阅读说明：本技术 山羊副流感病毒3型感染性cDNA克隆构建方法及其应用 (Goat parainfluenza virus type 3 infectious cDNA cloning construction method and application thereof ) 是由 李文良 李燕华 杨蕾蕾 毛立 钱晶 孙敏 刘茂军 张纹纹 程子龙 于 2021-09-17 设计创作，主要内容包括：本发明提供山羊副流感病毒3型感染性cDNA克隆构建方法及其应用,属于生物技术领域。该构建方法包括：以山羊副流感病毒3型的总RNA反转录产物为模板,扩增A、B、C和D片段；将片段A和B、片段C和D分别插入pCI-neo-1载体中,得到重组载体pCI-A-B、pCI-C-D；以pCI-A-B为模板,扩增片段A-B；以pCI-C-D为模板,扩增C-D片段；将线性化pYES1L载体、A-B和C-D片段混和,导入酵母内进行同源重组,得到山羊副流感病毒3型感染性cDNA克隆质粒。本发明构建方法效率高,得到的感染性cDNA克隆拯救的病毒感染细胞后能够快速产生细胞病变,病毒滴度高,且保持稳定。(The invention provides a construction method and application of goat parainfluenza virus type 3 infectious cDNA clone, belonging to the field of biotechnology. The construction method comprises the following steps: amplifying A, B, C and D segments by taking a total RNA reverse transcription product of the goat parainfluenza virus type 3 as a template; respectively inserting the fragments A and B and the fragments C and D into a pCI-neo-1 vector to obtain a recombinant vector pCI-A-B, pCI-C-D; amplifying the fragment A-B by taking pCI-A-B as a template; amplifying a C-D fragment by taking pCI-C-D as a template; the linearized pYES1L vector, the A-B and the C-D fragments are mixed and introduced into yeast for homologous recombination to obtain the goat parainfluenza virus type 3 infectious cDNA clone plasmid. The construction method of the invention has high efficiency, the obtained infectious cDNA clone can quickly generate cytopathy after rescued virus infects cells, the virus titer is high, and the virus titer is kept stable.)

1. The construction method of goat parainfluenza virus type 3 infectious cDNA clone is characterized by comprising the following steps:

(1) inserting the sequence shown in SEQ ID NO. 1 into pCI-neo plasmid to obtain recombinant plasmid pCI-neo-1;

(2) using a reverse transcription product of goat parainfluenza virus type 3 total RNA as a template, amplifying an A fragment by using primers AF1, AF2 and AR, amplifying a B fragment by using primers BF and BR, amplifying a C fragment by using primers CF and CR, and amplifying a D fragment by using primers DF, DR1 and DR 2; inserting the amplified fragments A and B into a pCI-neo-1 vector to obtain a recombinant vector pCI-A-B; inserting the amplified fragments C and D into a pCI-neo-1 vector to obtain a recombinant vector pCI-C-D;

(3) amplifying the fragment A-B by using primers AB-F and AB-R by using pCI-A-B as a template; amplifying a C-D fragment by using primers CD-F and CD-R by using pCI-C-D as a template; taking pYESlL plasmid as a template, and amplifying by using primers Vector-F and Vector-R to obtain a linearized pYES1L Vector;

(4) the linearized pYES1L vector, the A-B and the C-D fragments are mixed and introduced into yeast for homologous recombination to obtain the goat parainfluenza virus type 3 infectious cDNA clone plasmid pYES1L-CPIV 3.

2. The method of claim 1, wherein the amplification of the A fragment with primers AF1, AF2 and AR comprises two PCR amplification reactions; firstly, carrying out first PCR amplification by taking AF1 and AR as primers; then, the first PCR amplification product is used as a template, AF2 and AR are used as primers, and second PCR amplification is carried out to obtain an A fragment.

3. The method of claim 1 or 2, wherein the amplification of the D fragment with primers DF, DR1 and DR2 comprises two PCR amplification reactions; firstly, performing first PCR amplification by taking DF and DR1 as primers; and then carrying out second PCR amplification by taking the first PCR amplification product as a template and DF and DR2 as primers to obtain a D fragment.

4. Infectious cDNA clone plasmid of goat parainfluenza virus type 3 obtained by the method of any one of claims 1 to 3.

5. Use of the infectious cDNA clone of goat parainfluenza virus type 3 of claim 4 to rescue goat parainfluenza virus type 3.

6. The use according to claim 5, comprising the steps of: auxiliary plasmids for expressing goat parainfluenza virus type 3N, P, L protein are respectively constructed, and a transfection reagent Lipofectamine is utilized^TM2000 the infectious cDNA clone plasmid of claim 4 and helper plasmid were co-transfected into 293T cells, cell culture harvested and MDBK cells inoculated, cell culture harvested and rescued infectious goat parainfluenza virus type 3 rCPIV3 obtained.

7. The use of claim 6, wherein the helper plasmids are three helper plasmids, namely pCAGGS-N, pCAGGS-P and pCAGGS-L, obtained by cloning genes encoding goat parainfluenza virus type 3N, P and L protein into pCAGGS, respectively.

8. The use of claim 7, wherein the infectious cDNA cloning plasmid, helper plasmid pCAGGS-N, pCAGGS-P and pCAGGS-L are present in a mass ratio of 5: 5: 2: 1.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a construction method and application of goat parainfluenza virus type 3 infectious cDNA clone.

Background

Goat parainfluenza virus type 3 (CPIV 3) is a newly identified member of the genus Respirovirus of the family Paramyxoviridae, a enveloped mononegavirale RNA virus, that primarily infects goats and sheep. The CPIV3 genome has the size of 15624bp, and is similar to the virus of the same genus, and a 3 'end leader sequence, a nucleoprotein (N) gene, a phosphoprotein (P) gene, a matrix protein (M) gene, a fusion protein (F) gene, a hemagglutinin protein (HN) gene, a large transcription protein (L) gene and a 5' end sequence are arranged from the 3 'end to the 5' end in sequence. The virus is mainly spread through respiratory tract to cause respiratory tract diseases, and can cause obvious clinical symptoms under the conditions of stress, mixed or secondary infection with other pathogens and the like, thereby causing serious respiratory tract diseases and causing higher morbidity and mortality. Epidemiological studies indicate that domestic flocks have a high rate of viral infection. The virus is a newly discovered new pathogen, and although research has been conducted around the interaction of viral infection with type I interferon response, apoptosis, miRNA, and exosome, respectively, the research on the infection and pathogenic mechanism thereof is not deep yet. There is no report on the effective prevention and control method of the virus at home and abroad, and there is no vaccine capable of preventing the infection of the goat parainfluenza virus type 3.

The reverse genetic technology is an emerging important technology for directionally modifying or modifying viruses, has a very wide application prospect, and has been successfully and widely applied to vaccines of various viruses and research on pathogenic mechanisms. However, no infectious cDNA clone of goat parainfluenza virus type 3 has been constructed in the prior art.

Disclosure of Invention

The invention aims to provide a goat parainfluenza virus type 3 infectious cDNA cloning construction method and application thereof, the construction efficiency is high, the infectious cDNA clone rescued virus rCPIV3 can quickly generate cytopathic effect after infecting MDBK cells, and the virus titer can reach 10⁸TCID₅₀More than/mL, and is stable.

The purpose of the invention is realized by adopting the following technical scheme:

the construction method of goat parainfluenza virus type 3 infectious cDNA clone comprises the following steps:

(1) inserting the sequence shown in SEQ ID NO. 1 into pCI-neo plasmid to obtain recombinant plasmid pCI-neo-1;

(2) using a reverse transcription product of goat parainfluenza virus type 3 total RNA as a template, amplifying an A fragment by using primers AF1, AF2 and AR, amplifying a B fragment by using primers BF and BR, amplifying a C fragment by using primers CF and CR, and amplifying a D fragment by using primers DF, DR1 and DR 2; inserting the amplified fragments A and B into a pCI-neo-1 vector to obtain a recombinant vector pCI-A-B; inserting the amplified fragments C and D into a pCI-neo-1 vector to obtain a recombinant vector pCI-C-D;

(3) amplifying the fragment A-B by using primers AB-F and AB-R by using pCI-A-B as a template; amplifying a C-D fragment by using primers CD-F and CD-R by using pCI-C-D as a template; taking pYESlL plasmid as a template, and amplifying by using primers Vector-F and Vector-R to obtain a linearized pYES1L Vector;

(4) the linearized pYES1L vector, the A-B and the C-D fragments are mixed and introduced into yeast for homologous recombination to obtain the goat parainfluenza virus type 3 infectious cDNA clone plasmid pYES1L-CPIV 3.

In the present invention, the A fragment was amplified with primers AF1, AF2 and AR, involving two PCR amplification reactions; firstly, carrying out first PCR amplification by taking AF1 and AR as primers; then, the first PCR amplification product is used as a template, AF2 and AR are used as primers, and second PCR amplification is carried out to obtain an A fragment.

In the present invention, primers DF, DR1 and DR2 were used to amplify the D fragment, including two PCR amplification reactions; firstly, performing first PCR amplification by taking DF and DR1 as primers; and then carrying out second PCR amplification by taking the first PCR amplification product as a template and DF and DR2 as primers to obtain a D fragment.

The invention also provides the goat parainfluenza virus type 3 infectious cDNA clone plasmid obtained by the method and application thereof in rescuing the goat parainfluenza virus type 3.

In the present invention, the application comprises the following steps: auxiliary plasmids for expressing goat parainfluenza virus type 3N, P, L protein are respectively constructed, and a transfection reagent Lipofectamine is utilized^TM2000 the infectious cDNA clone plasmid of claim 4 and helper plasmid were co-transfected into 293T cells, cell culture harvested and MDBK cells inoculated, cell culture harvested and rescued infectious goat parainfluenza virus type 3 rCPIV3 obtained.

In the invention, the helper plasmids are three helper plasmids, namely pCAGGS-N, pCAGGS-P and pCAGGS-L, which are obtained by cloning genes encoding the goat parainfluenza virus type 3N, P and the L protein into pCAGGS.

In the preferred technical scheme, the mass ratio of the infectious cDNA clone plasmid, the helper plasmid pCAGGS-N, pCAGGS-P and pCAGGS-L is 5: 5: 2: 1.

compared with the prior art, the invention has the beneficial effects that: the invention takes a high-efficiency homologous recombination system, takes pYES1L as a skeleton vector, utilizes a yeast-based homologous recombination technology to firstly construct a goat parainfluenza virus type 3 strain JS14-2 strain whole genome cDNA infectious clone and 3 auxiliary plasmids, establishes a goat parainfluenza virus type 3 reverse genetic operation system, and can realize the stable and high-efficiency rescue of recombinant viruses by utilizing the system. The construction method of the invention has high efficiency. After MDBK cells are infected by the rescued virus rCPIV3, cytopathic effect is generated at 24-48h after infection, and the titer of the obtained rCPIV3 virus can reach 10⁸TCID₅₀More than mL, the virus propagation speed is higher than that of a wild strain, and the titer is the same as that of the wild strain IThus, the stability is maintained. Lays a foundation for the development of novel goat parainfluenza virus type 3 vaccines, multi-combination vaccines for sheep and the research on the pathogenic mechanism of the goat parainfluenza virus type 3 in the future.

Drawings

FIG. 1 shows a full-length cDNA fragment amplification strategy for goat parainfluenza virus type 3, wherein the number indicates the viral genome position (bp).

FIG. 2 construction strategy of full-length cDNA cloning plasmid of goat parainfluenza virus type 3.

FIG. 3 identification of goat parainfluenza virus type 3 helper plasmid constructs, in which Panel A: lane 1 is DNA marker DL2000, Lane 2 is BglII-cleaved pCAGGS-N, Lane 3 is BglII-cleaved pCAGGS-P, and Lane 4 is DNA marker 1kb plus; in the B diagram, lane 1 is DNA marker DL2000, lane 2 is HindIII-digested pCAGGS-L, and lane 3 is DNA marker 1kb plus.

FIG. 4 MDBK cytopathy of recombinant virus, wherein Panel A: normal MDBK cells, panel B: cytopathic 2 days after inoculation, panel C: and (3) inoculating the virus for 3 days.

FIG. 5 Indirect immunofluorescence assay of recombinant viruses, Panel A: normal cells, panel B: and (5) inoculating the virus cells.

FIG. 6 growth curves of recombinant viruses.

Detailed Description

The present invention is further described below in conjunction with specific examples, which are to be understood as being illustrative only and in no way limiting of the scope of the invention.

Example 1 construction of infectious cDNA cloning plasmid of goat parainfluenza Virus type 3 JS14-2 Strain

CDV-3 strain whole genome cloning and sequence determination

200 mu L of frozen goat parainfluenza virus 3 type JS14-2 strain (disclosed in Chinese patent ZL 201810926226.2, with the preservation number of CCTCC NO: V201832) cell culture suspension is taken, and total RNA is extracted according to the operational instructions of the Axygen nucleic acid extraction kit. According to a CPIV3 JS2013 reference sequence, a virus whole genome sequence is divided into 9 target fragments, 9 pairs of specific primers (shown in a table 1) are designed by a software Primer Premier5.0 to respectively amplify the target fragments 1-9, and the primers are sent to Nanjing engine Biotechnology limited company for synthesis. The extracted RNA is taken as a template, and each pair of specific primers are respectively used for RT-PCR amplification by a one-step RT-PCR kit (Beijing Quanyujin Biotechnology Co., Ltd.). The total volume of RT-PCR is 20 μ L, wherein 2 XR-Mix Buffer is 10 μ L, upstream and downstream primers are 0.5 μ L, E-Mix is 0.4 μ L, RNA template is 2 μ L, and RNase-free water is added to 20 μ L. And (3) amplification procedure: reverse transcription is carried out for 30min at the temperature of 45 ℃; 5min at 94 ℃; 30s at 94 ℃, 30s at 55-60 ℃ (annealing temperature is adjusted according to Tm values of different primer pairs), 2min at 72 ℃ and 35 cycles; 10min at 72 ℃. The target fragments 1-9 are recovered and purified, and the product is sent to Nanjing Optimus department Biotechnology Co. According to the sequencing result, the virus whole genome sequence is obtained by splicing, and the sequence is aligned to find that the homology of the sequence with the existing strain sequence is 99.6-99.9%, and has 15 nucleotide mutations compared with the AHQJ2015-1 strain (MF693177.1) with the highest homology (Table 2).

TABLE 1 primers for Whole genome amplification

TABLE 2JS14-2 vs AHQJ2015-1 Whole genome sequence differences

Construction of CPIV3 Whole genome cDNA recombinant plasmid

Software is used for analyzing the single enzyme cutting site of the whole genome sequence of the strain JS14-2, a proper position is selected as an engagement point, and the whole genome is divided into four segments A, B, C, D, which is shown in figure 1. First, sequence 1(SEQ ID NO:1) (TAGCCTCGAGAATTCACGCGTGGTACCTCTAGGTCGACGGTACCGTTTAAACGGGTTATTAATTAAATCATTCGCGGCCGCTTCC) was synthesized artificially and inserted between the XhoI and NotI cleavage sites of the pCI-neo plasmid (purchased from Promega) to obtain recombinant plasmid pCI-neo-1, which in turn contains cleavage sites XhoI-SalI-PmeI-PacI-NotI in the sequence.

A, B, C, D four fragments were amplified respectively using Primer premier5.0 design specific primers (see Table 3), wherein one end of the amplified A fragment was ligated with hammerhead ribozyme sequence and one end of the amplified D fragment was ligated with hepatitis delta ribozyme sequence.

The extracted total RNA of the JS14-2 strain virus is used as a template to prepare goat parainfluenza virus 3 type JS14-2 strain cDNA according to the specification of a reverse transcription kit (Beijing holotype gold Biotech Co., Ltd.).

Amplification of the A fragment involved two PCR amplifications. The first PCR amplification was performed in a total volume of 20. mu.L, 2 XPCR Buffer 10. mu.L, upstream and downstream primers (AF1, AR) each 0.5. mu.L, cDNA template 2. mu.L, and RNase-free water to 20. mu.L. And (3) amplification procedure: 5min at 94 ℃; 30s at 94 ℃, 30s at 58 ℃ and 6min at 72 ℃ for 35 cycles; 10min at 72 ℃. And performing second PCR amplification by using the first PCR amplification product as a template and AF2 and AR as primers to obtain an A fragment with one end connected with a hammerhead ribozyme sequence, wherein the PCR system is formed in the same way as the first PCR amplification, and the PCR program is formed in the same way as the first PCR amplification.

Amplifying the D fragment includes two PCR amplifications. For the first PCR amplification, the total volume of the PCR system is 20 muL, wherein 2 XPCR Buffer is 10 muL, the upstream primer and the downstream primer (DF, DR1) are respectively 0.5 muL, the cDNA template is 2 muL, and the RNase-free water is supplemented to 20 muL. And (3) amplification procedure: 5min at 94 ℃; 30s at 94 ℃, 30s at 58 ℃, 2min at 72 ℃ and 35 cycles; 10min at 72 ℃. And performing second PCR amplification by using the first PCR amplification product as a template and DF and DR2 as primers to obtain a D fragment with one end connected with a hepatitis delta ribozyme sequence, wherein the PCR system is formed in the same way as the first PCR amplification, and the PCR program is formed in the same way as the first PCR amplification.

When B fragment is amplified, primers are BF and BR, the total volume of PCR is 20 muL, wherein 2 XPCR Buffer is 10 muL, the upstream and downstream primers are 0.5 muL respectively, cDNA template is 2 muL, and RNase-free water is supplemented to 20 muL. And (3) amplification procedure: 5min at 94 ℃; 30s at 94 ℃, 30s at 58 ℃, 2min at 72 ℃ and 35 cycles; 10min at 72 ℃.

When the C fragment is amplified, the primers are CF and CR, the total volume of PCR is 20 muL, wherein 2 XPCR Buffer is 10 muL, the upstream primer and the downstream primer are 0.5 muL respectively, cDNA template is 2 muL, and RNase-free water is supplemented to 20 muL. And (3) amplification procedure: 5min at 94 ℃; 30s at 94 ℃, 30s at 58 ℃ and 6min at 72 ℃ for 35 cycles; 10min at 72 ℃.

The fragment A and the pCI-neo-1 with one end connected with a hammerhead ribozyme sequence are subjected to double enzyme digestion by XhoI and SalI enzymes respectively, then are connected by T4 DNA ligase, and the fragment A is inserted into the pCI-neo-1 to obtain pCI-A. And carrying out double enzyme digestion on the pCI-A and the fragment B by adopting SalI and PmeI enzymes respectively, then connecting by adopting T4 DNA ligase, and inserting the fragment B into the pCI-A to obtain the pCI-A-B.

Fragment C, pCI-neo-1 was double digested with PmeI and PacI enzymes, respectively, then ligated using T4 DNA ligase, and fragment C was inserted into pCI-neo-1 to give pCI-C. And carrying out double enzyme digestion on the pCI-C and the fragment D with the delta hepatitis ribozyme sequence connected to one end by adopting PacI and NotI respectively, then connecting by adopting T4 DNA ligase, and inserting the fragment D with the delta hepatitis ribozyme sequence connected to the pCI-neo-1 to obtain pCI-C-D.

Fragment A-B (fragment formed by connecting fragment A and fragment B) was amplified using pCI-A-B as a template and primers AB-F and AB-R. The total volume of PCR is 20. mu.L, wherein 2 XPCR Buffer is 10. mu.L, the upstream and downstream primers are 0.5. mu.L respectively, the template is 2. mu.L, and RNase-free water is supplemented to 20. mu.L. And (3) amplification procedure: 5min at 94 ℃; 30s at 94 ℃, 30s at 58 ℃ and 6min at 72 ℃ for 35 cycles; 10min at 72 ℃.

C-D fragment (fragment formed by connecting fragments C and D) was amplified using primers CD-F and CD-R with pCI-C-D as a template. The total volume of PCR is 20. mu.L, wherein 2 XPCR Buffer is 10. mu.L, the upstream and downstream primers are 0.5. mu.L respectively, the template is 2. mu.L, and RNase-free water is supplemented to 20. mu.L. And (3) amplification procedure: 5min at 94 ℃; 30s at 94 ℃, 30s at 58 ℃ and 6min at 72 ℃ for 35 cycles; 10min at 72 ℃.

The linearized pYES1L Vector was obtained by PCR amplification using pYESlL plasmid (purchased from Thermo Fisher scientific) as template and primers Vector-F and Vector-R.

The linearized pYES1L vector, fragments A-B and fragments C-D were mixed, added to MaV203 yeast competence (purchased from Thermo Fisher scientific), transformed into yeast cells using lithium acetate transformation, inoculated onto tryptophan (Trp) deficient plates (purchased from Thermo Fisher scientific), cultured at 30 ℃ for 3 days, and then subjected to colony PCR using primers CPIV3-F and CPIV3-R to select positive clones (cloning strategy shown in FIG. 2). Since the linearized pYES1L vector and fragment A-B, fragment A-B and fragment C-D, and the linearized pYES1L vector and fragment C-D, respectively, present terminal homology arms, recombinant plasmids carrying the CPIV3 full-length cDNA were assembled by a homologous recombination mechanism in the host cell yeast.

And (3) electrically transforming the lysate of the yeast colony positive to colony PCR screening into DH10B competent cells, screening positive recombinants through spectinomycin resistance, and purifying the recombinant plasmid carrying the CPIV3 full-length cDNA through a plasmid medium-amount extraction kit after proliferation, wherein the recombinant plasmid is named as pYES1L-CPIV 3. The recombinant plasmid pYES1L-CPIV3 was extracted in large quantities, split charged after the concentration was determined, and stored at-20 ℃.

TABLE 3 cloning of viral cDNA to construct amplification primers

Note: in Table 3, ribozyme sequences are underlined and the sites are in italics.

Example 2 construction and characterization of helper plasmids

Specific primers (containing homology arms, see table 4) were designed for the open reading frame region of N, P and L genes in CPIV3 using Primer premier5.0, and PCR amplification was performed using recombinant plasmid pYES1L-CPIV3 as a template to obtain N, P and L gene fragments. The N, P and L gene fragments after purification were recovered and cloned between XhoI and BglII sites of pCAGGS plasmid by homologous recombination method (refer to the kit instructions) using Clonexpress II One Step cloning kit of Nanjing Novowed Biotechnology Ltd, then recombinant plasmids carrying each gene were transformed into DH 5. alpha. Escherichia coli competent cells, and positive colony clones identified by PCR digestion (FIG. 3) were further sequenced and identified to screen out correct recombinant plasmids pCAGGS-N (recombinant plasmid carrying N gene), pCAGGS-P (recombinant plasmid carrying P gene) and pCAGGS-L (recombinant plasmid carrying L gene). Extracting plasmid according to the operation instruction of the endotoxin-free plasmid extraction kit, determining the concentration, subpackaging, and storing at-20 ℃.

TABLE 4N, P, L Gene amplification primers

Note: in Table 4, the portion of the primers drawn along the horizontal line is the cleavage site.

Example 3 rescue and biological characterization of goat parainfluenza Virus type 3 JS14-2 recombinant Virus

1. Rescue and amplification of recombinant viruses

In 24-well plates according to 5X 10⁴293T cells were seeded per well and transfection was initiated when the cells grew to 90% confluence, using the following plasmids per well: infectious cDNA cloning plasmid (recombinant plasmid pYES1L-CPIV3) 0.5. mu.g, helper plasmid pCAGGS-N0.5. mu.g, pCAGGS-P0.2. mu.g and pCAGGS-L0.1. mu.g, transfection reagent (Lipofectamine)^TM2000) 1.5. mu.L, specific transfection method according to Lipofectamine^TM2000 instructions to perform operations.

Transfected cells were incubated at 37 ℃ with 5% CO₂Culturing for 4-5 days under the condition, harvesting cell suspension, freezing and thawing for three times, centrifuging at 2000rpm for 5min, taking supernatant, inoculating MDBK cells, and observing cytopathic effect. As a result: the next day after inoculation, the MDBK cells developed typical cytopathies of goat parainfluenza virus type 3: syncytia, fusion, and shedding occurred (FIG. 4). Cell cultures were harvested to give the first generation of rescued virus, rciv 3. Rescued virus rCPIV3 was serially passaged and cultures were stored at-70 ℃ for each generation.

2. Identification of recombinant viruses

2.1 Indirect immunofluorescence detection

The harvested rescued virus, rCPIV3, was seeded into MDBK cells and cultured for 72 h. Discarding the culture solution, and fixing the cells for 15min by using an immunostaining fixing solution; adding immunostaining sealing liquid, sealing thoroughly at 37 deg.C for 30 min; adding a CPIV3 specific monoclonal antibody, and incubating for 1h at 37 ℃; PBS washing 3 times; FITC-labeled goat anti-mouse IgG fluorescent secondary antibody was added, incubated at 37 ℃ for 1h, and washed 3 times with PBS. Cells visibly infected with virus exhibited a specific green fluorescence when observed under a fluorescent microscope, whereas non-infected control cells did not (FIG. 5). The success of the recombinant virus rescue is demonstrated, and the recombinant virus has infectivity.

2.2RT-PCR identification

Respectively extracting recombinant virus (rescued virus rCPIV3) and parental virus (goat parainfluenza virus 3 type JS14-2 strain) RNA, using a one-step RT-PCR kit (Beijing Quanjin Biotechnology Co., Ltd.) and using primers CF and CR to carry out RT-PCR amplification, wherein the total volume of RT-PCR is 20 muL, wherein 2 XR-Mix Buffer is 10 muL, upstream and downstream primers are 0.5 muL respectively, E-Mix is 0.4 muL, RNA template is 2 muL, and RNase-free water is supplemented to 20 muL. And (3) amplification procedure: reverse transcription is carried out for 30min at the temperature of 45 ℃; 5min at 94 ℃; 30s at 94 ℃, 30s at 58 ℃, 2min at 72 ℃ and 35 cycles; 10min at 72 ℃.

The fragments were recovered after 1% agarose gel electrophoresis, and sequenced by Biotech Inc. of Nanjing Ongzhike to compare whether the sequence was consistent with the designed one. As a result: the amplified sequence was identical to the viral cDNA plasmid sequence.

3. Characterization of biological Properties

3.1TCID₅₀Measurement of

The third generation recombinant virus (rescued virus rCPIV3) was diluted in a 10-fold gradient with cell maintenance medium (DMEM medium containing 2% fetal bovine serum). Each dilution of virus solution was then inoculated into a 96-well cell culture plate full of a single layer of MDBK cells, 4 wells per dilution of virus solution, and 100 μ L per well. While a negative control without virus inoculation was set. Culturing at 37 deg.C for 5 days, observing cytopathic effect, and calculating TCID according to Reed-Muench method₅₀. The results show that: the titer of the third generation recombinant virus can reach 10⁸TCID₅₀More than mL.

3.2 growth Curve determination

Recombinant virus (rescued virus rciv 3) and parental virus (goat parainfluenza virus type 3 JS14-2 strain) were inoculated at multiplicity of infection (MOI ═ 1) into 24-well cell culture plates filled with a monolayer of MDBK cells, and cultured at 37 ℃. At 24h, 48h, 72h, 96h and 120h post-infection, cell culture supernatants were taken, and the TCID of the virus was determined according to the method in title 3.1₅₀And drawing a virus growth curve. The results show that the titer of the recombinant virus and the parental virusThe growth period is prolonged between 24 and 72 hours, the stability is achieved between 72 and 120 hours, and the titer of the recombinant virus is higher than that of the parent virus (0.5 log) in 24 to 48 hours₁₀) Indicating that the recombinant virus proliferated at a higher rate than the parental virus (FIG. 6).

3.3 stability assay

The titers of the various generations (third, fifth, eighth, and tenth) of the recombinant viruses were determined on MDBK cells according to the method described in title 3.1, and the results showed that the respective titers of the recombinant viruses of the respective generations were 10⁸TCID₅₀/mL、10^8.5TCID₅₀/mL、10^8.15TCID₅₀mL and 10^8.25TCID₅₀The virus growth titer can reach 10 per mL⁸TCID₅₀More than/mL and is stable.

SEQUENCE LISTING

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<223> DR2

<400> 11

tatgcggccg cctcccttag ccatccgagt ggacgtgcgt cctccttcgg atgcccaggt 60

cggaccgcga ggaggtggag a 81

<210> 12

<211> 33

<212> DNA

<213> artificial

<220>

<223> AB-F

<400> 12

cttgagctct aataggtctg tttggtctga tga 33

<210> 13

<211> 43

<212> DNA

<213> artificial

<220>

<223> AB-R

<400> 13

cataggttgg agggtgttta aactcttatg atttatttct atg 43

<210> 14

<211> 26

<212> DNA

<213> artificial

<220>

<223> CD-F

<400> 14

cataagagtt taaacaccct ccaacc 26

<210> 15

<211> 32

<212> DNA

<213> artificial

<220>

<223> CD-R

<400> 15

aactagaagg cacagctccc ttagccatcc ga 32

<210> 16

<211> 32

<212> DNA

<213> artificial

<220>

<223> Vector-F

<400> 16

ggatggctaa gggagctgtg ccttctagtt gc 32

<210> 17

<211> 35

<212> DNA

<213> artificial

<220>

<223> Vector-R

<400> 17

tcagaccaaa cagacctatt agagctcaag ctctg 35

<210> 18

<211> 18

<212> DNA

<213> artificial

<220>

<223> CPIV3-F

<400> 18

ggatgtataa caggagtc 18

<210> 19

<211> 17

<212> DNA

<213> artificial

<220>

<223> CPIV3-R

<400> 19

aggagaattc aaatggc 17