Recombinant canine measles virus expressing luciferase

A technology of measles virus and enzyme cleavage site, which can be applied to viruses, viral peptides, viruses/phages, etc., and can solve problems such as high-efficiency expression of NLuc

Pending Publication Date: 2020-10-02
QINGDAO AGRI UNIV
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  • Description
  • Claims
  • Application Information

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  • Recombinant canine measles virus expressing luciferase
  • Recombinant canine measles virus expressing luciferase
  • Recombinant canine measles virus expressing luciferase

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Embodiment 1

[0024] Embodiment 1, construction of rCMV-NLuc cDNAclone

[0025] The recombinant CMV nucleic acid fragment rCMV-NLuc cDNA clone used to rescue and express NLuc constructed in this example was distributed in "5'-N-P-NLuc-M-F-H-L-3'" ( figure 1 ); wherein, N, P, M, F, H, L are structural genes of the virus; the selected N, P, M, F, H, L genes of this embodiment are CMV strain 5804P (GenBank: AY386316.1) related genes; NLuc is The ORF of luciferase, the upstream of the ORF includes MGS and Not I restriction sites, and the downstream includes Pme I restriction sites and PGE.

[0026] In order to obtain an ideal rescue efficiency, the present invention inserts the exogenous NLuc gene between the P and M genes; at the same time, in order to increase the expression of NLuc, a Kozak sequence is inserted upstream of the NLuc ORF.

[0027] The nucleotide sequence of one rCMV-NLuc cDNA clone is SEQ ID NO:1.

[0028] The full-length sequence of the rCMV-NLuc cDNA clone whose nucleoti...

Embodiment 2

[0029] Embodiment 2, the rescue of rCMV-NLuc

[0030] Resuscitate BSR-T7 / 5 cells, passage twice in DMEM medium, DMEM medium contains penicillin (100U / mL), streptomycin (100μg / mL), amphotericin B (0.25μg / mL), G418 (500μg / mL) And 10% FBS (fetal bovine serum). The day before transfection, inoculate BSR-T7 / 5 cells in a 6-well plate, and when the cells reach 70% confluence the next day, clone rCMV-NLuc cDNA clone plasmids and eukaryotic expression plasmids expressing CMV N, P, and L proteins respectively BSR-T7 / 5 cells were co-transfected, and the transfection amounts of the four methods were 4 μg, 2 μg, 1 μg, and 1 μg per well, respectively. Prepare Solution I by adding 150 μL Opti-MEM and 16 μL Lipofectamine 2000 to each well. Add 150 μL Opti-MEM and 8 μg plasmid to each well to prepare solution II. Gently mix solutions I and II, and place at room temperature for 15 minutes to form liposome-DNA complexes. Aspirate the culture medium of the 6-well plate, add 1mL Opti-MEM to ea...

Embodiment 3

[0031] Embodiment 3, blind transmission and identification of rCMV-NLuc

[0032] The rescued rCMV-NLuc was blindly passed in VDS cells, and syncytial cell lesions could be observed in the second generation (P2) ( image 3 ). Continue to blindly pass to the fifth generation (P5), the virus solution is frozen and thawed once, and the supernatant is collected to extract total RNA, which is used for RT-PCR detection of the virus. The upstream primer sequence: 5'-gatcaaaagtatcacacatgcttaa-3', the downstream primer sequence: 5'-gatcgaagtcgtacacctcagtcat-3', amplifying a 799bp fragment. The RT-PCR reaction used High Fidelity OneStep RT-PCR Kit (Takara), and the reaction conditions were set as follows: 45°C for 10 minutes, 94°C for 2 minutes, and 30 cycles: 98°C (10s), 55°C (15s), 68°C ( 10s). In order to eliminate residual plasmid contamination, another PCR control was set up. The PCR reaction used 2×PrimeSTAR Max Premix (Takara), and the reaction conditions were set to 30 cycles...

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Abstract

The invention aims to provide a recombinant canine measles virus expressing luciferase NLuc. The structure of a recombinant CMV nucleic acid fragment for rescuing and expressing NLuc is 5'-N-P-NLuc-M-F-H-L-3', wherein N, P, M, F, H and L are structural genes of the virus; NLuc is ORF of luciferase, the upstream of the ORF contains an M gene initiation sequence and a Not I restriction enzyme cutting site, and the downstream contains a Pme I restriction enzyme cutting site and a P gene termination sequence. The rCMV-NLuc rescued by a reverse genetic technology has a high expression level on NLucin cells, and can emit macroscopic light blue fluorescence under the action of a luminescent substrate furimazine. The insertion of an exogenous gene does not significantly affect the growth kineticsof the virus, and the exogenous gene is relatively stable in the virus passage process.

Description

technical field [0001] The invention belongs to the technical field of recombinant virus construction, and specifically relates to an expression Luciferase from recombinant canine measles virus. Background technique [0002] Canine morbillivirus (CMV), formerly known as "canine distempervirus", belongs to the family Paramyxoviridae and a member of the genus Morbillivirus. CMV has a wide range of hosts, and canines, mustelids, and cats are all susceptible to infection. Within 24 hours after the virus invades the body, it mainly proliferates in the macrophages of the local tissue, and then spreads to the monocytes of the tonsils and bronchial lymph nodes, causing swelling of the tonsils and bronchial lymph nodes; it continues to replicate within 2 to 4 days after infection, and then spreads to other lymphoid organs. Clinical signs in sick animals are usually respiratory, gastrointestinal, epidermal, and central nervous system symptoms, accompanied by biphasic fever, genera...

Claims

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Application Information

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IPC IPC(8): C12N15/45C12N15/53C12N7/01C12N15/11C12Q1/70C12Q1/6869C12R1/93
CPCC07K14/005C12N7/00C12N9/0069C12Q1/701C12Q1/6869C12N2760/18421C12N2760/18422C12Q2531/113C12Q2535/101C12Q2565/125
Inventor 刘拂晓黄漪岚王迁迁王宁单虎
Owner QINGDAO AGRI UNIV
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