CN1974776A - Method of infecting silkworm with recombinant baculovirus - Google Patents
Method of infecting silkworm with recombinant baculovirus Download PDFInfo
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- CN1974776A CN1974776A CN 200610155054 CN200610155054A CN1974776A CN 1974776 A CN1974776 A CN 1974776A CN 200610155054 CN200610155054 CN 200610155054 CN 200610155054 A CN200610155054 A CN 200610155054A CN 1974776 A CN1974776 A CN 1974776A
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- 241000255789 Bombyx mori Species 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 20
- 241000701447 unidentified baculovirus Species 0.000 title claims abstract description 14
- 241000700605 Viruses Species 0.000 claims abstract description 28
- 239000002502 liposome Substances 0.000 claims abstract description 11
- 241000238631 Hexapoda Species 0.000 claims abstract description 10
- 238000011081 inoculation Methods 0.000 claims abstract description 10
- 108020004414 DNA Proteins 0.000 claims description 17
- 238000002347 injection Methods 0.000 claims description 9
- 239000007924 injection Substances 0.000 claims description 9
- 230000003612 virological effect Effects 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 6
- 230000002458 infectious effect Effects 0.000 claims description 5
- 238000007400 DNA extraction Methods 0.000 claims description 4
- 235000008708 Morus alba Nutrition 0.000 claims description 4
- 240000000249 Morus alba Species 0.000 claims description 4
- 108020005202 Viral DNA Proteins 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 208000024891 symptom Diseases 0.000 claims description 4
- 210000001015 abdomen Anatomy 0.000 claims description 3
- 239000008280 blood Substances 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 3
- 238000013016 damping Methods 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 238000003780 insertion Methods 0.000 claims description 3
- 230000037431 insertion Effects 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000005516 engineering process Methods 0.000 abstract description 7
- 238000011109 contamination Methods 0.000 abstract 1
- 208000015181 infectious disease Diseases 0.000 description 5
- 241000409811 Bombyx mori nucleopolyhedrovirus Species 0.000 description 3
- 238000013320 baculovirus expression vector system Methods 0.000 description 3
- 238000009792 diffusion process Methods 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 241001367049 Autographa Species 0.000 description 1
- 241000201370 Autographa californica nucleopolyhedrovirus Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000010297 mechanical methods and process Methods 0.000 description 1
- 230000005226 mechanical processes and functions Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 241000701366 unidentified nuclear polyhedrosis viruses Species 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses method of infecting silkworm with recombinant baculovirus. The method is percutaneous inoculation to silkworm by means of liposome mediation technology. The method can reach the same infecting effect as available direct virus infecting method, and is superior to traditional direct virus infecting method in that the method can avoid the latent risk of spread virus causing operation environment contamination and raise the biological safety of insect baculovirus expressing system.
Description
Technical field
The present invention relates to the virus infection technology, relate in particular to a kind of method of silkworm recombinant virus per oral inoculation.
Background technology
Baculovirus expression carrier system (BacuLovirus Expression Vector System, be called for short BEVS) be utilize the recombinant baculovirus that carries the external source goal gene do carrier in insect body or insect cell culture express a recombinant protein production system of production.Owing to having, this system can utilize individual or its culturing cell of insect to efficiently express advantage such as production on a large scale to become at present one of the most effective eukaryotic expression system.At present BEVS comprises that mainly American-European countries is just at autographa california nuclear polyhedrosis virus (the Autographa californica nuclear polyhedrosis virus of widespread use, be called for short AcNPV) and use more Bombyx mori nuclear polyhydrosis virus (Bombyx mori nuclearpolyhedrosis virus in China, Japan, be called for short BmNPV) two class expression systems (Smith et al., 1983; Maeda et al., 1984).The BmNPV expression system since the economic insects-silkworm that can utilize China's number of animals raised maximum as expression vector, thereby have the advantage that cost is low, be fit to large-scale production, have very much China's characteristic (Lv Hongsheng, 1998).Yet present inoculation technique all is to adopt recombinant virus to be injected directly into the intravital method of silkworm to infect expression, is easy to cause the diffusion of virus in actual mechanical process, thereby causes the pollution to environment.Therefore, for the diffusion and the pollution of strictness control operating environment recombinant virus, improve the biological safety of this system, it is very necessary that the infection method of exploitation safety seems.Silkworm baculovirus has the circular double stranded DNA of 130kb size, in the recombinant virus building process, use cationic-liposome-mediated method effectively its cotransfection to be imported insect cell culture, we consider that this technology also is suitable for the live body insect, so just changed the direct method of using virus, recombinant virus genomes DNA can be imported silkworm larva, and by optimizing inoculation condition raising efficiency of infection, to reach the infectious effect same with wild virus.
Summary of the invention
The object of the present invention is to provide a kind of method of infecting silkworm with recombinant baculovirus, set up a kind of without the recombinant virus direct infection, but the technology that adopts virus genom DNA and infect again after liposome mixes.
In order to achieve the above object, its step of the technical solution used in the present invention is as follows:
(1) extraction of virus genom DNA preparation: with recombinant silkworm baculovirus HyNPV-bFGF infected insect culturing cell Sf21, low-speed centrifugal collecting cell behind the 96h, add the PBS damping fluid suspend after centrifugal again 1 time; With the DNA Extraction Kit of STRATAGENE company extracting viral genome, virus genom DNA purity and concentration are measured with A260/280 in RNA digestion back;
(2) mixing of DNA and liposome: according to certain concentration blending ratio, the concentration of liposome is determined at 0.1 μ g/ μ L, viral DNA concentration 5ng/ μ L, and room temperature was placed 30 minutes before using;
(3) recombinant virus infects through skin: the percutaneous puncture inoculation mode of injection, in advance with five silkworm larva placements in age 10 minutes on ice, to contain the parallel insertion of the syringe needle insect silkworm belly intersegmental membrane place of the mixed solution of viral liquid or its DNA then, touch needle cylinder injection, note not hindering the internal.Every boss silkworm injection volume is 20 μ L; Inoculate back 30 minutes and give the mulberry leaf raising;
(4) infectious effect is measured: whether get the silkworm small amounts of blood after 72 hours and place on the slide glass, examining under a microscope has virus particle, if there is explanation to infect; And after 96 hours, observe disease symptom.
The present invention compares with background technology, and the beneficial effect that has is:
The result that present method is implemented shows, recombinant virus genomes DNA and cationic-liposome mix by a certain percentage after bark graft kind silkworm produces typical baculovirus infection symptom, prove that this new inoculation infection method has with using the identical infectious effect of viral direct inoculation.Its advantage has been to change traditional direct vaccination ways that uses virus, has avoided causing the potentially dangerous that operating environment pollutes after viral may the diffusion, has improved the insect baculovirus expression system biological safety.
Silkworm-baculovirus expression system might become a strong instrument that has independent intellectual property right in China's biotechnology industry; exploitation safely, inoculation technique has become a technical bottleneck that solves in the industrial scale fast; this patent provides the technology of a safety
Embodiment
1, material: viral DNA extraction agent box is purchased in Qiagene.DNA handles and the pcr amplification test kit purchase in Takara Biomedicals (Kyoto, Japan).Liposome is an Invitrogen company product.This test use silkworm hybrid, variety name be the autumn rich * white jade, the silkworm larva mulberry leaf are raised, temperature is 23～25 ℃.
2, workflow:
(1) extraction of virus genom DNA preparation: with recombinant silkworm baculovirus HyNPV-bFGF infected insect culturing cell Sf21, low-speed centrifugal collecting cell behind the 96h, add the PBS damping fluid suspend after centrifugal again 1 time; With the DNA Extraction Kit of STRATAGENE company extracting viral genome, virus genom DNA purity and concentration are measured with A260/280 in RNA digestion back;
(2) mixing of DNA and liposome: according to certain concentration blending ratio, the concentration of liposome is determined at 0.1g/L, viral DNA concentration 5ng/L, and room temperature was placed 30 minutes before using;
(3) recombinant virus infects through skin: the percutaneous puncture inoculation mode of injection, in advance with five silkworm larva placements in age 10 minutes on ice, to contain the parallel insertion of the syringe needle insect silkworm belly intersegmental membrane place of the mixed solution of viral liquid or its DNA then, touch needle cylinder injection, note not hindering the internal.Every boss silkworm injection volume is 20L; Inoculate back 30 minutes and give the mulberry leaf raising;
(4) infectious effect is measured: whether get the silkworm small amounts of blood after 72 hours and place on the slide glass, examining under a microscope has virus particle, if there is explanation to infect; And after 96 hours, observe disease symptom.
Claims (1)
1, a kind of method of infecting silkworm with recombinant baculovirus is characterized in that:
(1) extraction of virus genom DNA preparation: with recombinant silkworm baculovirus HyNPV-bFGF infected insect culturing cell Sf21, low-speed centrifugal collecting cell behind the 96h, add the PBS damping fluid suspend after centrifugal again 1 time; With the DNA Extraction Kit of STRATAGENE company extracting viral genome, virus genom DNA purity and concentration are measured with A260/280 in RNA digestion back;
(2) mixing of DNA and liposome: according to certain concentration blending ratio, the concentration of liposome is determined at 0.1g/L, viral DNA concentration 5ng/L, and room temperature was placed 30 minutes before using;
(3) recombinant virus infects through skin: the percutaneous puncture inoculation mode of injection, in advance with five silkworm larva placements in age 10 minutes on ice, the parallel insertion of the syringe needle insect silkworm belly intersegmental membrane place that to contain the mixed solution of viral liquid or its DNA then, touch needle cylinder injection, note not hindering the internal, every boss silkworm injection volume is 20L; Inoculate back 30 minutes and give the mulberry leaf raising;
(4) infectious effect is measured: whether get the silkworm small amounts of blood after 72 hours and place on the slide glass, examining under a microscope has virus particle, if there is explanation to infect; And after 96 hours, observe disease symptom.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610155054 CN1974776A (en) | 2006-12-07 | 2006-12-07 | Method of infecting silkworm with recombinant baculovirus |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610155054 CN1974776A (en) | 2006-12-07 | 2006-12-07 | Method of infecting silkworm with recombinant baculovirus |
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| CN1974776A true CN1974776A (en) | 2007-06-06 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101307315B (en) * | 2008-06-30 | 2010-06-02 | 浙江大学 | Method for expressing growth factor of human blood vessel in bombyx mori by Bac-to-Bac system |
| CN102286533A (en) * | 2010-06-18 | 2011-12-21 | 财团法人工业技术研究院 | Insect infection method for production of proteins |
| CN102885011A (en) * | 2012-10-29 | 2013-01-23 | 浙江大学 | Novel method for infecting silkworms with recombinant baculovirus |
| CN110079514A (en) * | 2019-04-12 | 2019-08-02 | 江苏大学 | A method of preparing protease 3 recombinant protein |
-
2006
- 2006-12-07 CN CN 200610155054 patent/CN1974776A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101307315B (en) * | 2008-06-30 | 2010-06-02 | 浙江大学 | Method for expressing growth factor of human blood vessel in bombyx mori by Bac-to-Bac system |
| CN102286533A (en) * | 2010-06-18 | 2011-12-21 | 财团法人工业技术研究院 | Insect infection method for production of proteins |
| CN102286533B (en) * | 2010-06-18 | 2014-09-03 | 财团法人工业技术研究院 | Insect infection method for production of proteins |
| CN102885011A (en) * | 2012-10-29 | 2013-01-23 | 浙江大学 | Novel method for infecting silkworms with recombinant baculovirus |
| CN110079514A (en) * | 2019-04-12 | 2019-08-02 | 江苏大学 | A method of preparing protease 3 recombinant protein |
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