Construction method and application of chicken anemia virus full-length gene infectious clone plasmid carrying EGFP (Enhanced Green Fluorescent Protein) tag

Abstract

The invention discloses a construction method and application of a chicken anemia virus full-length gene infectious clone plasmid carrying an EGFP tag, and belongs to the technical field of virus gene engineering. According to the invention, a cyclic genome of CAV is linearized by utilizing a reverse genetic technology, and the linearized CAV genome is connected with a cloning vector pcDNA3.1 (+) by virtue of a seamless cloning method, so that a CAV full-length infectious clone pcDNA3.1-CAV and a CAV full-length infectious clone pcDNA3.1-CAV-EGFP (Enhanced Green Fluorescent Protein) carrying an EGFP (Enhanced Green Fluorescent Protein) tag are successfully constructed; in addition, in-vitro virus rescue is realized on MDCC-CU147 cells, and pathogenicity consistent with that of CAV is realized in SPF chicken bodies. The invention provides a simple and rapid CAV reverse genetics operating system, and the CAV carrying the EGFP green fluorescent protein gene is successfully obtained. The method has wide application value in the aspects of research on a CAV pathogenic mechanism and a virus replication mechanism, research and development of CAV vaccines and diagnostic reagents and the like.

Description

Technical Field

[0001] The invention relates to the technical field of viral gene engineering, and in particular to a method for constructing an infectious clone plasmid of a full-length gene of a chicken anemia virus carrying an EGFP label and an application thereof. Background Art

[0002] Chicken anemia virus (CAV) belongs to the genus Gyrovirus of the Anelloviridae family. CAV is a single-stranded negative-strand DNA virus. Its genome is a single-stranded, circular, covalently linked closed DNA. The genome consists of about 2319 bp of bases, including three open reading frames (ORFs), which are 1350 bp, 609 bp and 366 bp, encoding the three proteins VP1, VP2 and VP3. CAV infection can cause anemia and immunosuppression in chickens. Therefore, the virus has a significant economic impact on the global poultry industry. After chickens are infected with CAV and develop infectious anemia, they usually show depression, emaciation, lying down, anorexia, fluffy and messy feathers, shrinking necks, slow growth and development, pale combs, wattles and mucous membranes, and reduced egg production during the laying period, and sometimes excrete yellow-green feces. Some sick chickens will have localized punctate bleeding of varying degrees on their skin, most of which can be seen on the wings, and sometimes on the head, neck, tail, chest, abdomen and legs. When the symptoms are severe, the skin turns blue-purple and breaks, with fluid oozing out, affecting growth and development. The entire course of the disease is relatively long, lasting about 7 to 30 days, with a mortality rate of 5% to 15%. The immunosuppression caused by CAV infection reduces the immunity of infected chickens, making it easy to cause secondary infections of immunosuppressive diseases such as Marek's disease, infectious bursal inflammation, and encephalomyelitis, leading to worsening of the disease, prolonged course of the disease, and a mortality rate of 60% to 80%.

[0003] Infectious clones are important tools in the field of viral reverse genetics and are widely used in basic and applied research such as virus rescue, viral gene structure and function, viral pathogenicity, virus-host interaction, and vaccine development.

[0004] EGFP is a classic fluorescent protein, the full name is Enhanced Green Fluorescent Protein. It is derived from the fluorescent protein (Green Fluorescent Protein, GFP) of the Aequorea victoria jellyfish. GFP has been improved through genetic engineering technology, which has enhanced its application value in biological research. EGFP is a single-chain protein composed of 238 amino acids, with a molecular weight of about 27 kilodaltons. Its fluorescence emission peak is located at about 509 nanometers. It can be excited by ultraviolet light (excitation wavelength is about 488 nanometers) to emit green fluorescence. EGFP is widely used in biological research, mainly for marking and tracking the localization and dynamic changes of proteins and cells. Using genetic engineering technology, the EGFP coding sequence can be connected to the target gene sequence, so that the target protein is expressed together with EGFP when expressed to form a fusion protein. By observing the fluorescent signal of the fusion protein, the localization, distribution and dynamic changes of the protein in the cell can be tracked in real time, so as to gain a deeper understanding of the mechanism and regulation of biological processes. Due to the high expression level, stable fluorescence performance and compatibility with many biological systems, EGFP has become one of the most commonly used fluorescent labeling tools in biological research.

[0005] In the process of constructing infectious clones, it is necessary to ensure that the CAV viral genome replicates and expresses normally in cells and produces infectious virus particles, while also ensuring the successful expression and correct positioning of the EGFP tag. There are many factors that affect the expression of the EGFP gene, so the selection of the insertion site is very important. It is not necessary to avoid the area with only high CG content. Even in the area with low CG content, incorrect selection of the insertion site may lead to rearrangement, deletion or mutation of the insertion area, affecting the stable expression of EGFP. On the other hand, the gene in the insertion site area may cause incorrect folding of EGFP, resulting in its abnormal function and inability to effectively emit green fluorescence. Therefore, ensuring the correct positioning of the EGFP tag is a key challenge for the construction of infectious clones carrying the tag in 13 viruses of the genus Cynovirus. Summary of the invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to provide a method for constructing an infectious clone plasmid of the full-length gene of chicken anemia virus carrying an EGFP tag and its application. The present invention inserts a Kozak sequence at the 5' of the EGFP tag, and designs parallel experiments to screen out the best insertion site of the EGFP tag in the full-length gene of CAV, and achieves virus rescue in MDCC-CU147 cells in vitro and in SPF chickens, filling the gap in the construction of infectious clones carrying EGFP tags for 13 viruses of the genus Cynovirus.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] In the first aspect of the present invention, a CAV full-length gene infectious clone plasmid carrying an EGFP tag is provided. The eukaryotic expression vector pcDNA3.1(+) and the CAV linear full genome are connected to construct the CAV full-gene recombinant plasmid pcDNA3.1-CAV, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0009] Furthermore, the CAV full-length gene infectious clone plasmid is composed of the CAV full-gene recombinant plasmid pcDNA3.1-CAV and the EGFP gene with a Kozak sequence, and the insertion site of the EGFP gene is located between the 1996th and 1997th nucleotides of CAV.

[0010] Furthermore, the nucleotide sequence of the CAV full-length gene infectious clone plasmid is shown in SEQ ID NO.8.

[0011] Furthermore, the construction method is as follows:

[0012] (1) Using reverse genetics technology, the CAV genome and the cloning vector pcDNA3.1(+) were linearized using linearization primer pair 1 and linearization primer pair 2, respectively, and the CAV linear full genome was connected to the cloning vector pcDNA3.1(+) through homologous recombination to construct the CAV full-length infectious clone pcDNA3.1-CAV;

[0013] (2) pcDNA3.1-CAV and EGFP were amplified using primer pair 1 and primer pair 2, respectively. The EGFP gene was inserted into the CAV non-structural protein region of pcDNA3.1-CAV using homologous recombination, and an infectious clone plasmid carrying the full-length CAV gene with an EGFP tag, namely pcDNA3.1-CAV-EGFP, was constructed.

[0014] Furthermore, in step (1), the nucleotide sequences of the homology arms used for homologous recombination are shown as SEQ ID NO.1 and SEQ ID NO.2.

[0015] Furthermore, in step (1), the nucleotide sequence of linearized primer pair 1 is shown as SEQ ID NOs. 9-10, and the nucleotide sequence of linearized primer pair 2 is shown as SEQ ID NOs. 11-12.

[0016] Furthermore, in step (2), the nucleotide sequences of the homology arms used for homologous recombination are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0017] Furthermore, in step (2), the nucleotide sequence of amplification primer pair 1 is shown as SEQ ID NOs. 13-14, and the nucleotide sequence of amplification primer pair 2 is shown as SEQ ID NOs. 19-20.

[0018] The second aspect of the present invention provides the use of the CAV full-length gene infectious clone plasmid carrying the EGFP tag in any one of the following (i)-(iii):

[0019] (i) CAV virus rescue and pathogenicity studies;

[0020] (ii) preparing anti-chicken anemia virus drugs;

[0021] (iii) Preparation of chicken anemia virus vaccine.

[0022] Beneficial effects of the present invention:

[0023] The present invention linearizes the circular genome of chicken anemia virus (CAV) by reverse genetics technology for the first time, connects the linearized CAV genome with the cloning vector pcDNA3.1(+) by seamless cloning, and successfully constructs the CAV full-length infectious clone pcDNA3.1-CAV. Subsequently, the same method is used to insert the EGFP gene with the Kozak sequence into the CAV non-structural protein region of pcDNA3.1-CAV, and the CAV full-length infectious clone pcDNA3.1-CAV-EGFP carrying the EGFP tag is constructed. This technology not only realizes the virus rescue and pathogenicity research in vitro and in SPF chickens, but also provides a platform for CAV pathogenicity and gene structure and function research. Finally, this technology provides a simple and fast circular virus reverse genetics operating system, which lays the foundation for studying the pathogenic mechanism of CAV and the genetic engineering vaccine of CAV. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram for constructing an infectious clone of the full-length genome of CAV carrying an EGFP tag;

[0025] Figure 2 is the test result, where Figure 2 Figure a is a schematic diagram of the results of linear PCR amplification of the CAV whole genome; Figure 2 Figure b is a schematic diagram of the linear PCR amplification results of the whole genome of pcDNA3.1; Figure 2 Middle c is a schematic diagram of the double enzyme digestion identification results of pcDNA3.1-CAV plasmid; Figure 2 Figure d in the figure is a schematic diagram of the linear PCR amplification results of the whole genome of pcDNA3.1-CAV;

[0026] Figure 3This is a schematic diagram of the PCR amplification results of the EGFP whole genome;

[0027] Figure 4 The image is of MDCC-CU147 transfected with pcDNA3.1-CAV-EGFP and observed under an inverted fluorescence microscope;

[0028] Figure 5 This is the in vitro growth curve of pcDNA3.1-CAV-EGFP and CAV-SDAU-1;

[0029] Figure 6 This is the blood viremia detection curve of experimental chickens;

[0030] Figure 7 These are the histopathological observation pictures of various organs of experimental chickens;

[0031] Figure 8 This is the green fluorescence co-localization image of CAV VP1 and pcDNA3.1-CAV-EGFP detected by IFA in various organs of experimental chickens (100×). DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0033] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0034] The experimental materials used in the examples of the present invention but not specifically described are all conventional experimental materials in the art and can be purchased through commercial channels.

[0035] The present invention provides a method for constructing a CAV full genome infectious clone plasmid carrying an EGFP tag, and the specific steps are as follows:

[0036] Utilizing the characteristics of the viral circular genome, primers were designed from the 343rd nucleotide, homology arms were added to the 5' ends of the upstream and downstream primers (SEQ ID NO.1: 5'-TAAGCTTGGTACCGAG-3', SEQ ID NO.2: 5'-CAGAATTCCACCACAC-3'), the CAV linear whole genome (SEQ ID NO.3) was amplified using a high-fidelity enzyme, and connected to pcDNA3.1(+) to construct a CAV full-gene recombinant plasmid pcDNA3.1-CAV (SEQ ID NO.4); primers were designed from the 2921st nucleotide of the recombinant plasmid pcDNA3.1-CAV, the recombinant plasmid pcDNA3.1-CAV was linearized, and then EGFP full-length primers were artificially designed, homology arms were added to the 5' ends of the upstream and downstream primers (SEQ ID NO.5: 5'-CCGAGTGGTTACTAT-3', SEQ ID NO.6: 5'-CAGGCTAGAATGGTGATGGA-3', and at the same time inserting the Kozak sequence (SEQ ID NO.7: 5'-GCCACC-3') at the upstream 5', homologously recombining with the linearized pcDNA3.1-CAV obtained above, and finally synthesizing the CAV full-length gene infectious clone pcDNA3.1-CAV-EGFP (SEQ ID NO.8) carrying the EGFP tag. The above scheme reselects the linearization site according to the characteristics of CAV and redesigns the primer sequence, which is significantly different from the existing conventional technology.

[0037] In addition, the inventors also provide the application of the above-mentioned CAV full-length gene infectious clone plasmid carrying the EGFP tag, and specifically provide a method for in vitro verification of infectious clones, including the following steps: culture of MDCC-CU147 cells; transfection of infectious clones; direct observation of pcDNA3.1-CAV-EGFP in vitro infection by inverted fluorescence microscope. Using the above-constructed CAV full-length gene infectious clone carrying the EGFP tag, in vivo virus rescue was performed on 1-day-old SPF chickens inoculated with muscle, and the viremia and tissue viral load of the inoculated chickens were detected at 7, 14, and 21 dpi. At 14 dpi, thymus, spleen, and bursa of Fabricius tissues were selected to observe the co-localization of pcDNA3.1-CAV-EGFP and CAV VP1 by immunofluorescence technology to verify the successful rescue of infectious viruses. At the same time, CAV cell isolation virus was inoculated in the same way as a positive control and PBS as a negative control.

[0038] The plasmid obtained above can be used for virus rescue and pathogenicity research in CAV, and the raw materials such as plasmid vectors involved are cheap and easy to obtain, which greatly shortens the cost and time of infectious clone construction. At the same time, the technical solution of this application fills the gap in this field, and the construction strategy of the present invention can be used for the construction of infectious clones and virus rescue for other circular viruses.

[0039] Example 1: PCR amplification of the entire genome of CAV and pcDNA3.1(+)

[0040] 1. Primer Design and Synthesis

[0041] According to the construction strategy ( Figure 1 ), according to the nucleic acid sequences named CAV-SDAU-1 (GenBank: OL448984.1) and pcDNA3.1(+) (GenBank: MN996867.1) in the NCBI GenBank database, the genes of CAV and pcDNA3.1(+) were linearized by reverse genetic technology using artificially designed primers, and 16 bp homology arms were designed upstream and downstream. The primer pair of CAV is CAV-F (SEQ ID NO.9) and CAV-R (SEQ ID NO.10), and the primer pair of pcDNA3.1(+) is pcDNA3.1(+)-F (SEQ ID NO.11) and pcDNA3.1(+)-R (SEQ ID NO.12). The primer sequences are shown in Table 1, and the underlined parts are homology arms.

[0042] Table 1 Primer sequences

[0043]

[0044] 2. Preparation of CAV Template DNA

[0045] CAV template DNA was prepared using SPF chicken tissues from the third regression of the CAV-SDAU-1 strain.

[0046] DNA from liver of CAV-infected chickens was extracted using a cell / tissue / blood DNA extraction kit (Tiangen, Beijing).

[0047] 3. PCR amplification and purification of CAV and pcDNA3.1(+) whole genome linearization

[0048] In order to accurately amplify the whole genome of CAV, two high-fidelity enzymes 2×Phanta Max Master Mix were tried, and the DNA samples containing CAV obtained above were used as templates for PCR reactions. The sample addition system and PCR reaction procedures are shown in Tables 2 and 3 below:

[0049] Table 2 PCR sample loading system

[0050]

[0051] Table 3 PCR reaction program

[0052]

[0053] The PCR reaction results were separated and purified by agarose gel electrophoresis. Figure 2 Middle a, Figure 2 The results in b show that the linear whole genome of CAV virus (SEQ ID NO.3, length is 2319 bp) and vector pcDNA3.1(+) (length is 5410 bp) were successfully amplified, and then gel recovery (Tiangen, Beijing) will be carried out for future use.

[0054] Example 2 Construction and identification of full-length gene recombinant plasmid pcDNA3.1-CAV

[0055] 1. Homologous recombination of pcDNA3.1(+) and CAV

[0056] 1) Calculation of the amount of vector fragment used:

[0057] Optimal amount of cloning vector used = [0.02 × number of base pairs of cloning vector] ng (0.03 pmol)

[0058] Optimal amount of insert fragment = [0.04 × number of base pairs of insert fragment] ng (0.06 pmol)

[0059] 2) Prepare the following reaction system on ice:

[0060] Table 4 Reaction system

[0061]

[0062] Note: X / Y is used to calculate the amount of vector and insert fragment.

[0063] Negative control-1 can be used to confirm whether there is any circular plasmid left in the linearized cloning vector, and is recommended.

[0064] Negative Control-2 is recommended when the insert amplification template is a circular plasmid with the same resistance as the cloning vector.

[0065] Positive control reactions can be used to exclude the influence of other experimental materials and operating factors.

[0066] Use a pipette to gently mix, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0067] 3) Incubate at 37°C for 30 min; cool to 4°C or immediately place on ice.

[0068] 2. Transformation of pcDNA3.1(+) and CAV homologous recombination products

[0069] The cells used for transformation were DH5α competent cells. After transformation, LB solid agar medium containing ampicillin was used to screen out cells that had been successfully transformed with and expressed the pcDNA3.1(+) plasmid containing the ampicillin resistance marker gene, and the cells were cultured continuously.

[0070] 3. Enzyme digestion and identification of pcDNA3.1-CAV positive clones

[0071] Pick a milky white, isolated and larger colony from each plate with a small white pipette tip, place it in 10 mL of LB liquid culture medium containing ampicillin, and place the test tube in a 37°C shaker at 220 rpm for 12-16 hours until the bacterial solution OD reaches 0. 600 When the concentration reached 2.0-3.0, the culture was stopped. According to the instructions, the plasmid was extracted from the bacterial solution using a plasmid extraction kit (Tiangen, Beijing), and the extracted plasmid pcDNA3.1-CAV was identified by double restriction digestion and agarose gel electrophoresis.

[0072] Figure 2 The results in c show that the bands after enzyme digestion are all correct bands. The agarose gel electrophoresis results show fragments of the same size (2185 bp and 5244 bp) for the two entries, proving that pcDNA3.1-CAV (SEQ ID NO. 4) was successfully constructed.

[0073] Example 3 Construction and identification of full-length gene recombinant plasmid pcDNA3.1-CAV-EGFP

[0074] 1. PCR amplification and purification of pcDNA3.1-CAV and EGFP whole genome

[0075] The PCR amplification and purification recovery of the whole genome of pcDNA3.1-CAV and EGFP were performed using the method of step 3 of Example 1. When amplifying pcDNA3.1-CAV, the template DNA used was the full-length gene recombinant plasmid pcDNA3.1-CAV obtained in Example 2, and the nucleotide sequence was shown in SEQ ID NO.13-SEQ ID NO.18; when amplifying EGFP, the template DNA used was EGFP, and the nucleotide sequence of the primer pair used was shown in SEQ ID NO.19-SEQ ID NO.24.

[0076] Results Figure 2 Middle d- Figure 3 , Figure 2 The results in Figure d show that the linearized whole genome of pcDNA3.1-CAV was successfully amplified. Figure 3 The results showed that the entire EGFP genome was amplified successfully.

[0077] Table 5 Primer sequences

[0078]

[0079] 2. Homologous recombination of pcDNA3.1-CAV and EGFP

[0080] Homologous recombination of pcDNA3.1-CAV and EGFP (pcDNA3.1-CAV-1 and EGFP-1 / , pcDNA3.1-CAV-2 and EGFP-2, pcDNA3.1-CAV-3 and EGFP-3) was performed using the method of step 1 of Example 2.

[0081] 3. Transformation of pcDNA3.1-CAV and EGFP homologous recombination products

[0082] The homologous recombination product of pcDNA3.1-CAV and EGFP was transformed using the method of step 2 of Example 2. The products pcDNA3.1-CAV-EGFP-1 (SEQ ID NO.8), pcDNA3.1-CAV-EGFP-2, and pcDNA3.1-CAV-EGFP-3 were obtained. The difference between them was that EGFP was inserted into different positions of the non-coding region of CAV. Taking the linear whole genome of CAV virus (SEQ ID NO.3) as an example, pcDNA3.1-CAV-EGFP-1 inserted EGPF between the 1996th and 1997th nucleotides of CAV; pcDNA3.1-CAV-EGFP-2 inserted EGPF between the 2028th and 2029th nucleotides of CAV; and pcDNA3.1-CAV-EGFP-3 inserted EGPF between the 2245th and 2246th nucleotides of CAV.

[0083] Example 4 pcDNA3.1-CAV-EGFP in vitro transfection verification

[0084] 1. Transfection of infectious clone pcDNA3.1-CAV-EGFP

[0085] Since most CAV strains can be stably propagated in MDCC-CU147, MDCC-CU147 was used for in vitro transfection verification of pcDNA3.1-CAV-EGFP. The plasmids used were empty vector pcDNA3.1(+), infectious clone plasmids pcDNA3.1-CAV-EGFP-1, pcDNA3.1-CAV-EGFP-2, and pcDNA3.1-CAV-EGFP-3. Fluorescence was captured under an inverted fluorescence microscope at 72 hours after transfection.

[0086] The results are as follows Figure 4As shown, green fluorescence was observed only in MDCC-CU147 cells transfected with pcDNA3.1-CAV-EGFP-1, and pcDNA3.1-CAV-EGFP-1 was used in subsequent experiments.

[0087] 2. In vitro growth curve of infectious clone pcDNA3.1-CAV-EGFP-1

[0088] The cells were collected at 12, 24, 36, 48, 60, and 72 hours after transfection in step 1, and DNA was extracted for quantitative PCR detection.

[0089] MDCC-CU147 cells were infected with CAV-SDAU-1 wild strain virus liquid, and the cells were collected at 12, 24, 36, 48, 60, and 72 hours after infection, and DNA was extracted for quantitative PCR detection.

[0090] The primer sequences, reaction system and reaction conditions for quantitative PCR detection are shown in Tables 6 to 8, and the probe is FAM-TATCGCTGTGTGGCTGCGCGAATGC (SEQ ID NO. 25)-BHQ2:

[0091] Table 6 CAV absolute fluorescence quantitative primer sequences

[0092]

[0093] Table 7 aqMan Real-time PCR amplification reaction system

[0094]

[0095] Table 8 TaqMan Real-time PCR amplification reaction conditions

[0096]

[0097] Results Figure 5 The pcDNA3.1-CAV-EGFP infectious clone can replicate in vitro, and the growth trend is consistent with that of CAV-SDAU-1 in vitro, reaching the peak of replication at the 60th hour of infection. This indicates that the CAV full-length gene infectious clone pcDNA3.1-CAV-EGFP carrying the EGFP tag can achieve in vitro virus rescue.

[0098] Example 5 In vivo virus rescue and establishment of SPF chicken infection model

[0099] 1. Establishment of Animal Infection Model

[0100] The infectious clone pcDNA3.1-CAV-EGFP-1 infection model was established by intramuscular inoculation of 1-day-old SPF chickens to ensure the full interaction of the infectious clone plasmid with the cells in vivo. 60 1-day-old SPF chickens were randomly divided into three groups, namely the normal control group (Mock), the CAV infectious clone group (pcDNA3.1-CAV-EGFP) and the CAV positive control group (CAV). The inoculation route was 1-day-old leg muscle inoculation. The CAV infectious clone group was inoculated with 3×100μLpcDNA3.1-CAV-EGFP-1, the CAV positive control group was inoculated with 3×100μL CAV-SDAU-1, and the normal control group was inoculated with 3×100μL PBS. 7, 14, and 21 days after intramuscular inoculation, 3 chickens were randomly selected from each group for blood sampling and dissection for rescue virus detection.

[0101] 2. Viremia detection and dynamic changes of viral load in various tissues

[0102] Viremia detection is an important indicator for testing whether the virus infectious clone is infectious. The blood virus load levels of 3 random chickens in each group were monitored at 7, 14, and 21 dpi (day post-infection).

[0103] The results are as follows Figure 6 As shown, during the experimental period, both the CAV infectious clone group and the positive control group had persistent viremia, among which the blood viral load of the CAV infectious clone group continued to increase, and the increase in the blood viral load of the CAV positive control group continued until 14 dpi, and then showed a downward trend. This shows that the CAV full-length gene infectious clone pcDNA3.1-CAV-EGFP carrying the EGFP tag can achieve in vivo virus rescue and effectively infect SPF chickens.

[0104] 3. Histopathological Observation

[0105] After all the chickens in the animal experiment were killed, fixed tissues of each group at 14 dpi were taken to make paraffin sections, which were stained with hematoxylin-eosin, and the pathological changes of each tissue were observed and photographed.

[0106] The results are as follows Figure 7As shown, on the 14th day after infection, in the thymus, the number of thymic cortical and medullary lymphocytes in the CAV infectious clone group and the positive control group was significantly reduced, the medulla area was enlarged, a large number of homogeneous red staining appeared, and the boundary between the cortex and medulla was unclear; in the spleen, the spleen of the CAV infectious clone group and the positive control group showed obvious lymphocytopenia; in the bursa of Fabricius, the number of bursal lymphoid follicles in the CAV infectious clone group and the positive control group was significantly reduced, the volume was reduced, and vacuoles appeared. This shows that the CAV full-length gene infectious clone pcDNA3.1-CAV-EGFP and CAV-SDAU-1 cell isolated virus carrying the EGFP tag both caused obvious lesions in the immune organs of experimental chickens.

[0107] 4. Colocalization Experiment

[0108] Mouse anti-CAV VP1 monoclonal antibody was used as the primary antibody, and enhanced enzyme-labeled goat anti-mouse IgG polymer was used as the secondary antibody. On the 14th day after infection, experimental samples were taken and the fluorescence of the sections was observed using a laser confocal microscope.

[0109] The results are as follows Figure 8 As shown, the EGFP tag of pcDNA3.1-CAV-EGFP can be expressed in the thymus, bursa of Fabricius, and spleen, and co-localized with CAV VP1 protein, with roughly the same position in the tissues. The results show that pcDNA3.1-CAV-EGFP can carry the EGPF tag for stable expression.

[0110] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A CAV full-length gene infectious clone plasmid carrying an EGFP tag, characterized in that: The eukaryotic expression vector pcDNA3.1(+) and the CAV linear whole genome were connected to construct the CAV whole gene recombinant plasmid pcDNA3.1-CAV, the nucleotide sequence of which is shown in SEQ ID NO.

4.

2. The CAV full-length gene infectious clone plasmid carrying an EGFP tag according to claim 1, characterized in that: The CAV full-length gene infectious clone plasmid is composed of the CAV full-gene recombinant plasmid pcDNA3.1-CAV and the EGFP gene with Kozak sequence, and the insertion site of the EGFP gene is located between the 1996th and 1997th nucleotides of CAV.

3. The CAV full-length gene infectious clone plasmid carrying an EGFP tag according to claim 2, characterized in that: The nucleotide sequence of the CAV full-length gene infectious clone plasmid is shown in SEQ ID NO.

8.

4. The CAV full-length gene infectious clone plasmid carrying an EGFP tag according to claim 1, characterized in that: The construction method is as follows: (1) Using reverse genetics technology, the CAV genome and the cloning vector pcDNA3.1(+) were linearized using linearization primer pair 1 and linearization primer pair 2, respectively, and the CAV linear full genome was connected to the cloning vector pcDNA3.1(+) through homologous recombination to construct the CAV full-length infectious clone pcDNA3.1-CAV; (2) pcDNA3.1-CAV and EGFP were amplified using primer pair 1 and primer pair 2, respectively. The EGFP gene was inserted into the CAV non-structural protein region of pcDNA3.1-CAV using homologous recombination, and an infectious clone plasmid carrying the full-length CAV gene with an EGFP tag, namely pcDNA3.1-CAV-EGFP, was constructed.

5. The CAV full-length gene infectious clone plasmid carrying an EGFP tag according to claim 4, characterized in that: In step (1), the nucleotide sequences of the homology arms used for homologous recombination are shown in SEQ ID NO.1 and SEQ ID NO.

2.

6. The infectious clone plasmid of the full-length gene of CAV carrying the EGFP tag according to claim 4, characterized in that: In step (1), the nucleotide sequence of linearized primer pair 1 is shown as SEQ ID NOs. 9-10, and the nucleotide sequence of linearized primer pair 2 is shown as SEQ ID NOs. 11-12.

7. The CAV full-length gene infectious clone plasmid carrying an EGFP tag according to claim 4, characterized in that: In step (2), the nucleotide sequences of the homology arms used for homologous recombination are shown in SEQ ID NO.5 and SEQ ID NO.

6.

8. The infectious clone plasmid of the full-length gene of CAV carrying the EGFP tag according to claim 4, characterized in that: In step (2), the nucleotide sequence of amplification primer pair 1 is shown as SEQ ID NOs. 13-14, and the nucleotide sequence of amplification primer pair 2 is shown as SEQ ID NOs. 19-20.

9. Use of the infectious clone plasmid of the full-length gene of CAV carrying the EGFP tag according to any one of claims 1 to 8 in any one of the following (i) to (iii): (i) CAV virus rescue and pathogenicity studies; (ii) preparing anti-chicken anemia virus drugs; (iii) Preparation of chicken anemia virus vaccine.

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