Porcine circovirus gene modified attenuated strain as well as construction method and application thereof

A porcine circovirus and genetic modification technology, applied in the field of biological genetic engineering, can solve the problems of restricted transcription or translation, inconvenient research on viral etiology and vaccine development, and low virus susceptibility.

Pending Publication Date: 2021-09-07
天康制药股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the genome structure of the virus itself, PCV2 itself does not encode replicase, and mainly relies on the enzymes and proteins expressed in the S phase of cell mitosis for rolling circle replication, which leads to the restriction of transcription or translation of related proteins of the virus and affects the efficiency of virion assembly; This is also the reason for the low viral titer
In addition, studies have shown that the susceptibility of the virus is not strong, the virus-infected cells do not produce cytopathic effect (CPE), and only 30% of the cells can infect, which brings inconvenience to the etiology research of the virus and the development of vaccines

Method used

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  • Porcine circovirus gene modified attenuated strain as well as construction method and application thereof
  • Porcine circovirus gene modified attenuated strain as well as construction method and application thereof
  • Porcine circovirus gene modified attenuated strain as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1. Construction of Porcine Ring Virus Gene Reconstruction Weaki Line (PCV2- △ CAP-GFP)

[0083] According to figure 1 The flow shown, constructs a pig ring virus type 2 modified weaker strain.

[0084] (1) Getting of the target fragment

[0085] According to the pathogens and epidemiological surveys of the current porcine circular viral disease, the current epidemic strain sequence was screened by the current epidemic strain virus all genome sequence.

[0086] The determined strain sequence is synthesized (including the inserted green fluorescent protein sequence), and the gene sequence is shown in SEQ ID NO.1, and inserted into the cloned carrier PMD18T, see figure 2 Sequence design. In addition, according to the PCV2 gene sequence, a pair of primers is designed using Primer5.0:

[0087] Primers 1: 5'-gaaggggccagttcgtcac-3 '(SEQ ID No.5);

[0088] Primers 2: 5'-tccaattggcgcgatgccctg-3 '(SEQ ID No.6).

[0089] This pair primer is used to identify ORF1 and GFP genes,...

Embodiment 2

[0149] Example 2 Porcine Circovirus Type 2 Cap protein recombinant plasmid (pcDNA3.1-PCV2-Cap) Construction of Embodiment

[0150] (1) PCV2 ORF2 gene synthesis

[0151] Existing in GenBank PCV2 genomic sequence, taken ORF2 gene by sequence analysis and codon optimized to obtain optimized ORF2 sequences, and inserted into BamH I and Not I restriction sites at both ends of the sequence, sequence by the company Shanghai Bioengineering synthesis.

[0152] (2) The recombinant plasmid (pcDNA3.1-PCV2-Cap) Construction

[0153] Recovery synthetic sequence: The recombinant plasmid by restriction enzymes BamH I and Not I digestion on a 1% agarose gel electrophoresis after digestion product recovered, the target band was cut into eppendorf tubes, production company Promga the recovered DNA fragment was recovered kit. The specific operation is as follows:

[0154] First the mass of gel was weighed accurately, a 1: 1 ratio was added to the sol (in a volume of 100μL sol gel solution is added 10...

Embodiment 3

[0168] Genetically engineered attenuated strain of type Example 3. Construction of infectious porcine circovirus type 2 embodiment

[0169] Since the transformation of porcine circovirus type gene attenuated strain (PCV2- △ Cap-GFP) non-infectious, pcDNA3.1-PCV2-Cap expression vector obtained in (or PCV2- △ Cap-GFP) were transfected comprising PCV2- △ Cap-GFP (or pcDNA3.1-PCV2-Cap expression vector) in PK-15 cells, the expression product by means of processing and modification of cells in the cell, complete the assembly of the virus, genetically modified so screened with infectious swine circovirus attenuated strain, named m 1 PCV2- △ Cap-GFP. The specific operation is as follows:

[0170] With pSK-PCV2- △ Cap-GFP transfection in logarithmic growth phase of PK-15 cells. After 24 hours, the cells were aspirated, the cells were washed three times with basal cell culture. Liposome transfection method pcDNA3.1-PCV2-Cap transfected cells described above. After 48 hours the cells were col...

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Abstract

The invention provides a porcine circovirus gene modified attenuated strain as well as a construction method and application thereof, and relates to the technical field of biological gene engineering. The genome of the attenuated strain is subjected to codon mutation or codon optimization or gene truncation and the like, so that the gene does not encode or encode part of capsid protein (Cap protein) finally, and a fluorescent protein gene is inserted into a nucleotide sequence deleted by gene truncation. On the basis, a genome of the gene modified attenuated strain is transfected into a donor cell capable of expressing capsid protein, and a complete virion capable of infecting a host cell is finally self-assembled by utilizing a replication system of the cell and the genome of the virus.

Description

Technical field [0001] The present invention relates to the field of genetic engineering, particularly to a porcine circovirus type genetically engineered attenuated strain, construction method and its application. Background technique [0002] Porcine circovirus type 2 (Porcine circovirus type 2, PCV2) belonging to the virus family ring, nonenveloped, a diameter of about 12-23nm, having a single-stranded circular DNA genome of approximately 1.7kb, porcine circovirus associated disease ( PCV2-associated disease, PCVAD) main pathogens, to the pig industry has caused tremendous economic losses. [0003] PCV2 genome has two major open reading frame (ORF). ORFl is the largest, is a Rep protein-encoding gene, a gene encoding a viral replication-associated protein Rep two Rep and Rep ', Rep and Rep' sequence capable of binding to a particular gene region located between the origin of replication (ori) within, for PCV2 roll start circle replication. Rep proteins can not start a single c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N15/70C12N15/85C12N1/21C12N5/10C12Q1/70A61K39/12A61P31/20C12R1/19
CPCC12N7/00C07K14/005C12N15/70C12N15/85C12Q1/70A61K39/12A61P31/20C12N2750/10021C12N2750/10022C12N2750/10034A61K2039/523
Inventor 贺笋张伟潘毅平李俊辉程兰玲候凤
Owner 天康制药股份有限公司
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