CN1637138A - Silkworm nuclear polyhedron virus and its prepn process and application in gene expression - Google Patents
Silkworm nuclear polyhedron virus and its prepn process and application in gene expression Download PDFInfo
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- CN1637138A CN1637138A CN 200410066139 CN200410066139A CN1637138A CN 1637138 A CN1637138 A CN 1637138A CN 200410066139 CN200410066139 CN 200410066139 CN 200410066139 A CN200410066139 A CN 200410066139A CN 1637138 A CN1637138 A CN 1637138A
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- 241000255789 Bombyx mori Species 0.000 title claims abstract description 46
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- 102000012410 DNA Ligases Human genes 0.000 claims description 2
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Abstract
本发明提供一种可以用于表达外源基因的改造的家蚕杆状病毒，在多角体基因两侧添加上Bsu 36I的酶切位点。这样的经过改造的家蚕杆状病毒以多角体添食家蚕或病毒粒子注射家蚕幼虫或蛹而获得大量的改造后的家蚕杆状病毒多角体，可以提取大量的经过改造的家蚕杆状病毒DNA。这样获得的DNA经Bsu 36I酶切后可以通过同源重组的方法用于表达外源基因。发生重组的家蚕杆状病毒可以通过显微镜观察，挑选没有多角体的发生重组的家蚕杆状病毒斑。简化了重组病毒筛选的过程。The invention provides a modified silkworm baculovirus which can be used to express exogenous genes, and Bsu 36I enzyme cutting sites are added on both sides of the polyhedron gene. Such modified silkworm baculovirus feeds silkworms with polyhedrons or injects virus particles into silkworm larvae or pupae to obtain a large amount of modified silkworm baculovirus polyhedrons, and a large amount of modified silkworm baculovirus DNA can be extracted. The DNA obtained in this way can be used to express foreign genes by means of homologous recombination after being digested with Bsu 36I. The recombined silkworm baculovirus can be observed through a microscope, and the recombined silkworm baculovirus plaques without polyhedrons can be selected. The process of recombinant virus screening is simplified.
Description
Technical field
The invention belongs to the production technique in viral organism reactor field, refer in particular to the improvement of the silkworm baculovirus DNA that is used for genetic expression, promptly improved polyhedrosis gene both sides contain Bombyx mori nuclear polyhydrosis virus and preparation method and its application in genetic expression of restriction enzyme site.
Background technology
Baculovirus has the history in 20 years as expression vector, has become one of expression system commonly used.Baculovirus has two kinds of main existence forms in the intravital developmental cycle of insect, a kind of is the viral ion that shuttles back and forth between insect cell and infect, and is called the virus of sprouting again, and another kind is the polyhedron form that virus particle is coated on one deck albuminous membranae.It is active that polyhedron can keep in environment, and by the insect food down, the cracking of polyhedron film discharges virus particle, causes infection.Because the polyhedron of virus is the nuclear internal packing at insect cell, is also referred to as nuclear polyhedrosis virus.Virus as expression vector is mainly alfalfa californica nuclear polyhedrosis virus and Bombyx mori nuclear polyhydrosis virus at present.Its principle is that the polyhedrosis gene of baculovirus has strong promoter, the expression amount height, and polyhedrosis gene is the virus replication dispensable gene, can lack from the genome of virus.The method of expression alien gene is exactly to utilize the method for homologous recombination, replaces polyhedrosis gene with foreign gene, constitutes one and contains foreign gene and the recombinant virus of polyhedrosis gene disappearance, and such recombinant virus can expression alien gene.The commercialization of alfalfa californica nuclear polyhedrosis virus virus system is fairly perfect, and the AcBacPAK6 virus of utilizing the homologous recombination mode and the Bac-to-Bac system that the swivel base reorganization takes place in bacterium are arranged.But the commercialization process of silkworm baculovirus is very slow, does not have business-like viral DNA for selling, and mainly is that the laboratory obtains by collecting virus particle.The silkworm baculovirus that is used to recombinate that uses is the virus that has replaced polyhedrosis gene with lac Z gene at present, and this virus can be expressed lac Z gene product, decomposes X-gal and forms blueness.Existing silkworm-baculovirus expression system has two shortcomings, and the one, viral DNA is difficult to obtain.The virus that contains lac Z gene can not form polyhedron, can only produce virus particle.Virus particle is very little, has only by ultracentrifugation and could collect.Method in common is to gather in the crops virus particle with the silkworm baculovirus particle that contains lac Z gene after infecting the BmN cell at present, carry out the ultracentrifugation purifying again, the viral DNA that this mode is obtained is less, also is unfavorable for preserving, and needs main equipment such as ultracentrifuge on equipment.Another shortcoming is when making plaque and analyze, add X-gal sometimes after, the blue spot that reorganization takes place also is difficult to differentiate, and the X-gal that adds also influences the growth conditions of bombyx mori cell, cause viral plaque not to be true to type, have only the expert who is engaged in this field for a long time can distinguish that just technical requirements is very high.
Summary of the invention
The purpose of this invention is to provide Bombyx mori nuclear polyhydrosis virus and preparation method and its application in genetic expression that a kind of improved polyhedrosis gene both sides contain restriction enzyme site, this silkworm baculovirus that is used for the foreign gene reorganization contains polyhedrosis gene, virus particle can be coated in the polyhedron, obtains Bombyx mori nuclear polyhydrosis virus DNA by separation, purifying polyhedron.
Described virus of the present invention is characterized in that:
, added Bsu 36I restriction enzyme site in the polyhedrosis gene both sides and formed through transforming by Bombyx mori nuclear polyhydrosis virus.
The preparation method of above-mentioned virus is:
1. design a pair of primer, the sequence that in primer 1 sequence, comprises one section polyhedron translation starting point, before this sequence, add the restriction enzyme site of Bsu 36I, in the primer 2 sequence, comprise the sequence of one section polyhedron translation termination point, after this sequence, add the restriction enzyme site of Bsu 36I;
2. amplify the polyhedrosis gene fragment with above-mentioned a pair of primer by the PCR reaction;
3. above-mentioned polyhedrosis gene fragment is cut with BamH I and EcoR I enzyme, and with transferring plasmid pBacPAK8 with BamH I and EcoR I enzyme chipping shape virus expression systems, connect with the T4 dna ligase.Connect liquid transformed competence colibacillus bacterium DH5 α, positive colony carries out enzyme and cuts evaluation;
4. the transferring plasmid and the linearizing BmPAK6 viral DNA cotransfection Bm N cell of reorganization polyhedrosis gene obtain to have polyhedrosis silkworm baculovirus strain isolated.
Polyhedrosis silkworm baculovirus strain isolated being applied as in genetic expression of above-mentioned acquisition:
1. contain polyhedrosis silkworm baculovirus strain isolated purified after, add the food silkworm larva or with virus particle liquid injection silkworm larva or pupa with polyhedron, the polyhedron of a large amount of amplicon virus;
2. behind the above-mentioned polyhedron purifying, use the lysate cracking, extract viral DNA;
3. viral DNA is behind Bsu 36I enzymolysis, and circumferential separation forms two DNA bands;
4. such viral DNA can produce recombinant virus as recombinating with foreign gene.
The invention has the advantages that:
Polyhedrosis gene is introduced in silkworm-baculovirus expression system again, and viral DNA can obtain in a large number by the mode of adding food silkworm larva or pupa with polyhedron; There is restriction enzyme site at two ends at polyhedrosis gene, can make the abundant linearizing of viral DNA, improve recombination fraction; During the viral plaque screening, the polyhedron spot that the virus of differentiating the spot of recombinant virus formation easily and taking place to recombinate forms.
Description of drawings
The improved polyhedrosis gene of Fig. 1 both sides contain the characteristic pattern of the silkworm baculovirus of restriction enzyme site.
The improved polyhedrosis gene of Fig. 2 both sides contain the electrophorogram of silkworm baculovirus DNA after Bsu 36 I enzymes are cut of restriction enzyme site.
The improved polyhedrosis gene of Fig. 3 both sides contain the polyhedron that forms behind the silkworm baculovirus infected silkworm cell of restriction enzyme site.
Embodiment
As shown in Figure 1, among the figure, 1 is illustrated in the characteristic sequence that an insertion is arranged before the polyhedrosis gene translation initiation codon,, comprise the restriction enzyme site of Bus 36 I; 2 are illustrated in the characteristic sequence that an insertion is arranged behind polyhedrosis gene translation stop codon, comprise the restriction enzyme site of Bus 36 I; The reading frame of 3 expression polyhedrosis genes begins to finish to taa from atg, amplifies the polyhedrosis gene fragment with above-mentioned a pair of primer by the PCR reaction, and reaction conditions is:: 94 ℃, sex change 4 minutes; 94 ℃, 50 seconds, 50 ℃ 40 seconds, 72 ℃, totally 3 circulations in 1 minute; 94 ℃, 50 seconds; 64 ℃ 30 seconds, 72 ℃, 1 minute, totally 25 circulations, last 72 ℃ were extended 10 minutes.
As shown in Figure 2, in silkworm baculovirus originally, do not contain Bsu 36 I restriction enzyme sites, can not be cut by its enzyme.The silkworm baculovirus DNA that contains restriction enzyme site through improved polyhedrosis gene both sides can be cut by Bsu 36 I enzymes, produces two fragments, about 750 bases of polyhedrosis gene fragment and another big fragment as homologous recombination.
The example that is configured to the recombinant silkworm baculovirus that contains green fluorescent protein:
1. the GFP gene fragment is inserted among the recombinant transfer plasmid BacPak8.
2. extract recombinant transfer plasmid BacPak8-GFP DNA in a large number.
3. collect the polyhedron particle that contains the Bombyx mori nuclear polyhydrosis virus of Bsu 36I restriction enzyme site through the polyhedrosis gene both sides of transformation.
4. above-mentioned polyhedron particle lysate cracking.Prescription is: 0.1mol/L Na
2CO
3, 0.15mol/L NaCl., 0.5mmol/LEDTA, pH 10.8.
5. lysate is after phenol/chloroform extracting, with 70% ethanol sedimentation viral DNA.
6. viral DNA dissolves with aqua sterilisa.
7. viral DNA 5 micrograms add 5 microlitre Bsu, 36 I enzyme liquid enzymolysis, and the reaction cumulative volume is 50 microlitres.
Viral DNA 1 microgram of recombinant transfer plasmid BacPak8-GFP DNA 5 micrograms and above-mentioned Bsu 36I enzymolysis liposome-mediated down, transfection silkworm BmN cell.Observe after 5 days, producing fluorescent substance in some cells is green fluorescent protein, does not have polyhedron to occur.
9. the silkworm Bm N cell that viral liquid transfection is fresh has a large amount of cells to send fluorescence, does not have polyhedron to occur.This viral DNA is described after enzyme is cut, recombination fraction closely reaches 100%.
As shown in Figure 3, what turn white look among the figure is the fluorescence that GFP protein sends, and shows that the GFP gene expresses, and does not observe polyhedrosis existence in cell.Illustrate and use technological method of the present invention can make the foreign gene recombination fraction reach nearly 100%.
Sequence table
Preparation method in the summary of the invention: wherein the sequence of primer 1 is: GGCGGATCCTTAGGATGCCGAATTATTCATACA, and the sequence of primer 2 is: GACGAATTCCTCAGGTTAATACGCCGGACCAGT;
The characteristic sequence of an insertion is arranged before the polyhedrosis gene translation initiation codon among the embodiment, be GGATCCTTAGG
The characteristic sequence of an insertion is arranged behind polyhedrosis gene translation stop codon, be CCTGAGGAATTC
Claims (4)
1. the Bombyx mori nuclear polyhydrosis virus that contains restriction enzyme site is characterized in that being transformed by the Bombyx mori nuclear polyhydrosis virus process, has added Bsu 36I restriction enzyme site in the polyhedrosis gene both sides and has formed.
2. the preparation method of the Bombyx mori nuclear polyhydrosis virus that contains restriction enzyme site of transformation according to claim 1 is characterized in that:
(1) designs a pair of primer, the sequence that in primer 1 sequence, comprises one section polyhedron translation starting point, before this sequence, add the restriction enzyme site of Bsu 36I, in the primer 2 sequence, comprise the sequence of one section polyhedron translation termination point, after this sequence, add the restriction enzyme site of Bsu 36I;
(2) amplify the polyhedrosis gene fragment with above-mentioned a pair of primer by the PCR reaction;
(3) above-mentioned polyhedrosis gene fragment is cut with BamH I and EcoR I enzyme, and with transferring plasmid pBacPAK8 with BamH I and EcoR I enzyme chipping shape virus expression systems, connect with the T4 dna ligase; Connect liquid transformed competence colibacillus bacterium DH5 α, positive colony carries out enzyme and cuts evaluation;
(4) transferring plasmid and the linearizing BmPAK6 viral DNA cotransfection Bm N cell of reorganization polyhedrosis gene obtain to have polyhedrosis silkworm baculovirus strain isolated.
3. the preparation method of the Bombyx mori nuclear polyhydrosis virus that contains restriction enzyme site of transformation according to claim 2, it is characterized in that: the sequence of described primer 1 is: GGCGGATCCTTAGGATGCCGAATTATTCATACA, the sequence of primer 2 is: GACGAATTCCTCAGGTTAATACGCCGGACCAGT.
4. realize the application of the Bombyx mori nuclear polyhydrosis virus that contains restriction enzyme site in genetic expression of the described transformation of claim 1, it is characterized by:
(1) contain polyhedrosis silkworm baculovirus strain isolated purified after, add the food silkworm larva or with virus particle liquid injection silkworm larva or pupa with polyhedron, the polyhedron of a large amount of amplicon virus;
(2) behind the above-mentioned polyhedron purifying, use the lysate cracking, extract viral DNA;
(3) viral DNA is behind Bsu 36I enzymolysis, and circumferential separation forms two DNA bands;
(4) such viral DNA can produce recombinant virus as recombinating with foreign gene.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200410066139 CN1637138A (en) | 2004-12-09 | 2004-12-09 | Silkworm nuclear polyhedron virus and its prepn process and application in gene expression |
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| CN 200410066139 CN1637138A (en) | 2004-12-09 | 2004-12-09 | Silkworm nuclear polyhedron virus and its prepn process and application in gene expression |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181405A (en) * | 2011-01-25 | 2011-09-14 | 湖州市农业科学研究院 | Recombinant virus and method for producing L-asparaginase II by using virus |
| CN108588122A (en) * | 2018-04-18 | 2018-09-28 | 西南大学 | The polygenes element and expression vector and application that two gene equivalent are expressed are realized based on 2A cleavage of peptide |
| CN110184243A (en) * | 2019-06-14 | 2019-08-30 | 辽宁省海洋水产科学研究院 | The method and its kit of Antheraea pernyi nuclear polyhedrosis virus genomic DNA linearisation |
| CN114847243A (en) * | 2022-04-27 | 2022-08-05 | 广西壮族自治区蚕业技术推广站 | Method for obtaining BmNPV disease pupae by feeding late-stage 5-year-old virus-inoculated silkworms or puncturing and virus-inoculated silkworms |
-
2004
- 2004-12-09 CN CN 200410066139 patent/CN1637138A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181405A (en) * | 2011-01-25 | 2011-09-14 | 湖州市农业科学研究院 | Recombinant virus and method for producing L-asparaginase II by using virus |
| CN108588122A (en) * | 2018-04-18 | 2018-09-28 | 西南大学 | The polygenes element and expression vector and application that two gene equivalent are expressed are realized based on 2A cleavage of peptide |
| CN110184243A (en) * | 2019-06-14 | 2019-08-30 | 辽宁省海洋水产科学研究院 | The method and its kit of Antheraea pernyi nuclear polyhedrosis virus genomic DNA linearisation |
| CN114847243A (en) * | 2022-04-27 | 2022-08-05 | 广西壮族自治区蚕业技术推广站 | Method for obtaining BmNPV disease pupae by feeding late-stage 5-year-old virus-inoculated silkworms or puncturing and virus-inoculated silkworms |
| CN114847243B (en) * | 2022-04-27 | 2023-02-07 | 广西壮族自治区蚕业技术推广站 | Method for obtaining BmNPV disease pupae by feeding late-stage 5-year-old virus-inoculated silkworms or puncturing and virus-inoculated silkworms |
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