CN113215117B - Duck tembusu virus attenuated live vaccine candidate strain and preparation method and application thereof - Google Patents

Abstract

The invention discloses a duck tembusu virus attenuated live vaccine candidate strain, which is a duck tembusu delta C64-96 mutant virus strain, wherein the duck tembusu delta C64-96 mutant virus strain is a gene deletion mutant virus strain, and the deleted gene sequence is a nucleotide sequence for coding 64 th-96 th amino acids of a duck tembusu virus Capsid protein. The attenuated live vaccine candidate strain has defects in the assembly process of virus particles, so that the virus is attenuated, the attenuation mechanism is clearly analyzed, and the use safety of the vaccine is effectively improved; and the weak live vaccine candidate strain can obviously activate the immune system of the infected duckling through single immunization of the duckling, and can provide a strong and effective immune protection effect.

Description

Duck tembusu virus attenuated live vaccine candidate strain and preparation method and application thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to a duck tembusu virus attenuated live vaccine candidate strain and a preparation method and application thereof.

Background

The duck tembusu virus disease rapidly spreads to a plurality of provinces and cities in China since the first outbreak in the coastal region of southeast China in 2010, and causes great economic loss to the duck breeding industry in China. The duck Tembusu disease is an acute infectious disease which is mainly characterized by the sudden and large drop of egg laying of laying ducks and breeding ducks and mainly characterized by hemorrhagic oophoritis. The current prevention and control measures for the disease are incomplete, and the disease still presents regional flow at present although commercial attenuated live vaccines and inactivated vaccines exist. The existing commercial inactivated vaccine has higher safety, but the inactivated vaccine has high production cost, strict requirements on transportation and storage conditions, and unsatisfactory immune effect, needs to enhance immunity for many times, and increases the stress response of duck groups. The existing attenuated live vaccines are subjected to traditional continuous passage, so that adaptive mutation is generated in the process of adapting viruses to heterologous host cells, and finally, an attenuated phenotype with immunogenicity is obtained. The molecular mechanism of viral attenuation of the attenuated vaccine candidate obtained in this manner is unclear or not completely resolved; the security risk is high because the lack of knowledge of its detailed mechanism would limit the assessment of the risk of its natural reversion to a virulent phenotype. Therefore, the current DTMUV vaccine still has a great room for improvement, and particularly, the development of a DTMUV vaccine with high safety and good immune effect is needed.

Disclosure of Invention

The invention aims to solve the problems of low safety and unsatisfactory immune effect of a duck tembusu virus attenuated vaccine in the prior art, and provides a duck tembusu virus attenuated live vaccine candidate strain which is a duck tembusu delta C64-96 mutant virus strain, wherein the duck tembusu delta C64-96 mutant virus strain has defects in the assembly process of virus particles, so that the virus is attenuated, the attenuation mechanism of the virus is clearly analyzed, and the use safety of the attenuated live vaccine candidate strain is effectively improved; and the single immunization of the duck Tembusu delta C64-96 mutant virus strain on the ducklings can obviously activate the immune system of the immunized ducklings, thereby greatly improving the immune effect.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a duck Tembusu virus attenuated live vaccine candidate strain is a duck Tembusu delta C64-96 mutant virus strain, the duck Tembusu delta C64-96 mutant virus strain is a gene deletion mutant virus strain, and a deleted gene sequence is a nucleotide sequence of 64 th-96 th amino acids of a coding duck Tembusu virus Capsid protein.

The invention also provides a preparation method of the duck tembusu attenuated live vaccine candidate strain, which comprises the following steps:

s1, carrying out PCR amplification by taking pACNR-FL-CQW1 plasmid of a duck Tembusu virus strain DTMUV-CQW1 strain as a template and taking sequences SEQ ID NO.1 and SEQ ID NO.2 as primers to obtain a fragment P1; taking the sequences SEQ ID NO.3 and SEQ ID NO.4 as primers, and carrying out PCR amplification to obtain a fragment P2; then, a fragment P1 and a fragment P2 are used as templates, sequences SEQ ID NO.1 and SEQ ID NO.4 are used as primers, a gene deletion mutant fragment P1P2 is obtained through fusion PCR amplification, and the gene sequence deleted by the fragment P1P2 is a nucleotide sequence of 64 th-96 th amino acids of a Capsid protein of a coded duck Tembusu virus;

s2, carrying out double enzyme digestion on the pACNR-FL-CQW1 plasmid by using restriction enzymes MluI and XhoI, and recovering and purifying to obtain a purified linear pACNR-FL-CQW1 plasmid vector;

s3, carrying out recombinant connection on the gene fragment P1P2 in the step S1 and the purified linear pACNR-FL-CQW1 plasmid vector in the step S2, then transforming escherichia coli competent cells, selecting positive colonies, sequencing and identifying to obtain a recombinant plasmid, and naming the recombinant plasmid as pACNR-FL-CQW 1-delta C64-96 plasmid;

s4, transfecting the plasmid pACNR-FL-CQW 1-delta C64-96 in the step S3 to BHK21 cells, and culturing for 5 days to obtain a supernatant, namely obtaining the duck tembusu delta C64-96 mutant virus strain.

Preferably, the nucleotide sequence of the fragment P1 is shown as SEQ ID NO. 5; the nucleotide sequence of the fragment P2 is shown as SEQ ID NO. 6; the nucleotide sequence of the fragment P1P2 is shown as SEQ ID NO. 7; the sequence of the pACNR-FL-CQW 1-delta C64-96 plasmid is shown in SEQ ID NO. 8.

Preferably, the conditions for PCR amplification are: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 15s, annealing at 63 ℃ for 15s, and extension at 72 ℃ for 15s/kb for 30 cycles; finally, stretching for 7min at 72 ℃.

The duck Tembusu attenuated live vaccine candidate strain prepared by the method is prepared into a duck Tembusu virus attenuated live vaccine immune duckling, and can bring safe and efficient immune protection for the duckling.

The capsid (C) protein is the smallest structural protein of flavivirus, and the mature C protein produced by cleavage with NS2B3 protease is only around 100 amino acids. The structure and function of the protein C are highly conserved in the flavivirus genus, and the structure of the protein C comprises 4 alpha helices, so that a dimer is formed through hydrophobic interaction, hydrogen bonds and other forces. The most important function of the C protein is to bind to viral genomic rna (vrna) via a dimeric structure, forming Nucleoscapid (NC) which participates in the assembly of virions, which form intact virions (Virion) after budding to obtain the viral envelope. Flavivirus studies have shown that the C protein can tolerate a greater degree of truncation mutations while maintaining viral infectivity, but results in increased production of Subviral particles (SVPs). SVPs do not contain NC, but are quite similar in conformation and immunogenicity to virion and are good immunogens. Thus, protein C is an ideal target for viral attenuation. The duck tembusu delta C64-96 mutant virus strain is obtained by gene deletion mutation, and a nucleotide sequence for coding 64 th-96 th amino acids of a duck tembusu virus Capsid protein is deleted, so that the duck tembusu delta C64-96 mutant virus strain cannot express the 64 th-96 th amino acids of the Capsid protein, and the deletion mutation directly influences the assembly process of virus particles, so that the duck tembusu delta C64-96 mutant virus strain is weakened.

The invention has the following beneficial effects:

firstly), the nucleotide sequence coding the 64 th-96 th amino acids of the duck Tembusu virus Capsid protein is deleted, so that the 64 th-96 th amino acids of the Capsid protein are deleted, and the duck Tembusu delta C64-96 mutant virus strain is obtained.

Secondly), the weakening mechanism of the duck tembusu delta C64-96 mutant virus strain is clearly analyzed, the mutant virus strain has obvious defects in the assembly process of virus particles, and the capability of packaging replicon RNA is reduced, so that the mutant virus strain is weakened, and the toxicity of the virus to duck embryos and ducklings is obviously reduced; and the mutant virus strain has genetic stability, so that the duck tembusu delta C64-96 mutant virus strain is prepared into the duck tembusu virus attenuated live vaccine immune duckling, and the safety of the vaccine can be effectively improved.

Thirdly), the duck tembusu delta C64-96 mutant virus strain can remarkably activate the immune system of the immunized ducklings, so that the expression levels of a plurality of immune-related cytokines are remarkably increased, and complete virus counteracting protection effect can be provided for the ducklings after single immunization, therefore, the duck tembusu delta C64-96 mutant virus strain is a safe and efficient attenuated live vaccine candidate virus strain.

Drawings

FIG. 1 is a plasmid map of pACNR-FL-CQW 1;

FIG. 2 is a schematic diagram of the construction of plasmid infectious clone pACNR-FL-CQW1- Δ C64-96;

FIG. 3 is a graph of the fluorescent signals of F0 and F1 generations after transfection of BHK21 cells with duck tembusu Δ C64-96 mutant virus strain in example 2;

FIG. 4 is a graph of the growth of the duck tembusu Δ C64-96 mutant virus strain of example 3 on BHK21 cells;

FIG. 5 is a graph showing the results of plaque assays at generations F1 and F10 of the duck Tembusu Δ C64-96 mutant virus strain of example 3;

FIG. 6 is a graph showing the results of the viral packaging test for the duck Tembusu Δ C64-96 mutant viral strain of example 3 (FIG. 6A is a diagram showing a trans-complementation packaging system, FIG. 6B is a diagram showing the effect of Δ C64-96 deletion mutations on viral packaging)

FIG. 7 shows the use of 2000TCID in example 5 ₅₀ Duck tembusu delta C64-96 mutant virus strains for duck embryo virulence test results;

fig. 8 is a graph showing the experimental results of a single injection immunization of the duck tembusu Δ C64-96 mutant virus strain against 5-day-old ducklings in example 6 (fig. 8A is a statistical graph of viremia titer, and fig. 8B is a statistical graph of ducklings death);

FIG. 9 is a graph showing the body weight changes of ducklings in the single post-injection challenge test of the duck Tembusu Δ C64-96 mutant virus strain of example 6 against 5-day-old ducklings;

FIG. 10 is a graph showing the results of the challenge test of the duck Tembusu Δ C64-96 mutant virus strain of example 6 after a single injection immunization of a duckling at the age of 5 days (FIG. 10A is a statistical graph of viremia titer, and FIG. 10B is a statistical graph of duckling death);

FIG. 11 is a graph showing vRNA loading of tissues of ducklings at 3 rd and 5 th days after immunization of 25-day-old ducklings with the duck Tembusu Δ C64-96 mutant virus strain of example 7;

FIG. 12 is a statistical chart showing the number of deaths of ducklings within 15 days after immunization of 25-day-old ducklings with the duck Tembusu Δ C64-96 mutant virus strain in example 7;

FIG. 13 is a graph showing the mRNA expression levels of IFN- α, IFN- β, IL-1 β and TNF- α in spleen tissue on day 5 after immunization of 25-day-old ducklings with the duck Tembusu Δ C64-96 mutant virus strain of example 7;

FIG. 14 is a graph showing data on T lymphocyte specific proliferation in peripheral blood of ducks immunized by the duck tembusu Δ C64-96 mutant virus strain of example 7 against 25-day-old ducklings;

FIG. 15 is a graph showing data on the expression levels of Th1 type cytokine IFN γ and Th2 type cytokine IL-4 in duck serum 14 days after the duck Tembusu Δ C64-96 mutant virus strain of example 7 was immunized against 25-day-old ducklings.

Detailed Description

The invention is further described with reference to the following figures and specific embodiments.

Plasmids, cells and experimental animals: the multiple resistances of the pACNR-FL-CQW1 plasmid vector, the BHK21 cell, the wild type duck Tembusu virus strain DTMUV-CQW1 and the anti-TMUV are all provided by the animal medical college avian disease control center of Sichuan agricultural university; SPF ducklings and SPF duck embryos were purchased from harbourine veterinary institute; the duck tembusu virus strain DTMUV-CHN-YC was gifted by the college of animal medicine of the university of agriculture in Huazhong.

The main kit comprises: clonexpress II One Step Cloning Kit was purchased from Nanjing Novozam; TransIntro EL transformation Reagent purchased from Beijing Quanyu gold organisms; phanta EVO HS Super-Fidelity DNApolymerase was purchased from Nanjing Novozam.

Example 1

1. Construction of duck tembusu virus low virulent strain namely duck tembusu delta C64-96 mutant virus strain

PCR amplification is carried out by taking pACNR-FL-CQW1 plasmid of duck tembusu virus strain DTMUV-CQW1 as a template and SEQ ID NO.1 and SEQ ID NO.2 as primers to obtain a fragment P1; taking the sequences SEQ ID NO.3 and SEQ ID NO.4 as primers, and carrying out PCR amplification to obtain a fragment P2; and then, the fragment P1 and the fragment P2 are used as templates, the sequences SEQ ID NO.1 and SEQ ID NO.4 are used as primers, the fragment P1P2 is obtained by fusion PCR amplification, the fragment P1P2 is a nucleotide sequence of gene deletion mutation, and the gene sequence of the fragment P1P2 deletion is a nucleotide sequence of 64 th-96 th amino acids of the Capsid protein of the coded duck Tembusu virus.

The two-step fusion PCR was performed using the high Fidelity enzyme Phanta EVO HS Super-Fidelity DNA Polymerase, and the reaction system of the PCR is detailed in Table 1:

5×EVOBuffer	10μL
		dNTPMix	1μL
enzyme	1μL
		Primer F	2μL
Primer R	2μL
		Form panel	1μL
ddH 2 O	Make up to 50. mu.L

The PCR amplification condition is pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 15s, annealing at 63 ℃ for 15s, and extension at 72 ℃ for 15s/kb for 30 cycles; finally extending for 7min at 72 ℃. And after each round of amplification is finished, carrying out gel electrophoresis identification, and carrying out liquid recovery or gel recovery. The primers used in the PCR amplification are detailed in Table 2.

TABLE 2PCR amplification primer sequence Listing

2. Construction of recombinant infectious clone plasmid pACNR-FL-CQW 1-delta C64-96

Carrying out double enzyme digestion on the pACNR-FL-CQW1 plasmid by using restriction enzymes MluI and XhoI, and recovering and purifying to obtain a purified linear pACNR-FL-CQW1 vector; the gene deletion mutant fragment P1P2 obtained above was subjected to recombinant ligation reaction with the linear pACNR-FL-CQW1 vector using the Cloneexpress II One Step Cloning Kit as described above, and then transformed into E.coli competent cells, after colonies were grown out, the cells were selected for PCR identification. Then, selecting a colony positive in PCR identification, and sending the colony to sequencing identification, wherein the plasmid confirmed by sequencing is named as pACNR-FL-CQW 1-delta C64-96 plasmid; and selecting a single colony with correct sequencing, performing amplification culture, storing, and extracting a plasmid for later use.

The plasmid map of pACNR-FL-CQW1 is shown in figure 1. Carrying out double enzyme digestion on the pACNR-FL-CQW1 plasmid by using restriction endonucleases MluI and XhoI, cutting a gene sequence of a coding Capsid protein on the pACNR-FL-CQW1 plasmid, and then carrying out recombination connection on a gene deletion mutant fragment P1P2 obtained by the fusion PCR and the double enzyme digested linear pACNR-FL-CQW1 plasmid to obtain a recombinant plasmid pACNR-FL-CQW 1-delta C64-96. A schematic diagram of the construction of pACNR-FL-CQW1- Δ C64-96 plasmid infectious clone is shown in FIG. 2.

Example 2

Transfection and rescue of duck tembusu delta C64-96 mutant virus strain

BHK21 cells in good growth state were inoculated into a 12-well plate and cultured for 16h, and when the cell confluence reached 70-90%, the following operations were performed according to the instructions of Transfection Reagent TransIntro EL Transfection Reagent. Setting a group Δ C64-96 and a group WT; the pACNR-FL-CQW1- Δ C64-96 plasmid prepared in example 1 was transfected into each well of Δ C64-96 group at 1.2 μ g, and the wild-type duck Tembusu virus strain DTMUV-CQW1 plasmid was transfected into each well of WT group at 1.2 μ g.

Cell climbing was harvested on day 5 post-transfection (F0 passage), IFA identification was performed with anti-TMUV polyclonal antibody, and the supernatant was simultaneously harvested to infect new BHK21 cells. Cell crawlers were harvested on the third day of infection (F1 passages) for IFA identification.

As shown in FIG. 3, the Δ C64-96 produced significant fluorescent signals at both F0 and F1, indicating that the duck Tembusu Δ C64-96 mutant strain produced infectious virions and was significantly weaker than the wild-type duck Tembusu strain DTMUV-CQW 1.

Example 3

Verifying weakening characteristics and weakening mechanism of duck tembusu delta C64-96 mutant virus strain

1. Verifying weakening characteristics of duck tembusu delta C64-96 mutant virus strain

The Δ C64-96 and WT groups were set. Group Δ C64-96 the duck Tembusu Δ C64-96 mutant virus strain rescued in example 2 was propagated and titer-determined in BHK21 and then used for determination of growth curves on BHK21 cells. WT group the wild-type duck Tembusu virus strain DTMUV-CQW1 was propagated in BHK21 and titer was determined for use, and its growth curve was determined on BHK21 cells. As shown in figure 4, the proliferation of the duck tembusu delta C64-96 mutant virus strain on BHK21 cells showed marked attenuation compared with wild-type duck tembusu virus DTMUV-CQW1, which is characterized by prolonged time for reaching peak value of titer and reduced peak value of titer.

The duck tembusu delta C64-96 mutant virus strain obtained by rescue in example 2 is continuously passaged on BHK21 cells for 10 times, and F1 generation and F10 generation of the duck tembusu delta C64-96 mutant virus strain are subjected to plaque test and are respectively marked as delta C64-96-F1 and delta C64-96-F10, and wild type duck tembusu virus DTMUV-CQW1 is used as a control and is marked as a WT group. As shown in FIG. 5, the plaque assay results showed that Δ C64-96-F1 and Δ C64-96-F10 formed only pinpoint-sized plaques; and the plaques formed by the deltaC 64-96-F10 after 10 passages have no obvious size difference compared with the F1 passage and are all far smaller than the plaques of the WT group. The results of the plaque test show that the proliferation capacity of the 10 th generation duck tembusu delta C64-96 mutant virus strain is almost unchanged compared with the first generation and is weaker than that of the wild type virus strain.

2. Verification of weakening mechanism of duck tembusu delta C64-96 mutant virus strain

Since the Capsid protein plays a key role in the assembly process of the duck tembusu virus, the Capsid protein and the genomic RNA of the virus jointly form a nucleocapsid, the alpha 4 helical structure of the Capsid protein is considered as a key stable point for combining with the viral genomic RNA, and the duck tembusu Δ C64-96 mutant virus strain just deletes the key region, the packaging experiment is carried out to verify whether the duck tembusu Δ C64-96 mutant virus strain has defects in the assembly process of the virus. As shown in FIG. 6A, a WT-type (or Δ C64-96) C-prM expression plasmid was co-transfected with a C protein-deleted subgenomic replicon plasmid carrying the Nluc luciferase gene into BHK21 cells to generate a single round of infectious virions in trans-complementation, denoted as WT group (or Δ C64-96 group). Supernatants were harvested on the third day post-transfection to infect new BHK21 cells and fluorescence values were determined 24 hours post-infection. The results show that the ability of ac 64-96 groups to package replicon RNA was significantly reduced compared to the WT group (fig. 6B).

In conclusion, the deletion mutation of the duck tembusu Δ C64-96 mutant virus strain causes defects in the assembly process of virus particles, which is the main reason for the weakness of the duck tembusu Δ C64-96 mutant virus strain in vitro. Thus, a defect in virion assembly is the molecular basis for the attenuation of the duck tembusu Δ C64-96 mutant virus strain.

Example 4

Verification of genetic stability of duck tembusu delta C64-96 mutant virus strain

Continuously passaging the duck tembusu delta C64-96 mutant virus strain obtained in the rescue in the example 2 on BHK21 cells for 10 times, and then carrying out whole genome sequencing on the virus; sequencing results show that after 10 passages, the gene deletion mutation of the duck tembusu delta C64-96 mutant virus still stably exists in the genome of the mutant virus, and the fact that the duck tembusu delta C64-96 mutant virus has genetic stability in deletion mutation of C protein from 64 th to 96 th is shown. Meanwhile, other adaptive mutations appear on the genome of the duck tembusu delta C64-96 mutant virus, as shown in Table 3.

TABLE 3 passage stability of Duck Tembusu Δ C64-96 mutant viruses

Example 5

Duck tembusu delta C64-96 mutant virus strain influence test on duck embryo toxicity

A delta C64-96-F1 group, a delta C64-96-F10 group, a CQW1-WT group and a Mock group were set, each group containing 10 duck embryos. After the duck tembusu delta C64-96 mutant virus strain obtained in example 2 was cultured for 10 generations continuously, duck tembusu delta C64-96 mutant virus strain F1 generation and duck tembusu delta C64-96 mutant virus strain F10 generation were obtained. Injecting 2000TCID into duck embryos of delta C64-96-F1 group, delta C64-96-F10 group and CQW1-WT group respectively ₅₀ The duck tembusu delta C64-96 mutant virus strain F1 generation, the duck tembusu delta C64-96 mutant virus strain F10 generation and the wild type duck tembusu virus strain DTMUV-CQW1 virus. Mock groups were injected with an equal dose of DMEM. Continuously observing for 7 days after the virus attack, and recording the death condition of the duck embryo every day. The results of the experiment are shown in FIG. 7, and the CQW1-WT group of duck embryosAll died at 4 days, compared to 70% of the duck embryos died from infection with Δ C64-96-F1, and the duck embryos of Δ C64-96-F10 were further reduced in virulence, with only 10% of the duck embryos died.

The experimental analysis shows that the duck tembusu delta C64-96 mutant virus strain mutation obviously reduces duck embryo virulence of the duck tembusu virus, and the virulence of the cell-adapted duck tembusu delta C64-96 mutant virus strain F10 is further reduced on the basis of F1.

Example 6

Immune protection of duck tembusu delta C64-96 mutant virus strain after immunization of 5-day-old ducklings

5-day-old ducklings were randomly divided into 3 groups, designated as CQW1 group, Δ C64-96 group and DMEM group, and the CQW1 group was inoculated with 200 μ L (10 μ L) by intramuscular injection ⁵ TCID ₅₀ ) The wild type duck tembusu virus DTMUV-CQW 1; 200 μ L (10) of the AC 64-96 group ⁵ TCID ₅₀ ) Immunizing duck tembusu delta C64-96 mutant virus; the DMEM group was injected with an equal dose of DMEM. Detecting the weight change, clinical symptoms and death conditions of 3 groups of ducklings 1-14 days after immunization, and determining the level of viremia on 3 days; on day 14 post-immunization, all surviving ducklings of 3 groups were challenged by intramuscular inoculation of 200 μ L (2 × 10) ⁶ TCID ₅₀ ) The duck tembusu virus strain DTMUV-CHN-YC, the weight change, clinical symptoms and death condition of each group of ducklings are monitored within 14 days after challenge, and the level of viremia is determined on the 3 rd day after challenge.

At day 3 post-immunization, we measured viremia in 3 groups of ducklings. As shown in FIG. 8A, the CQW1 group produced high levels of viremia with a virus titer of 10 ⁴ TCID ₅₀ Viremia was not detected in the 100. mu.L,. DELTA.C 64-96 and DMEM groups. From 3 days after immunization, the ducklings of the CQW1 group have clinical symptoms of mental depression, slow movement, unstable standing, even hind limb paralysis and the like; and death occurred from day 5, and by day 7, 60% of the ducklings died from infection with the wild-type duck tembusu virus DTMUV-CQW1 (fig. 8B). Whereas the ac 64-96 and DMEM groups did not show any significant clinical symptoms and death (fig. 8B). As shown in FIG. 9, on day 4 post immunizationInitially, the CQW1 group showed significantly lower levels of body weight gain than the DMEM group, while the ac 64-96 group was not significantly different from the DMEM group. These data indicate that the duck tembusu Δ C64-96 mutant virus has significantly reduced virulence for ducklings.

As shown in figure 9, the weight of DMEM was significantly lower than Δ C64-96 the next day after challenge, and clinical symptoms such as mental depression, bradykinesia, unstable stance and even hind limb paralysis began to appear. Viremia status at day 3 after challenge was detected and found to be non-viremic in neither the Δ C64-96 cohort nor the CQW1 cohort, except for the DMEM cohort, which produced significant viremia (FIG. 10A). As shown in fig. 10B, on days 5 and 7 after challenge, 1 duckling died in DMEM group, respectively; while the AC 64-96 and CQW1 groups did not show any mortality.

The data show that single intramuscular injection of the tembusu delta C64-96 mutant virus strain for the immune ducks can provide effective immune protection effect for the ducklings.

Example 7

Mechanism for inducing immune response in 25-day-old ducklings by duck tembusu delta C64-96 mutant virus strain

In order to further verify that the duck tembusu Δ C64-96 mutant virus strain provides immune protection for ducklings and induces an immune response mechanism in the ducklings, in the embodiment, 25-day-old ducklings are randomly divided into 3 groups, namely a CQW1 group, a DMEM group and a Δ C64-96 group, wherein each group comprises 11 ducklings. The CQW1 group is prepared by intramuscular injection of 200 mu L of wild type duck Tembusu DTMUV-CQW1 virus, the delta C64-96 group is prepared by intramuscular injection of 200 mu L of duck Tembusu delta C64-96 mutant virus, and the DMEM group is prepared by intramuscular injection of equivalent doses of DMEM. On the 3 rd and 5 th days after immunization, 3 ducklings were randomly dissected and killed in each group, and heart, liver, spleen, lung, kidney, brain and thymus tissue samples of the ducklings were taken. The remaining 5 of each group were observed continuously for 14 days and the death was recorded. On day 14 after immunization, the cellular immunity was determined.

As shown in fig. 11, on the third day after immunization, the CQW1 group detected high titers of vRNA load in each organ and further increased on day 5 after infection. In contrast, vRNA loading was consistently maintained at a minimum level in each of the organs of the ac 64-96 groups. As shown in fig. 12, one duckling in CQW1 group showed overt clinical symptoms and died on day 5 post-infection; in the Δ C64-96 group, none of the ducklings showed clinical symptoms and died.

To analyze the activation of duck tembusu Δ C64-96 mutant virus infection on innate immunity, the mRNA expression of IFN- α, IFN- β, IL-1 β and TNF- α in spleen tissue at day 5 after infection was further examined by RT-qPCR. As shown in FIG. 13, both CQW1 and Δ C64-96 groups activated upregulation of IFN- α, IFN- β, IL-1 β and TNF- α compared to DMEM.

In order to verify whether duck tembusu Δ C64-96 mutant virus infection can activate duck cell immunity, T lymphocyte specific proliferation test was performed on T lymphocytes isolated from duck peripheral blood in this example. As shown in FIG. 14, the stimulation coefficient of the CQW1 group reached 6.2 compared to the DMEM group, whereas the Δ C64-96 group was 4.8. This example further measured the expression levels of the Th1 type cytokine IFN γ and the Th2 type cytokine IL-4 in duck serum after 14 days post immunization by ELISA. As shown in FIG. 15, the expression levels of Th1 type cytokine IFN gamma and Th2 type cytokine IL-4 of Δ C64-96 were significantly up-regulated relative to the DMEM group. The data show that the duck tembusu delta C64-96 mutant virus infection can effectively activate cellular immunity.

In conclusion, the duck tembusu delta C64-96 mutant virus strain is obtained by gene deletion mutation, and a nucleotide sequence for coding amino acids 64 to 96 of a duck tembusu virus Capsid protein is deleted, so that amino acids 64 to 96 of the Capsid protein are deleted. The weakening mechanism of the virus strain is verified in the embodiment 3 of the invention, and the virus strain has defects in the assembly process of virus particles, namely after the nucleotide sequence of 64 th-96 th amino acids of the Capsid protein of duck Tembusu virus is deleted, the capability of packaging replicon RNA is obviously reduced, so that the mutant virus is weakened, and the toxicity of the virus on duck embryos and ducklings is obviously reduced. Example 4 demonstrates the genetic stability of the gene mutations of the duck tembusu Δ C64-96 mutant virus of the present invention. Example 6 and example 7 show that a single immunization of duckling with the duck tembusu delta C64-96 mutant virus can significantly activate the up-regulation of the expression levels of IFN-alpha, IFN-beta, IL-1 beta, TNF-alpha, Th1 type cytokine IFN gamma and Th2 type cytokine IL-4 of infected duckling, and provide effective immune protection for duckling.

The analysis shows that the duck tembusu delta C64-96 mutant virus strain has obvious weakening effect, clearly analyzed weakening mechanism and obvious genetic stability, and thus, the use safety of the duck tembusu delta C64-96 mutant virus strain can be effectively improved. When the virus strain is used for immunizing the ducklings, the immune system of the immunized ducklings can be effectively activated, the expression levels of multiple immune-related cytokines are obviously up-regulated, and efficient immune protection is provided for the ducklings. Therefore, the duck tembusu delta C64-96 mutant virus strain is a safe and efficient attenuated live vaccine candidate virus strain, and the duck tembusu virus attenuated live vaccine prepared from the duck tembusu virus candidate virus strain can be widely applied to prevention of duck tembusu related diseases.

The present specification and figures are to be regarded as illustrative rather than restrictive, and it is intended that all such alterations and modifications that fall within the true spirit and scope of the invention, and that all such modifications and variations are included within the scope of the invention as determined by the appended claims without the use of inventive faculty.

Sequence listing

<110> Sichuan university of agriculture

<120> duck tembusu virus attenuated live vaccine candidate strain and preparation method and application thereof

<141> 2021-05-17

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gtatcataca catacgacgc gttggagttc cgcgttacat aactt 45

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ggttggcctc agggctgtaa 20

<210> 3

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

acagccctga ggccaaccaa taagcggaag gcgaaacg 38

<210> 4

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tgtaaccttg atacttacct cgagcctgct gactgatcg 39

<210> 5

<211> 839

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gtatcataca catacgacgc gttggagttc cgcgttacat aacttacggt aaatggcccg 60

cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata 120

gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 180

cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac 240

ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg 300

cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc 360

aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc 420

aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc 480

gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct 540

ggtttagtga accgtagaag ttcatctgtg tgaacttatt ccaaacagtt ttttgggata 600

gtgcgtgtga acgtaaacac agtttgaacg ttttttggat agagacaact atgtctaaca 660

aaaaaccagg aagacccggc tcaggccggg ttgtcaatat gctaaagcgc ggaacgtccc 720

gcggaaatcc gctagcgcgg ataaagagga cgattgatgg ggtcctgaga ggagcaggac 780

ccataaggtt tgtgctggct ctactgactt tcttcaagtt tacagccctg aggccaacc 839

<210> 6

<211> 2306

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ttacagccct gaggccaacc aataagcgga aggcgaaacg tcgggggggg agttgctctt 60

gggtcatcat gttactcccg atagttgctg ggctgaaact tggaaattat aatggtagag 120

ttttggccac tttaaacaag actgatgtgt cagacttgct agtcattcca acaacggctg 180

gcagcaatgg atgcgtcgtg cgagctctag atgtgggact gatgtgtcag gatgacataa 240

cgtacctgtg cccaaagttg gagtacggct atgaacctga agacatagac tgctggtgca 300

atgagactga gatatacatt cattatggga gatgtacccc ttcacggcat ggacggaggt 360

ctaggaggtc ggtgaacgtg catcaccatg gagagagtct acttgaggcc aagaacacgc 420

cgtggatgga ttcgaccaaa gccactaaat atctcacaaa ggttgagaac tgggcgttga 480

gaaatcctgg gtacgccctt gctgccatct tcataggctg gaacttggga acgacgagaa 540

gccagaagat aattttcaca attatgttaa tgttaattgc cccagcgtac agcttcagct 600

gtctggggat gcagaaccga gactttgttg agggagtgaa tggtgttgag tggatcgatg 660

tcgttctgga aggaggctca tgcgtaacta ttacggcaaa agacaggccg accatagacg 720

tcaagatgat gaacatggag gctacggaat tagcggttgt gagatcttac tgctatgagc 780

cgaaagtgtc ggacgtgacg acagaatcca gatgcccaac catgggagag gctcataatc 840

ccaaggcaac ttatgctgaa tacatatgca aaaaagattt tgtggacagg ggttggggca 900

atggctgtgg cttgtttgga aaggggagca tccagacatg tgccaagttt gactgcacaa 960

agaaagcaga aggcaggatc gtgcagaagg aaaacgtcca gtttgaagtt gcagttttta 1020

tacatggttc cacggaagcg agcacctacc acaattattc agcccagcag tcgctgaaac 1080

atgccgctag attcgtgata acgcccaaaa gtcccgtcta cactgctgag atggaggatt 1140

atggtaccgt cacactcgaa tgcgaacccc gatctggggt tgacatgggg caattctacg 1200

tcttcaccat gaatacaaag agctggcttg ttaacagaga ctggtttcat gacctcaact 1260

taccatggac agggtcatca gcggggacgt ggcaaaacaa agagtcattg atagaatttg 1320

aggaggctca tgccaccaaa caatcagtgg tggctttggc atcacaagaa ggagccctcc 1380

atgcagcatt ggcgggagct attccagtga agtactctgg aaacaaattg gaaatgacct 1440

caggtcatct taaatgcagg gtcaaaatgc agggtttgaa gctgaaagga atgacctacc 1500

cgatgtgtag caatacattt tccctagtga agaatcctac cgacactggg catggcactg 1560

tcgtggtgga attgtcttat gcaggtaccg atgggccctg tagagttccc atatccatgt 1620

cggcagattt gaatgacatg acaccagttg gacgcttgat aacagtcaac ccatacgtgt 1680

cgacttcctc cacgggtgcc aagataatgg tggaagtgga acctccattc ggggattcat 1740

ttattttagt aggaagtgga aaaggacaga ttaggtacca gtggcataga agtgggagta 1800

caattggaaa agctttcacg tcaacactca aaggagcaca aaggatggtt gctttgggtg 1860

acactgcatg ggattttggt tcagttgggg gtgtactcac ttccattggg aaaggcattc 1920

atcaagtctt cggctcagca tttaaaagct tatttggagg aatgtcatgg attactcaag 1980

gcatgttagg ggcactgcta ttgtggatgg gcctgaatgc aagggacaga tccatttcta 2040

tgacctttct agtcgtagga ggaattttag tcttcttggc agtaaatgtc aatgccgaca 2100

cggggtgctc aatcgacttg gctaggaaag aattaaaatg tggacaaggc atgtttgtct 2160

tcaacgatgt tgaggcctgg aaggataatt acaagtacta tccatccaca ccaaggagac 2220

ttgccaaagt cgtggcaaaa gctcatgagg ctggaatttg tggcatacga tcagtcagca 2280

ggctcgaggt aagtatcaag gttaca 2306

<210> 7

<211> 3125

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gtatcataca catacgacgc gttggagttc cgcgttacat aacttacggt aaatggcccg 60

cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata 120

gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 180

cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac 240

ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg 300

cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc 360

aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc 420

aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc 480

gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct 540

ggtttagtga accgtagaag ttcatctgtg tgaacttatt ccaaacagtt ttttgggata 600

gtgcgtgtga acgtaaacac agtttgaacg ttttttggat agagacaact atgtctaaca 660

aaaaaccagg aagacccggc tcaggccggg ttgtcaatat gctaaagcgc ggaacgtccc 720

gcggaaatcc gctagcgcgg ataaagagga cgattgatgg ggtcctgaga ggagcaggac 780

ccataaggtt tgtgctggct ctactgactt tcttcaagtt tacagccctg aggccaacca 840

ataagcggaa ggcgaaacgt cgggggggga gttgctcttg ggtcatcatg ttactcccga 900

tagttgctgg gctgaaactt ggaaattata atggtagagt tttggccact ttaaacaaga 960

ctgatgtgtc agacttgcta gtcattccaa caacggctgg cagcaatgga tgcgtcgtgc 1020

gagctctaga tgtgggactg atgtgtcagg atgacataac gtacctgtgc ccaaagttgg 1080

agtacggcta tgaacctgaa gacatagact gctggtgcaa tgagactgag atatacattc 1140

attatgggag atgtacccct tcacggcatg gacggaggtc taggaggtcg gtgaacgtgc 1200

atcaccatgg agagagtcta cttgaggcca agaacacgcc gtggatggat tcgaccaaag 1260

ccactaaata tctcacaaag gttgagaact gggcgttgag aaatcctggg tacgcccttg 1320

ctgccatctt cataggctgg aacttgggaa cgacgagaag ccagaagata attttcacaa 1380

ttatgttaat gttaattgcc ccagcgtaca gcttcagctg tctggggatg cagaaccgag 1440

actttgttga gggagtgaat ggtgttgagt ggatcgatgt cgttctggaa ggaggctcat 1500

gcgtaactat tacggcaaaa gacaggccga ccatagacgt caagatgatg aacatggagg 1560

ctacggaatt agcggttgtg agatcttact gctatgagcc gaaagtgtcg gacgtgacga 1620

cagaatccag atgcccaacc atgggagagg ctcataatcc caaggcaact tatgctgaat 1680

acatatgcaa aaaagatttt gtggacaggg gttggggcaa tggctgtggc ttgtttggaa 1740

aggggagcat ccagacatgt gccaagtttg actgcacaaa gaaagcagaa ggcaggatcg 1800

tgcagaagga aaacgtccag tttgaagttg cagtttttat acatggttcc acggaagcga 1860

gcacctacca caattattca gcccagcagt cgctgaaaca tgccgctaga ttcgtgataa 1920

cgcccaaaag tcccgtctac actgctgaga tggaggatta tggtaccgtc acactcgaat 1980

gcgaaccccg atctggggtt gacatggggc aattctacgt cttcaccatg aatacaaaga 2040

gctggcttgt taacagagac tggtttcatg acctcaactt accatggaca gggtcatcag 2100

cggggacgtg gcaaaacaaa gagtcattga tagaatttga ggaggctcat gccaccaaac 2160

aatcagtggt ggctttggca tcacaagaag gagccctcca tgcagcattg gcgggagcta 2220

ttccagtgaa gtactctgga aacaaattgg aaatgacctc aggtcatctt aaatgcaggg 2280

tcaaaatgca gggtttgaag ctgaaaggaa tgacctaccc gatgtgtagc aatacatttt 2340

ccctagtgaa gaatcctacc gacactgggc atggcactgt cgtggtggaa ttgtcttatg 2400

caggtaccga tgggccctgt agagttccca tatccatgtc ggcagatttg aatgacatga 2460

caccagttgg acgcttgata acagtcaacc catacgtgtc gacttcctcc acgggtgcca 2520

agataatggt ggaagtggaa cctccattcg gggattcatt tattttagta ggaagtggaa 2580

aaggacagat taggtaccag tggcatagaa gtgggagtac aattggaaaa gctttcacgt 2640

caacactcaa aggagcacaa aggatggttg ctttgggtga cactgcatgg gattttggtt 2700

cagttggggg tgtactcact tccattggga aaggcattca tcaagtcttc ggctcagcat 2760

ttaaaagctt atttggagga atgtcatgga ttactcaagg catgttaggg gcactgctat 2820

tgtggatggg cctgaatgca agggacagat ccatttctat gacctttcta gtcgtaggag 2880

gaattttagt cttcttggca gtaaatgtca atgccgacac ggggtgctca atcgacttgg 2940

ctaggaaaga attaaaatgt ggacaaggca tgtttgtctt caacgatgtt gaggcctgga 3000

aggataatta caagtactat ccatccacac caaggagact tgccaaagtc gtggcaaaag 3060

ctcatgaggc tggaatttgt ggcatacgat cagtcagcag gctcgaggta agtatcaagg 3120

ttaca 3125

<210> 8

<211> 14327

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gtcgacgcgg ccgcgctagc gatgaccctg ctgattggtt cgctgaccat ttccgggtgc 60

gggacggcgt taccagaaac tcagaaggtt cgtccaacca aaccgactct gacggcagtt 120

tacgagagag atgatagggt ctgcatcagt aagccagatg ctacacaatt aggcttgtac 180

atattgtcgt tagaacgcgg ctacaattaa tacataacct tatgtatcat acacatacga 240

cgcgttggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 300

cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 360

tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 420

agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 480

gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 540

agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 600

gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 660

gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat 720

gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtag 780

aagttcatct gtgtgaactt attccaaaca gttttttggg atagtgcgtg tgaacgtaaa 840

cacagtttga acgttttttg gatagagaca actatgtcta acaaaaaacc aggaagaccc 900

ggctcaggcc gggttgtcaa tatgctaaag cgcggaacgt cccgcggaaa tccgctagcg 960

cggataaaga ggacgattga tggggtcctg agaggagcag gacccataag gtttgtgctg 1020

gctctactga ctttcttcaa gtttacagcc ctgaggccaa ccaataagcg gaaggcgaaa 1080

cgtcgggggg ggagttgctc ttgggtcatc atgttactcc cgatagttgc tgggctgaaa 1140

cttggaaatt ataatggtag agttttggcc actttaaaca agactgatgt gtcagacttg 1200

ctagtcattc caacaacggc tggcagcaat ggatgcgtcg tgcgagctct agatgtggga 1260

ctgatgtgtc aggatgacat aacgtacctg tgcccaaagt tggagtacgg ctatgaacct 1320

gaagacatag actgctggtg caatgagact gagatataca ttcattatgg gagatgtacc 1380

ccttcacggc atggacggag gtctaggagg tcggtgaacg tgcatcacca tggagagagt 1440

ctacttgagg ccaagaacac gccgtggatg gattcgacca aagccactaa atatctcaca 1500

aaggttgaga actgggcgtt gagaaatcct gggtacgccc ttgctgccat cttcataggc 1560

tggaacttgg gaacgacgag aagccagaag ataattttca caattatgtt aatgttaatt 1620

gccccagcgt acagcttcag ctgtctgggg atgcagaacc gagactttgt tgagggagtg 1680

aatggtgttg agtggatcga tgtcgttctg gaaggaggct catgcgtaac tattacggca 1740

aaagacaggc cgaccataga cgtcaagatg atgaacatgg aggctacgga attagcggtt 1800

gtgagatctt actgctatga gccgaaagtg tcggacgtga cgacagaatc cagatgccca 1860

accatgggag aggctcataa tcccaaggca acttatgctg aatacatatg caaaaaagat 1920

tttgtggaca ggggttgggg caatggctgt ggcttgtttg gaaaggggag catccagaca 1980

tgtgccaagt ttgactgcac aaagaaagca gaaggcagga tcgtgcagaa ggaaaacgtc 2040

cagtttgaag ttgcagtttt tatacatggt tccacggaag cgagcaccta ccacaattat 2100

tcagcccagc agtcgctgaa acatgccgct agattcgtga taacgcccaa aagtcccgtc 2160

tacactgctg agatggagga ttatggtacc gtcacactcg aatgcgaacc ccgatctggg 2220

gttgacatgg ggcaattcta cgtcttcacc atgaatacaa agagctggct tgttaacaga 2280

gactggtttc atgacctcaa cttaccatgg acagggtcat cagcggggac gtggcaaaac 2340

aaagagtcat tgatagaatt tgaggaggct catgccacca aacaatcagt ggtggctttg 2400

gcatcacaag aaggagccct ccatgcagca ttggcgggag ctattccagt gaagtactct 2460

ggaaacaaat tggaaatgac ctcaggtcat cttaaatgca gggtcaaaat gcagggtttg 2520

aagctgaaag gaatgaccta cccgatgtgt agcaatacat tttccctagt gaagaatcct 2580

accgacactg ggcatggcac tgtcgtggtg gaattgtctt atgcaggtac cgatgggccc 2640

tgtagagttc ccatatccat gtcggcagat ttgaatgaca tgacaccagt tggacgcttg 2700

ataacagtca acccatacgt gtcgacttcc tccacgggtg ccaagataat ggtggaagtg 2760

gaacctccat tcggggattc atttatttta gtaggaagtg gaaaaggaca gattaggtac 2820

cagtggcata gaagtgggag tacaattgga aaagctttca cgtcaacact caaaggagca 2880

caaaggatgg ttgctttggg tgacactgca tgggattttg gttcagttgg gggtgtactc 2940

acttccattg ggaaaggcat tcatcaagtc ttcggctcag catttaaaag cttatttgga 3000

ggaatgtcat ggattactca aggcatgtta ggggcactgc tattgtggat gggcctgaat 3060

gcaagggaca gatccatttc tatgaccttt ctagtcgtag gaggaatttt agtcttcttg 3120

gcagtaaatg tcaatgccga cacggggtgc tcaatcgact tggctaggaa agaattaaaa 3180

tgtggacaag gcatgtttgt cttcaacgat gttgaggcct ggaaggataa ttacaagtac 3240

tatccatcca caccaaggag acttgccaaa gtcgtggcaa aagctcatga ggctggaatt 3300

tgtggcatac gatcagtcag caggctcgag gtaagtatca aggttacaag gcaggtttaa 3360

ggagaccaat agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca 3420

cctattggtc ttactgacat ccactttgcc tttctctcca cagcacaata tgtgggtaag 3480

catcaaacat gagttgaatg cgatcttgga agacaatgcc attgatttga ctgtggtggt 3540

tgaagaaaat cctggaagat acaggaaaac caatcagagg ctgccgaacg ttgatggaga 3600

gctcacgtac ggatggaaga aatgggggaa aagtattttt agcagcccga agatgtcaaa 3660

taatacattt gtcattgatg gaccaaaaac tagagagtgc ccagatgaga gaagagcatg 3720

gaatagcatg aaaattgaag actttgggtt tggagtgttg tccacaaagg tatggatgga 3780

aatgcgaaca gaaaatacaa ctgattgtga caccgcagta atgggcacag caattaaagg 3840

aaatagagct gtgcacagtg acctgagcta ttggatagag agcaagaata atggaagctg 3900

gaaactggag agggctgtgt tgggcgaggt gaagtcatgc acatggccag aaacccacac 3960

cctgtggagt gacagcgttg tggagagtga actcatcata cctaagacat tgggaggacc 4020

gaagagtcat cacaacacga ggacaggata cagggttcag agttccggac cgtgggatga 4080

gaaagagatt gcaatagact tcgactactg tcctggaaca actgtcacag taacgagctc 4140

gtgccgcgac agagggcctt cagctaggac aacaacagcg agtgggaaat tgataacaga 4200

ttggtgttgt aggtcttgca ccatcccacc actgagattt gttacaaaaa gtggatgctg 4260

gtatgggatg gaaattcggc caattgttca cggagacgac atgttgatca aatcaaaggt 4320

catggctttt caagggggtg gcatggaacc tatgcaatta gggatgctcg ttatgattgt 4380

agcagcccag gagattttga gaaggcgcat gacggctcca attgcttggt cagcgctgct 4440

gttgctgatg gctttggtcc tgtttggagg aatcacgtac agtgatctgg tcaagtacgt 4500

catcctagtg gcagctgcat ttgctgagag caatacaggt ggtgacattg tgcacttggc 4560

catggtggct gcttttaaca ttcagccagg tttactgatt ggatttttac tgaggaggaa 4620

gtggagcaat caggaaagca gattgcttgg cgttgcgtta gcactcataa cagtggcgat 4680

gagagacttg aacatgagta taccaacatt actaaactcc ggagccatgg cctggctctt 4740

gctgagagcc gtgtttgaag ggacggttag ctcctttgcc ctgccgcttg ttagcttgct 4800

ggctccagga ctcagaatag tggggataga tgtggtgagg ataggtgtgc taaccctggg 4860

gatcctctca ctattgaaag agaggagcaa cgcaatggca aaaaagaagg gaggcatgct 4920

cctgggagtg gcatgcgcta ccgctggaat cgctagccct ttggtgtttg ctggtctgca 4980

catggtgctg aagccagtga cacggagagg gtggccagtc agtgaggctt tgactgctgt 5040

gggattgaca ttcgcgttgg caggaggaat agcccagttt gatgacagca gcatggcgat 5100

tccattagcc gttggcggga tcatgctggt ggtggcagtg gtgacaggct tctctacaga 5160

cttatggcta gagaaagcga gcgacatctc gtggagtgag gaggcgaggg tgactggagc 5220

atcacagaga tttgatgtgg aaattgatca ggacggcaat atgagattgc tgaacgatcc 5280

tggtgtgtcg ctcggcgttt gggcctttcg aactgggctt attctgctat cttcatacaa 5340

cccatatttc ctgccattga ctctggcagg ttactggatg acaactaagg caaaacaacg 5400

aggaggagtc atctgggatg tgccagctcc aaaggaaagg aagagagccg aagtaggcaa 5460

tggagttttc cgaattatgg caagaggact gttaggaaaa taccaggctg gggtgggagt 5520

catgcatgag ggagtgtttc acaccatgtg gcacgtgacg aacggggccg ttatccaagc 5580

aggagaagga acactggtcc catattgggc gagtgtacgc aatgatctga tttcctatgg 5640

tggaccatgg aaattgggga agcaatggaa tggtgtagat gaagtgcaag tcatcgtcgt 5700

gcaaccaggc aaagaggtca taaacgtgca gactcagcca ggaattttca agactcaata 5760

tggtgaagtt ggagctgtgt ccctcgatta cccaacggga acctctggat cacctattat 5820

tgacaaggaa ggacaggtgg ttggcctcta tggtaatgga attctggtgg gttcaggcga 5880

ttttgtcagc atgattactc aaggggagaa gaaggaggaa gaagttcctc aggtgtttga 5940

cgaaaacatg ctgcggaaaa ggcaactgac agttctggac ctacatccag gttcaggaaa 6000

gaccagaaag gtcctcccca tgattctgaa gagcgccatt gacaaacgat taagaacagc 6060

tgtcttggct ccgacgcggg tggtggccgc tgaaatagcg gaagcactga aaggactccc 6120

aatacggtat ctgactccgg cagtaaagag ggagcatact ggaacagaga taatagatgt 6180

gatgtgtcac gcgactttga cagcgcggct gctcacacct cagcgagtgc cgaattacaa 6240

cctgttcatt atggatgagg ctcacttcac agaccctgcc agcattgctg ccagaggata 6300

catatcaaca aaggtggaac tgggagaggc agctgcaata ttcatgacag ccacacctcc 6360

aggtacaact gaggcatttc cggactccaa ctcgccaata acagacattg aagagcaaat 6420

ccctgacaga gcttggaatt ctgggtatga gtggataaca gactttcaag gaaagactgt 6480

atggtttgtc cccagcgtga agtctggtaa tgagatcgcc gtgtgcttga caaaggccgg 6540

taagaaggta attcagttaa ataggaagag ttttgactca gagtatccta agtgcaagag 6600

tggagaatgg gatttcgtga taaccactga catctcagaa atgggagcga actttggagc 6660

gcaacgggtc atagatagtc ggaagtgcat taaaccagtg attattgagg atggagaagg 6720

aagtgtgcaa atgaatggac cagttccaat aacatcagcc agtgcagccc agcgtcgtgg 6780

acgggttgga agggatgtga cacaaattgg agatgagtac cactactcag gaccaaccag 6840

cgaggatgat catgatttcg ctcattggaa agaggccaag atactgctgg acaacattaa 6900

catgccagat gggctggttg cccagttgta cggcccagag cgggacaagg ttgacgcaat 6960

tgatggggaa ttcagactga ggactgagca gaggaaacac tttgtggagt atctgaggac 7020

aggagacctc cctgtctgga tatcgtacaa ggtcgctgaa gctgggataa gttacaatga 7080

ccggcggtgg tgctttgatg gaccctcatg caatactgtt ctggaggaca ataacccagt 7140

ggagttatgg acaaagtcag gtgagaagaa aatcttgaag ccccggtgga gagatggaag 7200

attgtgggca gatcaccagg ccttaaaagc cttcaaggat tttgcgagtg gaaagagatc 7260

agcgataggg atccttgagg tcttcaggat gcttcccgat cacttcgctc acagaatgac 7320

agaatccatg gacaacatat acatgctgac tacagctgag aaagggagta gggcccacag 7380

agaagccctg gaggaactgc ctgagacact tgaaacattt ttactggtgt tcatgatgac 7440

agtcgcctct atgggggtgt tcttgttctt tgttcagagg agaggtttag ggaagacagg 7500

tcttggagcc atggtcatgg ccacagtcac ggttttgtta tggatagcag aagtcccagc 7560

ccagaagatt gccggtgtgc tcctagtttc tctattgctg atgattgttc tgatcccaga 7620

accagagaga cagagatcac agacggatag tcacttggct gttttcatga ttgttgtctt 7680

gttagtggtg ggtgctgtgg cgtcaaatga aatgggttgg ctagagcaaa caaagaagga 7740

cttgtcagct ctgtttggga gaaaaagcga aagccatcaa gaaacctgga gtatgccttg 7800

gccggatttg agaccagcga cggcatgggc ggcctacgca ggagctacaa catttctgac 7860

tcccttgcta aaacacctca taataacaga gtatgtgaat ttttcactca tggcaatgac 7920

ggcgcaggct ggagcactat ttggactagg gaaaggcatg ccttttgtca aagcagactt 7980

gtcagtaccc ctgctactct tagggtgttg gggacagttc acaatgacaa caacggtctc 8040

ggcagtcatg atggtcatac tgcattatgc atttttggtg ccaggttggc aagcagaagc 8100

catgaggtcg gcccagagga gaactgctgc aggtgtgatg aaaaatcccg tggttgatgg 8160

catagtggct acagatgttc cagaccttga ggccagcact cctattacag aaaagaaatt 8220

gggtcaatgc gtgctagtgg gaatagcctt ggtggcggtg tttctaacac caaacacgct 8280

aactttgact gagtttggaa tgttgacctc tgccgcttcg gtgacattaa ttgagggagc 8340

tgcaggtcgt atttggaacg caaccacagc cgttgctatg tgccatctgt tgaggaaaaa 8400

ctggttggct ggggcctctc tagcatggac tataactcgg aatctccagg cagggacctt 8460

gcgtcgagga ggaggaactg gcagaacttt gggggaagca tggaaggccc agcttaacca 8520

actgacccgg caagagttta tggaataccg gaaagacggg attattgaag tagatagagc 8580

tgctgcaaaa agagcccgcc gtgaaggaaa tgtgacagga gggcacccag tttcacgagg 8640

cacggcaaag ttgaggtggc tcgtggagcg tgggtttctc aaaccaagag gcaaagttgt 8700

ggatttaggc tgcggcagag gaggctggag ttactactgt gctacattaa agcaggttca 8760

ggaagtgaga ggttacacaa aaggagggcc agggcatgag gaaccagtga tgacccagag 8820

ctatggctgg aacattgtga cgttaaagag tggggttaat gttcatttca agccgactga 8880

accatctgac acactgctat gtgacatagg tgaagcttca cccgtcccag aaattgaatc 8940

tgccagaaca atcagggtgc tgcaaatggc cgaggaatgg ttagctaggg gcgttgaaga 9000

gttctgcata aaagtgcttt gtccctacat gccagcggtc ataaaagaac tggaaagact 9060

gcagctgaaa tggggaggtg gtttggtcag agtgccactc tcgcgtaatt caacgcatga 9120

gatgtactgg gtgagcggct caagtgggaa tgtgacaaat agtattaata cagtgagcca 9180

aatgctgatc aacaggatgc acaaaaccaa ccgtaatgga cccaggtatg aagaagatgt 9240

ggacttgggt tcagggacca gagctgtgag ctgcacaaga cagaggactg actggggaat 9300

ggtcgctgat agggtgaaga atttggccag agaatatgct ccgtcttggc attatgacca 9360

agacaatcct tacaagactt ggaactatca tggaagttac gaagtgaaag ccacaggctc 9420

agccagctca atggttaatg gggtagttag gatactgtca aaaccttggg acaccttgca 9480

aaacgtggtg aatatggcca tgacggacac tactcctttt gggcaacagc gcgtatttaa 9540

agaaaaggtt gataccaaag ccccagaacc acctgcagga acagctaggg ttatgaacat 9600

cgtggcaaga tggatgtgga actttgttgg caggaacaaa caaccaagga tgtgcacaaa 9660

agaagagttc atagagaagg tgaatagtaa cgcagccctg ggggccatgt ttgaggagca 9720

acacaaatgg gccagcgcca gggaagcggt tgaggatcct gaattttgga gtcttgttga 9780

cagagagaga gaactgcact tgcaagggaa gtgcgagacc tgcatttaca acatgatggg 9840

aaagcgagaa aagaagatgg gagagttcgg gaaagcaaaa ggtagcagag ctatttggta 9900

catgtggctc ggggccagat tcctagagtt cgaagccttg ggcttcttga acgaggatca 9960

ctggatgagc agggaaaaca ctaaaggagg cgttgaagga cttggactcc aaaagttggg 10020

gtatgtgctg cgtgacattt cggccaaaga aggaggactt atgtacgcag acgacacggc 10080

cggatgggac actagaataa ccaaggctga tttggaaaac gaagccatca tcttggaaaa 10140

gatggaacca atgcacagag ctgttgcaga accactcatt aaatttgcct acatgaataa 10200

ggtggtgaag gtgatgcgac cgggacgtga tgggaagaca gttatggatg tcatctcgcg 10260

ggaagaccag aggggaagtg gacaggttgt gacctatgct ctcaacactt tcacgaacct 10320

gtgtgtccag ctcattagat gtatggaagg ggaggagctg ctgctccccg aggaaacaga 10380

gcgtctaaaa aaaggaaagg agaagcgcat ccaagaatgg ctccaaaaga atggagagaa 10440

caggttgtca gccatggcag tcagtgggga tgactgtgtg gtgaaaccag cggatgacag 10500

attcgccaca gcactgcact tcctcaatag tatgtctaag gtgaggaaag atactcagga 10560

atggaagccc tcaaccggtt ggagaaactg gcaagaagtc cccttttgct cacaccattt 10620

ccacgagctg caaatgaaag atggcagaaa gattgtggtt ccatgtcgag accaggatga 10680

gctaattgga agagccaggc tctctccagg gtctggctgg tcactaacag aaacagcatg 10740

cctgagcaaa gcatatgctc agatgtggtt attgatgtac ttccacagga gggacctcag 10800

actaatggca aacgccatct gctcatctgt ccctgtctca tgggtcccca caggaaggac 10860

aacgtggtca atccatggaa aaggcgagtg gatgacttct gaagacatgc tggcagtgtg 10920

gaacagggtg tggattgaag aaaatgaaca catggaagac aaaaccccag tgacttcatg 10980

gaacgaagtg ccataccttg gaaagaggga agatggctgg tgtggtagtc tgattggaca 11040

ccgagccaga tctacctggg ccgagaacat atacactcca attatgcaga tcagagctct 11100

cattggccct gagcactatg tagattatat gccaactcta aataggttca aacccattga 11160

aagctggagt gaaggtgttt tgtaaatata tgaggtaggt gtaaaaatgt atgtaaagta 11220

gtgttagtct agagtagata aatatataaa ttagcatttg tttgaataga taggaagagg 11280

aagtcaggcc agggaatccc tgccaccgga tgttggatga cggtgctgtc tgcgttccaa 11340

ccccaggagg actgggttaa caaatctggg tgcatggagg agctaagcgt tcaataccgc 11400

ctcggagaac tccctggctc acgaagtgcc ctggaccagt gtcgggccac aggttttgtg 11460

ccactagcgt gcagtgcagc ccggacaaaa gacacgcccc aggaggactg ggaaaacaaa 11520

gccgaaatgg cccccacggc ctgaaatgat ggagctggtg tgaccatcat ggagggacta 11580

gaggttagag gagaccccgt ggaaagaaag caaggcccaa cctagagtca agctgtaact 11640

ctaggggaag gactagaggt tagaggagac cccttgcgag tgagcaccac aagaaacagc 11700

atattgacac ctgggataga ctaggagacc ctctgtccta acaacaccag ccacttggca 11760

cagatcgccg aaagtgtggc tggtggtggt agaacacagg atctgggtcg gcatggcatc 11820

tccacctcct cgcggtccga cctgggctac ttcggtaggc taagggagaa gaacttgttt 11880

attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca 11940

tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcgcgac 12000

aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag 12060

acagttttat tgttcatgat gatatatttt tatcttgtgc aatgtaacat cagagatttt 12120

gagacacaac gtggctttgt tgaataaatc gaacttttgc tgagttgaag gatcagatca 12180

cgcatcttcc cgacaacgca gaccgttccg tggcaaagca aaagttcaaa atcaccaact 12240

ggtccaccta caacaaagct ctcatcaacc gtggctccct cactttctgg ctggatgatg 12300

gggcgattca ggcctggtat gagtcagcaa caccttcttc acgaggcaga cctcagcgct 12360

agcggagtgt atactggctt actatgttgg cactgatgag ggtgtcagtg aagtgcttca 12420

tgtggcagga gaaaaaaggc tgcaccggtg cgtcagcaga atatgtgata caggatatat 12480

tccgcttcct cgctcactga ctcgctacgc tcggtcgttc gactgcggcg agcggaaatg 12540

gcttacgaac ggggcggaga tttcctggaa gatgccagga agatacttaa cagggaagtg 12600

agagggccgc ggcaaagccg tttttccata ggctccgccc ccctgacaag catcacgaaa 12660

tctgacgctc aaatcagtgg tggcgaaacc cgacaggact ataaagatac caggcgtttc 12720

ccctggcggc tccctcgtgc gctctcctgt tcctgccttt cggtttaccg gtgtcattcc 12780

gctgttatgg ccgcgtttgt ctcattccac gcctgacact cagttccggg taggcagttc 12840

gctccaagct ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc gccttatccg 12900

gtaactatcg tcttgagtcc aacccggaaa gacatgcaaa agcaccactg gcagcagcca 12960

ctggtaattg atttagagga gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa 13020

aggacaagtt ttggtgactg cgctcctcca agccagttac ctcggttcaa agagttggta 13080

gctcagagaa ccttcgaaaa accgccctgc aaggcggttt tttcgttttc agagcaagag 13140

attacgcgca gaccaaaacg atctcaagaa gatcatctta ttaaggggtc tgacgctcag 13200

tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 13260

tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 13320

tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 13380

cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 13440

ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 13500

tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 13560

gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 13620

agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt 13680

atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 13740

tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 13800

gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 13860

agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 13920

cgaccgagtt gctcttgccc ggcgtcaaca cgggataata ccgcgccaca tagcagaact 13980

ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 14040

ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 14100

actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 14160

ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 14220

atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 14280

caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgt 14327

Claims (2)

1. A duck Tembusu virus attenuated live vaccine candidate strain is characterized by being a duck Tembusu delta C64-96 mutant virus strain, wherein the duck Tembusu delta C64-96 mutant virus strain is a gene deletion mutant virus strain, and the deleted gene sequence is a nucleotide sequence encoding 64 th-96 th amino acids of a duck Tembusu virus Capsid protein.

2. The use of the duck tembusu virus attenuated live vaccine candidate strain of claim 1 in the preparation of a duck tembusu virus attenuated live vaccine.

Images