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Full-length infectious DNA (deoxyribonucleic acid) clone of porcine parvoviruses, method for constructing full-length infectious DNA and application thereof

A parvovirus and infectious technology, applied in the field of the full-length infectious clone of porcine parvovirus and its construction, the virus infectious clone and its construction, and the establishment of the reverse genetics operating system of porcine parvovirus, which can solve the difficult problem. Duplication, no virus rescue pathogenic mechanism, influence on the establishment of reverse genetics operation platform, etc.

Inactive Publication Date: 2018-08-17
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although PPV has been around for more than 50 years, few examples of full-length clones of PPV have been studied, especially full-length infectious clones
At present, scholars at home and abroad have used different methods to sequence the PPV genome, but some methods are more complicated and not easy to repeat, and some amplifications cannot accurately obtain complex structural sequences at both ends.
Although it has been reported that most of the viral genome of PPV can be cloned and sequenced, there is no research on the rescue of the virus and its molecular pathogenic mechanism.
Due to the special genome structure of autonomously replicating parvovirus, it is difficult to clone its full-length genome, which seriously affects the establishment of its reverse genetics operation platform
So far, there have been no research reports on the establishment of PPV reverse genetic manipulation platform, which has greatly affected the in-depth study of its molecular pathogenic mechanism and its interaction with host cells
[0011] At present, in the research and application of porcine parvovirus, the pathogenic mechanism of PPV and the pathogenicity of the main antigen protein of the virus are not completely clear, and this problem still needs to be solved urgently

Method used

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  • Full-length infectious DNA (deoxyribonucleic acid) clone of porcine parvoviruses, method for constructing full-length infectious DNA and application thereof
  • Full-length infectious DNA (deoxyribonucleic acid) clone of porcine parvoviruses, method for constructing full-length infectious DNA and application thereof
  • Full-length infectious DNA (deoxyribonucleic acid) clone of porcine parvoviruses, method for constructing full-length infectious DNA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction of mutant porcine parvovirus vector

[0039] (1) Transformation vector pBluscript II SK(+) (purchased from Bao Biological Engineering Co., Ltd.):

[0040] Add two restriction sites, namely Afe I and Stu I, between the Kpn I and Sac I restriction sites in the vector pBluscript II SK(+), to obtain a new multi-cloning restriction site Kpn I-Afe I -Stu I-Sac I, the modified vector is named pBSK-Modified;

[0041] (2) Synthesis of full-length infectious DNA of porcine parvovirus

[0042] Synthesize a 114bp fragment (fragment I) at the 3' end of the PPV genome, and clone it into the transformed pBSK-Modified vector through the Kpn I / Afe I restriction site to obtain pBSK-FY1; synthesize PPV genome 3 linked to fragment I The fragment (fragment II) with a length of 65 bp at the 'end was ligated with fragment I by Infusion cloning technology to obtain pBSK-FY; the vector containing the 3' end fragment of the PPV genome was linearized with Stu I; it was de...

Embodiment 2

[0045] The preparation of embodiment 2 rescue virus

[0046] PK-15 cells were inoculated and placed in a six-well plate, and transfected when the cell density was 80%-90%. 5ug of plasmid pPPV and 10ul of Lipofectamine 2000 (purchased from Invitrogen) transfection reagent were added to each well, shaken and mixed in 500ul of Opti-MEM, and transfected according to the instructions of the transfection reagent. And observe the cytopathic changes day by day. Cytopathic changes (CPE) appeared on the third day. On the fifth day, the cell transfection was collected and frozen and thawed three times and blindly passed. The virus cell culture of the seventh generation was harvested and frozen and thawed repeatedly three times to obtain the rescued virus pPPV, which was stored at -70°C for future use.

Embodiment 3

[0047] The identification of embodiment 3 rescue virus

[0048] The rescue virus prepared in Example 2 was tested for titer with the parental virus, and the results showed that there was no significant difference between the parental virus PPV and the rescued virus pPPV TCID50 and hemagglutination titer, and the multi-step growth curve was measured at the same time, and the results showed that 8h after infection After the start of replication, the replication peak was reached at 60h, and the difference between the wild virus and the rescued virus was not significant ( image 3 ).

[0049] The results show that the present invention uses the reverse genetic operating system to obtain a full-length infectious clone with similar growth characteristics to the wild virus, and at the same time shows that the introduction of biogenetic markers into the virus genome does not affect the growth of the entire virus. The genetic marker EcoRv introduced into the full-length infectious clo...

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Abstract

The invention discloses full-length infectious DNA (deoxyribonucleic acid) clone of porcine parvoviruses, a method for constructing the full-length infectious DNA and application thereof. Nucleotide sequences of the full-length infectious DNA of the porcine parvoviruses are shown as SEQ ID NO.1. The full-length infectious DNA clone, the method and the application have the advantages that rescue viruses with growth characteristics similar to growth characteristics of wild PPV (porcine parvoviruses) can be rescued by the aid of the full-length infectious DNA, and genetic markers EcoRv are introduced during full-length infectious clone, still can stably exist even after passage is carried out, and can be used as reliable genetic markers for identifying the wild porcine parvoviruses and the rescue viruses; a porcine parvovirus reverse genetics operating system is further established and can be used for analyzing the virulence of porcine parvovirus proteins, and novel inactivated vaccine orattenuated vaccine can be correspondingly prepared; platforms can be provided to the aspects such as research on porcine parvovirus gene structures and functions and research on the pathogenicity, and a foundation can be laid for research on vaccine and the like for porcine parvovirus diseases.

Description

technical field [0001] The invention relates to a virus infectious clone and a construction method thereof, in particular to a full-length porcine parvovirus infectious clone and a construction method thereof, and also relates to the establishment of a porcine parvovirus reverse genetics operating system. The invention belongs to the field of biotechnology. Background technique [0002] Porcine parvovirus (PPV) is a member of the Parvovirus genus in the family Parvoviridae, and it is a pathogen that causes reproductive disorders in sows. In 1966, Mayr discovered PPV while performing tissue culture of classical swine fever virus. Since Cartwright and Huck first isolated porcine parvovirus from the organs of aborted pigs in the UK in 1967, it has been found in Belgium (1967), Germany (1968), the United States (1972), Japan (1972), and the Netherlands (1972). , Australia (1973), Finland (1979), France (1977) and Canada (1978) and other countries have successively reported. I...

Claims

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Application Information

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IPC IPC(8): C12N15/35C12N15/63C12N15/66C12N5/10C12N7/00
CPCC07K14/005C12N7/00C12N15/63C12N15/66C12N2750/14021C12N2750/14022
Inventor 崔尚金张玲玲
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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