WO2014032456A1 - 一种双标记重组家蚕杆状病毒及其制备方法和应用 - Google Patents

一种双标记重组家蚕杆状病毒及其制备方法和应用 Download PDF

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WO2014032456A1
WO2014032456A1 PCT/CN2013/077518 CN2013077518W WO2014032456A1 WO 2014032456 A1 WO2014032456 A1 WO 2014032456A1 CN 2013077518 W CN2013077518 W CN 2013077518W WO 2014032456 A1 WO2014032456 A1 WO 2014032456A1
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gene
rfp
baculovirus
egfp
seq
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张耀洲
王俊祥
陈剑清
舒特俊
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天津耀宇生物技术有限公司
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/20—Vector systems having a special element relevant for transcription transcription of more than one cistron

Definitions

  • the invention relates to the field of biological genetic engineering technology, in particular to a double-labeled recombinant silkworm baculovirus and a preparation method and application thereof. Background technique
  • Baculovirus is a kind of DNA virus that specifically infects arthropods in nature.
  • the virions are rod-shaped, the genome is a double-stranded circular DNA molecule, and the DNA is compressed and packaged in a rod-shaped capsid in a supercoiled manner.
  • Bombyx mori nuclear polyhedrosis virus is the pathogen of silkworm pus
  • most of the research on baculovirus is concentrated in the genus Helicoverpa armigera nuclear polyhedrosis virus (AcMNPV), and for the Bombyx mori nuclear polyhedrosis virus (BmNPV, also known as
  • AcMNPV Helicoverpa armigera nuclear polyhedrosis virus
  • BmNPV Bombyx mori nuclear polyhedrosis virus
  • the baculovirus surface display system was developed based on a deep understanding of the structure and function of the viral genome.
  • the baculovirus surface display system uses a baculovirus as a vector, and the foreign gene fragment is inserted between the signal peptide of the viral capsid protein and the mature protein, and is expressed by fusion with the capsid protein or with a specific anchor site.
  • the signal peptide is excised to form an N-terminal fusion protein, which is stably expressed by baculovirus and displayed on the surface of infected cells or virions, and a rod expressing a specific target protein is screened.
  • Virus particles are examples of viruses.
  • the foreign protein or polypeptide can also be fused to the capsular glycoprotein of the baculovirus, and the baculovirus is used as a carrier to obtain an effective display on the surface of the infected cell or the capsule of the virion.
  • the envelope glycoprotein gp64 used for fusion with foreign proteins is a structural protein unique to type I budding viruses such as AcMNPV and BmNPV, and is linked to disulfide bonds to homodimer and trimer.
  • the form of a body or a tetramer is present in the envelope and on the surface of infected cells or virions, mediating the fusion and infestation of viruses and insect cells.
  • the gp64 gene in AcMNPV is about 512 bp in length and is a type I transmembrane glycoprotein. It belongs to the phospho-glycosylated protein and contains two highly hydrophobic regions.
  • the N-terminus is a signal peptide sequence with an endoplasmic reticulum processing. It is a hydrophobic transmembrane domain (TM).
  • TM transmembrane domain Connected to the TM is a hydrophilic domain that links the glycoproteins in the viral envelope to the glycoproteins in the cell membrane.
  • the foreign protein is fused to the N-terminus of gp64 at the C-terminus of the signal peptide, and the fusion protein is passed through.
  • the signal peptide is excised, and the formed N-terminal fusion protein is stably expressed on the surface of the baculovirus to form a baculovirus "pseudovirus".
  • the fusion of the protein of interest into the membrane anchoring domain of gp64 that is, the insertion of the gene of interest into the ORF of the gp64 gene, which disrupts the integrity of gp64, can achieve expression expression of the target protein.
  • the technical problem to be solved by the present invention is that no model organism in the prior art can be used to study the invasion mechanism of the nuclear polyhedrosis virus to the host and the subsequent development of the virus for controlling the virus.
  • the present invention provides a double-labeled recombinant silkworm baculovirus comprising a baculovirus envelope protein gp64 signal peptide SP gene, an enhanced green fluorescent protein EGFP gene, and a baculovirus envelope protein gp64.
  • the nucleotide sequence of the tandem gene SP-EGFP-TM is as shown in SEQ ID NO: 1: the nucleotide sequence of the tandem gene RFP-VP39 is as shown in SEQ ID NO: 2; The nucleotide sequence of -RFP is shown as SEQ ID NO: 3.
  • the preparation method of the above double-labeled recombinant silkworm baculovirus is as follows:
  • Recombinant baculovirus transposition vector was transformed into E. coli competent cells, and cultured on LB medium containing kanamycin, gentamicin, tetracycline, X-gal and IPTG for 40 to 48 hours to pick white spots. After 20 to 24 hours of culture, the genome was extracted for PCR identification, and the correct double-labeled recombinant silkworm baculovirus was identified.
  • the amplification primer of the step (1) baculovirus envelope protein gp64 signal peptide SP gene is SEQ IDNO: 4 ⁇ 5
  • the amplification primer for the baculovirus envelope protein gp64 transmembrane domain TM gene is SEQ ID NO: 6 to 7
  • the amplification primer for the enhanced green fluorescent protein EGFP gene is SEQ ID NO: 8 to 9, red fluorescent protein.
  • the amplification primers for the RFP gene are SEQ ID NO: 10-11 or SEQ ID NOs: 12 to 13
  • the amplification primers for the baculovirus capsid protein VP39 gene are SEQ ID NO: 14-15 or SEQ ID NOs: 16 to 17.
  • the viral expression vector of the step (2) is pFastBac-Dual.
  • the tandem gene SP-EGFP-TM is inserted into the multiple cloning site of the vector pFastBac-Dual PplO promoter; the tandem gene RFP-VP39 or VP39-RFP is inserted into the vector pFastBac-Dual Multiple cloning site after the PpH promoter.
  • the tandem gene SP-EGFP-TM is inserted through a Sma l ⁇ WXho l double restriction site; the tandem gene RFP-VP39 or VP39-RFP is inserted through a HI and EcoR I double restriction sites.
  • the Escherichia coli according to the step (2) is DH10Bac.
  • the amplification primers of the PCR of the step (3) are as shown in SEQ ID NOs: 18 to 19.
  • the dual-labeled recombinant silkworm baculovirus of the present invention is used as a silkworm nuclear polyhedrosis virus model organism. Compared with the prior art, the present invention has the following beneficial effects:
  • GP64 is the envelope glycoprotein of wild-type silkworm baculovirus.
  • the wild-type virus displays the surface of GP64 protein on the capsule.
  • the virus modified by the present invention continues to use GP64 signal peptide (SP) and transmembrane region (TM).
  • SP signal peptide
  • TM transmembrane region
  • the intermediate display protein changed the original GP64 to the green fluorescently labeled protein EGFP, thus displaying the EGFP surface on the envelope.
  • the red fluorescent protein RFP is expressed on the nucleocapsid.
  • the viral strain Bmgp64EGFP-RFPVP39 genome differs from the wild BmNPV genome in that the gene sequences between the two transposons Tn7L and Tn7R are different, and in the recombinant vector pFastBac-Dual-gp64-EGFP-RFP-VP39 of the present invention, the transposon The gene sequence between Tn7L and Tn7R is homologously recombined with the gene sequence between the transposon Tn7L and Tn7R of wild BmNPV. Then, the target genes SP-EGFP-TM and RFP-VP39 (or VP39-RFP) of the present invention. On the wild BmNPV genome, the resulting virus is a recombinant virus with a fluorescent protein marker.
  • the recombinant virus of the invention uses the two-color fluorescent protein to fluorescently label the viral capsid and the envelope protein, and can more clearly observe the whole process of baculovirus infection and replication, using the world advanced microscopic equipment (Leica three-channel laser) Confocal microscope, electron microscope) invasion, expression, assembly of viruses Real-time monitoring for a more intuitive understanding of the virus infection mechanism.
  • the virus can be used to study the expression and processing of other display proteins in host cells.
  • Figure 1 is a schematic diagram showing the structure of the recombinant transfer plasmid pFastBac-Dual-gp64-EGFP-RFP-VP39.
  • Figure 2 is a schematic diagram of the original plasmid pFastBac-Dual map.
  • Figure 3 is the result of the overlap and identification of the tandem gene SP-EGFP-TM, wherein the left panel M: DNA standard molecular weight, 1: SP-EGFP overlapping gene, 2: SP-EGFP-TM overlapping gene; right panel M: DNA standard molecular weight , 1 : PCR amplification product of SP-EGFP-TM, 2: double digestion product of pFastBac-Dual-SP-EGFP-TM.
  • Figure 4 is the overlap and identification results of VP39 and RFP
  • M is the DNA standard molecular weight
  • left panel 1 VP39 product obtained by amplification of primer pair VP39.1, 2: RFP product amplified by primer pair RFP.1 , 3: use primer pair RFP.2 product, 4: VP39.2 PCR product
  • middle panel 1 VP39-RFP overlapping gene
  • 2 RFP-VP39 overlapping gene
  • right panel 1 RFP-VP39 overlapping gene PCR amplification Product
  • 2 RFP-VP39 overlapping gene double digested product.
  • Figure 5 is a laser confocal micrograph of the recombinant baculovirus strain Bmgp64EGFP-RFPVP39 of the silkworm, in which red (A) shows the red fluorescent protein RFP-labeled baculovirus capsid protein VP39, and green (B) shows enhanced green fluorescence. Protein EGFP-tagged baculovirus envelope protein gp64 transmembrane domainTM. detailed description
  • Biomaterial sources pFastBac-Dual vector, E. coli TG1 competent cells and E. coli DHlOBac were purchased from Invitrogen; wild Bombyx mori baculovirus BmNPV, EGFP-containing recombinant vector, and plasmid containing red fluorescent protein RFP gene were saved by our laboratory. .
  • the vector for providing the above gene such as wild silkworm baculovirus BmNPV, EGFP-containing recombinant vector, and the plasmid containing the red fluorescent protein RFP gene, can be replaced with any other biological material containing the above gene.
  • primers P1 and P2 were used for PCR to amplify the signal peptide (SP) gene sequence of gp64.
  • SP signal peptide
  • the PCR reaction parameters were set to: pre-denaturation at 98 °C for 5 min, denaturation at 98 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s, 30 cycles, and total extension at 72 °C for 5 min.
  • the amplified fragment is identified by electrophoresis, and the target fragment is recovered by gelatinization.
  • TM transmembrane region
  • primers P3 and P4 were used for PCR to amplify the transmembrane region (TM) gene sequence of gp64.
  • the PCR reaction parameters were set to: pre-denaturation at 98 °C for 5 min, denaturation at 98 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s, 30 cycles, and total extension at 72 °C for 5 min.
  • P3 CGAGCTGTACAAGATGGCTGAAGGCGAATTGGC ( SEQ ID NO: 6 );
  • P4 CTGCTCGAGTTAATATTGTCTACTATTACGG (SEQ ID NO: 7).
  • the amplified fragment is identified by electrophoresis, and the target fragment is recovered by gelatinization.
  • Amplification of EGFP gene fragment is based on the EGFP-containing recombinant vector constructed in our laboratory (the EGFP protein gene sequence is a known sequence, so other vectors or cells containing the EGFP protein gene sequence can also be used, etc.)
  • P5 and P6 were used as primers for PCR amplification.
  • the PCR parameters were set to: pre-denaturation at 98 °C for 5 min, denaturation at 98 °C for 30 s, annealing at 63 °C for 30 s, 72 °. C extends for 30 s, 30 cycles, and a total extension of 5 minutes at 72 °C.
  • P5 TGCCTTTGCGGCGGTGAGCAAGGGCGAGGAGC (SEQ ID NO: 8); P6: ATTCGCCTTCAGCCATCTTGTACAGCTCGTCC (SEQ ID NO: 9).
  • the amplified fragment is identified by electrophoresis, and the target fragment is recovered by gelatinization.
  • PCR reaction parameters were set to: pre-denaturation at 98 °C for 5 min, denaturation at 98 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 30 s, 30 cycles, and total extension at 72 °C for 5 min.
  • the amplified fragment was identified by electrophoresis, and the target fragment SP-EGFP was recovered by gelatinization.
  • Overlapping extension PCR of tandem fragment SP-EGFP and TM Take the recovered product of tandem fragment SP-EGFP and the recovered product of TM gene into PCR tube without primer (for the same reason as above), perform overlap extension PCR, and set the PCR parameters. : 98°C pre-denaturation for 5min, 98 °C for 30s, 54°C for 30s, 72°C for 30s, 25 cycles, 72°C for 5min.
  • the electrophoresis was carried out, and the overlapping fragment SP-EGFP-TM (small amount) was recovered by gel-cutting, and the overlapping fragment SP-EGFP-TM was used as a template, P1 was used as the upstream primer, and P4 was the downstream primer.
  • the overlapping fragment SP-EGFP-TM was added.
  • the PCR reaction parameters were set to: pre-denaturation at 98 °C for 5 min, denaturation at 98 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 30 s, 30 cycles, total extension at 72 °C for 5 min. .
  • the water bath was digested at 37 °C for 30 min, and the digested products were identified by electrophoresis.
  • the vector plasmid and the target fragment were each subjected to gel extraction, ligation, transformation into E. coli TG1 competent cells, and LB culture plates containing ampicillin and gentamicin.
  • the culture was carried out, cultured at 37 °C for 12 hours, spotted, and shaken for 8 hours, and the plasmid was identified.
  • RFP gene The plasmid containing the red fluorescent protein RFP gene constructed in our laboratory is used as a template (RFP gene is known, any biological material containing the gene such as vector or cell can be used as a template), P7 and P8 was PCR amplified by primers.
  • the PCR parameters were set to: pre-denaturation at 98 °C for 5 min, denaturation at 98 °C for 30 s, annealing at 57 °C for 30 s, extension at 72 °C for 30 s, 30 cycles, and total extension at 72 °C for 5 min. Dd3 ⁇ 40 23.5 ⁇ L 98°C pre-denaturation for 5min
  • the amplification of the VP39 gene can also use the following primer pair VP39.1 (P13 and P14): P13: ATCAGGATCC ATGGCGCTAGTGC (SEQ ID NO: 16);
  • RFP gene and VP39 gene overlap extension PCR Take the recovered fragment RFP and VP39 into the PCR tube without primers (due to the design of the primers, each of the amplified products has a sequence overlapping, and the two products can be mutually primer-interacting For the template), the overlap extension PCR was carried out.
  • the PCR reaction parameters were set to: pre-denaturation at 98 °C for 5 min, denaturation at 98 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 30 s, 25 cycles, total extension at 72 °C for 5 min. .
  • the electrophoresis was carried out, and the overlapping fragment RFP-VP39 (small amount) was recovered by cutting, and the overlapping fragment RFP-VP39 was used as a template, P7 was used as the upstream primer, P12 was the downstream primer, and the overlapping fragment RFP was amplified. -VP39.
  • the PCR reaction parameters were set to: pre-denaturation at 98 °C for 5 min, denaturation at 98 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 30 s, 30 cycles, and total extension at 72 °C for 5 min.
  • the electrophoresis was carried out, and the overlapping fragment RFP-VP39 was recovered by cutting.
  • the primers RFP.1 and VP39.1 products RFP and VP39 since the primers were designed with overlapping portions, overlap extension PCR can be performed without adding primers, and the resulting product is VP39-RFP, amplified.
  • the overlapping fragment VP39-RFP can use P13 as the upstream primer and P10 as the downstream primer.
  • the 3Q ⁇ water bath was digested at 37 °C for 30 min, identified by electrophoresis, and the vector and the target fragment were each subjected to gel extraction, ligation, transformation into E. coli TG1 competent cells, and cultured on an LB plate containing ampicillin and gentamicin, 37 Incubate for 12 hours at °C, select spots, shake the bacteria for 8 hours, and extract the plasmid for identification.
  • the overlapping fragment VP39-RFP was also digested with the restriction enzyme site S « HI A&coR I into the upstream and downstream ends of the multiple cloning site of the P PH promoter of the recombinant vector P FastBac-Dual-gp64-EGFP.
  • - VP39 the description of which is not repeated here, and the constructed plasmid was named pFastBac-Dual-gp64-EGFP-VP39-RFP.
  • Primer (M13 upstream primer sequence: CGCCAGGGTTTTCCCAGTCACGAC ⁇ SEQ ID NO: 18>; M13 downstream primer sequence: CAGGAAACAGCTATGACC ⁇ SEQ ID NO: 19>) PCR identification was performed.
  • the successfully identified recombinant baculovirus genome was transfected into BmN cells (purchased from Invitrogen) by liposome-mediated method. After the onset (microscopic observation), a generation of virus suspension was obtained, stored at 4 ° C, and the viral genome was extracted with M13.
  • the silkworm recombinant baculovirus strain Bmgp64EGFP-RFPVP39 was obtained by primer identification.
  • the recombinant transfer plasmid pFastBac-Dual-gp64-EGFP-VP39-RFP can also be obtained by the above transformation, screening and transfection, and the recombinant silkworm virus can be obtained by the same method.
  • the resulting virus was named Bmgp64EGFP-VP39RFP.
  • Example 2 Application of Bombyx mori recombinant baculovirus strain Bmgp64EGFP-RFPVP39 as a model organism Before the silkworm recombinant baculovirus strain Bmgp64EGFP-RFPVP39 infects the cells, the green fluorescent protein is displayed on the surface of the capsule, and the red fluorescent protein is displayed on the capsid.
  • red and green fluorescence can be observed simultaneously.
  • the capsule is fused with the cell membrane, and the nucleocapsid enters the cell, and the red fluorescence can be observed to observe the direction of the viral nucleocapsid until the entire infection pathway and process enter the nucleus. (See Figure 5)
  • the virus Bmgp64EGFP-VP39RFP has the same function as Bmgp64EGFP-RFPVP39, and the specific experimental procedure will not be repeated here.

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Abstract

提供了一种双标记重组家蚕杆状病毒及其制备方法和应用。该病毒含有杆状病毒囊膜蛋白gp64的信号肽SP基因、增强型绿色荧光蛋白EGFP基因和杆状病毒囊膜蛋白gp64的跨膜结构域TM基因形成的串联基因SP-EGFP-TM，以及杆状病毒衣壳蛋白VP39基因和红色荧光蛋白RFP基因形成的串联基因RFP-VP39或VP39-RFP。该病毒的制备方法是将两段串联基因插入到病毒表达载体的多克隆位点，构建成重组杆状病毒载体，在转化大肠杆菌感受态细胞，经过蓝白斑筛选和PCR鉴定后，得到该病毒。该病毒可作为模式生物用于单一病毒粒子的原位示踪。

Description

一种双标记重组家蚕杆状病毒及其制备方法和应用
技术领域
本发明涉及生物学基因工程技术领域， 具体涉及一种双标记重组家蚕杆 状病毒及其制备方法和应用。 背景技术
杆状病毒是一类在自然界中专一性感染节肢动物的 DNA病毒，病毒粒子呈 杆状， 基因组为双链环状 DNA分子， DNA以超螺旋形式压縮包装在杆状衣壳 内， 大小在 90〜180 kb之间。 家蚕核型多角体病毒是家蚕脓病的病原体， 对于 杆状病毒的研究大多数集中在苜蓿银紋夜蛾核型多角体病毒（AcMNPV), 而对 于家蚕核型多角体病毒 （BmNPV, 又称家蚕杆状病毒） 的研究的报道甚少， 所 以开展家蚕核型多角体病毒的侵染机制的研究对于防治家蚕脓病具有重要的意 义。
杆状病毒表面展示系统是在对病毒基因组结构和功能深刻认识的基础上 发展起来的。 杆状病毒表面展示系统以杆状病毒为载体， 外源基因片段插入 到病毒衣壳蛋白的信号肽与成熟蛋白之间， 与衣壳蛋白融合表达或与特异性 的锚定部位结合表达。融合蛋白经过真核细胞内质网加工后，信号肽被切除， 形成 N端融合蛋白， 借助杆状病毒稳定地表达并展示于感染细胞或病毒粒子 的表面， 筛选得到表达有特异目的蛋白的杆状病毒粒子。 外源蛋白或多肽也 可与杆状病毒的囊膜糖蛋白融合， 利用杆状病毒作为载体而在感染细胞的表 面或病毒粒子的囊膜上获得有效的展示。 在杆状病毒表面展示中， 用于与外 源蛋白发生融合的囊膜糖蛋白 gp64是 AcMNPV、BmNPV等 I型出芽病毒特 有的结构蛋白， 与二硫键相连以同源二聚体、 三聚体或四聚体的方式存在于 囊膜内及被感染细胞或病毒粒子的表面， 介导病毒和昆虫细胞的融合与侵染 过程。 AcMNPV中 gp64基因全长约 512 bp, 是 I型跨膜糖蛋白， 属于磷酸 糖基化蛋白， 含有 2个高度疏水区， N端为带有一个内质网加工的信号肽序 列， 近 C端是一个疏水的跨膜结构域 (transmembrane domain,TM), 与 TM相 连的是亲水性结构域， 可以把病毒囊膜内的糖蛋白与细胞膜内的糖蛋白连接 在一起。 外源蛋白在信号肽的 C端与 gp64的 N端发生融合， 融合蛋白经过 真核细胞内质网加工后， 信号肽被切除， 形成的 N端融合蛋白可稳定地在杆 状病毒表面进行表达， 形成杆状病毒 "假病毒"。 另外， 也有将目的蛋白融合 于 gp64的膜锚定结构域中， 即将目的基因插入到 gp64基因的 ORF中， 这种 做法虽然破坏了 gp64的完整性， 但可以实现目的蛋白的表达展示。 发明内容
本发明要解决的技术问题是现有技术中还没有一种模式生物可以用于研究 核型多角体病毒对宿主的侵入机制及后续防治该病毒药物的研制。 为了解决上述技术问题， 本发明提供了一种双标记重组家蚕杆状病毒， 该 病毒含有杆状病毒囊膜蛋白 gp64信号肽 SP基因、 增强型绿色荧光蛋白 EGFP 基因和杆状病毒囊膜蛋白 gp64跨膜域 TM基因形成的串联基因 SP-EGFP-TM, 以及杆状病毒衣壳蛋白 VP39 基因和红色荧光蛋白 RFP 基因形成的串联基因 RFP-VP39或 VP39-RFP。 优选地， 所述串联基因 SP-EGFP-TM的核苷酸序列如 SEQ ID ΝΟ: 1所示； 所述串联基因 RFP-VP39的核苷酸序列如 SEQ ID NO:2所示； 所述 VP39-RFP 的核苷酸序列如 SEQ ID NO:3所示。 上述双标记重组家蚕杆状病毒的制备方法， 歩骤如下：
( 1 )依次串联杆状病毒囊膜蛋白 gp64信号肽 SP基因、 增强型绿色荧光蛋 白 EGFP 基因和杆状病毒囊膜蛋白 gp64 跨膜域 TM 基因， 形成串联基因 SP-EGFP-TM;串联杆状病毒衣壳蛋白 VP39基因序列和红色荧光蛋白 RFP基因 序列， 形成串联基因 RFP-VP39或 VP39-RFP;
(2)将串联基因 SP-EGFP-TM和 RFP-VP39,或 SP-EGFP-TM和 VP39-RFP 插入到病毒表达载体的多克隆位点， 构建成重组杆状病毒转座载体；
(3 ) 重组杆状病毒转座载体转化大肠杆菌感受态细胞， 在含有卡那霉素、 庆大霉素、 四环素、 X-gal和 IPTG的 LB培养基上培养 40〜48小时， 挑取白斑， 培养 20〜24小时后抽提基因组进行 PCR鉴定， 鉴定正确的为双标记重组家蚕杆 状病毒。 优选地，歩骤（1 )杆状病毒囊膜蛋白 gp64信号肽 SP基因的扩增引物为 SEQ IDNO:4〜5， 杆状病毒囊膜蛋白 gp64跨膜域 TM基因的扩增引物为 SEQIDNO: 6〜7， 增强型绿色荧光蛋白 EGFP基因的扩增引物为 SEQIDNO: 8〜9， 红色荧光 蛋白 RFP基因的扩增引物为 SEQIDNO: 10-11或 SEQ ID NO:12〜13，杆状病毒 衣壳蛋白 VP39基因的扩增引物为 SEQIDNO: 14-15或 SEQ ID NO:16〜17。 优选地， 歩骤 （2) 所述病毒表达载体为 pFastBac-Dual。 优选地， 歩骤 （2) 所述串联基因 SP-EGFP-TM插入到载体 pFastBac-Dual 的 PplO启动子后的多克隆位点； 串联基因 RFP-VP39或 VP39-RFP插入到载体 pFastBac-Dual的 PpH启动子后的多克隆位点。 优选地， 串联基因 SP-EGFP-TM是通过 Sma l ^WXho l双酶切位点插入的； 串联基因 RFP-VP39或 VP39-RFP是通过 a H I和 EcoR I双酶切位点插入的。 优选地， 歩骤 （2) 所述大肠杆菌为 DH10Bac。 优选地， 歩骤 （3) 所述 PCR的扩增引物如 SEQIDNO:18〜19所示。 本发明所述的双标记重组家蚕杆状病毒作为家蚕核型多角体病毒模式生物 的应用。 与现有技术相比， 本发明具有以下有益效果：
GP64是野生型家蚕杆状病毒的囊膜糖蛋白， 野生型的病毒将 GP64蛋白表 面展示在囊膜上，本发明改造的病毒继续用 GP64的信号肽 (SP)和跨膜区（TM), 中间的展示蛋白将原先的 GP64改成绿色荧光标记蛋白 EGFP, 这样就将 EGFP 表面展示在囊膜上。 同理， 红色荧光蛋白 RFP被表达在核衣壳上。 病毒株 Bmgp64EGFP-RFPVP39基因组与野生 BmNPV基因组的区别在于两 个转座子 Tn7L、 Tn7R 之间的基因序列不同， 在本发明的重组载体 pFastBac-Dual-gp64-EGFP-RFP-VP39中， 转座子 Tn7L、 Tn7R之间的基因序列 与野生 BmNPV的转座子 Tn7L、 Tn7R之间的基因序列发生了同源重组， 那么， 本发明的目的基因 SP-EGFP-TM和 RFP-VP39 (或 VP39-RFP) 就交换到了野生 BmNPV基因组上， 产生的病毒就是带有荧光蛋白标记的重组病毒。
本发明的重组病毒是利用双色荧光蛋白对病毒衣壳及囊膜蛋白进行荧光标 记， 能够更清楚地观察到杆状病毒侵染复制的全过程， 采用全球先进的显微设 备 （莱卡三通道激光共聚焦显微镜， 电子显微镜） 对病毒的入侵， 表达， 组装 进行实时监控， 更直观的理解病毒的侵染机制。 可以通过该病毒研究其他展示 蛋白在宿主细胞中的表达、 加工展示过程情况。 附图说明
图 1是重组转移质粒 pFastBac-Dual-gp64-EGFP-RFP-VP39结构示意图。 图 2是原始质粒 pFastBac-Dual图谱示意图。 图 3是串联基因 SP-EGFP-TM的重叠及鉴定结果， 其中左图 M: DNA标准 分子量， 1 : SP-EGFP重叠基因， 2: SP-EGFP-TM重叠基因； 右图 M: DNA标 准分子量， 1 : SP-EGFP-TM的 PCR扩增产物， 2: pFastBac-Dual- SP-EGFP-TM 的双酶切产物。
图 4是 VP39与 RFP的重叠及鉴定结果， M为 DNA标准分子量，其中左图 1： 使用引物对 VP39.1扩增所得的 VP39产物， 2: 使用引物对 RFP.1扩增所得 的 RFP产物， 3 : 使用引物对 RFP.2产物， 4: VP39.2PCR产物； 中间图中 1 : VP39-RFP重叠基因， 2: RFP-VP39重叠基因； 右图中 1 : RFP-VP39重叠基因 PCR扩增产物， 2: RFP-VP39重叠基因双酶切产物。
图 5是家蚕重组杆状病毒株 Bmgp64EGFP-RFPVP39激光共聚焦显微图，其 中红色 (A)显示为红色荧光蛋白 RFP标记的杆状病毒衣壳蛋白 VP39, 绿色 (B)显 示为增强型绿色荧光蛋白 EGFP标记的杆状病毒囊膜蛋白 gp64跨膜域 TM。 具体实施方式
下面结合附图和具体实施例对本发明作进一歩说明， 以使本领域的技术人 员可以更好的理解本发明并能予以实施， 但所举实施例不作为对本发明的限定。
生物材料来源： pFastBac-Dual载体、 大肠杆菌 TG1感受态细胞和大肠杆菌 DHlOBac购自 Invitrogen公司； 野生家蚕杆状病毒 BmNPV、 含 EGFP的重组载 体、 含有红色荧光蛋白 RFP基因的质粒均由本实验室保存。 因 gp64信号肽 （SP ) 基因序列 （SEQ ID NO:20)、 gp64跨膜区 （TM) 基 因序列（SEQ ID NO:21 )、杆状病毒衣壳蛋白 VP39基因（SEQ ID NO:22)、 EGFP 蛋白的基因 （SEQ ID NO:23 ) 和 RFP的基因 （SEQ ID NO:24 )均为现有已知基 因序列， 所以本发明使用的提供上述基因的载体如野生家蚕杆状病毒 BmNPV、 含 EGFP的重组载体何含有红色荧光蛋白 RFP基因的质粒可以更换成任何其他 含有上述基因的生物材料。
实施例 1家蚕重组杆状病毒株 Bmgp64EGFP-RFPVP39的制备
( 1 ) gP64信号肽 （SP ) 基因的扩增
以野生家蚕杆状病毒 BmNPV的基因组为模板， 分别用引物 Pl、 P2进行 PCR,扩增 gp64的信号肽（SP)基因序列。 PCR反应参数设为： 98°C预变性 5min， 98°C变性 30s， 58°C退火 30s， 72°C延伸 30s， 30个循环， 72°C总延伸 5min。
P 1： ACACCCGGGATGGTAGGCGCTATTG ( SEQ ID NO:4 ) ;
P2: CCTCGCCCTTGCTCACCGCCGCAAAGGCAGAATG ( SEQ ID NO:5 )。 反应体系如下：
dd¾0 23.5 μL 98°C预变性 5min
5 HF Buffer 10 μL 98°C变性 30s
dNTP 5 μL 58°C退火 30s
PI 5 μL 72°C延伸 30s
Figure imgf000006_0001
P2 5 μL 72 °C 5min 模板 1 μL 4°C 一 酶 0.5 μL
50 μL
待反应结束后， 电泳鉴定扩增片段， 同时切胶回收目的片段。
(2) gp64跨膜区 （TM) 基因的扩增 以野生家蚕杆状病毒 BmNPV的基因组为模板， 用引物 P3、 P4进行 PCR, 扩增 gp64的跨膜区 （TM) 基因序列。 PCR反应参数设为： 98°C预变性 5min， 98°C变性 30s， 56°C退火 30s， 72°C延伸 30s， 30个循环， 72°C总延伸 5min。
P3： CGAGCTGTACAAGATGGCTGAAGGCGAATTGGC ( SEQ ID NO:6 ) ; P4: CTGCTCGAGTTAATATTGTCTACTATTACGG ( SEQ ID NO:7 )。
dd¾0 23.5 μL 98°C预变性 5min
5 HF Buffer 10 μL 98°C变性 30s dNTP 5 μL 56°C退火 30s
P3 5 μL 72°C延伸 30s
Figure imgf000007_0001
P4 5 μL 72 °C 5min 模板 1 μL 4°C 一 酶 0.5 μL
50 μL
待反应结束后， 电泳鉴定扩增片段， 同时切胶回收目的片段。
(3 ) EGFP的基因片段扩增 以本实验室构建的含 EGFP的重组载体为模板（EGFP蛋白基因序列为已知 序列， 所以也可以使用其它包含 EGFP 蛋白基因序列的载体或细胞等， 此处为 了方便， 使用本实验室构建的重组载体）， P5和 P6为引物进行 PCR扩增， PCR 反应参数设为： 98°C预变性 5min， 98°C变性 30s， 63°C退火 30s， 72°C延伸 30s， 30个循环， 72°C总延伸 5min。
P5: TGCCTTTGCGGCGGTGAGCAAGGGCGAGGAGC ( SEQ ID NO:8 ) ; P6: ATTCGCCTTCAGCCATCTTGTACAGCTCGTCC ( SEQ ID NO:9 )。
dd¾0 23.5 μL 98°C预变性 5min
5 HF Buffer 10 μL 98°C变性 30s dNTP 5 μL 63°C退火 30s
P5 5 μL 72°C延伸 30s
Figure imgf000007_0002
P6 5 μL 72 °C 5min 模板 1 μL 4°C —— 酶 0.5
50 μ 待反应结束后， 电泳鉴定扩增片段， 同时切胶回收目的片段。
(4) SP基因与 EGFP基因的重叠延伸 PCR 取回收的 SP基因扩增产物和回收的 EGFP基因扩增产物， 加入到 PCR管， 不加引物 （由于引物设计时， 各自扩增出来的产物有一段序列是重叠的， 两产 物可以互为引物互为模板）， 直接进行重叠延伸 PCR, PCR反应参数设为： 98°C 预变性 5min， 98°C变性 30s， 63 °C退火 30s， 72°C延伸 30s， 25个循环， 72°C总 延伸 5min。
ddH20 8.5 μL 98°C预变性 5min
5 HF Buffer 6 μL 98°C变性 30s dNTP 5 μL 60°C退火 30s
SP基因扩增产物 5 μL 72°C延伸 30s
Figure imgf000008_0001
EGFP基因扩增产物 5 μL 72 °C 5min 酉每 0.5 μL 4°C 一
30 μL 待反应结束后， 走电泳鉴定， 同时切胶回收重叠片段 SP-EGFP (少量）， 再 以重叠片段 SP-EGFP为模板， 以 P1为上游引物， P6为下游引物， 扩增重叠片 段 SP-EGFP, PCR反应参数设为： 98°C预变性 5min， 98°C变性 30s， 60°C退火 30s, 72°C延伸 30s， 30个循环， 72°C总延伸 5min。
dd¾0 23.5 μL 98°C预变性 5min
5 HF Buffer 10 μL 98°C变性 30s dNTP 5 μL 60°C退火 30s
PI 5 μL 72°C延伸 30s
Figure imgf000008_0002
P6 5 μL 72 °C 5min 模板 1 μΐ^ 4°C ― 酶 0.5 μ
50 μ 待反应结束后， 电泳鉴定扩增片段， 同时切胶回收目的片段 SP-EGFP。
( 5 ) 串联片段 SP-EGFP与 TM的重叠延伸 PCR 取串联片段 SP-EGFP的回收产物和 TM基因的回收产物加入 PCR管，不加 引物（理由同上）， 进行重叠延伸 PCR, PCR参数设为： 98°C预变性 5min， 98 °C 变性 30s， 54°C退火 30s， 72°C延伸 30s， 25个循环， 72°C总延伸 5min。
dd¾0 8.5 μL 98°C预变性 5min
5 HF Buffer 6 μL 98°C变性 30s dNTP 5 μL 54°C退火 30s
SP-EGF扩增产物 5 μL 72°C延伸 30s
Figure imgf000009_0001
TM扩增产物 5 μL 72 °C 5min 酶 0.5 μL 4°C 一
30 μL 待反应结束后，走电泳鉴定， 同时切胶回收重叠片段 SP-EGFP-TM (少量）， 再以重叠片段 SP-EGFP-TM为模板， 以 P1为上游引物， P4为下游引物， 扩增 重叠片段 SP-EGFP-TM, PCR反应参数设为： 98°C预变性 5min， 98°C变性 30s， 54°C退火 30s， 72°C延伸 30s， 30个循环， 72°C总延伸 5min。
dd¾0 23.5 μL 98°C预变性 5min
5 HF Buffer 10 μL 98°C变性 30s dNTP 5 μL 54°C退火 30s
PI 5 μL 72°C延伸 30s
Figure imgf000009_0002
P4 5 μL 72 °C 5min 模板 1 μL 4°C 一 酶 0.5
50 μ 待反应结束后， 走电泳鉴定， 同时切胶回收重叠片段 SP-EGFP-TM。
( 6 ) 重组转移质粒 pFastBac-Dual-gp64-EGFP构建 将上述 PCR扩增获得的重叠片段 SP-EGFP-TM通过限制性内切酶
Sma I IXho I (购自 Fermentas公司） 双酶切插入 pFastBac-Dual载体 （购自 Invitrogen公司） 的 PplQ启动子后的多克隆位点的上下游两端， 构建成重组的杆 状病毒表面展示载体 pFastBac-Dual-gp64-EGFP。 反应体系如下：
质粒 pFastBac-Dual 20μΙ^
SP-EGFP-TM 2(^L
lO Fast Digest Buffer 3
酶 Sma I 1
M Xho I 1 ddH20 5 μΐ,
水浴锅 37°C酶切 30min， 酶切产物用电泳鉴定， 载体质粒和目的片段各自 切胶回收， 连接， 转化大肠杆菌 TG1感受态细胞， 在含有氨苄霉素， 庆大霉素 的 LB培养板上培养， 37 °C倒置培养 12小时， 挑斑， 摇菌 8小时， 抽提质粒鉴 定。
( 7 ) RFP基因的扩增 以本实验室构建的含有红色荧光蛋白 RFP基因的质粒为模板 （RFP基因为 已知， 可以选用任何含有该基因的生物材料例如载体或细胞作为模板）， P7 和 P8为引物进行 PCR扩增， PCR反应参数设为： 98°C预变性 5min， 98°C变性 30s， 57°C退火 30s， 72°C延伸 30s， 30个循环， 72°C总延伸 5min。 dd¾0 23.5 μL 98°C预变性 5min
5 HF Buffer 10 μL 98°C变性 30s dNTP 5 μL 57°C退火 30s
P9 5 μL 72°C延伸 30s
Figure imgf000011_0001
P10 5 μL 72 °C 5min 模板 1 μL 4°C 一 酶 0.5 μL
50 μL 待反应结束后， 走电泳鉴定， 结果见图 4左图泳道 3， 同时切胶回收目的片 段 RFP。 该歩骤 RFP基因的扩增还可以使用以下引物对 RFP. l (P9和 P10):
P9: TGTAGCCGCCGGAGGAGGAGGAGGATCGATGGTGCGCTCC ( SEQ ID NO: 12);
P10: GTGGACAAGGACATCCTTAAGACGC ( SEQ ID NO： 13 )。 所得扩增产物的电泳结果如图 4左图的泳道 2。
( 10) VP39基因的扩增 以野生家蚕杆状病毒 BmNPV的基因组为模板， P11和 P12为引物， 扩增杆 状病毒衣壳蛋白 VP39基因序列。 PCR反应参数设为： 98°C预变性 5min， 98 °C 变性 30s， 52°C退火 30s， 72°C延伸 30s， 30个循环， 72°C总延伸 5min。
Pl l :
P12: CCTGAATTCTTAGGCGGCTACACTT(SEQ ID NO: 15)。 dd¾0 23.5 μL 98°C预变性 5min
5 HF Buffer 10 μL 98°C变性 30s dNTP 5 μL 52°C退火 30s
P7 5 μL 72°C延伸 30s
Figure imgf000012_0001
P8 5 μL 72 °C 5min 模板 1 μL 4°C 一 酶 0.5 μL
50 μL 待反应结束后， 走电泳鉴定， 结果见图 4左图泳道 4， 同时切胶回收目的片 段 VP39。 该歩骤 VP39基因的扩增还可以使用以下引物对 VP39.1(P13和 P14): P13: ATCAGGATCC ATGGCGCTAGTGC ( SEQ ID NO:16);
TG ( SEQ ID NO: 17)。 所得扩增产物的电泳结果如图 4左图的泳道 1。
( 11 ) RFP基因与 VP39基因重叠延伸 PCR 取回收片段 RFP和 VP39加入 PCR管， 不加引物 （由于引物设计时， 各自 扩增出来的产物有一段序列是重叠的， 两产物可以互为引物互为模板）， 进行重 叠延伸 PCR, PCR反应参数设为： 98°C预变性 5min， 98°C变性 30s， 54°C退火 30s, 72°C延伸 30s， 25个循环， 72°C总延伸 5min。
dd¾0 8.5 μL 98°C预变性 5min
5 HF Buffer 6 μL 98°C变性 30s dNTP 5 μL 54°C退火 30s
RFP扩增产物 5 μL 72°C延伸 30s
Figure imgf000012_0002
VP39扩增产物 5 μL 72 °C 5min 酶 0.5 4°C ―
30 μ 待反应结束后， 走电泳鉴定， 同时切胶回收重叠片段 RFP-VP39 (少量）， 再以重叠片段 RFP-VP39为模板， 以 P7为上游引物， P12为下游引物， 扩增重 叠片段 RFP-VP39。 PCR反应参数设为： 98°C预变性 5min， 98°C变性 30s， 54 °C 退火 30s， 72°C延伸 30s， 30个循环， 72°C总延伸 5min。
dd¾0 23.5 μL 98°C预变性 5min
5 HF Buffer 10 μL 98°C变性 30s dNTP 5 μL 52°C退火 30s
P9 5 μL 72°C延伸 30s
Figure imgf000013_0001
P8 5 μL 72 °C 5min 模板 1 μL 4°C 一 酶 0.5 μL
50 μL 待反应结束后， 走电泳鉴定， 同时切胶回收重叠片段 RFP-VP39。 使用引物对 RFP.1和 VP39.1所得的产物 RFP和 VP39, 因为引物设计时已 经添加了重叠部分， 因此可以在不添加引物的情况下进行重叠延伸 PCR, 所得 产物为 VP39-RFP, 扩增重叠片段 VP39-RFP可以使用 P13为上游引物， P10为 下游引物。
( 12) 重组转移质粒 pFastBac-Dual-gp64-EGFP-RFP-VP39的构建 将上述 PCR扩增获得的重叠片段 RFP-VP39通过酶切位点 Bamli I /EcoR I 双酶切插入重组载体 pFastBac-Dual-gp64-EGFP的 Pra启动子后的多克隆位点上 下 游 两 端 ， 构 建 成 重 组 的 杆 状 病 毒 转 座 载 体 pFastBac-Dual-gp64-EGFP-RFP-VP39。 反应体系：
pFastBac-Dual-gp64EGFP 20μL 目的片段 RFP-VP39 20μ lOxFast Digest Buffer 3 μL
酶 Sma I 1 Xho I Ι μ dd¾0 5
3Qμ 水浴锅 37°C酶切 30min， 电泳鉴定， 载体和目的片段各自切胶回收， 连接， 转化大肠杆菌 TG1感受态细胞， 在含有氨苄霉素， 庆大霉素的 LB培养板上培 养， 37°C倒置培养 12小时， 挑斑， 摇菌 8小时， 抽提质粒鉴定。 重叠片段 VP39-RFP 也以酶切位点 S« H I A&coR I双酶切插入重组载体 PFastBac-Dual-gp64-EGFP的 PPH启动子后的多克隆位点上下游两端， 构建方式 同 RFP-VP39 ， 此 处不 再重 复 说 明 ， 构 建成 的 质 粒命名 为 pFastBac-Dual-gp64-EGFP-VP39-RFP。
( 13 ) 家蚕重组杆状病毒 Bmgp64EGFP-RFPVP39的获得 鉴定成功的重组转移质粒 pFastBac-Dual-gp64-EGFP-RFP-VP39转化大肠杆 菌 DHlOBac感受态细胞， 在含有卡那霉素、 庆大霉素、 四环素、 X-gal和 IPTG 的 LB培养板上进行蓝白斑筛选， 避光培养 40〜48小时后挑取白斑， 培养 20〜24 小时后用异丙醇抽提重组杆状病毒基因组， 并用 M13通用引物（M13 上游引物 序列： CGCCAGGGTTTTCCCAGTCACGAC< SEQ ID NO: 18>; M13 下游引物 序列： CAGGAAACAGCTATGACC <SEQ ID NO: 19>) 进行 PCR鉴定。 鉴定成 功的重组杆状病毒基因组通过脂质体介导法转染家蚕 BmN细胞 (购自 Invitrogen 公司）， 发病后 （显微镜观察） 获得一代病毒悬液， 4°C保存， 提取病毒基因组 用 M13通用引物鉴定， 获得所述家蚕重组杆状病毒株 Bmgp64EGFP-RFPVP39。
重组转移质粒 pFastBac-Dual-gp64-EGFP-VP39-RFP经过上述转化、 筛选、 转染歩骤也可以得到重组家蚕病毒， 方法同上。 所得病毒命名为 Bmgp64EGFP-VP39RFP。 实施例 2 家蚕重组杆状病毒株 Bmgp64EGFP-RFPVP39作为模式生物的应用 家蚕重组杆状病毒株 Bmgp64EGFP-RFPVP39侵染细胞前，由于其囊膜表面 展示绿色荧光蛋白， 衣壳上展示了红色荧光蛋白在激光共聚焦显微镜下可以同 时观察到红、 绿色荧光， 侵入细胞时， 其囊膜与细胞膜发生融合， 核衣壳进入 细胞， 可以继续观察红色荧光从而观察病毒核衣壳走向， 直至进入细胞核内的 整个侵染途径和过程。 （参见图 5 )
病毒 Bmgp64EGFP-VP39RFP与 Bmgp64EGFP-RFPVP39具有同样的功能， 此处不再重复说明具体实验过程。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例， 本发明的 保护范围不限于此。 本技术领域的技术人员在本发明基础上所作的等同替代或 变换， 均在本发明的保护范围之内。 本发明的保护范围以权利要求书为准。

Claims

权利要求书
1 . 一种双标记重组家蚕杆状病毒， 其特征在于， 该病毒含有杆状病毒囊膜 蛋白 gp64信号肽 SP基因、 增强型绿色荧光蛋白 EGFP基因和杆状病毒囊膜蛋 白 gp64跨膜域 TM基因形成的串联基因 SP-EGFP-TM, 以及杆状病毒衣壳蛋白 VP39基因和红色荧光蛋白 RFP基因形成的串联基因 RFP-VP39或 VP39-RFP。
2. 根据权利要求 1所述的双标记重组家蚕杆状病毒， 其特征在于， 所述串 联基因 SP-EGFP-TM 的核苷酸序列如 SEQ ID ΝΟ: 1 所示； 所述串联基因 RFP-VP39的核苷酸序列如 SEQ ID NO:2所示；所述 VP39-RFP的核苷酸序列如 SEQ ID NO:3所示。
3. 权利要求 1或 2所述的双标记重组家蚕杆状病毒的制备方法， 其特征在 于， 歩骤如下：
( 1 )依次串联杆状病毒囊膜蛋白 gp64信号肽 SP基因、 增强型绿色荧光蛋 白 EGFP 基因和杆状病毒囊膜蛋白 gp64 跨膜域 TM 基因， 形成串联基因 SP-EGFP-TM;串联杆状病毒衣壳蛋白 VP39基因序列和红色荧光蛋白 RFP基因 序列， 形成串联基因 RFP-VP39或 VP39-RFP;
(2)将串联基因 SP-EGFP-TM和 RFP-VP39,或 SP-EGFP-TM和 VP39-RFP 插入到病毒表达载体的多克隆位点， 构建成重组杆状病毒转座载体；
(3 ) 重组杆状病毒转座载体转化大肠杆菌感受态细胞， 在含有卡那霉素、 庆大霉素、 四环素、 X-gal和 IPTG的 LB培养基上培养 40〜48小时， 挑取白斑， 培养 20〜24小时后抽提基因组进行 PCR鉴定， 鉴定正确的为双标记重组家蚕杆 状病毒。
4. 根据权利要求 3所述的双标记重组家蚕杆状病毒的制备方法， 其特征在 于歩骤 （1 ) 杆状病毒囊膜蛋白 gp64 信号肽 SP 基因的扩增引物为 SEQ ID
NO:4〜5，杆状病毒囊膜蛋白 gp64跨膜域 TM基因的扩增弓 1物为 SEQ ID NO: 6〜7， 增强型绿色荧光蛋白 EGFP基因的扩增引物为 SEQ ID NO: 8〜9， 红色荧光蛋白
RFP基因的扩增引物为 SEQ ID NO: 10-11或 SEQ ID NO:12〜13， 杆状病毒衣壳 蛋白 VP39基因的扩增引物为 SEQ ID NO: 14〜15或 SEQ ID NO: 16〜17。
5. 根据权利要求 3所述的双标记重组家蚕杆状病毒的制备方法， 其特征在 于歩骤 （2 ) 所述病毒表达载体为 pFastBac-Dual。
6. 根据权利要求 5所述的双标记重组家蚕杆状病毒的制备方法， 其特征在 于歩骤 （2 ) 所述串联基因 SP-EGFP-TM插入到载体 pFastBac-Dual的 PplQ启动 子后的多克隆位点；串联基因 RFP-VP39或 VP39-RFP插入到载体 pFastBac-Dual 的 PpH启动子后的多克隆位点。
7. 根据权利要求 6所述的双标记重组家蚕杆状病毒的制备方法， 其特征在 于串联基因 SP-EGFP-TM是通过 Sma l ^W Xho l双酶切位点插入的； 串联基因 RFP-VP39或 VP39-RFP是通过 Bamli I和 EcoR I双酶切位点插入的。
8. 根据权利要求 3所述的双标记重组家蚕杆状病毒的制备方法， 其特征在 于歩骤 （2 ) 所述大肠杆菌为 DH10Bac。
9. 根据权利要求 3所述的双标记重组家蚕杆状病毒的制备方法， 其特征在 于歩骤 （3 ) 所述 PCR的扩增引物如 SEQ ID NO: 18〜19所示。
10. 权利要求 1或 2所述的双标记重组家蚕杆状病毒作为家蚕核型多角体病 毒模式生物的应用。
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