Recombinant parainfluenza virus type 5 for expressing double report tags

A parainfluenza and virus technology, applied in the field of recombinant parainfluenza virus type 5

Pending Publication Date: 2021-06-25
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, PIV5 can infect a variety of cell lines, such as Vero cells, BHK cells, MDBK cells, etc., but usually does not cause or only mild cytopathic changes, which limits the application of PIV5 in many ways, such as virus titer determination

Method used

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  • Recombinant parainfluenza virus type 5 for expressing double report tags
  • Recombinant parainfluenza virus type 5 for expressing double report tags
  • Recombinant parainfluenza virus type 5 for expressing double report tags

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1, construction of rPIV5-GN cDNA clone

[0033] In this embodiment, the eGFP-T2A-NLuc fusion protein sequence is first constructed, the upstream includes the M gene start sequence (M GS) and the Pme I restriction site, and the downstream includes the Not I restriction site and the V / P gene termination sequence (V / P GE). eGFP-T2A-NLuc fusion protein sequence as figure 1 , the arrow indicates the self-cleavage site. On this basis, the recombinant PIV5 nucleic acid fragment rPIV5-GN cDNA clone used to rescue co-expression of eGFP and NLuc was constructed. The fragment is distributed in "5'-NP-V / P-eGFP-T2A-NLuc-M-F-SH-HN-L-3'" ( figure 2 ). Among them, NP, V / P, M, F, SH, HN, and L genes are related genes of PIV5 strain (GenBank: NC_006430.1); eGFP is enhanced green fluorescent protein ORF; T2A is a self-cleaving peptide sequence; NLuc ORF for NanoLuc luciferase.

[0034] The nucleotide sequence of the rPIV5-GN cDNA clone constructed in this example is SEQ...

Embodiment 2

[0036] Example 2, Rescue of rPIV5-GN

[0037] 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, BSR-T7 / 5 cells were inoculated in 12-well plates, and the next day when the cells reached 70% confluence, the rPIV5-GN cDNA clone plasmid and three auxiliary plasmids ( image 3) co-transfected BSR-T7 / 5 cells, and the transfection amounts of the four were 2 μg, 1 μg, 0.5 μg, and 0.5 μg per well, respectively. Solution I was prepared by adding 75 μL Opti-MEM and 8 μL Lipofectamine2000 to each well. Then add 75 μL Opti-MEM and 4 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. Add the liposome-DNA mixture dropwise to the cell wells. Place the 12-well plate in CO 2 After transfection in the incu...

Embodiment 3

[0038] Embodiment 3, identification of rPIV5-GN

[0039] The rescued rPIV5-GN was continuously blindly passed to the seventh passage in MDBK cells. With the blind passage of the virus, eGFP reached the desired expression level, but no cytopathic changes were observed throughout the passaging process ( Image 6 ). At the same time, 48 hours after the seventh-generation rPIV5-GN infected cells, NLuc could also be highly expressed ( Figure 7 ). The 7th passage virus solution was frozen and thawed once, and the supernatant was collected to extract total RNA, which was used for RT-PCR detection of the virus. Upstream primer sequence: 5'-GCAATCATCCGCAGTGCAATTTGA-3', downstream primer sequence: 5'-TGCGGGAATGCTGATGGATGGCAT-3', preamplified 1578bp fragment. The RT-PCR reaction adopts HighFidelity One Step RT-PCR Kit (Takara), and the reaction conditions are set as follows: 45°C for 10min, 94°C for 2min, and 30 cycles: 98°C (10s), 55°C (15s), 68°C ( 16s). In order to eliminate re...

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Abstract

The invention aims to provide a recombinant parainfluenza virus type 5 (PIV5) for co-expressing enhanced green fluorescent protein (eGFP) and NanoLuc luciferase (NLuc) The structure of a recombinant PIV5 nucleic acid fragment for rescuing the co-expression of the eGFP and the NLuc is 5'-NP-V/P-eGFP-T2A-NLuC-M-F-SH-HN-L-3', wherein NP, V/P, M, F, SH, HN and L are self genes of PIV5; eGFP-T2A-NLuc is used as a fusion sequence; T2A is a self-cleavage peptide sequence and is responsible for cutting off eGFP and NLuc and separating the eGFP and the NLuc. The recombinant PIV5 rescued by a reverse genetic technology cannot cause MDBK cytopathic effect and cannot induce plaque formation, but the expression level of eGFP and NLuc is relatively high. The growth kinetics of the recombinant PIV5 is positively correlated with the expression kinetics of NLuc. The gene of the eGFP-T2A-NLuc fusion sequence is relatively stable in the continuous 20 times of passage processes of the recombinant PIV5.

Description

technical field [0001] The invention belongs to the technical field of recombinant virus construction, and in particular relates to a recombinant parainfluenza virus type 5 expressing double reporter tags. Background technique [0002] Parainfluenza virus type 5 (parainfluenza virus 5, PIV5), formerly known as "simian virus type 5", belongs to the family Paramyxoviridae and a member of the genus Orthorubulavirus. The PIV5 genome is a single-stranded, negative-strand, linear RNA with a full length of 15246 nt and contains seven non-overlapping genes in the order of 3′-NP-V / P-M-F-SH-HN-L-5′, where the P gene contains V Gene, corresponding to the coding V protein (non-structural protein). After purification, the shape of the virus under the electron microscope is indeterminate, but most of them are spherical or ellipsoidal, covered with a lipid capsule, and the outer surface of the capsule is embedded with rivet-like fibrils. The inner surface of the capsule is adjacent to th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/45C12N7/01C12N15/62C12N15/11
CPCC12N7/00C07K14/005C07K14/43595C12N9/0069C12N2760/18721C12N2760/18743C07K2319/00
Inventor 刘拂晓王迁迁单虎
Owner QINGDAO AGRI UNIV
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