CN1654658A - Combined application of gp64 gene and p35 gene of baculovirus - Google Patents
Combined application of gp64 gene and p35 gene of baculovirus Download PDFInfo
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- CN1654658A CN1654658A CN 200410061433 CN200410061433A CN1654658A CN 1654658 A CN1654658 A CN 1654658A CN 200410061433 CN200410061433 CN 200410061433 CN 200410061433 A CN200410061433 A CN 200410061433A CN 1654658 A CN1654658 A CN 1654658A
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Abstract
The present invention is the joint application of baculovirus gp64 gene and p35 gene. The gp64 gene and p35 gene of baculovirus AcMNPV, or Autogrpha California multiple nuclear polyhedrosis virus, are jointly expressed in the joint application. The gp64 gene and p35 gene of baculovirus AcMNPV have index number in GenBank of NC001623. The gp64 gene and p35 gene combination is used in the transfer vector. The present invention provides important theoretic base for constituting recombinant virus with expanded host domain and provides one hopeful way for developing recombinant virus pesticide with wide spectrum pesticidal activity, and improving and developing genetically the expression vector system.
Description
Technical field
The present invention is that a kind of baculovirus is the combined utilization of the gp64 and the p35 gene of autographa californica nuclear polyhedrosis virus (AcMNPV).
Background technology
Baculovirus is the single infection and the arthropods that causes death, it mainly is the pathogenic virus of lepidopterous insects, because baculovirus is to the person poultry harmless, no environment is detained, insect is difficult for producing advantages such as resistance, and 1973 is the control of harmful insect by the WHO promotion, has been used widely so far, be the ideal substitute of chemical pesticide, have extensive market prospects.But because that baculovirus wild-type virus sterilant has an insecticidal spectrum is narrow, the slow-footed shortcoming of desinsection, scientific research persons have renderd a service the desinsection of wild-type virus by gene engineering method and have improved.But the improvement of recombinant virus also mainly concentrates on the desinsection speed aspect that improves virus at present, and to transforming the narrow shortcoming of host range, the transformation that promptly influences baculovirus commercialized development application inferior position also is in the starting stage.Simultaneously, another important use of baculovirus is the expression that is used for recombinant exogenous protein, because it has the modification system of eukaryotic gene expression, it is the external source eukaryotic gene expression system of tool important value, same improvement to this system, particularly improve its host domain for expand baculovirus in the expression of external source eukaryotic protein with regard to the tool significance.
Our research and a large amount of data show that all autographa californica nuclear polyhedrosis virus AcMNPV can infect the cell of multiple different genera, has relatively large host domain, supposition is to be positioned the glycoprotein gp64 on the virus envelope and to have the wide spectrum anti-cell to transfer the active p35 gene of dying to bring into play vital role (Wang Hualin, Ph D dissertation) in the host domain decision of AcMNPV virus.So far the gp64 gene of AcMNPV and the research of p35 gene also are in the stage that function is further resolved both at home and abroad, are not applied to the report of recombinant virus improvement, the example that more above-mentioned two genes are carried out coupling.Thereby we unite the gp64 gene of AcMNPV and p35 gene two committed steps in the virus infection are combined among the present invention, in the hope of obtaining the recombinant virus of tool broad-spectrum insecticidal activity.
Envelope protein gp64 has the recognizing cells acceptor usually and invades the function of sensitive cells with inducing virus in the course of infection of virus, determined identification and the infection of virus to the host.Discover, gp64 class glycoprotein can both cause very strong low pH inductive cytogamy phenomenon during transient expression in various kinds of cell, illustrate that its inducing cell film fusion has tangible high efficiency and broad spectrum, supposition is a kind of target gene that can be used for improveing the recombinant virus insecticidal spectrum.The coded product of p35 gene is a kind of albumen that anti-cell is transferred the function of dying that has.The p35 gene is present in that (AcMNPV, BmNPV OpMNPV), have very high homology each other in the multiple baculovirus.Because p35 brings into play function by interacting with caspase, all works in various kinds of cell, has the inhibition natural death of cerebral cells activity of broad spectrum, we infer that this function of p35 gene also is the target gene that possible be used for improveing recombinant virus.
Summary of the invention
Technical problem to be solved by this invention is: utilize two broad spectrum gene gp64 and p35 with outstanding performance among the baculovirus AcMNPV, with " invasion " of baculovirus and these two relatively independent molecular events of repulsion ability of virus " resisting " host cell, combine dexterously by the form that makes up " pseudovirus ", in the hope of disclosing the mechanism of action of restriction host domain, acquisition has the recombinant baculovirus of wide spectrum infection activity, lays the foundation for making up recombinant virus and the exogenous protein expression system that novel host domain enlarged.
The technical solution adopted in the present invention is as follows:
Baculovirus AcMNPV obtains expressing in application the gp64 and the uniting of p35 gene of autographa californica nuclear polyhedrosis virus jointly, and wherein: the gp64 gene of AcMNPV and the call number of p35 gene in GenBank are NC_001623.
The gp64 of AcMNPV and p35 gene are united the purposes in transfer vector.
We are these two genes among the present invention, promptly encode the AcMNPV membrane glycoprotein the gp64 gene and the coding anti-apoptotic the p35 gene, carried out combined utilization, carried out combination these two relatively independent molecular events of opposing of the invasion of virus and viral pair cell rejection are ingenious from brand-new angle.Unite the effect that the reorganization pseudovirus of expressing these two genes has been analyzed these two gene couplings by structure, find that the gp64 of AcMNPV and the combined utilization of p35 gene can significantly improve the infectivity of HaSNPV virus and the effect of host domain, have the significant application value that is used for recombinant virus exploitation and protein expression system exploitation.
Main effect of the present invention is: gp64 gene and the p35 gene of baculovirus (for example autographa california virus) are carried out coupling, can be used in structure and unite the recombinant virus of expressing gp64 and p35 gene efficiently, the recombinant virus HZ8-P35-GP64-EGFP that is obtained has tangible raising and improvement aspect viral infection and the host's subject range, thereby is that a new way is walked out in the genetic improvement and the exploitation of opening up recombinant virus sterilant and expression vector system.With the bollworm is example, and bollworm is the important pests in the agriculture production, and annual generation area in China is up to more than 100,000,000 mu, and the loss that causes thus accounts for 15～20% of cotton ultimate production, sometimes even surpass 50%, nearly 10,000,000,000 yuan of year financial loss.Therefore, the exploitation of the recombinant virus of high-efficiency broad spectrum just has important social benefit and economic implications, unite the development of the recombinant virus of expressing gp64 and p35 gene and exploitation because it can efficient extn virus host territory and infection activity, all have its great application prospect will be infinitely wide.
Based on above-mentioned effect, the present invention provides the most important theories foundation for making up the recombinant virus that host domain expands, and provides an approach likely for developing the recombinant virus sterilant with broad-spectrum insecticidal activity and the genetic improvement and the exploitation of more effective protein expression carrier system.
Description of drawings
Fig. 1 is the gp64 gene nucleotide sequence of AcMNPV.
Fig. 2 is the p35 gene nucleotide sequence of AcMNPV.
Fig. 3 is gp64 gene and a kind of associating synoptic diagram of p35 gene in this patent of AcMNPV.
Fig. 4 is the signal that contains the transfer vector pFastBac-P35-GP64-EGFP of the p35 of AcMNPV and the associating of gp64 gene, and the structure synoptic diagram of uniting the recombinant virus HZ8-P35-GP64-EGFP of the p35 that expresses AcMNPV and gp64 gene.
Fig. 5 is the infectious graphic representation of uniting the recombinant virus HZ8-P35-GP64-EGFP of the gp64 gene of expressing AcMNPV and p35 gene.
Embodiment
The present invention expresses autographa california virus (Autographa californica MNPV by uniting, AcMNPV) gp64 gene and p35 gene, the recombinant virus of the found coupling gp64 gene of AcMNPV and p35 gene infers that increasing significantly aspect the infectivity of virus and the host's subject range and improveing gp64 gene and the p35 gene of BmNPV and OpMNPV may also have similar effect.
The invention will be further described below in conjunction with example and accompanying drawing.
The gp64 of one .AcMNPV and p35 gene
Gp64 and p35 gene nucleotide series are seen Fig. 1, Fig. 2 respectively, and its call number at the GenBank database is NC_001623.Wherein: gp64 gene coding region total length 1501 Nucleotide, the membrane glycoprotein product that size is 58.5kDa of encoding; P35 gene coding region total length 841 Nucleotide, the anti-cell that the size of encoding is 34.8kDa is transferred the protein product of dying.
The combined utilization of the gp64 of two .AcMNPV and p35 gene
The associated form of two genes as shown in Figure 3, the purposes of its combined utilization (hereinafter to be referred as coupling) in transfer vector, comprise the structure that is used for the modified form recombinant virus, the purposes of the improvement exploitation of recombinant virus comprises the genetic improvement and the exploitation of recombinant virus sterilant or protein expression carrier system.In the process that makes up the modified form recombinant virus, the sequence of the polymerase chain reaction primer of gp64 gene, p35 gene and the op166 promotor of the AcMNPV that is used to increase is as follows:
op166For： 5’-gag?ctc?gcc?gct?cga?gcg?gaa?att?atc?gca?aga?taa?ggc?gca-3’
op166Rev： 5’-gaa?ttc?gga?tcc?ggt?ctt?gta?ggt?ctt?gta?gtg?ttc-3’
AcMNPV?gp64For：5’-aag?ctt?gcc?tca?atg?cta?cta?gta?aat?c-3’
AcMNPV?gp64Rev：5’-aag?ctt?gtg?agt?tca?agt?ctc?gcc-3’
AcMNPV?p35For：?5’-atg?tgt?gta?att?ttt?ccg?gta?gaa?atc?gac?g-3’
AcMNPV?p35Rev：?5’-aat?att?aca?ttt?ttg?ttg?agt?gca?cta?gtt?aca-3’。
Above-mentioned op166 promotor is transformed OpMNPV gp64 gene promoter, and the GenBank call number of its original series is NC_001875.The total length of op166 promotor is 199 nucleotide bases, wherein nucleic acid base 1-6 position is the SacI restriction enzyme site, and the 11-16 position is the XhoI restriction enzyme site, and the 20-185 position is original OpMNPV gp64 promoter sequence, the 188-193 position is the BamHI restriction enzyme site, and the 194-199 position is the EcoRI restriction enzyme site.
The nucleotide sequence of improved op166 promotor is:
1. gagctcgccg?ctcgagcgga?aattatcgca?agataaggcg?cacgttgatt?gggtcacccg 60
61.?gtgtacgttg?ataaagtcac?gtgggcaccc?aacgcgttga?taagcatggg?tatataaggg 120
121.cctacagtgt?tctggtaaat?cagttgcact?gtgctcttca?caggaacact?acaagaccta 180
181.caagaccgga?tccgaattc 199
Above-mentioned transfer vector can be pFastBac-P35-GP64-EGFP, and its construction step is as follows:
1. forward primer (AcMNPV gp64For) and the reverse primer (AcMNPV gp64Rev) with AcMNPV gp64 is template with the AcMNPV virus genom DNA, the PCR condition is for (beginning circulation behind 94 ℃ of sex change 3min, 94 ℃ of sex change 1min, 53 ℃ of annealing 1min, 72 ℃ are extended 2min, and 10Min is extended in 30 circulations at last) amplify the gp64 gene of AcMNPV, product cloning is to pGEM-T-Easy carrier (Promega company product), and the sequence verification sequence is correct.Amplify the op166 promotor with op166For and op166Rev primer from the OpMNPV genome, product cloning is to pGEM-T-Easy carrier (Promega company product), and the sequence verification sequence is correct.By multistep clone construct and contain the intermediate transfer carrier PFBOp166-gp64-egfp that the op166 promotor starts AcMNPV gp64 gene thereafter, concrete steps referring to the Wang Shu master thesis (Chinese Academy of Sciences Wuhan virus Master degree candidate's academic dissertation in 2002, pp16-20).
2. with PCR method middle transfer vector PFBOp166-gp64-egfp is increased the transformation of Bsu36I restriction enzyme site, on the Bsu36I restriction enzyme site that adds by enzyme cutting clone on the expression cassette of hsp70 promoters driven AcMNPV p35 genetic expression, promptly obtain the p35 of the AcMNPV of containing shown in Figure 3 and the transfer vector pFastBac-P35-GP64-EGFP of gp64 gene associating.
In addition, also can be to make up in the coupling of the gp64 of AcMNPV and p35 gene with other any-mode, common feature be exactly in application this two gene obtain jointly expressing, two expression of gene products play a role jointly.
The example of the gp64 of three .AcMNPV and p35 gene combined utilization
Unite the structure of the recombinant virus HZ8-P35-GP64-EGFP of the p35 gene of expressing AcMNPV and gp64 gene, method is as follows:
1. utilize the Bac-to-Bac system of bollworm nucleopolyhedrosis virus to carry out the structure of recombinant virus.
As shown in Figure 4, its concise and to the point construction step comprises:
(1) constructs the transfer vector pFastBac-P35-GP64-EGFP of the gp64 that contains transposon and AcMNPV and the associating of p35 gene as above-mentioned mode, extract the plasmid of transfer vector.
(2) utilize HaBamid transposon system among the intestinal bacteria DH10B, transfer vector pFastBac-P35-GP64-EGFP is transformed among the intestinal bacteria DH10B that contains the HaBamid transposon system, by swivel base the fragment swivel base of entrained AcMNPV p35 gene of transfer vector and the associating of gp64 gene to the HaBacmid genome, obtain to unite the p35 that expresses AcMNPV and the viral HZ8-P35-GP64-EGFP of reorganization HaSNPV of gp64 gene.
(3) carrying out PCR and genomic HindIII enzyme with the Auele Specific Primer of the p35 of AcMNPV and gp64 gene cuts the exactness of recombinant virus HZ8-P35-GP64-EGFP is verified.Make up with reference to routine techniques by containing transposon transfer system construction of recombinant virus.
Swivel base in this specification sheets is meant under the effect of transposase, and the gene fragment between transposon homology exchange arm is transferred to the viral Bacmid that contains transposon receiving sequence (att) from transfer vector.Conversion among Fig. 4 is meant and utilizes electric conversion instrument, and the genetic material of plasmid or virus is imported to bacterial cell or insect cell.
2. the recombinant virus HZ8-P35-GP64-EGFP of the p35 of coupling AcMNPV and gp64 gene can induce the fusion of extremely strong HzAM1 cell under the low pH condition of pH=5.2, and fusion faculty is better than contrast virus far away.
3. the recombinant virus HZ8-P35-GP64-EGFP of the p35 of coupling AcMNPV and the gp64 gene accent that can suppress the SeUCR cell of HaSNPV virus induction is died.Use recombinant virus HZ8-P35-GP64-EGFP and two viral HZ8-GP64-EGFP of contrast and HZ8-EGFP to infect respectively, the result shows, these three kinds of virus infection SeUCR carefully can cause the apoptosis of SeUCR cell to some extent, but the HZ8-P35-GP64-EGFP that infects under the infection multiplicity of MOI=1 only causes 50% left and right sides cell generation apoptosis, contrast viral HZ8-EGFP and then cause 90% above cell generation apoptosis, the apoptosis that HZ8-GP64-EGFP causes also has 70%-80%.
4. the infectivity of the recombinant virus HZ8-P35-GP64-EGFP of the p35 of coupling AcMNPV and gp64 gene is significantly improved.With the p35 gene of not expressing AcMNPV and gp64 gene or only two recombinant viruses of single expression AcMNPV gp64 gene be contrast, measured p35 gene and the recombinant virus virus HZ8-P35-GP64-EGFP of gp64 gene and the growth curve (see figure 5) of viral HZ8-GP64-EGFP of contrast and HZ8-EGFP of coexpression AcMNPV in the sensitive cell line HzAm1 of HaSNPV cell, infecting used virus titer all is 5MOI.Measurement result shows: the virus titer of the p35 gene of coexpression AcMNPV and the recombinant virus of gp64 gene was respectively viral HZ8-GP64-EGFP of contrast and HZ8-EGFP 9 times and 25 times (referring to subordinate list) at metainfective 72 hours.
5. the recombinant virus HZ8-P35-GP64-EGFP of the p35 of coupling AcMNPV and gp64 gene can infect non-sensitive cell.
(1) to the infection of non-sensitive cell SeUCR
Use recombinant virus HZ8-P35-GP64-EGFP and two viral HZ8-GP64-EGFP of contrast and HZ8-EGFP to infect the SeUCR cell respectively, the result shows, these three kinds of viruses can infect the SeUCR cell, but three kinds of viruses can cause the apoptosis of SeUCR cell to some extent, infect with MOI=1, HZ8-EGFP can cause 90% above cell generation apoptosis, the apoptosis that HZ8-GP64-EGFP causes also has 70%-80%, and HZ8-P35-GP64-EGFP only causes 50% left and right sides cell generation apoptosis.And if infect with identical MOI, it is most effective that HZ8-P35-GP64-EGFP infects.
We have also investigated the propagation of virus in this non-sensitive cell.48 hours supernatant behind the extraction virus infection, thereafter infect the HzAm1 cell, the result shows that HZ8-P35-GP64-EGFP and HZ8-GP64-EGFP can breed out, viewed virus multiplication situation is consistent with viewed apoptosis phenomenon, the propagation that promptly causes the more obvious HZ8-GP64-EGFP virus of apoptosis is compared much lower with HZ8-P35-GP64-EGFP virus, explanation is in the SeUCR cell, host cell has limited the infection of the non-virus of assigning by apoptotic pathways, the proteic existence of p35 has suppressed host cell and has entered the approach of dying of transferring, thereby make virus obtain certain duplicating, promptly Bing Du host range is expanded accordingly.
(2) to the infection of non-sensitive cell sf21 and sf9
Use recombinant virus HZ8-P35-GP64-EGFP and two viral HZ8-GP64-EGFP of contrast and HZ8-EGFP to infect sf9 and sf21 cell respectively, the result shows, these three kinds of viruses can infect the sf cell, but two kinds of contrast viruses is infectious relatively poor, and recombinant virus HZ8-P35-GP64-EGFP then can form stronger infection.Also present on apoptosis-induced and infect the similar situation of SeUCR cell, but, the quantity of sf cell generation apoptosis that infects these three kinds of recombinant viruses is not a lot, has only 20%-30% at most, and infected the back 24-72 hour, the fluorescence volume showed increased, but As time goes on, fluorescence weakens again gradually, illustrates that virus removed by host cell in the later stage of infecting.
Result of study show among the present invention the gp64 gene of autographa california virus and p35 gene carried out coupling after, the coexpression gp64 gene that is obtained and the recombinant virus of p35 gene have viral infection and host's subject range of obvious raising and improvement.
6. recombinant virus HZ8-P35-GP64-EGFP infectable infection bollworm 4 instar larvaes of the p35 of coupling AcMNPV and gp64 gene relatively can shorten the desinsection time 30% with contrast virus, have the insecticidal effect of obvious raising.
The array mode of the gp64 of four .AcMNPV and p35 gene different do not influence the effect of above-mentioned two gene combined utilization improvement recombinant virus.The promotor that changes gp64 and p35 gene becomes other to have the effect that stable promotor gene expression promoter (IE1/hr5, hsp70 etc.) does not influence two gene combined utilization improvement recombinant virus yet.
Five. subordinate list
The infectious measurement result of recombinant virus HZ8-P35-GP64-EGFP
Unit: pfu/ml 0h p.i. 12h p.i. 24h p.i. 36h p.i. 48h p.i. 72h p.i.
HZ8-EGFP 3.5*10
3 3.5*10
3 2.0*10
4 5.0*10
4 7.9*10
4 2.0*10
5
HZ8-GP64-EGFP 2.0*10
3 2.0*10
3 7.9*10
4 2.0*10
5 5.0*10
5 6.3*10
5
HZ8-P35-GP64-EGFP 2.0*10
3 2.0*10
3 7.9*10
4 6.3*10
5 1.1*10
6 5.0*10
6
Claims (6)
1. the combined utilization of gp64 of a baculovirus and p35 gene, it is characterized in that: baculovirus AcMNPV obtains expressing in application the gp64 and the uniting of p35 gene of autographa californica nuclear polyhedrosis virus jointly, and the gp64 gene of AcMNPV and the call number of p35 gene in GenBank are NC 001623.
2.AcMNPV gp64 and p35 gene unite purposes in transfer vector.
3. purposes according to claim 2 is characterized in that described transfer vector is pFastBac-P35-GP64-EGFP.
4. purposes according to claim 2 is characterized in that being used for the structure of modified form recombinant virus.
5. purposes according to claim 2 is characterized in that in the process that makes up the modified form recombinant virus, and the sequence of the polymerase chain reaction primer of gp64 gene, p35 gene and the op166 promotor of the AcMNPV that is used to increase is as follows:
op166For：5’-gag?ctc?gcc?gct?cga?gcg?gaa?att?atc?gca?aga?taa?ggc?gca-3’
op166Rev：5’-gaa?ttc?gga?tcc?ggt?ctt?gta?ggt?ctt?gta?gtg?ttc-3’
AcMNPV?gp64For：5’-aag?ctt?gcc?tca?atg?cta?cta?gta?aat?c-3’
AcMNPV?gp64Rev：5’-aag?ctt?gtg?agt?tca?agt?ctc?gcc-3’
AcMNPV?p35For：5’-atg?tgt?gta?att?ttt?ccg?gta?gaa?atc?gac?g-3’
AcMNPV?p35Rev：5’-aat?att?aca?ttt?ttg?ttg-gca?cta?gtt?aca-3’。
6. purposes according to claim 5 is characterized in that: the op166 promotor is transformed OpMNPVgp64 gene promoter, and the GenBank call number of original series is NC_001875,
The nucleotide sequence of improved op166 promotor is:
1. gagctcgccg?ctcgagcgga?aattatcgca?agataaggcg?cacgttgatt?gggtcacccg 60
61.?gtgtacgttg?ataaagtcac?gtgggcaccc?aacgcgttga?taagcatggg?tatataaggg 120
121.cctacagtgt?tctggtaaat?cagttgcact?gtgctcttca?caggaacact?acaagaccta 180
181.caagaccgga?tccgaattc 199
Above-mentioned op166 promotor is transformed OpMNPV gp64 gene promoter, and the GenBank call number of its original series is NC_001875; The total length of op166 promotor is 199 nucleotide bases, wherein nucleic acid base 1-6 position is the SacI restriction enzyme site, and the 11-16 position is the XhoI restriction enzyme site, and the 20-185 position is original OpMNPV gp64 promoter sequence, the 188-193 position is the BamHI restriction enzyme site, and the 194-199 position is the EcoRI restriction enzyme site.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102382803A (en) * | 2011-11-08 | 2012-03-21 | 中国科学院武汉病毒研究所 | Construction method of novel gene engineering recombinant virus for expressing gp64 gene |
| CN114075614A (en) * | 2020-08-12 | 2022-02-22 | 四川大学华西医院 | Method for detecting DNA content of baculovirus |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102382803A (en) * | 2011-11-08 | 2012-03-21 | 中国科学院武汉病毒研究所 | Construction method of novel gene engineering recombinant virus for expressing gp64 gene |
| CN114075614A (en) * | 2020-08-12 | 2022-02-22 | 四川大学华西医院 | Method for detecting DNA content of baculovirus |
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