Minigenome system for studying NDV RNA editing properties and methods for differentially expressing two exogenous genes

Abstract

The present invention belongs to the field of gene engineering technology, and specifically relates to a mini-genome system for studying the editing characteristics of NDV RNA and a method for differentially expressing two exogenous genes. The recombinant gene provided by the present invention can produce multiple transcripts in NDV, and make the expression of two target genes produce a constant ratio difference, and the ratio of the target gene transcript upstream of the editing site to the target gene transcript downstream of the editing site is about 4:1.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a mini-genome system for studying NDV RNA editing characteristics and a method for differentially expressing two exogenous genes. Background Art

[0002] Newcastle disease (ND) is an acute, highly contagious disease caused by Newcastle disease virus (NDV) that mainly infects poultry. The disease is mainly characterized by digestive and respiratory tract lesions, with high morbidity and mortality, which has brought serious harm to the poultry industry. The NDV genome encodes six structural proteins, namely nucleocapsid protein (NP), phosphorylated protein (P), matrix protein, fusion protein, hemagglutinin-neuraminidase protein and RNA-dependent RNA polymerase (L) and two non-structural proteins, namely V protein and W protein. The production of its non-structural proteins is mainly due to the short pause of viral RNA polymerase (RdRp) at the editing site (Editing site, ES, whose specific sequence is "AAAAAGGG" (A5G3)) during the transcription of the P gene, and the non-template-dependent insertion of one guanine (+1G) and two guanines (+2G). At present, the detection means of RNA editing phenomenon is mainly through high-throughput sequencing. This method has high sensitivity, but its cost is also high and inconvenient. Therefore, the use of a mini-gene system can be observed by a fluorescent microscope to see whether there is RNA editing phenomenon, which can be used to study the characteristics of RNA editing sites. The constant ratio of differential expression of multiple exogenous gene technologies using its RNA editing function on NDV vectors has important application value in the fields of vaccines, tumors or gene therapy and exogenous gene expression. However, the prior art mainly realizes differential expression of exogenous genes by inserting two or more exogenous genes into different positions of the NDV genome, but the difference is not obvious, and the difference between the two is not a constant ratio. Summary of the invention

[0003] The purpose of the present invention is to solve the deficiencies of the prior art, to provide a mini-genome system for studying the editing properties of NDV RNA and a method for differentially expressing two exogenous genes using the editing function of NDV RNA, and to differentially express different proteins at a constant ratio.

[0004] The present invention provides a recombinant gene, comprising gene I, an editing site, an extra base a, a stop codon, an extra base b, gene II and an extra base c;

[0005] The gene I, the editing site, the extra base a, the stop codon, the extra base b, the gene II and the extra base c are connected in sequence;

[0006] The gene I and gene II encode different proteins;

[0007] The nucleotide sequence of the editing site is 5'-AAAAAGGG-3'.

[0008] Preferably, a 6 nt flanking sequence is inserted between the downstream of gene I and the upstream of the editing site; the nucleotide sequence of the 6 nt flanking sequence is 5'-GTCATC-3' or 5'-AATGCT-3'.

[0009] Preferably, the gene I or gene II includes a fluorescent gene or a luciferase gene; the fluorescent gene includes eGFP, RFP, mCherry, DsRed or BFP; the luciferase gene includes Fluc or Rluc.

[0010] The present invention also provides the use of the recombinant gene described in the above technical solution in constructing a gene differential expression system.

[0011] The present invention also provides a mini-genome system for studying the characteristics of NDV RNA editing sites, wherein the elements of the mini-genome system include Promoter, Leader, GS and 3'UTR, the recombinant gene described in the above technical solution, 5'UTR, GE, Trailer and Terminator.

[0012] The present invention also provides a method for differentially expressing two exogenous genes, comprising the following steps:

[0013] The mini-genome system described in the above technical solution is mixed with the auxiliary plasmid and co-transfected into cells.

[0014] Preferably, the helper plasmids include pCAGGS-T7, pCAGGS-NP, pCAGGS-P and pCAGGS-L; and the cells include BHK-21 cells.

[0015] The present invention also provides the use of the recombinant gene described in the above technical solution or the mini-genome system described in the above technical solution in preparing an element for differentially expressing two exogenous genes.

[0016] The present invention also provides a recombinant virus, wherein the recombinant gene described in the above technical solution is inserted between the P gene and the M gene of the NDV genome.

[0017] The present invention provides a recombinant gene, comprising gene I, an editing site, an additional base a, a stop codon, an additional base b, gene II and an additional base c; the gene I, the editing site, the additional base a, the stop codon, the additional base b, the gene II and the additional base c are connected in sequence; the gene I and the gene II encode different proteins; the nucleotide sequence of the editing site is shown in SEQ ID NO. 1. The editing site of the present invention is a recognition site of viral RNA polymerase (RdRp) during the transcription process of Newcastle disease virus (NDV), and a short pause occurs at the editing site of the present invention during the transcription process, and one guanine (+1G) and two guanines (+2G) are inserted non-template-dependently, which can generate multiple transcripts, and the expression of different target genes produces a constant ratio difference, and the ratio of the target gene transcript upstream of the editing site to the target gene transcript downstream of the editing site is about 4:1.

[0018] Furthermore, the present invention limits the 6nt nucleotide sequence flanking the upstream of the editing site, which can further increase the editing frequency. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0020] Figure 1 Schematic diagram of the construction of the MG plasmid expressing the dual fluorescence reporter gene in Example 1-1;

[0021] Figure 2 The results of the conservation analysis of the upstream flanking bases of the ES of the P gene of different genotypes of NDV in Example 2-1;

[0022] Figure 3 This is the fluorescence detection result of test example 1;

[0023] Figure 4 This is the result of quantitatively detecting the effect of 6 nt upstream of ES on RNA editing frequency in test example 2, where ** indicates extremely significant difference, P < 0.01, and *** indicates extremely significant difference, P < 0.001;

[0024] Figure 5 It is a map of the reverse genetics system of the NDV virulent strain JS / 17 in Example 3;

[0025] Figure 6 JS-Δ6P in Example 3 HA -eGFP, rJS-P HA -eGFP proliferation ability on BHK-21 cells;

[0026] Figure 7 is rJS-P in Example 3HA -eGFP and rJS-Δ6P HA -Schematic diagram of eGFP plasmid construction;

[0027] Figure 8 The expression of P / V / W after the recombinant virus infected the cells in Example 3;

[0028] Fig. 9 The results of constructing the recombinant virus expressing the foreign gene containing ES and flanking bases in Example 4;

[0029] Figure 10-1 and Figure 10-2 The effect of ES flanking bases on the differential expression of exogenous genes in Example 4;

[0030] Fig.11 The conditions of each transcript at different time points after virus infection in Example 5;

[0031] Fig.12 is the expression ratio of each transcript in Example 5. DETAILED DESCRIPTION

[0032] The present invention provides a recombinant gene, comprising gene I, an editing site, an additional base a, a stop codon, an additional base b, gene II and an additional base c; the gene I, the editing site, the additional base a, the stop codon, the additional base b, gene II and the additional base c are connected in sequence; the gene I and gene II encode different proteins; the nucleotide sequence of the editing site is 5'-AAAAAGGG-3'. The editing site of the present invention is an ES sequence.

[0033] In the present invention, the gene I and gene II are preferably any two genes that need to be differentially expressed, and gene I or gene II preferably includes a fluorescent gene or a luciferase gene; when the gene I and gene II are recombined, the gene I does not contain a stop codon, and the gene II does not contain a start codon. The fluorescent gene preferably includes eGFP, RFP, mCherry, DsRed or BFP; the luciferase gene includes Fluc or Rluc. In the specific implementation of the present invention, the recombinant gene is recorded as gene I-ES-gene II, and eGFP is gene I and RFP is gene II as an example, or Fluc is gene I and Rluc is gene II as an example to obtain the recombinant gene eGFP-ES-RFP or Fluc-ES-Rluc, but it cannot be understood as the entire protection scope of the present invention. The present invention is preferably explained by taking the fluorescent gene as an example, which is simple to operate and can directly quantify the fluorescent protein for easy observation; taking the luciferase gene as an example to explain, the operation is simple, and the quantitative detection of the protein can be achieved. The genes on both sides of the editing site of the present invention encode different proteins to achieve differential expression.

[0034] In the present invention, the additional base b preferably consists of 2 bases, the types of the 2 bases may be the same or different, preferably TC; the additional base b enables normal encoding of gene II after non-template-dependent insertion of the base at the editing site.

[0035] The present invention preferably determines the number of bases contained in the extra bases a and c according to the length of gene I and gene II. Specifically: the sum of the base numbers of the gene I, the editing site and the extra base a is preferably 3n, where n is a positive integer; the sum of the base numbers of the gene I, the editing site, the extra base a, the stop codon, the extra base b, the gene II and the extra base c is preferably 6n, where n is a positive integer. The extra base a of the present invention causes the normal transcript to terminate at the stop codon; the extra base c satisfies the "six-base principle".

[0036] In the specific implementation process of the present invention, eGFP is preferably used as gene I and RFP is used as gene II as an example to illustrate the number of bases contained in the additional bases a, b and c, but it cannot be understood as the entire scope of the protection required by the present invention. Specifically, when gene I is eGFP and gene II is RFP, the additional base a is preferably 1 base, which can be A, T, C or G, preferably C; the additional base b is preferably composed of 2 bases, the types of the 2 bases can be the same or different, preferably TC; the additional base c is preferably composed of 4 bases, the types of the 4 bases can be the same or different, preferably AGCA. The additional base a causes the normal transcript of gene I to terminate at the stop codon. When a guanine (+1G) is inserted non-template-dependently, the base behind the additional base b is shifted, so that gene II can be encoded normally, and the additional base c enables the recombinant gene to meet the "six-base principle". When the gene I is eGFP and the gene II is RFP, the recombinant gene is preferably as shown in SEQ ID NO.1, specifically 5'-eGFP sequence (delete the stop codon) + ES sequence + C (extra base a) + TAA + TC (extra base b) + RFP sequence (delete the start codon) + AGCA (extra base c)-3', denoted as G-A5G3-R

[0037] The present invention preferably inserts 6nt on the flank between the downstream of gene I and the upstream of the editing site. The nucleotide sequence of the 6nt on the flank is preferably 5'-GTCATC-3' or 5'-AATGCT-3', and more preferably 5'-GTCATC-3. After the 6nt is inserted in the present invention, a gene fragment is formed in which gene I, 6nt, editing site, additional base a, stop codon, additional base b, gene II and additional base c are sequentially connected, which is recorded as gene I-6nt-ES-gene II. In one embodiment of the present invention, the recombinant gene is preferably as shown in SEQ ID NO.2 or SEQ ID NO.3, and more preferably as shown in SEQ ID NO.3, specifically:

[0038] SEQ ID NO.2: 5'-eGFP sequence (stop codon deleted) + AATGCT (6nt) + ES sequence + C (additional base a) + TAA + TC (additional base b) + RFP sequence (start codon deleted) + AGCA (additional base c) -3', denoted as G-6nt-A5G3-R.

[0039] SEQ ID NO.3: 5'-eGFP sequence (delete stop codon) + GTCATC (6nt) + ES sequence + C (additional base a) + TAA + TC (additional base b) + RFP sequence (delete start codon) + AGCA (additional base c) -3', recorded as G-m6nt-A5G3-R. The 6nt nucleotides of the present invention enhance the frequency of RNA editing.

[0040] The present invention also provides the use of the target gene in constructing a gene differential expression system. The gene differential expression system constructed using the recombinant gene of the present invention can achieve differential expression of different proteins, so that the expression of two target genes produces a constant ratio difference, and the ratio of the target gene transcript upstream of the editing site to the target gene transcript downstream of the editing site is about 4:1.

[0041] The present invention provides a mini-genome system for studying the editing characteristics of NDV RNA, wherein the elements of the mini-genome system include Promoter, Leader, GS and 3'UTR, the recombinant gene described in the above technical solution, 5'UTR, GE, Trailer and Terminator. The gene differential expression system described in the present invention is based on the classic MG system, and the reporter gene in the classic MG system is replaced by the recombinant gene described in the present invention, retaining the simplest transcription and replication elements of the virus, and constructing a mini-genome system of recombinant genes containing P gene editing sites, differentially expressing different target genes at a constant ratio, simulating the production of V protein, and the constructed plasmid is recorded as pUC-Mini-A5G3-R. The present invention has no strict requirements on the source of the elements of the gene differential expression system and the construction method of the gene differential expression system, and can be obtained by methods well known in the art.

[0042] The present invention provides a method for differentially expressing two exogenous genes, comprising the following steps:

[0043] The mini-genome system described in the above technical solution is mixed with the auxiliary plasmid and co-transfected into cells.

[0044] In the present invention, the auxiliary plasmid preferably includes pCAGGS-T7, pCAGGS-NP, pCAGGS-P and pCAGGS-L; the mass ratio of pCAGGS-T7, pCAGGS-NP, pCAGGS-P and pCAGGS-L is preferably 5:2:2:1. The cells described in the present invention preferably include BHK-21 cells. The auxiliary plasmids pCAGGS-T7, pCAGGS-NP, pCAGGS-P and pCAGGS-L described in the present invention respectively provide the T7 RNA polymerase required for gene expression and the NP, P, and L required to constitute the viral polymerase. The co-transfection method described in the present invention is preferably liposome transfection, and the Turbofect in vitro transfection reagent used is Thermo's Turbofect. During the co-transfection process, the mass of each plasmid and the volume ratio with TurboFect are preferably 1ng:2μL. After the cells are co-transfected with the present invention, the mini-genome system transcribes vRNA(-) under the action of T7 RNA polymerase, and vRNA(-) can be replicated to generate cRNA(+) under the action of viral polymerases NP, P and L, and cRNA(+) can also be replicated to generate vRNA(-), and vRNA(-) can also be transcribed under the action of viral polymerase to generate mRNA. When the viral polymerase recognizes the editing site in the mRNA and inserts one base at the editing site, the base behind the additional base b shifts, so that gene II can be normally encoded, so that translation produces a fusion protein of two fluorescent genes, and expresses two different proteins; when the mRNA is not edited or two bases are inserted at the editing site, only the target gene I upstream of the editing site can be expressed, and one protein is expressed.

[0045] The present invention also provides the use of the recombinant gene or the mini-genome system in preparing an element for differentially expressing two exogenous genes. The element of the present invention preferably includes an expression cassette, an expression vector or a cell. The recombinant gene or the mini-genome gene system of the present invention simulates the production of Newcastle disease virus V protein with a fluorescent group (pUC-Mini-A5G3-R).

[0046] The present invention also provides a recombinant virus, in which the recombinant gene described in the above technical solution is inserted between the P gene and the M gene of the NDV genome. In the specific implementation process of the present invention, the recombinant gene described in the above technical solution is inserted between the P gene and the M gene of the NDV genome as an independent transcription unit, preferably inserted into MssⅠ between the P gene and the M gene of the NDV genome. The present invention preferably connects the recombinant gene described in the above technical solution with the GE-IGS-GS-Kozak sequence to obtain an independent transcription unit (denoted as GE-IGS-GS-Kozak-recombinant gene) and then inserts it between the P gene and the M gene of the NDV genome; the nucleotide sequence of the independent transcription unit is preferably as shown in any one of SEQ ID NO.4 to 6, wherein SEQ ID NO.4 does not contain 6nt; SEQ ID NO.5 contains 6nt, and the nucleotide sequence of 6nt contained in SEQ ID NO.5 is 5'-AATGCT-3'; SEQ ID NO.6 contains 6nt, and the nucleotide sequence of 6nt in SEQ ID NO.6 is 5'-GTCATC-3', specifically:

[0047] SEQ ID NO.4: 5′-TTAGAAAAAA (GE) + C (IGS) + ACGGGTAGAA (GS) + CGCCACC (Kozak) + eGFP sequence (stop codon deleted) + ES sequence + C (additional base a) + TAA + TC (additional base b) + RFP sequence (start codon deleted) + AGCA (additional base c) -3′;

[0048] SEQ ID NO.5: 5'-TTAGAAAAAA(GE)+C(IGS)+ACGGGTAGAA(GS)+CGCCACC(Kozak)+eGFP sequence (stop codon deleted)+AATGCT(6nt)+ES sequence+C(additional base a)+TAA+TC(additional base b)+RFP sequence (start codon deleted)+AGCA(additional base c)-3'.

[0049] SEQ ID NO.6: 5'-TTAGAAAAAA(GE)+C(IGS)+ACGGGTAGAA(GS)+CGCCACC(Kozak)+eGFP sequence (stop codon deleted)+GTCATC(6nt)+ES sequence+C(additional base a)+TAA+TC(additional base b)+RFP sequence (start codon deleted)+AGCA(additional base c)-3'.

[0050] In the specific implementation of the present invention, it is preferred to add a restriction enzyme site Mss I at the 5' and 3' ends of the independent transcription unit, and insert the independent transcription unit between the P gene and the M gene of the NDV genome by enzyme ligation. The restriction enzyme sites connected to the 5' and 3' ends are preferably Mss I restriction enzyme sites.

[0051] The recombinant virus of the present invention performs RNA editing on the target gene in the recombinant gene, encodes different proteins, and causes the transcripts of the upstream gene I and the downstream gene II of the editing site to have a constant 4:1 (100%:26.22%) difference in ratio.

[0052] In order to further illustrate the present invention, a dual fluorescence reporter gene and a dual luciferase reporter gene system and applications thereof provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0053] Unless otherwise specified, the specific operating steps in the following embodiments are routine operations in the art.

[0054] Example 1-1

[0055] 1. Construction of a dual fluorescent reporter gene system simulating NDV P gene editing: Using pEGFP-1 as a template, using the sequences shown in SEQ ID NO.7 (5'-CCATCGATATGGTGAGCAAGGGCGAGGAG-3') and SEQ ID NO.8 (5'-CTCGCTGATTAGCCCTTTTTCTTGTACAGCTCGTCCATGC-3') as amplification primers, amplification obtained product 1; using pCD513B-2 as a template, using the sequences shown in SEQ ID NO.9 (5'-GAAAAAGGGCTAATCAGCGAGCTGATCAAGGAGAACATG-3') and SEQ ID NO.10 (5'-ACATGCATGCTGCTTCATCTGTGCCCCAGTTTGCTAG-3') as amplification primers, amplification obtained product 2; using product 1 and product 2 as templates, using the sequences shown in SEQ ID NO.7 and SEQ ID NO.10 as amplification primers, Overlap PCR technology was used to amplify to the region containing the restriction site Cla I / Sph I, namely eGFP-A5G3-RFP, the nucleotide sequence of which is shown in SEQ ID NO.1. The fragment was cloned into the mini-genome basic backbone plasmid pUC19-Mini by restriction digestion and ligation. The specific map is shown in Figure 1 shown.

[0056] 2. It was co-transfected into BHK-21 cells with the helper plasmids pCAGGS-T7, pCAGGS-NP, pCAGGS-P and pCAGGS-L (which express viral T7 RNA polymerase, NP, P, and L viral polymerases, respectively).

[0057] Example 1-2

[0058] Construction of the MG system containing the upstream flanking 6nt and the dual fluorescent reporter gene of ES

[0059] 1. By analyzing the flanking bases of ES of different NDV genotypes (the accession numbers of Class Ⅰ and Ⅰ to VI are DQ097393.1, AY562991.1, KJ019841.1, EF201805.1, AY741404.1, MK214318.1, AJ880277.1 and AF431744.3, respectively), it was found that the upstream flanking 6nt (AATGCT) was relatively conservative. The analysis results are as follows Figure 2 shown.

[0060] 2. Overlap PCR technology was used to amplify the above 6nt (AATGCT) into an eGFP-6nt-ES-RFP fragment containing restriction sites Cla I / Sph I, the nucleotide sequence of which was as shown in SEQ ID NO.3, and the fragment was cloned into the minigene genome basic backbone plasmid pUC19-Mini by restriction ligation to obtain a recombinant plasmid, recorded as pUC-Mini-G-6nt-A5G3-R.

[0061] 3. The pUC-Mini-G-6nt-A5G3-R obtained in step 2 and the auxiliary plasmids pCAGGS-T7, pCAGGS-NP, pCAGGS-P and pCAGGS-L (expressing viral T7 RNA polymerase, NP, P, L viral polymerase, respectively) were co-transfected into BHK-21 cells using the method of Example 1-1.

[0062] Examples 1-3

[0063] Same as Example 1-2, except that the upstream flanking 6nt nucleotide sequence is GTCATC, and the eGFP-m6nt-ES-RFP fragment containing the restriction site Cla I / Sph I is amplified, and the nucleotide sequence is as SEQ ID NO.2.

[0064] Comparative Example 1-1

[0065] Same as Example 1-1, except that the auxiliary plasmids pCAGGS-T7, pCAGGS-NP and pCAGGS-P were co-transfected, and pCAGGS-L was not transfected.

[0066] Comparative Example 1-2

[0067] Same as Example 1-1, except that, in step 2, Overlap PCR technology is used to amplify the eGFP-RFP fragment containing the restriction site Cla I / Sph I, and the nucleotide sequence is as shown in SEQ ID NO.11, specifically: 5'-eGF P sequence+RFP sequence (delete the start code)-3'.

[0068] Test Example 1

[0069] The fluorescence of Example 1-1 (denoted as A5G3), Example 2-1 (denoted as 6nt-A5G3), Example 2-2 (denoted as m6nt-A5G3), Comparative Example 1-1 (denoted as △L), and Comparative Example 1-2 (denoted as No ES) was observed using a fluorescence microscope. The results are as follows Figure 3 As shown. Figure 3 It can be seen that after the MG system containing the dual fluorescent reporter gene of the upstream flanking 6nt and ES was co-transfected with the auxiliary plasmid, its red light expression level increased, that is, the flanking 6nt played a significant role in the RNA editing frequency.

[0070] Example 2-1

[0071] In order to verify the effect of the flanking bases of the editing site on the RNA editing frequency by quantitative detection, the fragments Fluc-A5G3-Rluc, Fluc-6nt-A5G3-Rluc, Fluc-No ES-Rluc, and Fluc-Rluc were amplified by Overlap PCR using pNF-κB-luc and pRL-SV40-N as templates. Their nucleotide sequences are shown in SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15. The fragment was then cloned into the minigenome basic backbone plasmid pUC19-Mini by enzyme digestion (Cla I / Sph I) ligation method to obtain four different plasmids: pUC19-Mini-Fluc-A5G3-Rluc (denoted as -Fluc-A5G3-Rluc), pUC19-Mini-Fluc-6nt-A5G3-Rluc (denoted as -Fluc-6nt-A5G3-Rluc), pUC19-Mini-Fluc-No ES-Rluc (denoted as -No ES), and pUC19-Mini-Fluc-A5G3-Rluc (denoted as Throughtout). Then, these four plasmids were co-transfected with auxiliary plasmids (pCAGGS-T7, pCAGGS-NP, pCAGGS-P, pCAGGS-L) into cells (pCAGGS-L was not transfected as a negative control, denoted as △L). 48 hours after transfection, the quantitative expression of luciferase reporter gene after each plasmid transfection was detected by multi-function microplate reader. The luciferase reporter gene detection process was operated according to the instructions of Biyuntian RG027. The results are as follows Figure 4 As shown in Table 1. Figure 4 From Table 1, we can see that after the MG system containing the upstream flanking 6nt and ES luciferase reporter gene was co-transfected with the auxiliary plasmid, the expression of Rluc increased, that is, the flanking 6nt played a significant role in the RNA editing frequency. The editing efficiency of inserting +1G at the editing site after plasmid transfection was analyzed according to the following formula. Figure 4 As shown in Table 2. It can be seen that the flanking 6nt has a significant enhancing effect on the RNA editing frequency.

[0072] SEQ ID NO.12: 5'-Fluc sequence (delete stop codon) + ES sequence + C (additional base a) + TAA + TC (additional base b) + Rluc sequence (delete start codon) + G (additional base c) -3';

[0073] SEQ ID NO.13: Fluc sequence (delete stop codon) + AATGCT (6 nt) + ES sequence + C (additional base a) + TAA + TC (additional base b) + Rluc sequence (delete start codon) + G (additional base c) -3;

[0074] SEQ ID NO.14: 5'-Fluc sequence + Rluc sequence (start codon deleted)-3';

[0075] SEQ ID NO.15: 5'-Fluc sequence (stop codon deleted)+Rluc sequence (start codon deleted)-3'.

[0076] Table 1 Luciferase quantitative detection results

[0077]

[0078]

[0079] Table 2 Analysis results of editing frequency

[0080]

[0081] According to Tables 1-2 and Figure 4 It can be seen that the editing frequency of the MG system + 1G containing only the dual luciferase reporter gene of ES is about 0.6%. When there is an upstream flanking 6nt, its RNA editing frequency is significantly increased to about 30%.

[0082] Example 3

[0083] The reverse genetics system of the strong NDV strain JS / 17 (pBR322-JS, map as shown in Figure 5 Based on the results shown in Figure 2, a recombinant virus (denoted as rJS-P) with an HA tag attached to the N-terminus of the P gene and expressing eGFP was constructed and rescued according to conventional steps. HA -eGFP, such as Figure 6 ), and a recombinant virus lacking 6 nt upstream of ES and attached with HA tag at the N-terminus and expressing eGFP was successfully constructed and rescued (denoted as rJS-△6P HA -eGFP, such as Figure 6 ).

[0084] BHK-21 cells were infected with the recombinant virus at a dose of 0.01 MOI, and the proliferation ability of BHK-21 cells was observed. Figure 7 As shown. Figure 7 It can be seen that rJS-Δ6P HA -eGFP proliferation ability on BHK-21 cells was significantly lower than that of rJS-P HA-eGFP.

[0085] Western Blot was used to detect rJS-P HA -eGFP and rJS-Δ6P HA -eGFP infected cells after the expression of P, V, W proteins, the results are as follows Figure 8 As shown. Figure 8 It can be seen that rJS-P HA -eGFP showed obvious expression of P protein and V protein, but no expression of W protein was detected, while rJS-Δ6P HA After -eGFP infected cells, only the expression of P protein was detected, and the expression of V protein and W protein was not detected. The 6nt upstream of ES plays a crucial role in RNA editing and significantly affects the replication of the virus.

[0086] Example 4

[0087] GE, IGS, GS, Kozak, eGFP-ES-RFP obtained in Example 1-1 and GE were sequentially connected to obtain an independent transcription unit, which was inserted between the P and M genes of the NDV genome to obtain a recombinant virus, which was recorded as rJS-G-A5G3-R. Fig. 9 As shown;

[0088] GE, IGS, GS, Kozak, eGFP-6nt-ES-RFP obtained in Example 2-1 and GE were sequentially connected to obtain an independent transcription unit, which was inserted between the P and M genes of the NDV genome to obtain the recombinant virus rJS-G-6nt-A5G3-R. Fig. 9 As shown;

[0089] GE, IGS, GS, Kozak, eGFP-m6nt-ES-RFP obtained in Example 2-2 and GE were sequentially connected to obtain an independent transcription unit, which was inserted between the P and M genes of the NDV genome to obtain the recombinant virus rJS-G-m6nt-A5G3-R. Fig. 9 As shown;

[0090] The differential expression of the two exogenous genes in BHK-21 cells infected with the recombinant virus was detected by fluorescence microscopy and Western Blot. Figure 10-1 and Figure 10-2 .according to Figure 10-1 and Figure 10-2It can be seen that the eGFP+RFP (protein produced by inserting +1G at the ES editing site) band produced after rJS-G-6nt-A5G3-R infected cells is thicker, while the eGFP+RFP bands produced after infection with the other two recombinant viruses are weaker, and the effect of rJS-G-m6nt-A5G3-R is better than that of rJS-G-A5G3-R. The specific flanking 6nt upstream of ES is essential for effective differential expression.

[0091] Example 5

[0092] The method of Example 1-1 was used to infect cells with rJS-G-6nt-A5G3-R obtained in Example 4, and then NGS was used to collect cell samples at different infection time points, extract RNA, and amplify using corresponding primers (for specific steps, refer to ChenX, JiaY, Ren S, Chen S, Wang X, Gao X, Wang C, Adam F, Wang X, YangZ. 2020. Identification of Newcastle disease virus P-gene editing using next-generation sequencing. Journal of Veterinary Medical Science, 82(8): 1231-1235). The detection primers and the detection results are shown in Tables 3 to 5 and Figures 11-12 .

[0093] Table 3 Detection primers

[0094]

[0095]

[0096] Table 4 Editing effect of P gene in rJS-G-6nt-A5G3-R

[0097]

[0098] Table 5 Editing effect of exogenous genes in rJS-G-6nt-A5G3-R

[0099]

[0100] According to Tables 4-5 and Figures 11-12It was found that +1G, +2G, and +3G to +6G existed at the ES editing site, but only accounted for a small part. The editing frequency changed slightly with viral infection, indicating that the recombinant virus edited the RNA of the two exogenous genes, resulting in a constant 4:1 (100%:26.22%) ratio difference between the transcripts of eGFP and RFP.

[0101] It can be seen from the above examples that the dual fluorescence reporter gene provided by the present invention can produce multiple transcripts in Newcastle disease virus and make the expression of two fluorescent genes produce a constant ratio difference, and the ratio of the transcripts is about 4:1.

[0102] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A minigenome system for studying the properties of NDV RNA editing sites, characterized in that: The elements of the minigenome system include Promoter, Leader, GS, 3'UTR, recombinant gene, 5'UTR, GE, Trailer and Terminator connected in sequence; The recombinant gene includes gene I, an editing site, an extra base a, a stop codon, an extra base b, gene II and an extra base c; The gene I, the editing site, the extra base a, the stop codon, the extra base b, the gene II and the extra base c are connected in sequence; The gene I and gene II encode different proteins, the gene I does not contain a stop codon, and the gene II does not contain a start codon; The nucleotide sequence of the editing site is 5'-AAAAAGGG-3'; The sum of the base numbers of the gene I, the editing site and the additional base a is 3n, where n is a positive integer; the sum of the base numbers of the gene I, the editing site, the additional base a, the stop codon, the additional base b, the gene II and the additional base c is 6n, where n is a positive integer.

2. The minigenome system according to claim 1, characterized in that: The recombinant gene further includes a 6 nt flank; the 5' end of the 6 nt flank is connected to the 3' end of the gene I, and the 3' end is connected to the 5' end of the editing site; The 6 nt flanking nucleotide sequence is 5'-GTCATC-3' or 5'-AATGCT-3'.

3. The minigenome system according to claim 1, characterized in that: The gene I or gene II includes a fluorescent gene or a luciferase gene; the fluorescent gene includes eGFP, RFP, mCherry, DsRed or BFP; the luciferase gene includes Fluc or Rluc.

4. A method for differentially expressing two exogenous genes, characterized in that: The steps include: The minigenome system according to any one of claims 1 to 3 is mixed with an auxiliary plasmid and co-transfected into cells.

5. The method according to claim 4, characterized in that The auxiliary plasmids include pCAGGS-T7, pCAGGS-NP, pCAGGS-P and pCAGGS-L; and the cells include BHK-21 cells.

6. Use of the minigenome system according to any one of claims 1 to 3 in preparing a recombinant virus that differentially expresses two exogenous genes.

7. A recombinant virus, characterized in that Based on the minigenome system according to any one of claims 1 to 3, a recombinant fragment is inserted between the P gene and the M gene of the NDV genome; The recombinant fragment includes GS, 3'UTR, recombinant gene, 5'UTR and GE connected in sequence; The recombinant gene includes gene I, an editing site, an extra base a, a stop codon, an extra base b, gene II and an extra base c; The gene I, the editing site, the extra base a, the stop codon, the extra base b, the gene II and the extra base c are connected in sequence; The gene I and gene II encode different proteins, the gene I does not contain a stop codon, and the gene II does not contain a start codon; The nucleotide sequence of the editing site is 5'-AAAAAGGG-3'; The sum of the base numbers of the gene I, the editing site and the additional base a is 3n, where n is a positive integer; the sum of the base numbers of the gene I, the editing site, the additional base a, the stop codon, the additional base b, the gene II and the additional base c is 6n, where n is a positive integer.

8. The recombinant virus according to claim 7, characterized in that The recombinant gene further includes a 6 nt flank; the 5' end of the 6 nt flank is connected to the 3' end of the gene I, and the 3' end is connected to the 5' end of the editing site; The 6 nt flanking nucleotide sequence is 5'-GTCATC-3' or 5'-AATGCT-3'.

9. The recombinant virus according to claim 7, characterized in that The gene I or gene II includes a fluorescent gene or a luciferase gene; the fluorescent gene includes eGFP, RFP, mCherry, DsRed or BFP; the luciferase gene includes Fluc or Rluc.

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