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Dual-luciferase reporter gene carrier based on porcine CD163 gene

A dual-luciferase and reporter gene technology is applied in the field of dual-luciferase reporter gene vectors based on porcine CD163 gene, which can solve problems such as inability to regulate candidate miRNAs

Active Publication Date: 2013-12-25
湖北丰美禾生态牧业有限公司
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AI Technical Summary

Problems solved by technology

Most miRNAs regulate gene expression through specific recognition of the 3'UTR region of the target gene. However, the 3'UTR sequence of the porcine CD163 gene is not complete at present, and it is impossible to identify candidate miRNAs that regulate porcine CD163 gene expression. Therefore, obtaining the complete sequence of the 3'UTR region of porcine CD163 gene and constructing its dual luciferase reporter vector can be used to screen for candidate miRNAs that regulate CD163 gene expression, and can also be used as candidate miRNAs for inhibiting the proliferation of PRRSV infection for further research

Method used

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  • Dual-luciferase reporter gene carrier based on porcine CD163 gene
  • Dual-luciferase reporter gene carrier based on porcine CD163 gene
  • Dual-luciferase reporter gene carrier based on porcine CD163 gene

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Embodiment 1

[0034] 1. 3' RACE sequencing of porcine CD163 gene

[0035] 1.1 Primer design

[0036] Refer to the cDNA sequence of porcine CD163 gene GenBank (Accession No.: NM_213976.1) to design the outer and inner two upstream specific primers, combined with the outer and inner downstream anchor primers of the 3' RACE kit of Treasure Bioengineering Dalian Co., Ltd., using Slot touchdown PCR amplifies the 3'UTR region fragment. The primer sequences are as follows:

[0037] The upstream primer of the outer primer: 5'-CAGGTCGCTCATCTTTTGTTGC-3' (SEQ ID NO: 1 in the sequence listing)

[0038] Downstream primer of outer primer: 5'-TACCGTCGTTCCACTAGTGATTT-3'

[0039] The upstream primer of the internal primer: 5'-GCCTGAAAGCAGATGAAACGGA-3' (SEQ ID NO: 2 in the sequence listing)

[0040] Downstream primer of inner primer: 5'-CGCGGATCCTCCACTAGTGATTTCACTATAGG-3'

[0041] 1.2 Extraction and detection of total RNA from porcine tissue

[0042] Trizol reagent (purchased from Invitrogen) was used ...

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Abstract

The invention belongs to the technical field of livestock gene engineering, and relates to construction and an application of a dual-luciferase reporter gene carrier based on a porcine CD163 gene. A key receptor gene, namely the porcine CD163 gene, of a PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) is amplified and sequenced to obtain a complete fragment of a 3' UTR (Untranslated Region) of the gene, candidate microRNAs (micro Ribonucleic Acids) and an action target of the 3' UTR of the porcine CD163 gene are predicted and regulated, and a sequence of the porcine CD163 gene containing the action target is amplified, and inserted into a dual-luciferase carrier to construct the dual-luciferase reporter gene carrier psi-CHECK2-CD163-3UTR of the 3' UTR of the porcine CD163 gene. The carrier can be used for detecting regulation activity of the candidate microRNAs on porcine CD163 gene expression, and screening the microRNAs inhibiting the porcine CD163 gene expression, and the like.

Description

technical field [0001] The invention belongs to the technical field of livestock genetic engineering, and in particular relates to a dual-luciferase reporter gene carrier based on porcine CD163 gene, which can be used to screen and detect candidate microRNAs that regulate the expression of porcine CD163 gene, and explore its effect on inhibiting PRRSV infection and proliferation , so as to indirectly screen microRNAs against porcine reproductive and respiratory syndrome virus infection, and further be used in the research of microRNAs against porcine reproductive and respiratory syndrome. Background technique [0002] Porcine reproductive and respiratory syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS), due to the cyanosis of the pig's ears, is purple-red plaques, also known as "blue ear disease", which is caused by porcine reproductive and respiratory syndrome virus (PRRSV) infection. It is a very harmful infectious disease, which mainly leads to reproductive ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/11C12N15/66
Inventor 吴俊静梅书棋彭先文乔木武华玉刘贵生宋忠旭孙华李良华李明波董斌科
Owner 湖北丰美禾生态牧业有限公司
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