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Gene sequence for effectively inhibiting Type II PRRSV (porcine reproductive and respiratory syndrome virus) infection and application thereof

A technology of gene sequence and base sequence, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of increased antibody dependence, fast mutation of virus strains, etc., and achieve the effect of reducing huge losses

Active Publication Date: 2018-12-21
重庆吉棠生物技术研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the rapid mutation of PRRSV strains; there is an antibody-dependent enhancement phenomenon between PRRSV and the host; these factors have led to the inability of vaccine immune protection mechanisms to effectively provide immune protection to pigs worldwide

Method used

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  • Gene sequence for effectively inhibiting Type II PRRSV (porcine reproductive and respiratory syndrome virus) infection and application thereof
  • Gene sequence for effectively inhibiting Type II PRRSV (porcine reproductive and respiratory syndrome virus) infection and application thereof
  • Gene sequence for effectively inhibiting Type II PRRSV (porcine reproductive and respiratory syndrome virus) infection and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Preparation of gene sequence capable of effectively inhibiting type II PRRSV infection as shown in SEQ ID 1

[0025] 1) Call out the porcine CD163 gene sequence in the NCBI gene bank;

[0026] 2) Use the Primer5 software to design the upstream primer F1 and downstream primer R1 with base sequences as shown in SEQ ID 3 and SEQ ID 4 respectively;

[0027] 3) Utilize the PCR in vitro amplification method to obtain the target gene sequence whose base sequence is shown in SEQ ID 1;

Embodiment 2

[0029] 1-1. Design of sgRNA sequence and construction of PX330 expression vector

[0030] Two sgRNA sequences targeting porcine CD163 exon7 were designed and synthesized. The sgRNA sequence designed above was synthesized; the DNA sequences of the four single-stranded sgRNAs were annealed to form two oligonucleotide chains targeting different sites of the porcine CD163 exon7 oligonucleotides; and then the oligonucleotides were connected into the PX330 plasmid vector.

[0031] The sequences of these two sgRNAs and the sequences of their action sites are:

[0032] SgRNA-1 sequence: 5-GGAAACCCAGGCTGGTTGGA-3

[0033] The sequence of the sgRNA-1 action site: 5-TCCAACCAGCCTGGGTTTCC-3

[0034] SgRNA-2 sequence: 5-GAGTAGCACCCCCGCCCTGAC-3

[0035] The sequence of the sgRNA-2 action site: 5-GTCAGGGCGGGGTGCTACTC-3

[0036] Among them, the present invention involves

[0037] The RNA sequence of sgRNA-1 is: 5-GGAAACCCAGGCUGGUUGGA-3

[0038] The complementary sequence of the RNA seque...

Embodiment 3

[0045] Construction of homologous targeting vector

[0046] According to the screened high-efficiency sgRNA and construct a homologous targeting vector matching the sgRNA (sequence shown in SEQ ID 19), the main components of the targeting vector are: the upstream homology arm whose sequence is shown in SEQ ID 7, pig The sequence is the fifth exon of CD163 shown in SEQ ID1, the sequence is the downstream homology arm shown in SEQ ID 10 and the backbone vector for prokaryotic expression. The site-specific integration targeting plasmid and the screened sgRNA can be used to carry out specific genetic modification on the porcine CD163 exon7 site, and then combined with PCR and RFLP methods can easily analyze the integration and expression of foreign genes at the sgRNA recognition site feasibility. The base sequence of the site-directed integration targeting vector constructed in the present invention is consistent with SEQ ID 19.

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Abstract

The invention discloses a gene sequence for effectively inhibiting Type II PRRSV (porcine reproductive and respiratory syndrome virus) infection, particularly a gene sequence for effectively inhibiting Type II PRRSV infection after replacing of a seventh exon of pig CD163, and also discloses application of the gene sequence in disease-resistant study. The gene sequence has the advantages that a CRISPR / Cas9 technique is adopted; the gene sequence of a fifth exon of pig CD163 shown in the SEQ ID NO:1 is used for successfully replacing the gene sequence of the seventh exon of pig CD163, and the gene sequence of CD163, such as the alveolar macrophages of positive cloning pig shown in SEQ ID NO:2, is obtained; a theoretical foundation is laid for the illumination of the molecule mechanism of PRRSV infection, and the subsequent obtaining of pig population with inhibiting of PRRSV infection, thereby reducing the huge loss of the pig culture industry by PRRSV.

Description

technical field [0001] The invention discloses a gene sequence that can effectively inhibit type II PRRSV infection, which is a gene sequence that can effectively inhibit type II PRRSV infection after replacing the seventh exon of porcine CD163, and also discloses the use of the sequence in disease resistance research The application belongs to the field of bioengineering technology. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), also known as porcine blue ear disease, is a worldwide infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). Abortion, pneumonia in piglets and fattening pigs, accompanied by respiratory disorders, slow growth of piglets, etc.; in addition, severe immunosuppression and persistent infection. The disease was first discovered in my country in 1996. Since 2006, the emergence of highly pathogenic variants has caused a large-scale outbreak of the disease in China, causing serious ec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/10C12N15/85A01K67/027
CPCA01K67/0276A01K2207/15A01K2227/108A01K2267/02C07K14/70596C12N15/10C12N15/85
Inventor 欧阳红生袁泓明逄大欣
Owner 重庆吉棠生物技术研究院有限公司
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