CN1241642C - Novel pestilence DNA vaccine and its usage - Google Patents

Abstract

The DNA vaccine of Newcastle disease involved in the present invention is made up of NDV-LasotaF gene and eukaryotic expression vector pVAX1, and the construction of this DNA vaccine is through the following steps: extraction and purification of total RNA of NDV Lasota virus, design of specific primers, through TDRT- The full-length cDNA gene sequence of NDV LasotaF was obtained by PCR amplification and cloned, and the purified LasotaF full-length cDNA fragment was recombined into pVAX1 eukaryotic expression plasmid. Compared with the currently commonly used vaccines, the amount required for the nucleic acid vaccine to achieve the same immune effect is small, and it can simultaneously and efficiently stimulate humoral immunity and cellular immunity. It is more effective than other immune methods, and is safe, long-acting, stable, Easy to operate. The Newcastle disease DNA vaccine can be used as medicine for preventing and treating chicken Newcastle disease.

Description

A DNA vaccine for Newcastle disease and its application

technical field

The invention relates to a DNA vaccine for Newcastle disease, in particular to a Newcastle disease virus PVAX1-LasotaFDNA vaccine and its application.

technical background

Chicken Newcastle disease, also known as Newcastle disease virus (Newcastle disease virus, hereinafter referred to as NDV) is the pathogenic agent, and it is widely present in the world. It is one of the important infectious diseases that endanger the world's chicken industry. An important restrictive factor for the development of the vast rural poultry industry. Data statistics confirm that rapid-onset NDV is the prevalent NDV strain in rural chicken flocks in many developing countries. The disease is characterized by rapid onset, with a morbidity rate of almost 100% and a mortality rate of over 80%. Generally, a global pandemic will occur every four or five years. The prevalence of NDV often leads to huge economic losses, so the whole world is working hard to control and eliminate NDV. After chickens are infected by NDV virus, there is no special treatment drug at present. Most of them adopt the traditional inoculation of live attenuated NDV virus vaccine or inactivated bacteria as the preventive vaccine. Chick vaccination is the main preventive measure. However, the NDV virus is easy to mutate, resulting in immune evasion, which is one of the main reasons for the uncontrollable disease and the prevalence of NDV.

According to different antigen components, vaccines can be divided into live attenuated vaccines, inactivated bacterial vaccines and recombinant vaccines. Compared with the currently commonly used attenuated live vaccines or inactivated bacterial vaccines, recombinant vaccines, that is, DNA vaccines, have obvious advantages: 1) The amount of immunity required is small, 20-100 micrograms is enough; 2) DNA vaccines can simultaneously Efficiently stimulate humoral immunity and cellular immunity, which is more effective than other immunization methods; 3) It not only has the effect of preventing diseases but also has the effect of treating diseases; 4) Gene immunity is safe and long-lasting (the immune ability has a long-term memory), Stable and easy to operate. Therefore, DNA vaccines have potential advantages that other immunization methods cannot match. Its application prospect is very attractive.

In the work of a variety of NDV virus DNA vaccines developed against Newcastle disease virus (NDV), Germany's Meszsaros used NDV-6/10 virus variants to construct a vaccine that produced a strong resistance to NDV infection in chickens through spray inoculation. However, the presence of humoral immunity cannot be detected, and although direct spray inoculation of DNA vaccine can produce mucosal immunity, the immune effect is far less good than that of intramuscular microinjection.

To construct a DNA vaccine that can not only produce high-titer antibodies but also activate cellular immunity, it is very important to screen for protein genes with antigenic activity. The F protein of NDV virus is the molecular determinant protein of NDV pathogenicity, and the amino acid sequence of its F protein cleavage site determines the pathogenicity of NDV virus. The specific cleavage mediated by a tryptase leads to the splitting of the fusion protein _F0 to produce active _F1 and _F2 polypeptides, and the _F1 polypeptides fuse with host cells to infect the cells of the body. Therefore, by stimulating an effective immune response against the F protein, it is possible to achieve the purpose of preventing and treating chicken Newcastle disease. The virus recombinant vaccine prepared by Belgium's Letellier using the NDV-F protein gene can stimulate high-titer humoral immunity and resist NDV infection, but the antibody can not play a good role in neutralizing NDV virus.

Contents of the invention

The purpose of the present invention is to overcome the defect that the prior art NDV virus DNA vaccine cannot simultaneously stimulate humoral immunity, the immune effect is poor, and the ability of the antibody produced to neutralize the NDV virus is weak, thereby providing a pVAX1- that can prevent and treat Newcastle disease LasotaF DNA vaccine, which selects the complete gene sequence of the F protein of Lasota _E4 , contains both highly conserved homologous sequences and antigenic determinant gene sequences. In addition, its eukaryotic expression vectors pVAX1 and pcDNA3.1 are both It is a carrier plasmid that has been continuously improved in recent years, so it can stimulate cellular immunity and humoral immunity at the same time, and the immune effect is good.

Another object of the present invention is to provide the construction of the DNA vaccine for Newcastle disease.

Another object of the present invention is to provide the application of the DNA vaccine of Newcastle disease.

The purpose of the present invention is achieved by the following technical solutions:

The present invention provides a DNA vaccine for Newcastle disease, which is composed of NDV-LasotaF gene and eukaryotic expression vector pVAX1, characterized in that: the NDV-LasotaF gene is the pathogenic determinant gene of NDV virus, which is NDV virus Nucleotides 4541 to 6223 in the Lasota E ₄ gene have the nucleic acid sequence shown in FIG. 1 .

The present invention provides a kind of construction of described Newcastle disease DNA vaccine, comprises the steps:

1) Extraction and purification of NDV Lasota virus total RNA: inject NDV Lasota E ₄ virus strain into chicken embryos hatched for 14 days, after the death of chicken embryos, collect allantoic fluid, and extract NDV Lasota virus total RNA according to conventional methods;

2) design specific primers: design a pair of specific primers according to the Lasota virus F protein gene sequence analysis,

Upstream primer: 5′-AAGATGGGCTCCAGACCTTCTACC-3′;

Downstream primer: 5′-CCTTCGTTCCTCATCTGTGTTCAC-3′;

3) Cloning the full-length cDNA gene sequence of NDV LasotaF by temperature drop and reverse transcription-polymerase chain amplification method;

4) The LasotaF amplified in step 3) is recovered and purified by the Wizard® PCR Preps.DNA purification system kit to recover and purify the LasotaF cDNA fragment;

5) The full-length cDNA fragment of LasotaF was recombined into pVAX1 eukaryotic expression plasmid to construct a DNA vaccine against Newcastle disease—pVAX1-LasotaF DNA vaccine against NDV.

The temperature drop reverse transcription-polymerase chain amplification method in the step 3) includes: the reverse transcription reaction is reacted at 48° C. for 45 minutes, followed by inactivation at 95° C. for 5 minutes, and the temperature drop reverse transcription-polymerase chain amplification method is started. Amplification method Amplification reaction: polymerase is added in the first denaturation step, the denaturation temperature is 94°C, lasts for 1 minute, the annealing temperature starts at 68°C, lasts for 1 minute, and then decreases by 1°C every four cycles Decrease to 56°C for 1 minute, extend at 68°C for 1.5 minutes; finally continue amplification at an annealing temperature of 58°C/min for 15 cycles; finally extend at 72°C for 10 minutes.

The eukaryotic expression vector in step 5) can also be pcDNA3.1.

The present invention further provides the use of the Newcastle disease DNA vaccine in the preparation of medicines for preventing chicken Newcastle disease.

The invention also provides the application of the Newcastle disease DNA vaccine in preparing medicine for treating chicken Newcastle disease.

The advantages of the Newcastle disease DNA vaccine provided by the present invention are: 1) It has the effect of highly effective prevention and treatment of chicken Newcastle disease; 2) The required amount of immunity is small, just 20-50 micrograms; 3) The nucleic acid vaccine can simultaneously Efficiently stimulate humoral immunity and cellular immunity, which is more effective than other immunization methods; 4) Gene immunization is safe, long-acting (the immune ability has a long-term memory), stable, and easy to operate.

Description of drawings

Fig. 1 is the nucleotide sequence of NDV-LasotaF gene.

Detailed ways

The three yellow chickens used in the examples of the present invention were incubated by Beijing Changping Chicken Farm, and the Laihang chickens were incubated by Beijing Animal Experiment Center.

Embodiment 1. The construction of Newcastle disease DNA vaccine of the present invention—pVAX1-LasotaF DNA vaccine

The construction steps of pVAX1-LasotaF DNA vaccine of the present invention are as follows:

1) Extraction and purification of NDV virus (Virus) total RNA:

The RNAgents® Total RNA Isolation System kit of Promega Company was used to extract the total RNA of NDV Lasota virus. The specific operation was to inject the NDV Lasota E ₄ virus strain provided by Beijing China Veterinary Medicine Supervision Institute into the chicken embryo hatched for 14 days, and observe it under the light. After finding that the chicken embryo is dead, collect 20ml of allantoic fluid, and centrifuge the collected allantoic fluid at a speed of 700rpm for 15 minutes; add 20ml of 16wt% PEG-0.525M NaCl to the supernatant, and ice-bath for 1 hour; centrifuge at a speed of 10000rpm Precipitate virus particles at 4°C for 5 minutes; dissolve the virus pellet with 800 μl denaturing solution, transfer to 1.5 ml EP tube; add 80 μl sodium acetate to mix, add 800 μl phenol: chloroform: isoamyl alcohol (volume ratio 1:1:1 ) and mix well, vibrate for 8-12 seconds, and bathe in water for 15 minutes; centrifuge at 4°C for 15 minutes at a speed of 10,000 rpm, and take out the supernatant; centrifuge at 4°C for 10 minutes at a speed of 10,000 rpm; Nuclease water, stored at -80°C;

2) design specific primers: design a pair of specific primers according to the Lasota virus F protein gene sequence analysis,

Upstream primer: 5′-AAGATGGGCTCCAGACCTTCTACC-3′;

Downstream primer: 5′-CCTTCGTTCCTCATCTGTGTTCAC-3′;

3) Cloning and amplifying the full-length cDNA of LasotaF gene by TDRT-PCR (temperature drop reverse transcription-polymerase chain reaction):

The 50 μL reaction system consists of 10 μL AMV/Tf1 5× reaction buffer, 2 mM dNTPs mixture, 3 mM MgSO ₄ , 1 μM upstream primer and downstream primer, 0.1 U/μL AMV reverse transcriptase, 0.1 U/μL Tf1 DNA polymerase, 2 μg of total RNA and nuclease-free water; the reverse transcription reaction was reacted at 48°C for 45 minutes, followed by inactivation at 95°C for 5 minutes, and the temperature drop reverse transcription-polymerase chain amplification method (TDRT -PCR) amplification reaction; in the stage of landing TD-PCR amplification, the denaturation temperature is 94°C for 1 minute, the annealing temperature starts from 68°C and lasts for 1 minute, and then drops to 1°C every four cycles. 56°C for 1 minute, the extension temperature is 68°C for 1.5 minutes; finally continue to amplify for 15 cycles at the annealing temperature of 58°C for 1 minute; finally extend at 72°C for 10 minutes;

4) by the LasotaF gene cDNA amplified by TDRT-PCR, the method is the same as 3), utilize the Wizard PCR Preps.DNA purification system kit to reclaim and purify the LasotaF gene cDNA fragment;

5) Recombine the LasotaF full-length cDNA fragment into the pVAX1 eukaryotic expression plasmid to construct an anti-NDV pVAX1-LasotaF DNA vaccine: select the eukaryotic plasmid expression vector pVAX1 containing the eukaryotic promoter CMV according to the instructions of the pVAX1 vector kit, It can efficiently express foreign proteins in eukaryotic cells, and recombine the full-length cDNA fragment of LasotaF into the pVAX1 eukaryotic expression plasmid: transform the competent cell Top10F' with the ligation reaction solution, and take 80ul of the transformed bacteria solution to coat LB (containing ampicillin Penicillin) plate, cultivated overnight at 37°C; pick colonies and shake overnight, extract plasmid DNA by alkaline lysis method, that is, the pVAX1-LasotaF DNA vaccine of this embodiment, carry out enzyme digestion identification and sequence the positive recombinant plasmid.

Embodiment 2.pVAX1-LasotaF DNA vaccine exogenous LasotaF gene in vitro transient expression and identification of expression content

In vitro mRNA expression of LasotaF gene: establishment of transient expression system for HeLa cells in vitro, the eukaryotic expression plasmid pVAX1-LasotaF constructed in Example 1 was transfected into HeLa cells cultured in 24-well plates through liposomes, collected in 36 hours Cells, the total RNA of the transfected HeLa cells collected at 36 hours was extracted, and the expression of LasotaF gene in vitro was analyzed by RT-PCR. The LasotaF cDNA band was amplified by RT-PCR, and the results showed that the pVAX1-LasotaF eukaryotic expression plasmid could be efficiently expressed at the mRNA level in the Hela cell in vitro transient expression system, and the expression level of the mRNA level was higher than that of the control group (empty plasmid ) was 138% higher.

In vitro protein expression of exogenous LasotaF: The expression of LasotaF protein in transfected HeLa cells was detected by immunofluorescence. The HeLa cells transfected for 36 hours were fixed in 4wt% phosphate buffer-paraformaldehyde (PH7.4) for 1 hour at room temperature, washed 3 times with phosphate buffer (for convenience, hereinafter referred to as PBS), and the ratio of 1:50 (For convenience, 1: x in all embodiments of this patent all refers to the chicken anti-NDV protein antiserum (0.5mg/mL) 37 ℃ of 37 ℃ incubation 3 hours of diluting x times with PBS material, after PBS rinses, Samples were incubated at 1:100 diluted goat anti-chicken IgG-FITC for 1 hour at 4°C, rinsed with PBS, stained with 20 μg/mL propidium iodide (PI) at 4°C for 2 minutes, rinsed with PBS, and placed on a laser confocal microscope (Confocal) at 488nm and 564nm excitation wavelength for observation and analysis. Immunofluorescence results confirmed that pVAX1-LasotaF can produce biologically active LasotaF protein in Hela cells in vitro transient expression system, and the expression level of the protein level is 196% higher than that of the control group (empty plasmid).

Example 3. Identification of exogenous LasotaF gene expression and expression content of pVAX1-LasotaF DNA vaccine in vivo

In vivo expression detection of exogenous LasotaF gene: Three yellow chickens incubated in Changping chicken factory in Beijing were randomly divided into 2 groups, 18 in each group, one group was the experimental group, and 20 μg pVAX1-LasotaF constructed in Example 1 was intramuscularly injected into the three yellow chickens Plasmid DNA; the other group was the control group, and 20 μg pVAX1 empty plasmid DNA was injected intramuscularly into Sanhuang chickens. Three weeks after the injection of the plasmid DNA, the muscle, liver and spleen tissues of the three-yellow chickens immunized with pVAX1-LasotaF were extracted, and the total RNA of the muscle tissue, liver and spleen tissues collected from the three-yellow chickens immunized with pVAX1-LasotaF was extracted, and the exogenous LasotaF gene was detected in the three-yellow chickens by RT-PCR expression. It can be seen from the test results that after immunization with pVAX1-LasotaF DNA vaccine, LasotaF is expressed at the mRNA level in muscle, liver and spleen tissue cells, and the average expression level of mRNA level is 369% higher than that of the control group (empty plasmid).

The detection of embodiment 4.pVAX1-LasotaF DNA vaccine in vivo immune response

ELISA detection of humoral immune response of three yellow chickens: experimental grouping, pVAX1-LasotaF DNA vaccine injection method and site are the same as in Example 3. Use 1:50-fold dilution of normal serum (without injecting any plasmid) and 1:50-fold dilution of serum injected with 20 μg of pVAX1 empty plasmid DNA as controls respectively, and collect the serum of pVAX1-LasotaF DNA vaccine immunized three yellow chickens three weeks later by 1: The ratios of 100 and 1:500 were diluted, and 50 μL/well of 1 μg/mL LasotaE ₄ standard protein was used as the coating antigen, and the standard ELISA method was used to detect the humoral immune response. It can be seen from the test results that after immunization with pVAX1-LasotaF DNA vaccine, the body can produce high-titer antibodies (antibody titer > 1:2600). Example 5. Safety assessment of pVAX1-LasotaF DNA vaccine

For the safety test of immunized animals: test different doses (20μg～300μg) of pVAX1-LasotaF DNA vaccine to immunize 40 Sanhuang chickens, and none of them died after 180 days; the toxicology and routine of different doses of pVAX1-LasotaF DNA vaccine injection animals Observation and measurement of physiological indicators, using histochemical method to determine the pathological changes of animal tissues after injection of the vaccine: Frozen sections of corresponding tissues and organs were observed under a microscope, and no pathological changes were found in animal tissues.

Example 6. Evaluating whether pVAX1-LasotaF DNA vaccine immunizes poultry and whether it affects other animals.

15 three-day-hatched Sanhuang chickens and 15 three-day-hatched Laihang chickens were mixed, and 6 were randomly selected to be injected with 20 μg pVAX1-LasotaF DNA vaccine and marked. After 6 weeks, RT-PCR was used to detect the other 24 chickens Whether there is expression of exogenous LasotaF gene of pVAX1-LasotaF DNA vaccine in Sanhuang chickens or Laihang chickens without any plasmid injection, the test results of 24 chickens showed that no expression of exogenous LasotaF gene was detected at the mRNA level, which indicated that pVAX1-LasotaF Poultry immunized with LasotaF DNA vaccine has no infection effect on other unimmunized animals.

Embodiment 7.pVAX1-LasotaF DNA vaccine is in the curative effect experiment-I of actual prevention and treatment Newcastle disease in flock of chickens

40 white Laihang chicken chicks were hatched in Beijing Animal Experiment Center. After one week, the both male and female chicks were randomly divided into four groups with 10 chicks in each group. The injected Newcastle disease virus NDV Lasota E ₄ was 100 μl of allantoic fluid containing Lasota E ₄ virus diluted 100 times.

Group 1 was not inoculated with any plasmid, and was injected with Newcastle disease virus NDV Lasota E ₄ after 3 weeks;

The second group was inoculated with 100 μl of normal saline, and injected with Newcastle disease virus NDV Lasota E ₄ after 3 weeks;

The third group was inoculated with 20 μg pVAX1 empty plasmid, and injected with Newcastle disease virus NDV Lasota E ₄ after 3 weeks;

The fourth group was inoculated with 20 μg pVAX1-LasotaF DNA vaccine, and injected with Newcastle disease virus NDV Lasota E ₄ after 3 weeks;

Observation started on the third day after injection of Newcastle disease virus NDV Lasota E ₄ , and the results were:

Group 1, Group 2, and Group 3: After 3 days, 30 chickens showed different degrees of chicken plague symptoms, and 4 of them died; after 4 days, 26 chickens showed worse symptoms of chicken plague, and 12 of them died; 5 days later, 14 chickens Only 2 chicks survived; 2 chicks also died after 6 days;

Group 4: After 3 days, 10 chickens were healthy and lively without any symptoms of chicken plague; after 4 days, 10 chickens were healthy and lively without any symptoms of chicken plague; after 5 days, 10 chickens were healthy and lively without any symptoms of chicken plague; after 6 days, 1 chicken The chicks showed mild symptoms of chicken plague, and the remaining 9 were healthy and lively; after 7 days, 2 chickens showed mild symptoms of chicken plague, and the remaining 8 were healthy and lively; after 120 days, none of the 10 chickens had any symptoms of chicken plague, and they were healthy and lively;

From the experimental results, it can be seen that the pVAX1-LasotaF DNA vaccine has a highly effective anti-Newcastle disease virus infection effect, and compared with the three groups of control groups, the anti-Newcastle disease virus infection effect is significant (p＜0.01).

Example 8. Efficacy experiment of pVAX1-LasotaF DNA vaccine in actual prevention and treatment of chicken Newcastle disease in chicken flocks-II

Incubate 90 three-yellow chicken chicks in Beijing Changping Chicken Farm. One week later, 90 chicks of both sexes were randomly divided into four groups, with 20 chicks in each group from the first to third groups, and 30 chicks in the fourth group. The injected Newcastle disease virus NDV Lasota E ₄ was 100 μl of allantoic fluid containing Lasota E ₄ virus diluted 100 times.

Group 1 was not inoculated with any plasmid, and was injected with Newcastle disease virus NDV Lasota E ₄ after 3 weeks;

The second group was inoculated with 100 μl of normal saline, and injected with Newcastle disease virus NDV Lasota E ₄ after 3 weeks;

Group 3 was inoculated with 20 μg pVAX1 empty plasmid, and injected with Newcastle disease virus NDV Lasota E ₄ 3 weeks later

The fourth group was inoculated with 20 μg pVAX1-LasotaF DNA vaccine, and injected with Newcastle disease virus NDV Lasota E ₄ after 3 weeks;

Observation started on the third day after injection of Newcastle disease virus NDV Lasota E ₄ , and the results were:

Group 1, Group 2, and Group 3: After 3 days, none of the 60 chicks showed symptoms of chicken plague, and they were healthy and lively; after 4 days, none of the 60 chickens showed symptoms of chicken plague, and they were healthy and lively; Symptoms of chicken plague began to appear, and the severity of symptoms varied; after 6 days, the symptoms of chicken plague in 60 chickens became worse, and 25 of them died; after 7 days, the symptoms of chicken plague in the remaining 35 chickens became worse, and 32 chickens died; after 10 days, finally 3 chicks died one after another;

Group 4: After 3 to 5 days, 30 chickens had no symptoms of chicken plague and were healthy and lively; after 6 to 7 days, 4 of the 30 chickens showed mild symptoms of chicken plague, and the other chickens were healthy and lively; after 8 to 9 days 7 of the 30 chickens had mild symptoms of chicken plague, and the other chickens were healthy and lively; after 10 days, 1 of the 30 chickens died and the other 29 chickens had no symptoms of chicken plague;

After 20 days, the 29 surviving chickens were re-injected with 100 μl of 200-fold diluted allantoic fluid containing Lasota E ₄ virus; after 24 days, 5 of the 29 chickens showed mild symptoms of chicken plague, and the other chickens were healthy and lively; after 28 days The symptoms of fowl plague in 5 chickens disappeared, and all 29 chickens were healthy and lively;

After 30 days, these 29 chickens were injected for the third time with 100 μl of 100-fold diluted allantoic fluid containing Lasota E ₄ virus; after 34 days, 29 chickens were healthy and lively, and no fowl plague symptoms occurred; after 38 days, all 29 chickens were healthy and lively;

After 40 days, these 29 chickens were injected for the fourth time with 100 μl of diluted 50-fold allantoic fluid containing Lasota E ₄ virus; after 44 days, 29 chickens were healthy and lively, and no fowl plague symptoms occurred; after 120 days, 29 chickens were all healthy and lively.

Chicken group experiments once again proved that the pAX1-LasotaF DNA vaccine has a highly effective anti-Newcastle disease virus infection effect, and the effect is very significant compared with the three groups of controls (p<0.01). Chicken flocks were not infected by the virus NDV Lasota E ₄ strain, which proved that the anti-virus effect of pVAX1-LasotaF DNA vaccine was stable and reliable.

Example 9. Curative effect experiment of pVAX1-LasotaF DNA vaccine in the actual treatment of chicken Newcastle disease in flocks

Thirty three-yellow chicken chicks were hatched in Changping chicken factory in Beijing, and they were divided into three groups of 10 chicks in each group.

The first group: 10 chicks were injected with Newcastle disease virus NDV Lasota E ₄ 100 μl (1:200) 5 days later, and the chicks showed mild fowl plague symptoms after 4 to 5 days, and at this time, 20 μg of pVAX1-LasotaF DNA vaccine was injected into each chick; The symptoms of Newcastle disease in chickens weakened in the second to three days after vaccination; after the sixth day, 2 chickens died of the disease, and the symptoms of the other 8 chickens did not change significantly; after the tenth day, 5 chickens recovered, and the rest 5 died.

Treatment experiments show that pVAX1-LasotaF DNA vaccine has a certain effect on the treatment of fowl plague. For chicks within one week after hatching, inject pVAX1-LasotaF DNA vaccine after strong virus challenge and infection of fowl plague, and the curative effect reaches 50%.

The second group: 10 chicks were injected with Newcastle disease virus NDV Lasota E ₄ 100 μl (1:100) 15 days later, and the chicks showed symptoms of chicken plague after 4 to 5 days. 3 to 4 days after the vaccination, the symptoms of Newcastle disease in the chickens were significantly weakened; on the seventh day, except for 2 chickens died, the symptoms of the other 8 chickens were weakened; after the tenth day, 7 chickens recovered completely, and the remaining 3 died ; One week after recovery, the 7 chickens were re-injected with 100 μl (1:100) of Newcastle disease virus NDV Lasota E _4. After 90 days, the 7 chickens were healthy and active, and no symptoms of fowl plague appeared.

The second group of treatment experiments showed that the pVAX1-LasotaF DNA vaccine had a curative effect of 70% on chickens infected with fowl plague by strong virus challenge 15 days after hatching, and it also had a good preventive effect while treating fowl plague.

The third group: 10 chicks were injected with Newcastle disease virus NDV Lasota E ₄ 100μl (1:100) 30 days later. After 4 to 5 days, the chickens showed symptoms of chicken plague. After 3 to 4 days, the symptoms of Newcastle disease in chickens were obviously weakened; on the eighth day, except for 2 chickens died, the symptoms of other 8 chickens were weakened; after the tenth day, 8 chickens recovered completely; the recovered 8 chickens One week later, 100 μl (1:100) of Newcastle disease virus NDV Lasota E ₄ was injected again, and 8 chickens were healthy and lively after 90 days, and none of them showed symptoms of fowl plague.

The third group of treatment experiments showed that pVAX1-LasotaF DNA vaccine had an 80% curative effect on chicks infected with fowl plague by strong virus challenge 30 days after hatching.

These three groups of experiments show that the curative effect of pVAX1-LasotaF DNA vaccine has a certain correlation with the age of chickens.

Sequence Listing

SEQUENCE LISTING

<110> Institute of Zoology, Chinese Academy of Sciences

<120> A DNA vaccine for Newcastle disease and its use

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<170>PatentIn version 3.1

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gaa 1683

Claims (6)

1, a kind of DNA vaccine of Newcastle disease, this vaccine is made up of NDV-LasotaF gene and eukaryotic expression vector pVAX1, it is characterized in that: described NDV-LasotaF gene is the pathogenic determinant gene of NDV virus, and it is the gene of NDV virus The 4541st to 6223rd nucleotides in the Lasota E ₄ gene have the nucleotide sequence shown below: aagatgggct ccagaccttc taccaagaac ccagcaccta tgatgctgac tatccgggtt 60 gcgctggtac tgagttgcat ctgtccggca aactccattg atggcaggcc tcttgcagct 120 gcaggaattg tggttacagg agacaaagcc gtcaacatat acacctcatc ccagacagga 180 tcaatcatag ttaagctcct cccgaatctg cccaaggata aggaggcatg tgcgaaagcc 240 cccttggatg catacaacag gacattgacc actttgctca ccccccttgg tgactctatc 300 cgtaggatac aagagtctgt gactacatct gcaccccgga gacaggggcg ccttataggc 360 gccattattg gcggtgtggc tcttggggtt gcaactgccg cacaaataac agcggccgca 420 gctctgatac aagccaaaca aaatgctgcc aacatcctcc gacttaaaga gagcattgcc 480 gcaaccaatg aggctgtgca tgaggtcact gacggattat cgcaactagc agtggcagtt 540 gggaagatgc agcagtttgt taatgaccaa tttaataaaa cagctcagga attagactgc 600 atcaaaattg cacagcaagt tggtgtagag ctcaacctgt acctaaccga attgactaca 660 gtattcggac cacaaatcac ttcacctgct ttaaacaagc tgactattca ggcactttac 720 aatctagctg gtggaaatat ggattactta ttgactaagt taggtgtagg gaacaatcaa 780 ctcagctcat taatcggtag cggcttaatc accggtaacc ctattctata cgactcacag 840 actcaactct tgggtataca ggtaactcta ccttcagtcg ggaacctaaa taatatgcgt 900 gccacctact tggaaacctt atccgtaagc acaaccaggg gatttgcctc ggcacttgtc 960 cccaaagtgg tgacacaggt cggttctgtg atagaagaac ttgacacctc atactgtata 1020 gaaactgact tagatttata ttgtacaaga atagtaacgt tccctatgtc ccctggtatt 1080 tattcctgct tgagcggcaa tacgtcggcc tgtatgtact caaagaccga aggcgcactt 1140 actacaccat acatgactat caaaggttca gtcatcgcca actgcaagat gacaacatgt 1200 agatgtgtaa accccccggg tatcatatcg caaaactatg gagaagccgt gtctctccta 1260 gatccacaat catgcaatgt tttatcctta ggcgccataa ctttaaggct cagtggggaa 1320 ttcgatgtaa cttatcagaa gaatatctca atacaagatt ctcaagtaat aataacaggc 1380 aatcttgata tctcaactga gcttgggaat gtcaacaact cgatcagtaa tgctttgaat 1440 aagttagagg aaagcaacag aaaactagac aaagtcaatg tcaaactgac tagcacatct 1500 gctctcatta cctatatcgt tttgactatc atatctcttg tttttggtat acttagcctg 1560 attctagcat gctacctaat gtacaagcaa aaggcgcaac aaaagacctt attatggctt 1620 gggaataata ctctagatca gatgagagcc actacaaaaa tgtgaacaca gatgaggaac 1680 gaa 1683 2. The construction of a DNA vaccine of Newcastle disease according to claim 1, comprising the steps of: 1) Extraction and purification of NDV Lasota virus total RNA: inject NDV Lasota E ₄ virus strain into chicken embryos hatched for 14 days, after the death of chicken embryos, collect allantoic fluid, and extract NDV Lasota virus total RNA according to conventional methods; 2) design specific primers: design a pair of specific primers according to the Lasota virus F protein gene sequence analysis, Upstream primer: 5′-AAGATGGGCTCCAGACCTTCTACC-3′; Downstream primer: 5′-CCTTCGTTCCTCATCTGTGTTCAC-3′; 3) Cloning the full-length cDNA gene sequence of NDV LasotaF by temperature drop and reverse transcription-polymerase chain amplification method; 4) using the LasotaF amplified in step 3) to recover and purify the LasotaF cDNA fragment using the Wizard® PCR Preps.DNA purification system kit; 5) The full-length cDNA fragment of LasotaF was recombined into pVAX1 eukaryotic expression plasmid to construct a DNA vaccine against Newcastle disease—pVAX1-LasotaF DNA vaccine against NDV. 3. The construction of the DNA vaccine of Newcastle disease as claimed in claim 2, characterized in that: the temperature drop reverse transcription-polymerase chain amplification method in the step 3) comprises: the reverse transcription reaction is reacted at 48° C. for 45 minutes , then inactivate at 95°C for 5 minutes, and start the amplification reaction of the temperature drop reverse transcription-polymerase chain amplification method: add polymerase at the first denaturation step, the denaturation temperature is 94°C, last for 1 minute, and anneal The temperature starts at 68°C and lasts for 1 minute, then drops to 56°C at a rate of 1°C every four cycles, and lasts for 1 minute. 15 cycles of amplification; final extension at 72°C for 10 minutes. 4. The construction of DNA vaccine for Newcastle disease according to claim 2, characterized in that: the eukaryotic expression vector in step 5) is pcDNA3.1. 5. The application of the DNA vaccine of Newcastle disease according to claim 1 in the preparation of medicine for preventing Newcastle disease in chickens. 6. The application of the DNA vaccine of Newcastle disease according to claim 1 in the preparation of medicine for treating chicken Newcastle disease.

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