Novel pestilence DNA vaccine and its construction and application

A technology of DNA vaccine and Newcastle disease, which is applied in the field of DNA vaccine and its construction and application, can solve the problems of low antibody titer, weak preventive effect, and poor immune effect, and achieve strong immune effect, safe genetic immunity, and convenient operation Effect

Inactive Publication Date: 2006-02-15
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Abstract
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Problems solved by technology

[0005] The purpose of the present invention is to overcome existing traditional inoculation NDV virus live attenuated vaccine or inactivated bacteria as preventive vaccine, the antibody titer that produces is low, immune effect is poor, easily produces immune evasion, has only weak preventive effect and no treatment Function and other defects of Newcastle disease, thereby providing a pVAX1-LasotaHN DNA vaccine capable of preventing and treating Newcastle disease

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  • Novel pestilence DNA vaccine and its construction and application

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Embodiment 1

[0024] Embodiment 1. Newcastle disease DNA vaccine of the present invention---the construction of pVAX1-LasotaHN DNA vaccine

[0025] The construction steps of pVAX1-LasotaHN DNA vaccine of the present invention are as follows:

[0026] 1) Extraction and purification of NDV virus (Virus) total RNA:

[0027] The RNAgents Total RNA Isolation System kit of Promega Company was used to extract the total RNA of NDV Lasota virus, and its specific operation was to use the NDV Lasota E 4 The virus strain was injected into chicken embryos incubated for 14 days. After the death of the chicken embryos was observed under the light, 20ml of allantoic fluid was collected, and the collected allantoic fluid was centrifuged at 700rpm for 15 minutes; the supernatant was added to 20ml of 16wt% PEG-0.525MNaCl, ice bath for 1 hour; centrifuge at 10,000rpm for 5 minutes, precipitate virus particles at 4°C; dissolve the virus pellet with 800μl denaturation solution, transfer to 1.5ml EP tube; add 80...

Embodiment 2

[0035] Example 2. Identification of exogenous LasotaHN gene in vitro transient expression and expression content of pVAX1-LasotaHN DNA vaccine

[0036] In vitro mRNA expression of LasotaHN gene: establishment of transient expression system for HeLa cells in vitro, the eukaryotic expression plasmid pVAX1-LasotaHN 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 transfected HeLa cells collected at 36 hours was extracted, and the expression of LasotaHN gene in vitro was analyzed by RT-PCR method. The LasotaHN cDNA band was amplified by RT-PCR, and the results showed that the pVAX1-LasotaHN 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 157% higher.

[0037] In vitro protein expression of exo...

Embodiment 3

[0038] Example 3. Identification of exogenous LasotaHN gene expression and expression content of pVAX1-LasotaHN DNA vaccine in vivo

[0039] In vivo expression detection of exogenous LasotaHN gene: Three yellow chickens incubated in Beijing Changping chicken factory were randomly divided into 2 groups, each with 18 chickens, one group was the experimental group, and 20 μg of the pVAX1-LasotaHN plasmid constructed in Example 1 was injected intramuscularly into the three yellow chickens DNA; the other group was the control group, and 20 μg pVAX1 empty plasmid DNA was injected intramuscularly into the three yellow chickens. Three weeks after the injection of the plasmid DNA, the muscle, liver and spleen tissues of the three-yellow chicken immunized with pVAX1-LasotaHN were extracted, and the total RNA of the muscle tissue, liver and spleen tissue collected from the three-yellow chicken immunized with pVAX1-LasotaHN was extracted, and the exogenous LasotaHN gene was detected in the...

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Abstract

The invention relates to an animal epidemic DNA vaccine, which is consisted of NDV-Laso610taHN gene and eukayon expression vector. The preparing process of the DNA vaccine includes following steps: extracting and purifying NDV Lasota virus complete RNA, designing specific primer, and obtaining complete cDNA gene sequence by TDRT-PCR amplify cloning, recombining purified LasotaHN complete cDNA sequence to PCR 3.1 eukayon expression vector. The small dosage of the nucleic vaccine can leads to high immune competence, in addition its safe, stable, convenient and have longer acting time.

Description

technical field [0001] The invention relates to a DNA vaccine and its construction and application, in particular to a Newcastle disease virus pVAX1-LasotaHN DNA vaccine and its construction and application. technical background [0002] In 1990, Dr. John Woffer and Dr. Feigner accidentally discovered that plasmid DNA can be transformed and expressed in muscle cells, and speculated that injecting DNA into the body may produce an immune response. In 1992, the Robinson Laboratory, Johoston Laboratory, and David Laboratory in the United States simultaneously developed and researched the prototype of the DNA vaccine, and reported it at the vaccinology new progress conference held in September 1992. In 1992, Tang reported in Nature that the plasmid DNA containing the human growth hormone gene was introduced into mice, and specific anti-human growth hormone antibodies were detected in the serum of the mice. Since then, the new technology of DNA vaccines has come out, and has immed...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/17A61K48/00A61P31/14
Inventor 彭景楩陈云杨颖孙泉红王金玲
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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