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

Abstract

The present invention discloses a candidate strain of duck tembusu virus attenuated live vaccine, the candidate strain is duck tembusu ΔC64-96 mutant virus strain, and the duck tembusu ΔC64-96 mutant virus strain is a gene deletion mutation A virus strain whose deleted gene sequence is the nucleotide sequence encoding the amino acids 64-96 of the Capsid protein of duck Tembusu virus. The attenuated live vaccine candidate strain has defects in the virion assembly process, which leads to the weakening of the virus, and its weakening mechanism has been clearly analyzed, which effectively improves the safety of the vaccine; A single immunization can significantly activate the immune system of infected ducklings and provide strong and effective immune protection.

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 candidate strain of duck Tembusu virus attenuated live vaccine and a preparation method and application thereof.

Background technique

Since the first outbreak of duck Tembusu virus disease in the southeastern coastal areas of China in 2010, it has rapidly spread to many provinces and cities across the country, causing huge economic losses to my country's duck industry. Duck Tembusu disease is an acute infectious disease characterized by a sudden and sharp drop in egg production in laying ducks and breeding ducks, and hemorrhagic oophoritis as the main lesion. At present, the prevention and control measures of the disease are not perfect. Although there are commercialized live attenuated vaccines and inactivated vaccines, the disease is still showing a regional trend. The existing commercial inactivated vaccines are relatively safe, but the production cost of inactivated vaccines is high, the transportation and storage conditions are strict, and the immunization effect is not ideal. reaction. However, the existing live attenuated vaccines are all traditionally serially passaged, so that the virus produces adaptive mutations in the process of adapting to heterologous host cells, and finally obtains an attenuated immunogenic phenotype. The molecular mechanisms of virus attenuating of the attenuated vaccine candidates obtained in this way are unclear or not fully resolved; the lack of understanding of their detailed mechanisms will limit the risk of their natural reversion to a virulent phenotype assessment, and therefore a higher security risk. Therefore, the current DTMUV vaccine still has a lot of room for improvement, and it is especially necessary to develop a DTMUV vaccine with high safety and good immune effect.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the problems of low safety and unsatisfactory immune effect of the duck Tembusu virus attenuated vaccine existing in the prior art, and to provide a duck Tembusu virus attenuated live vaccine candidate strain, and the candidate strain is a duck Tembusu ΔC64-96 mutant virus strain, duck Tembusu ΔC64-96 mutant virus strain has defects in the assembly process of virions, resulting in weakening of the virus, and its weakening mechanism has been clearly analyzed, effectively improving the weakening of live vaccine The use of the candidate strain is safe; and a single immunization of ducklings with the duck Tembusu ΔC64-96 mutant virus strain can significantly activate the immune system of the immunized ducklings and greatly improve the immune effect.

In order to solve the above-mentioned technical problems, the technical solution adopted in the present invention is as follows: a candidate strain of a duck tembusu virus attenuated live vaccine, the candidate strain is a duck tembusu ΔC64-96 mutant virus strain, and the duck tembusu ΔC64 The -96 mutant virus strain is a gene deletion mutant virus strain, and the deleted gene sequence is the nucleotide sequence encoding amino acids 64-96 of Capsid protein of duck Tembusu virus.

The present invention also provides a method for preparing the above-mentioned candidate strain of the duck tembusu attenuated live vaccine, comprising the following steps:

S1. Using the pACNR-FL-CQW1 plasmid of the duck Tembusu virus strain DTMUV-CQW1 as a template, and using the sequences of SEQ ID NO.1 and SEQ ID NO.2 as primers, PCR amplification was performed to obtain fragment P1; .3 and SEQ ID NO.4 are primers, and PCR amplification obtains fragment P2; then take fragment P1 and fragment P2 as templates, take sequence SEQ ID NO.1 and SEQ ID NO.4 as primers, and obtain by fusion PCR amplification The fragment P1P2 of the gene deletion mutation, the gene sequence of the fragment P1P2 deletion is the nucleotide sequence encoding the 64th-96th amino acid of the Capsid protein of duck Tembusu virus;

S2. The pACNR-FL-CQW1 plasmid is double digested with restriction enzymes MluI and XhoI, and recovered and purified to obtain a purified linear pACNR-FL-CQW1 plasmid vector;

S3. Recombinantly connect the gene fragment P1P2 in step S1 with the purified linear pACNR-FL-CQW1 plasmid vector in step S2, then transform E. coli competent cells, select positive colonies for sequencing and identification, and obtain a recombinant plasmid, which is named as pACNR-FL-CQW1-ΔC64-96 plasmid;

S4. The pACNR-FL-CQW1-ΔC64-96 plasmid in step S3 was transfected into BHK21 cells, and the supernatant was harvested after culturing for 5 days to obtain the duck tembusu ΔC64-96 mutant virus strain.

Preferably, the nucleotide sequence of the fragment P1 is shown in SEQ ID NO. 5; the nucleotide sequence of the fragment P2 is shown in SEQ ID NO. 6; the nucleotide sequence of the fragment P1P2 is shown in SEQ ID NO. ID NO.7; the sequence of the pACNR-FL-CQW1-ΔC64-96 plasmid is shown in SEQ ID NO.8.

Preferably, the PCR amplification conditions are: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 15s, annealing at 63°C for 15s, extension at 72°C for 15s/kb, a total of 30 cycles; and a final extension at 72°C for 7 min.

The duck Tembusu attenuated live vaccine candidate strain prepared by the above method is prepared into ducklings immunized with duck Tembusu virus attenuated live vaccine, which can bring safe and efficient immune protection to the ducklings.

Capsid (C) protein is the smallest structural protein of flavivirus, and the mature C protein produced by NS2B3 protease cleavage is only about 100 amino acids. The structure and function of protein C are highly conserved among flaviviruses, and its structure contains four α-helices, which can form dimers through hydrophobic interactions, hydrogen bonds, and other forces. The most important function of protein C is to bind viral genomic RNA (vRNA) through a dimer structure to form Nucleocapsid (NC) and participate in the assembly of virions. NC forms complete virions (Virion) after budding to obtain the viral envelope. Studies on flaviviruses have found that protein C can tolerate a greater degree of truncation mutation and still maintain the infectivity of the virus, but it will lead to an increase in the production of subviral particles (SVPs). SVPs do not contain NC, but their conformation and immunogenicity are very similar to virion, making them good immunogens. Therefore, protein C is an ideal target for viral attenuation. The duck tembusu ΔC64-96 mutant virus strain in the present invention is obtained by gene deletion mutation, and it lacks the nucleotide sequence encoding the 64th-96th amino acids of the duck tembusu virus Capsid protein. The Busu ΔC64-96 mutant strain could not express amino acids 64-96 of the Capsid protein. This deletion mutation directly affected the assembly process of the virion, resulting in the weakening of the duck Tembusu ΔC64-96 mutant strain.

The beneficial effects that the present invention has:

1) In the present invention, the nucleotide sequence encoding the 64th-96th amino acid of the Capsid protein of duck Tembusu virus is deleted, and the 64th-96th amino acid of the Capsid protein is deleted to obtain duck tembusu ΔC64-96 mutant virus strains.

2) The weakening mechanism of the duck Tembusu ΔC64-96 mutant virus strain in the present invention is clearly analyzed, and the mutant virus strain has obvious defects in the assembly process of the virion, which reduces the ability to package the replicon RNA, thereby making the The weakening of the mutant virus strain significantly reduces the virulence of the virus to duck embryos and ducklings; and the mutant virus strain is genetically stable, so the duck Tembusu ΔC64-96 mutant virus strain was prepared into a weakened duck Tembusu virus. Immunizing ducklings with live vaccines can effectively improve the safety of vaccine use.

3) The duck Tembusu ΔC64-96 mutant virus strain in the present invention can significantly activate the immune system of the immunized ducklings, significantly up-regulate the expression levels of multiple immune-related cytokines, and can provide the ducklings after a single immunization. Complete challenge protection effect, therefore, duck Tembusu ΔC64-96 mutant virus strain is a safe and efficient attenuated live vaccine candidate virus strain.

Description of drawings

Fig. 1 is the plasmid map of pACNR-FL-CQW1;

Figure 2 is a schematic diagram of the construction of an infectious clone of the pACNR-FL-CQW1-ΔC64-96 plasmid;

Figure 3 is a graph of the fluorescence signals of the F0 and F1 generations after the duck Tembusu ΔC64-96 mutant virus strain was transfected into BHK21 cells in Example 2;

Fig. 4 is the growth curve diagram of duck Tembusu ΔC64-96 mutant virus strain on BHK21 cells in Example 3;

5 is a graph showing the plaque test results of the F1 generation and the F10 generation of the duck Tembusu ΔC64-96 mutant virus strain in Example 3;

Figure 6 is a graph showing the results of the virus packaging test of the duck Tembusu ΔC64-96 mutant virus strain in Example 3 (Fig. 6A is a diagram of a trans-complementary packaging system, and Fig. 6B is a graph of the effect of the ΔC64-96 deletion mutation on viral packaging)

Fig. 7 is the result diagram of duck embryo virulence test using 2000TCID ₅₀ duck Tembusu ΔC64-96 mutant virus strain in Example 5;

Fig. 8 is a graph showing the results of a single injection of duck Tembusu ΔC64-96 mutant virus strain in Example 6 to immunize 5-day-old ducklings with a single injection (Fig. 8A is a graph of viremia titer statistics, and Fig. 8B is a graph of the death statistics of ducklings) picture);

Figure 9 is a graph showing the weight change of ducklings in the challenge test after a single injection of duck Tembusu ΔC64-96 mutant virus strain in Example 6 to 5-day-old ducklings;

Fig. 10 is a graph showing the results of a challenge test after a single injection of duck Tembusu ΔC64-96 mutant virus strain in Example 6 to 5-day-old ducklings (Fig. 10A is a graph of viremia titers, and Fig. 10B is a graph of ducklings) death chart);

Figure 11 is the vRNA load map of each tissue of the ducklings on the 3rd and 5th days after the duck Tembusu ΔC64-96 mutant virus strain was immunized to 25-day-old ducklings in Example 7;

Figure 12 is a statistical graph of the number of dead ducklings within 15 days after the duck Tembusu ΔC64-96 mutant virus strain was immunized to 25-day-old ducklings in Example 7;

Figure 13 shows the mRNA expressions of IFN-α, IFN-β, IL-1β and TNF-α in the spleen tissue on the 5th day after immunization of 25-day-old ducklings with duck Tembusu ΔC64-96 mutant virus strain in Example 7 situation diagram;

Figure 14 is a graph showing the specific proliferation data of T lymphocytes in the peripheral blood of ducks after the duck Tembusu ΔC64-96 mutant virus strain in Example 7 was immunized with 25-day-old ducklings;

15 is a graph showing the expression levels of Th1-type cytokine IFNγ and Th2-type cytokine IL-4 in duck serum 14 days after immunization of 25-day-old ducklings with duck Tembusu ΔC64-96 mutant virus strain in Example 7.

Detailed ways

The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

Plasmids, cells and experimental animals: pACNR-FL-CQW1 plasmid vector, BHK21 cells, wild-type duck Tembusu virus strain DTMUV-CQW1, and polyclonal antibodies against anti-TMUV were provided by the Center for Avian Disease Control, School of Veterinary Medicine, Sichuan Agricultural University; SPF ducklings and SPF duck embryos were purchased from Harbin Veterinary Research Institute; duck Tembusu virus strain DTMUV-CHN-YC was donated by the School of Veterinary Medicine of Huazhong Agricultural University.

Main kits: ClonExpress II One Step Cloning Kit was purchased from Nanjing Novozymes; TransIntroTM EL Transfection Reagent was purchased from Beijing Quanzhijin Bio; Phanta EVO HS Super-Fidelity DNAPolymerase was purchased from Nanjing Novozymes.

Example 1

1. Construction of duck tembusu virus attenuated strain, namely duck tembusu ΔC64-96 mutant virus strain

Using the pACNR-FL-CQW1 plasmid of the duck Tembusu virus strain DTMUV-CQW1 strain as the template, and using the sequence SEQ ID NO.1 and SEQ ID NO.2 as the primers, the fragment P1 was obtained by PCR amplification; and SEQ ID NO.4 as primers, PCR amplification to obtain fragment P2; then with fragment P1 and fragment P2 as templates, with sequence SEQ ID NO.1 and SEQ ID NO.4 as primers, by fusion PCR amplification to obtain fragment P1P2 , the fragment P1P2 is the nucleotide sequence of the gene deletion mutation, and the gene sequence of the fragment P1P2 deletion is the nucleotide sequence of the 64th-96th amino acid of the Capsid protein of the duck Tembusu virus.

Two-step fusion PCR was performed using the high-fidelity enzyme Phanta EVO HS Super-Fidelity DNA Polymerase. The PCR reaction system is shown in Table 1:

5×EVOBuffer 10μL dNTPMix 1μL enzyme 1μL Primer F 2μL Primer R 2μL template 1μL ddH₂O Make up 50 μL

PCR amplification conditions were pre-denaturation at 95 °C for 1 min; denaturation at 95 °C for 15 s, annealing at 63 °C for 15 s, and extension at 72 °C for 15 s/kb, a total of 30 cycles; and a final extension at 72 °C for 7 min. After each round of amplification, gel electrophoresis was performed for identification, and liquid recovery or gel recovery was performed. The primers used in PCR amplification are detailed in Table 2.

Table 2 PCR amplification primer sequence list

2. Construction of recombinant infectious cloning plasmid pACNR-FL-CQW1-ΔC64-96

The pACNR-FL-CQW1 plasmid was double digested with restriction enzymes MluI and XhoI, and recovered and purified to obtain a purified linear pACNR-FL-CQW1 vector; using the ClonExpress II One Step Cloning Kit, according to the instructions, the obtained The gene deletion mutant fragment P1P2 was recombined with the linear pACNR-FL-CQW1 vector, and then transformed into E. coli competent cells. After the colonies grew, the bacteria were picked for PCR identification. Then, the positive colonies identified by PCR were selected and sent for sequencing identification, and the plasmid confirmed by sequencing was named pACNR-FL-CQW1-ΔC64-96 plasmid; single colonies with correct sequencing were selected for expansion and culture, and the plasmid was stored and extracted for future use.

The plasmid map of pACNR-FL-CQW1 is shown in Figure 1. The pACNR-FL-CQW1 plasmid was double digested with restriction enzymes MluI and XhoI, and the gene sequence encoding the Capsid protein on the pACNR-FL-CQW1 plasmid was excised. The digested linear pACNR-FL-CQW1 plasmid was recombined and ligated to obtain the recombinant plasmid pACNR-FL-CQW1-ΔC64-96. A schematic diagram of the construction of an infectious clone of the pACNR-FL-CQW1-ΔC64-96 plasmid is shown in FIG. 2 .

Example 2

Transfection and rescue of duck Tembusu ΔC64-96 mutant virus strain

Inoculate BHK21 cells with good growth status into 12-well plates and culture for 16 hours. When the cell confluence reaches 70-90%, perform the following operations according to the instructions of TransIntroTM EL Transfection Reagent. Set ΔC64-96 group and WT group; 1.2 μg of pACNR-FL-CQW1-ΔC64-96 plasmid prepared in Example 1 was transfected in each well of ΔC64-96 group, and wild-type duck tembo was transfected in each well of WT group Plasmid 1.2 μg of Su virus strain DTMUV-CQW1.

On the 5th day after transfection (F0 generation), the cell slides were harvested and identified by IFA with anti-TMUV polyclonal antibody. At the same time, the supernatant was harvested to infect new BHK21 cells. Cell slides were harvested on the third day of infection (F1 generation) for IFA identification.

As shown in Figure 3, ΔC64-96 produced obvious fluorescent signals in both the F0 generation and F1, indicating that the duck tembusu ΔC64-96 mutant virus strain can produce infectious virions, which are significantly weaker than wild-type duck tannins. Busu virus strain DTMUV-CQW1.

Example 3

Validation of the weakening characteristics and weakening mechanism of duck Tembusu ΔC64-96 mutant virus strain

1. Verify the weakening properties of the duck Tembusu ΔC64-96 mutant virus strain

Set ΔC64-96 group and WT group. In the ΔC64-96 group, the duck tembusu ΔC64-96 mutant virus strain rescued in Example 2 was propagated in BHK21 and the titer was determined for use, and its growth curve was determined on BHK21 cells. In the WT group, the wild-type duck Tembusu virus strain DTMUV-CQW1 was propagated in BHK21 and the titer was determined for use, and its growth curve was determined on BHK21 cells. As shown in Figure 4, the proliferation of duck tembusu ΔC64-96 mutant virus strain on BHK21 cells was significantly weakened compared with that of wild-type duck tembusu virus DTMUV-CQW1, and the specific manifestation was that the titer reached a peak. The time was prolonged, and the peak titer decreased.

The duck tembusu ΔC64-96 mutant virus strain rescued in Example 2 was serially passaged 10 times on BHK21 cells, and plaque assays were performed on the F1 and F10 generations of the duck tembusu ΔC64-96 mutant virus strain, respectively. Denoted as ΔC64-96-F1 and ΔC64-96-F10, with wild-type duck Tembusu virus DTMUV-CQW1 as the control, denoted as WT group. As shown in Figure 5, the plaque test results showed that ΔC64-96-F1 and ΔC64-96-F10 only formed plaques the size of pinpoints; and after 10 passages, the plaques formed by ΔC64-96-F10 were similar to those of F1 passage. In contrast, there was no significant difference in size, and the plaques were much smaller than those in the WT group. The results of the plaque assay showed that the proliferation ability of the 10th generation duck Tembusu ΔC64-96 mutant strain had little change compared with the first generation, and was weaker than that of the wild-type strain.

2. Verify the weakening mechanism of the duck Tembusu ΔC64-96 mutant virus strain

Since the Capsid protein plays a key role in the assembly process of duck Tembusu virus, it forms a nucleocapsid together with the viral genomic RNA, and its α4 helix structure is considered to be a key stable point for binding to the viral genomic RNA, while duck The Tempusu ΔC64-96 mutant virus strain just deleted this key region, so this example uses packaging experiments to verify whether the duck Tempusu ΔC64-96 mutant virus strain has defects in the virus assembly process. As shown in Fig. 6A, the WT type (or ΔC64-96) C-prM expression plasmid was co-transfected into BHK21 cells with a subgenomic replicon plasmid lacking C protein and carrying the Nluc luciferase gene. A single round of infectious virions was generated in a complementary manner, denoted as the WT group (or ΔC64-96 group). The supernatant was harvested on the third day after transfection to infect new BHK21 cells, and the fluorescence value was measured 24 hours after infection. The results showed that the ability of the ΔC64-96 group to package replicon RNA was significantly reduced compared with the WT group (Fig. 6B).

In conclusion, the deletion mutation of the duck tembusu ΔC64-96 mutant virus strain caused defects in the assembly process of the virion, which was the main reason for the weakening of the duck tembusu ΔC64-96 mutant virus strain in vitro. Therefore, defects in virion assembly are the molecular basis for the weakening of the duck Tembusu ΔC64-96 mutant strain.

Example 4

Validation of the genetic stability of the duck Tembusu ΔC64-96 mutant virus strain

The duck Tembusu ΔC64-96 mutant virus strain rescued in Example 2 was serially passaged on BHK21 cells for 10 times, and then the virus whole genome was sequenced; the sequencing results showed that after 10 passages, duck Tembusu ΔC64-96 The gene deletion mutation of the mutant virus still exists stably in the genome of the mutant virus, indicating that the deletion mutation at the 64th-96th position of the C protein of the duck Tembusu ΔC64-96 mutant virus has genetic stability. At the same time, other adaptive mutations appeared in the genome of duck Tembusu ΔC64-96 mutant virus, as shown in Table 3.

Table 3 Passaging stability of duck Tembusu ΔC64-96 mutant virus

Example 5

Experiment on the effect of duck Tembusu ΔC64-96 mutant virus strain on duck embryo virulence

The ΔC64-96-F1 group, the ΔC64-96-F10 group, the CQW1-WT group and the Mock group were set, with 10 duck embryos in each group. After the duck tembusu ΔC64-96 mutant virus strain obtained in Example 2 was continuously cultured for 10 generations, the F1 generation of the duck tembusu ΔC64-96 mutant virus strain and the F10 generation of the duck tembusu ΔC64-96 mutant virus strain were obtained. Duck embryos of ΔC64-96-F1 group, ΔC64-96-F10 group and CQW1-WT group were injected with 2000 TCID ₅₀ of duck tembusu ΔC64-96 mutant virus strain F1 generation and duck tembusu ΔC64-96 mutant virus strain F10, respectively Generation, wild-type duck Tembusu virus strain DTMUV-CQW1 virus. The Mock group was injected with the same dose of DMEM. After the challenge, the animals were observed for 7 consecutive days, and the death of duck embryos was recorded every day. The test results are shown in Figure 7. All duck embryos in the CQW1-WT group died within 4 days. In contrast, 70% of the duck embryos died of ΔC64-96-F1 infection, and ΔC64-96-F10 embryos died. The virulence of duck embryos was further reduced, and only 10% of duck embryos died.

The above experimental analysis showed that the mutation of the duck tembusu ΔC64-96 mutant virus strain significantly reduced the duck embryo virulence of duck tembusu virus, and the duck tembusu ΔC64-96 mutant virus strain F10 after adaptation to cells was in the F1 On the basis, the virulence is further reduced.

Example 6

Immune protection of duck Tembusu ΔC64-96 mutant virus strain after immunization of 5-day-old ducklings

The 5-day-old ducklings were randomly divided into 3 groups, designated as CQW1 group, ΔC64-96 group and DMEM group, respectively. The CQW1 group was inoculated with 200 μL (10 ⁵ TCID ₅₀ ) of wild-type duck Tembusu virus DTMUV- CQW1; ΔC64-96 group was inoculated with 200 μL (10 ⁵ TCID ₅₀ ) of duck Tembusu ΔC64-96 mutant virus for immunization; DMEM group was injected with the same dose of DMEM. On the 1st to 14th day after immunization, the weight changes, clinical symptoms and death of the three groups of ducklings were detected, and the level of viremia was determined on the 3rd day; on the 14th day after immunization, all surviving chicks in the three groups were tested Ducks were challenged by intramuscular inoculation of 200 μL (2×10 ⁶ TCID ₅₀ ) of duck Tembusu virus strain DTMUV-CHN-YC, and the weight changes, clinical symptoms and In case of death, the level of viremia was determined on the 3rd day post-challenge.

On the 3rd day after immunization, we measured the viremia of 3 groups of ducklings. As shown in Fig. 8A, the CQW1 group developed a high level of viremia, and the virus titer reached 10 ⁴ TCID _50/100 μL, and neither the ΔC64-96 group nor the DMEM group detected viremia. From the 3rd day after immunization, the ducklings in the CQW1 group developed clinical symptoms such as depression, slow movement, unsteady standing and even hindlimb paralysis; and died from the 5th day, and by the 7th day, 60% of the ducklings Died from infection with wild-type duck Tembusu virus DTMUV-CQW1 (FIG. 8B). However, the ΔC64-96 and DMEM groups did not experience any obvious clinical symptoms and death (Fig. 8B). As shown in Fig. 9, starting from the 4th day after immunization, the level of body weight gain in the CQW1 group was significantly lower than that in the DMEM group, while the ΔC64-96 group had no significant difference compared with the DMEM group. These data indicate that the virulence of duck Tembusu ΔC64-96 mutant virus to ducklings is significantly reduced.

As shown in Figure 9, on the second day after challenge, the body weight of DMEM was significantly lower than that of ΔC64-96, and clinical symptoms such as depression, slow movement, unsteady standing and even hind limb paralysis began to appear. Viremia was detected on day 3 post-challenge and found that the ΔC64-96R and CQW1 groups did not produce viremia except for the DMEM group which produced obvious viremia (Fig. 10A). As shown in FIG. 10B , on the 5th and 7th days after the challenge, 1 duckling died in the DMEM group, respectively; while there was no death in the ΔC64-96 group and the CQW1 group.

The above data show that a single intramuscular injection of immunized duck Tembusu ΔC64-96 mutant virus strain can provide effective immune protection for ducklings.

Example 7

Mechanism of immune response induced by duck Tembusu ΔC64-96 mutant virus strain in 25-day-old ducklings

In order to further verify the mechanism by which the duck Tembusu ΔC64-96 mutant virus strain provides immune protection for ducklings and induces an immune response in the ducklings, in this example, the 25-day-old ducklings were randomly divided into three groups, namely the CQW1 group, DMEM group, ΔC64-96 group, 11 in each group. The CQW1 group received 200 μL of wild-type duck tembusu DTMUV-CQW1 virus intramuscularly, the ΔC64-96 group received 200 μL of duck tembusu ΔC64-96 mutant virus intramuscularly, and the DMEM group received the same dose of DMEM by intramuscular injection. On the 3rd and 5th days after immunization, 3 ducklings in each group were sacrificed at random, and the heart, liver, spleen, lung, kidney, brain and thymus tissue samples of the ducklings were collected. The remaining 5 animals in each group were observed for 14 consecutive days, and the death was recorded. On the 14th day after immunization, the cellular immunity was measured.

As shown in Fig. 11 , on the third day after immunization, the load of vRNA with high titer in each organ of the CQW1 group could be detected, and it further increased on the fifth day after infection. In contrast, the vRNA load of each organ in the ΔC64-96 group was always kept at the lowest level. As shown in Figure 12, on the 5th day after infection, one duckling in the CQW1 group developed obvious clinical symptoms and died; while in the ΔC64-96 group, no duckling developed clinical symptoms and died.

In order to analyze the activation of innate immunity caused by the infection of duck tembusu ΔC64-96 mutant virus, RT-qPCR was used to further detect IFN-α, IFN-β, IL-1β in spleen tissue on the 5th day after infection and TNF-α mRNA expression. As shown in Figure 13, compared with the DMEM group, both the CQW1 group and the ΔC64-96 group could activate the up-regulation of IFN-α, IFN-β, IL-1β and TNF-α.

In order to verify whether the infection of duck Tembusu ΔC64-96 mutant virus can activate the cellular immunity of ducks, in this example, T lymphocytes from the peripheral blood of ducks were isolated for T cell specific proliferation test. As shown in Figure 14, compared with the DMEM group, the stimulation coefficient of the CQW1 group reached 6.2, while that of the ΔC64-96 group was 4.8. In this example, the expression levels of Th1-type cytokine IFNγ and Th2-type cytokine IL-4 in duck serum on the 14th day after immunization were further measured by ELISA. As shown in FIG. 15 , the expression levels of Th1-type cytokine IFNγ and Th2-type cytokine IL-4 in ΔC64-96 were significantly up-regulated relative to the DMEM group. All the above data indicated that the infection of duck Tembusu ΔC64-96 mutant virus could effectively activate cellular immunity.

To sum up, the duck Tembusu ΔC64-96 mutant virus strain in the present invention is obtained by gene deletion mutation, and the nucleotide sequence encoding the 64th-96th amino acids of the Capsid protein of duck Tembusu virus is deleted, so that the Capsid The 64th-96th amino acid of the protein is deleted. The weakening mechanism of the virus strain was verified in Example 3 of the present invention. The virus strain has defects in the assembly process of the virus particles, that is to say, it deletes the 64th to 96th positions of the Capsid protein encoding duck Tembusu virus. After the nucleotide sequence of amino acids, the ability to package the replicon RNA was significantly reduced, thereby weakening the mutant virus and significantly reducing the virulence of the virus on duck embryos and ducklings. Example 4 proves that the gene mutation of the duck Tembusu ΔC64-96 mutant virus in the present invention has genetic stability. Example 6 and Example 7 show that a single immunization of ducklings with duck Tembusu ΔC64-96 mutant virus can significantly activate IFN-α, IFN-β, IL-1β, TNF-α in infected ducklings , Th1-type cytokine IFNγ and Th2-type cytokine IL-4 expression levels were up-regulated, providing effective immune protection for ducklings.

The above analysis shows that duck tembusu ΔC64-96 mutant virus strain has obvious weakening effect, its weakening mechanism has been clearly analyzed, and it has obvious genetic stability, so it can effectively improve the safety of its use. Using this virus strain to immunize ducklings can effectively activate the immune system of the immunized ducklings, significantly up-regulate the expression levels of multiple immune-related cytokines, and provide efficient immune protection for the ducklings. Therefore, the duck tembusu ΔC64-96 mutant virus strain in the present invention is a safe and efficient attenuated live vaccine candidate virus strain, and it can be widely used in duck tambusu virus by preparing it into a duck tembusu virus attenuated live vaccine Prevention of Busu-related diseases.

The description and drawings of the present invention are considered to be illustrative rather than restrictive. On the basis of the present invention, those skilled in the art can make some technical features without creative work according to the disclosed technical content. Some replacements and modifications are within the protection scope of the present invention.

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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

<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 catgggagg 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

<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

<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 acgtttttttg 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 gaaagggggag 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 aaaagttgggg 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.

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