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Newcastle disease virus heat resistant live vaccine vector system and application thereof

A technology of Newcastle disease virus and vector system, which is applied in the field of Newcastle disease virus heat-resistant live vaccine vector system and live virus vector system to achieve the effects of convenient use, high infection rate of the same group, and reduced transportation cost

Active Publication Date: 2014-09-24
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example: the document "Newcastle Disease LaSota Vaccine Strain Reverse Genetic Operating System and Its Application" with patent number 200510097997.8 discloses a non-heat-resistant live vaccine carrier based on Newcastle disease virus LaSota vaccine strain, but does not involve heat-resistant live vaccine carrier ; Patent No. 200610075781.6 document "Recombinant Newcastle Disease LaSota Attenuated Vaccine Strain Expressing Avian Influenza Virus H5 Subtype HA Protein" discloses the avian influenza and Newcastle disease dual genetically engineered live vaccine constructed with the Newcastle Disease LaSota vaccine strain carrier, also Heat resistant live vaccine vectors not involved

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  • Newcastle disease virus heat resistant live vaccine vector system and application thereof
  • Newcastle disease virus heat resistant live vaccine vector system and application thereof
  • Newcastle disease virus heat resistant live vaccine vector system and application thereof

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Embodiment 1

[0034] Construction of Newcastle Disease Virus Heat-resistant Live Vaccine Vector System

[0035] The heat-resistant live vaccine vector system of Newcastle disease virus mainly consists of a transcription plasmid capable of expressing the full-length genome sequence of a heat-resistant strain of Newcastle disease virus, and a tripartite protein capable of expressing nucleoprotein, phosphoprotein and large polymerase protein of a heat-resistant strain of Newcastle disease virus respectively. The construction of the transcription plasmid and the three auxiliary plasmids is mainly described below.

[0036] 1. Construction of transcriptional plasmids

[0037] Strategies for constructing transcription plasmids such as figure 1 As shown, firstly, the whole genome sequence is amplified by fragments, which are divided into 7 fragments A-G to amplify the whole genome sequence. Wherein, a T7 promoter is added upstream of the A segment, and a hepatitis D virus ribozyme sequence an...

Embodiment 2

[0049] Artificially Obtaining Recombinant Newcastle Disease Virus Heat-resistant Strains Using Newcastle Disease Virus Heat-resistant Live Vaccine Vector System

[0050] Co-transfect the transcriptional plasmid and helper plasmid in the Newcastle disease virus heat-resistant live vaccine vector system into a host cell with a replication-permitted Newcastle disease virus heat-resistant strain, culture the transfected host cells, harvest the cell fluid, and inoculate the host cells or SPF The heat-resistant strain of recombinant Newcastle disease virus can be artificially obtained by subculture and multiplication on chicken embryos.

[0051] 1. Artificial Rescue of Recombinant Newcastle Disease Virus Heat-resistant Strains

[0052] Adjust the state of BHK-21 cells to the best, transfer them to 6-well plates, and the cells grow to 80-90% after 1 day of passage; the cell nutrient solution is replaced with DMEM medium with 2% newborn bovine serum; the vaccinia virus vTF7-3 Inf...

Embodiment 3

[0058] Biological Characteristic Test of Recombinant Newcastle Disease Virus Heat-resistant Strain

[0059] After artificially obtaining the recombinant Newcastle disease virus heat-resistant strain, in order to verify whether it has similar biological characteristics to the TS09-C parent strain, the biological characteristics test of the recombinant virus was carried out.

[0060] 1. Cell Growth Curve Test of Recombinant Newcastle Disease Virus Heat-resistant Strain

[0061] The diluted virus liquid was inoculated into BHK-21 cells that had grown into a dense monolayer, and the supernatant of the culture liquid was drawn at 6, 12, 24, 48, 72, and 96 hours after infection, and the virus titer was measured. The method of determination is as follows: the virus liquid is serially diluted 10 times, and the dilution range is 10 -1 to 10 -8 , 100ml of each dilution was added to a 96-well plate containing a monolayer of BHK-21 cells, and each dilution was repeated 5 times. Aft...

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Abstract

The invention belongs to the field of virus genetic operation, and in particular to a Newcastle disease virus (NDV) heat resistant live vaccine vector system and application thereof. The Newcastle disease virus (NDV) heat resistant live vaccine vector system comprises a) a transcription plasmid, b) three auxiliary plasmids and c) host cells. The transcription plasmid is obtained by cloning genomic full-length cDNA of a NDV heat resistant vaccine strain to pBR322 vector; and the three auxiliary plasmids are obtained by cloning nucleoprotein, phosphoprotein and large polymerase protein gene of the NDV heat resistant vaccine strain to pcDNA3.1 vector. The artificial recombinant Newcastle disease virus has the characteristic of heat resistance, and the Newcastle disease virus (NDV) heat resistant live vaccine vector system is established for the first time. The artificial recombinant Newcastle disease virus has great application prospect in the aspects of research and development of multiple (multivalent) heat resistant genetic engineering live vaccines of the NDV, avian influenza and other major diseases of poultry, research on virus heat-resistant mechanism, and the like.

Description

technical field [0001] The invention belongs to the field of virus genetic manipulation, and relates to a live virus carrier system, more specifically, the invention relates to a Newcastle disease virus heat-resistant live vaccine carrier system and its application. Background technique [0002] Newcastle Disease (ND), also known as Asian chicken plague, is a highly contagious and devastating disease caused by Newcastle Disease Virus (NDV). OIE) is listed as one of the two Class A poultry diseases (the other is avian influenza), which belongs to the national compulsory immunization disease. Since the first outbreak of Newcastle disease in Java, Indonesia and New Town, England in 1926, it has been prevalent all over the world except Oceania, causing huge economic losses to the world. In my country, Newcastle disease is also one of the most serious poultry diseases, and the disease has always been prevalent in many areas of our country. The control of Newcastle disease is ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N7/01C12R1/93
CPCC12N7/00C12N2760/18143C12N15/86C12N2760/18151C12N2760/18121C12N2760/18122
Inventor 温国元邵华斌杨峻王红琳罗青平张蓉蓉艾地云罗玲商雨郭静陈晨
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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