | Method for synthesizing and secreting rabies virus nucleoprotein in middle silkworm silk-gland cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101235867A CN102242147B (en) | 2011-05-13 | 2011-05-13 | Method for synthesizing and secreting rabies virus nucleoprotein in middle silkworm silk-gland cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102242147A CN102242147A (en) | 2011-11-16 |
| CN102242147B true CN102242147B (en) | 2012-11-21 |
Family
ID=44960392
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011101235867A Expired - Fee Related CN102242147B (en) | 2011-05-13 | 2011-05-13 | Method for synthesizing and secreting rabies virus nucleoprotein in middle silkworm silk-gland cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102242147B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016125885A1 (en) * | 2015-02-06 | 2016-08-11 | 国立研究開発法人農業生物資源研究所 | Cell death-inducing vector and site-specific cell death-inducing silkworm system having same |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321651A (en) * | 2011-09-09 | 2012-01-18 | 遵义医学院附属医院 | Fusion gene containing influenza A virus nucleoprotein, its construction and its expression method |
| CN106480199B (en) * | 2016-10-31 | 2019-11-05 | 广西壮族自治区蚕业技术推广总站 | The homozygous method for genetic of single copy transgenic bombyx mori strain |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100723565B1 (en) * | 2002-03-06 | 2007-06-04 | 도레이 가부시끼가이샤 | Process for Producing Physiologically Active Protein Using Genetically Modified Silkworm |
| FR2852325B1 (en) * | 2003-03-13 | 2005-06-03 | NUCLEIC ACID DIRECTING THE EXPRESSION OF A POLYPEPTIDE OF INTEREST IN POST-LEPIDOPTERA SERICIGENIC GLANDS, AND USES THEREOF | |
| CN101423842A (en) * | 2008-08-29 | 2009-05-06 | 浙江理工大学 | Method for synthesizing protein by using cultivated silkworm middle silk gland vitro expression system |
-
2011
- 2011-05-13 CN CN2011101235867A patent/CN102242147B/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016125885A1 (en) * | 2015-02-06 | 2016-08-11 | 国立研究開発法人農業生物資源研究所 | Cell death-inducing vector and site-specific cell death-inducing silkworm system having same |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102242147A (en) | 2011-11-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20150166615A1 (en) | Method and uses for Bombyx mori silk fibroin heavy chain mutation sequence and mutant | |
| CN102226202B (en) | Method for synthetizing secretion lysozyme by middle silkgland cell of silkworm | |
| CN104846011A (en) | Method for synthesizing royal jelly main protein 1 by using bombyx mori posterior silkgland | |
| CN102242147B (en) | Method for synthesizing and secreting rabies virus nucleoprotein in middle silkworm silk-gland cells | |
| CN106148406B (en) | Pig ApoE gene knockout carrier and its construction method and application | |
| CN102604953B (en) | BmCP274 promoter of bombyx mori cuticular protein, recombinant expression vector and application of the promoter | |
| CN102286529B (en) | Method for synthesizing silkworm silk gland cells capable of secreting rabies virus glucoprotein | |
| CN103725676A (en) | Target sequence suitable for transgene fixed point insertion of silkworm W-chromosome and locus and application thereof | |
| CN105331632A (en) | Method for synthesizing secretion calcium ion binding protein through bombyx mori posterior silkgland | |
| CN105400817B (en) | Utilize the method for the silkworm simultaneously synthesizing traction of secretion latrodectus mactans silk-fibroin 1 and albumen 2 | |
| JP5997772B2 (en) | Mutated sequences of silkworm fibroin heavy chain gene, and methods and applications for inducing mutations | |
| CN110468132A (en) | A kind of sgRNA and transgene expression vector, expression strain, screening technique | |
| CN105463022B (en) | The method for synthesizing secretion latrodectus mactans traction silk-fibroin 2 using domestic natural silk gland bioreactor | |
| CN106978416A (en) | A kind of assignment of genes gene mapping integrant expression system and its application | |
| CN101993895A (en) | Construction and application method of efficient double promoter PLEGFP-N1-spMyoD1 green fluorescence retrovirus vector | |
| CN102618528A (en) | Deleting system for long fragments in genome based on TALEN (transcription activator-like effector Nuclease) and ssODNs (single stranded oligonucleotides) and application thereof | |
| CN103952424B (en) | Method for producing double-muscular trait somatic cell cloned pig with MSTN (myostatin) bilateral gene knockout | |
| CN105969801A (en) | Bombyx mori middle silkgland bioreactor universal plasmid for expressing T4 ligase as well as application and method of universal plasmid | |
| CN1234871C (en) | Construction method using detoxiase gene as stable expression system in silkworm | |
| KR101570783B1 (en) | Transgenic silkworms producing antimicrobial peptide | |
| US20120054880A1 (en) | Method for enhancing production of pathogen-resistant proteins using bioreactor | |
| CN102559749A (en) | Integration cattle NRAMPl macrophage specificity expression vector/cattle fibroblast and method thereof | |
| Rajoriya et al. | Transgenic animals: Prospects for improving livestock productivity | |
| CN101412999A (en) | A kind of gene targeting locus-specific transgenic method and application thereof | |
| CN106337049A (en) | Breeding method of anti-influenza transgenic animals expressing double genes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20160708 Address after: Xuanwu District of Nanjing City, Jiangsu province 210014 Wei Tong Road No. 4 506 Patentee after: Nanjing Shiji Animal Health Management Co., Ltd. Patentee after: Zhejiang University Address before: 310018, No. 7, No. 266, No. 10, Hangzhou economic and Technological Development Zone, Hangzhou, Zhejiang, Jianggan District Province, 2 floors Patentee before: Zhejiang Tongdian Biological Technology Co.,Ltd. Patentee before: Zhejiang University |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20121121 Termination date: 20180513 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |