阅读说明：本技术 一种表达荧光素酶的重组犬麻疹病毒 (Recombinant canine measles virus expressing luciferase ) 是由 刘拂晓 黄漪岚 王迁迁 王宁 单虎 于 2020-07-01 设计创作，主要内容包括：本发明的目的是提供一种表达<Image he="67" wi="270" file="DDA0002564502030000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>荧光素酶NLuc的重组犬麻疹病毒,其中用于拯救表达NLuc的重组CMV核酸片段的结构为5′-N-P-NLuc-M-F-H-L-3′；其中,N、P、M、F、H、L为病毒的结构基因；NLuc为<Image he="59" wi="275" file="DDA0002564502030000012.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>荧光素酶的ORF,该ORF上游包含M基因起始序列和Not I酶切位点,下游包含Pme I酶切位点及P基因终止序列。本发明通过反向遗传技术拯救的rCMV-NLuc,在细胞内对于NLuc的表达水平较高,在发光底物furimazine作用下,可发出肉眼可见的淡蓝色荧光。外源基因的插入并未显著影响病毒的生长动力学,且外源基因在病毒传代过程中相对较为稳定。(It is an object of the present invention to provide an expression Recombinant canine measles virus of luciferase NLuc, wherein the recombinant CMV nucleic acid fragment for rescuing the expressed NLuc has a structure of 5 '-N-P-NLuc-M-F-H-L-3'; wherein N, P, M,F. H, L structural genes of the virus; NLuc is An ORF for luciferase comprising an M gene start sequence and a Not I cleavage site upstream of the ORF and a Pme I cleavage site and a P gene termination sequence downstream of the ORF. The rCMV-NLuc rescued by the reverse genetic technology has higher expression level to the NLuc in the cell, and can emit light blue fluorescence which can be seen by naked eyes under the action of a luminescent substrate furimazine. The insertion of the exogenous gene does not significantly affect the growth kinetics of the virus, and the exogenous gene is relatively stable in the virus passage process.)

1. A nucleic acid fragment for rescuing recombinant canine measles virus expressing NLuc, wherein the structure of the nucleic acid fragment is 5 '-N-P-NLuc-M-F-H-L-3', wherein N, P, M, F, H, L is the structural gene of canine measles virus; NLuc is

2. The nucleic acid fragment of claim 1, wherein the sequence of the nucleic acid fragment is SEQ ID No. 1.

3. Use of the nucleic acid fragment of claim 1 or 2 for the intracellular rescue of recombinant canine measles virus.

4. The use of claim 3, wherein said cells are BSR-T7/5 cells and Vero-Dog-SLAM cells.

5. A recombinant canine measles virus capable of expressing NLuc protein, wherein the recombinant canine measles virus was rescued in cells using the nucleic acid fragment of claim 1 or 2.

6. A primer pair for detecting the recombinant canine measles virus of claim 5, wherein the sequence of the upstream primer is 5'-gatcaaaagtatcacacatgcttaa-3' and the sequence of the downstream primer is 5'-gatcgaagtcgtacacctcagtcat-3'.

Technical Field

The invention belongs to the technical field of recombinant virus construction, and particularly relates to expression

Recombinant canine measles virus of luciferase.

Background

Canine Measles Virus (CMV), formerly known as "canine distemper virus", belongs to the family Paramyxoviridae (Paramyxoviridae), the genus Morbillivirus (Morbillivirus). CMV is widespread and susceptible to infection by canines, ferrets, felines, and the like. The virus invades the body for 24 hours, mainly proliferates in local tissue macrophages, and then spreads to tonsil and bronchial lymph node monocytes to cause tonsil and bronchial lymph node enlargement; replication continued for 2-4 days after infection and subsequent spread to other lymphoid organs. The clinical symptoms of the affected animal are usually respiratory, gastrointestinal digestive, epidermal mucosa and central nervous system symptoms, accompanied by biphasic fever, systemic discomfort and viremia.

The CMV genome is a single, negative-strand, linear RNA, 15690 nt in length, encoding six structural proteins (N, P, M, F, H, L proteins) and two non-structural proteins (C, V proteins). The virus is purified and observed under an electron microscope to be in an indefinite shape, but is mostly in a spherical or ellipsoidal shape, a lipid envelope is coated outside, and rivet-shaped fiber protrusions are embedded on the outer surface of the envelope. The inner surface of the envelope is closely adjacent to matrix protein, and the interaction between the monomers forms the outer skeleton of the whole virus, and the inner part contains the herring bone-shaped spiral nucleocapsid. CMV has oncolytic activity and is an ideal oncolytic virus. The research on the oncolytic activity of the oncolytic virus can be aided by luminous images in the bodies of the small animals, so that the observation of the oncolytic effect is more visual. Therefore, the construction of the recombinant oncolytic virus expressing luciferase (rather than fluorescent protein) lays a good foundation for the research of the downstream oncolytic activity.

Luciferase (F: (

luciferase, NLuc) is a small molecule enzyme engineered with a molecular weight of 19.1kD, and is a novel bioluminescent reporter protein. NLuc catalyzes a novel substrate, furimazine, resulting in high intensity, glow-type luminescence. The NLuc-catalyzed bioluminescence reaction does not depend on ATP, the self-luminous background is low, the optical signal is obviously enhanced compared with the traditional fluorescein, the background luminescence is inhibited, and the extremely high detection sensitivity can be obtained. NLuc is used as a fluorescent tracing protein (Genbank accession number: MH037010), and the Open Reading Frame (ORF) is shorter (516bp), so that the gene operation is greatly facilitated. However, there is currently no efficient method for expressing NLuc with CMV.

Disclosure of Invention

The object of the present invention is to provide an expressionRecombinant canine measles virus (rCMV-NLuc) of luciferase (NLuc) to make up for the deficiencies of the prior art.

The invention provides a recombinant CMV nucleic acid fragment (rCMV-NLuc cDNAclone) for rescuing and expressing NLuc, wherein the structure of the nucleic acid fragment is as follows: 5 '-N-P-NLuc-M-F-H-L-3'. Wherein N, P, M, F, H, L is the structural gene of the virus; NLuc is

An ORF for luciferase comprising an M gene start sequence (M GS) and Not I cleavage site upstream and a Pme I cleavage site and a P gene termination sequence downstream of the ORF: (P GE)。

As a specific description of an example, the sequence of the nucleic acid fragment is SEQ ID NO 1;

the nucleic acid fragment provided by the invention is used for rescuing the recombinant canine measles virus capable of expressing NLuc in cells;

the cells are BSR-T7/5 cells and Vero-Dog-SLAM (VDS) cells which are respectively used for rescuing and passaging the recombinant viruses;

in a further aspect of the present invention there is provided a recombinant canine measles virus capable of expressing NLuc, said virus being rescued in cells using a recombinant plasmid expressing a recombinant CMV nucleic acid fragment of NLuc.

The rCMV-NLuc rescued by the reverse genetic technology has higher expression level to the NLuc in the cell, and can emit light blue fluorescence which can be seen by naked eyes under the action of a luminescent substrate furimazine. The insertion of the exogenous gene does not significantly affect the growth kinetics of the virus, and the exogenous gene is relatively stable in the virus passage process.

Drawings

FIG. 1: rCMV-NLuc cDNA clone pattern diagram.

FIG. 2: fluorescence analysis was performed 72h after co-transfection of the four plasmids.

FIG. 3: rCMV-NLuc blindly transmitted the second generation induced syncytial lesions.

FIG. 4: RT-PCR identification of rCMV-NLuc in the fifth generation.

FIG. 5: and (3) carrying out test strip detection on the rCMV-NLuc in the fifth generation.

FIG. 6: fluorescence analysis of fifth generation rCMV-NLuc.

FIG. 7: cytopathic effects of rCMV-NLuc of the seventh generation and wild-type virus induced at different infection times.

FIG. 8: comparison of rCMV-NLuc and wild-type virus growth curves.

FIG. 9: rCMV-NLuc fluorescence analysis of each generation.

FIG. 10: RT-PCR identifies rCMV-NLuc of different generations.

Detailed Description

The invention constructs rCMV-NLuc cDNA clone containing NLuc, and cotransfects BSR-T7/5 cells with eukaryotic expression plasmids respectively expressing CMV N and P, L proteins, successfully saves rCMV-NLuc, and further identifies and analyzes characteristics of recombinant viruses.

The present invention is further illustrated by the following examples, which are to be understood, however, as being merely illustrative and not limiting of the scope of the invention.