Porcine reproductive and respiratory syndrome virus recombinant vaccine strain PRRSV-SP as well as preparation method and application thereof

A PRRSV-SP, respiratory syndrome technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problem that the precise transformation of the virus cannot be carried out, affect the research and development of high-efficiency broad-spectrum vaccines, and hinder the interaction between the virus and the host. role research, etc.

Active Publication Date: 2020-03-24
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The whole genome sequencing and comparison of PRRSV vaccine strain SP has been completed, but its infectious clone has not been established before this work, which greatly hinders the research on the interaction between the virus and the host, making it impossible to carry out precise artificial transformation of the virus. This will seriously affect the development of a new generation of high-efficiency broad-spectrum vaccines

Method used

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  • Porcine reproductive and respiratory syndrome virus recombinant vaccine strain PRRSV-SP as well as preparation method and application thereof
  • Porcine reproductive and respiratory syndrome virus recombinant vaccine strain PRRSV-SP as well as preparation method and application thereof
  • Porcine reproductive and respiratory syndrome virus recombinant vaccine strain PRRSV-SP as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] The construction of the infectious clone of embodiment 1 porcine reproductive and respiratory syndrome virus recombinant vaccine strain PRRSV-SP

[0108] At first, construct 4 plasmids, these plasmids comprise 5 fragments A, B1, B2, C and D (B1 and B2 are the two parts of Bm fragment, and Bm fragment contains BsmBI enzyme internally across whole recombinant vaccine strain PRRSV-SP genome cleavage site, so it is cleaved into B1 and B2 fragments). Such as figure 1 shown. A BsmBI and NotI restriction site and a T7 promoter were respectively introduced at the beginning of fragment A, and T7 RNA polymerase was used to perform in vitro transcription. The specific steps ( figure 1 )as follows:

[0109] (1) Infect Marc-145 cells (purchased from ATCC, USA) with commercialized vaccine strain SP (purchased from Schering-Plough Animal Health Company), and the inoculation method is shown in the literature "Lv Yonggang, et al. Different inoculation methods for pigs Comparing the ...

Embodiment 2

[0125] Example 2 Construction of chimeric strain vSP-Hub2 infectious clone

[0126] First, construct 6 plasmids containing 7 fragments (A, B1, B2, C, D1, Hub2, N) spanning the whole chimeric strain vSP-Hub2 genome, such as image 3 shown. A BsmBI and NotI restriction site and a T7 promoter were respectively introduced at the beginning of fragment A. Utilize T7RNA polymerase to carry out in vitro transcription, specific steps ( image 3 )as follows:

[0127] (1) See Example 1 for the construction of plasmids pTOPO-SP-A, pTOPO-SP-Bm, and pXL-SP-C.

[0128] (2) Taking the full-length cDNA of the recombinant vaccine strain PRRSV-SP obtained in Example 1 as a template, carry out polymerase chain reaction by using specific primers (Table 4) and a high-fidelity DNA polymerase (High Fidelity PCR) kit , amplify and obtain the chimeric strain vSP-Hub2 nucleotide fragments D1 (10576-13902nt) and N (15004-15520nt), and then clone them into the corresponding plasmid pGEM respectively t...

Embodiment 3

[0141] Example 3 Recombinant Virus Sequencing Verification

[0142] (1) Verification of recombinant vaccine strain PRRSV-SP.

[0143] Infect Marc-145 or BHK21 cells with the recombinant vaccine strain PRRSV-SP obtained by electroporation in step (6) of Example 1. See the document "Lv Yonggang, et al. Effects of Different Inoculation Methods on the Proliferation of Porcine Reproductive and Respiratory Syndrome Virus" for the inoculation method Comparison." After inoculation, the culture was continued for 3 to 4 days, and when more than 50% of the cells were apoptotic, they were stored in a -80°C refrigerator. Freeze-thaw the cells together with the medium three times, centrifuge, take the supernatant, and repeat the infection twice. The supernatant after the third infection was collected, which was the recombinant vaccine strain PRRSV-SP, and was frozen. Total RNA was extracted from infected cells. Using this RNA as a template, RT-PCR was performed with specific primers (PRR...

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Abstract

The invention provides a porcine reproductive and respiratory syndrome virus recombinant vaccine strain PRRSV-SP as well as a preparation method and application thereof. The T3448A site of a vaccine strain SP is mutated to obtain the recombinant vaccine strain PRRSV-SP. According to the method, genes ORF5 and ORF6 of main structural proteins GP5 and M of a highly pathogenic porcine reproductive and respiratory syndrome virus Hubei isolate strain HUB2 are put into corresponding coding regions of the recombinant vaccine strain PRRSV-SP for expression, so that a chimeric toxic strain vSP-Hub2 isobtained. The recombinant vaccine strain PRRSV-SP and the chimeric toxic strain vSP-Hub2 provided by the invention have no obvious pathogenicity, can be used as the basic material for further studyingthe mutation mechanism of an attenuated vaccine and a PRRSV toxic strain, can construct a novel attenuated vaccine strain through an infectious cloning system, and can be used for preparing a novel,efficient and broad-spectrum vaccine against PRRSV homologous and heterologous toxic strains.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain PRRSV-SP and a preparation method and application thereof. Background technique [0002] Porcine Reproductive and Respiratory Syndrome (PRRS) is a viral infectious disease caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection. high rate features. PRRSV infection causes huge economic losses to the global swine industry every year. At present, inactivated vaccines and attenuated vaccines are mainly used to control PRRSV infection. However, the inactivated vaccine has poor immune effect on heterologous strains, the inoculation dose is large, the number of immunizations is high, and the cost is high. It takes a certain time to develop immunity; The strain effect was not significant. Therefore, it is particularly important to develop a vaccine with high efficiency and broad-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N7/01C12N15/40C12N15/85A61K39/12A61P31/14
CPCC12N7/00C07K14/005C12N15/85A61K39/12A61P31/14C12N2770/10021C12N2770/10022A61K2039/5254C12N2770/10034A61K2039/552
Inventor 刘定祥黄梅冯涛声代过
Owner SOUTH CHINA AGRI UNIV
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