A marc-145 cell line that knocks down pkr

A cell line, cell technology, applied in the field of molecular biology and cell biology, to achieve the effect of promoting proliferation

Active Publication Date: 2022-04-15
SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that PKR may have an inhibitory effect on the proliferation of PRV in theory.

Method used

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  • A marc-145 cell line that knocks down pkr
  • A marc-145 cell line that knocks down pkr
  • A marc-145 cell line that knocks down pkr

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Design and screening of embodiment 1 siRNA and shRNA

[0031] 1. PKR gene cloning of Marc-145

[0032] Primers were designed according to the published Marc-145 related species PKR gene (GeneBank accession number: XM_015112081), the specific sequence is shown in Table 1. The total RNA of Marc-145 cells was extracted, cDNA was obtained by reverse transcription, and the target gene was amplified by high-fidelity enzyme Q5.

[0033] Table 1 Primers for PKR gene amplification in Marc-145 cells

[0034]

[0035] PCR products were electrophoresed on 1% agarose ( figure 1 ), the product whose size fits the target gene (1653 bp) was recovered, base "A" was added to the 3' end, cloned into pMD18-T vector and sequenced, the sequence is shown in SEQ ID NO:7. Save the plasmid with correct sequencing for future use.

[0036] 2. Construction of PKR fusion EGFP expression vector

[0037] According to the sequences in Table 2, primers PKR+C1-F and PKR+C1-FR were synthesized re...

Embodiment 2

[0045] Example 2 Construction of shRNA stably expressing Marc-145 cell line

[0046] Synthesized according to Table 4 containing Bam H I and Hind The DNA sequence of the restriction site III used to generate shRNA, and set irrelevant shRNA templates as negative control (NC) and positive control PKR 1870S1C1 (Sigma-Aldrich, TRCN0000196400) templates. convert shRNA to Bam H I+ Hin d III double enzyme digestion, use T4 DNA ligase to connect to the same double digestion pSilencer™ 2.1-U6 hygro vector, transform into Amp resistance screening, pick a single colony and culture it overnight, extract the plasmid, and sequence the correct ones and name them pShRNA - PKR NC, pShRNA-PKR 143, pShRNA-PKR 295 and pShRNA-PKR 1870S1C1.

[0047] Table 4 DNA sequence of PKR gene shRNA in Marc-145 cells

[0048]

[0049] Select the pShRNA-PKR constructed above to transfect Marc-145, and select with 300 µg / mL hygromycin B. After 7 days, most of the cells die, but as time goes on, a si...

Embodiment 3

[0050] Example 3 Effect of Marc-145∧143 on proliferation of NDV, PRRSV and PRV

[0051] According to Tang Na, et al. (Tang Na, Yang Hui, Liu Lei, et al. Construction and Identification of Recombinant Porcine Reproductive and Respiratory Syndrome Virus Expressing EGFP[J]. Chinese Journal of Animal Infectious Diseases, 28(4):8.) EGFP PRRSV XZ06a-EGFP recombinant virus.

[0052] 1. PRRSV infection

[0053] PRRSV XZ06a-EGFP was respectively infected into Marc-145 cells, Marc-145∧143, Marc-145∧295, Marc-145∧1870S1C1 and Marc-145∧NC F3 generation cells, the infection dose was MOI=0.1, by observing the virus Cytopathic effect (CPE), Western Blot detection of PRRSV main protein and EGFP expression, analysis of PKR down-regulated expression on PRRSV proliferation.

[0054] The results showed that when the constructed cell line was infected with 0.1 MOI PRRSV XZ06a-EGFP, no obvious CPE appeared in Marc-145∧143 and Marc-145∧1870S1C1 cells after 72 h to 96 h, while Marc-145 and the cont...

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Abstract

The invention belongs to the fields of molecular biology and cell biology, and in particular relates to a PKR gene knockdown Marc-145 cell line. The present invention provides a forward and reverse sequence of siRNA for knocking down PKR gene by RNAi as shown in SEQ ID NO:1-2; the sequence of shRNA is shown as SEQ ID NO:3. The expression vector containing the gene sequence of interest shown in SEQ ID NO:3 is transfected into Marc-145 cell line, and after screening, a cell line with reduced expression of PKR protein capable of stable inheritance can be obtained: Marc-145∧143, the cell line The expression of PKR protein was reduced by not less than 90%. The siRNA, shRNA or Marc‑145∧143 cell line can be used to produce medicament or vaccine for preventing or treating NDV, PRRSV or PRV.

Description

technical field [0001] The invention belongs to the fields of molecular biology and cell biology, and in particular relates to a PKR gene knockdown Marc-145 cell line. Background technique [0002] Interferon-induced, double-stranded (ds) RNA-activated kinase (PKR) is a serine-threonine kinase that can be activated by double-stranded RNA ( dsRNA) or synthetic analogues (such as polyI:C), play an important role in the host antiviral defense process. Its antiviral activity is mainly through the combination of monomeric PKR and double-stranded RNA (dsRNA) produced during viral replication, forming a dimer and autophosphorylation, and then catalyzing its substrate-eukaryotic translation initiation factor 2α (alpha Subunit of the eukaryotic initiation factore, eIF2α) phosphorylation, resulting in inhibition of viral protein synthesis, resulting in antiviral effects. [0003] Research has shown that [1] , Down-regulating the expression of PKR in Hela cells by siRNA transfection...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85C12N5/10A61K35/22A61P31/14A61P31/22
CPCC12N15/1137C12Y207/11001C12N15/85C12N5/0686C12N9/12A61K35/22A61P31/22A61P31/14C12N2310/14C12N2310/531C12N2800/107C12N2510/02
Inventor 肖跃强杨慧王文秀魏凤唐娜李峰沈志强
Owner SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY
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