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
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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.
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| 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 |
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