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Recombinant MARC-145 cell, and construction method and application thereof

A construction method and technology of marc-145pcd163, applied in the field of genetic engineering, can solve the problems of limitation, inability to separate PRRSV, high cost, etc., and achieve the effect of rapid proliferation

Pending Publication Date: 2021-03-19
LONGYAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The technology described by this patented describes how certain genes can be used to make mice that are resistive against viruses like measles virus or influenza bacteria. By comparing these genes' levels between different strains of rats infected with them, researchers have been able to determine which factors affect their ability to fight off disease caused by specific types of microorganisms such as those causing respiratory illness. This allows us to develop better ways to protect individuals from future exposure to harmful germs called MRSA (methicillin-resistant Staphylococcus A) due to previous epidemiological evidence showing it has become more commonplace over time.

Problems solved by technology

This patents discusses how different types of animal pathogenesis involve certain proteins called Prion or CRMNepidum necroticis associated with specific bacteria like Actinosynnema nodosae or Salmonella enteritis. These organelles play crucial roles during these processes because prions bind directly to their own DNA without being transmitted through blood vessels. However, despite advances made towards understanding them, no successful way exists at present to produce large amounts of purified mature Mammals containing sufficient quantities of these enzymatic components needed for effective therapy against various forms of rhinovirus infections.

Method used

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  • Recombinant MARC-145 cell, and construction method and application thereof
  • Recombinant MARC-145 cell, and construction method and application thereof
  • Recombinant MARC-145 cell, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042]Example 1 Extraction of total RNA and amplification of pCD163 and pSiglec-10 genes

[0043] (1) Extraction of total RNA

[0044] According to the instructions of the RNA extraction kit, the total RNA of alveolar macrophages (PAMs) cells was extracted, and the Superscript III reverse transcriptase product instructions were followed. The reverse transcription reaction system was 20 μL, RNA 8 μL, oligo (dT) 201 μL, dNTP (10 mmol / L ) 1 μL, ddH 2 O 2 μL, mix well, bathe in 65°C water for 5 min, place on ice for 2 min; add 5×First-Strand Buffer 4 μL, 0.1M DTT 1 μL, RNaseOUT TM Recombinant RNase Inhibitor 1 μL, SuperScript TM III RT (200units / μL) 2μL, mix well, after 1h in 55°C water bath, 15min in 70°C water bath to inactivate reverse transcriptase, store the synthesized cDNA at -20°C or use for PCR amplification.

[0045] (2) PCR amplification of pCD163 gene and pSiglec-10 gene

[0046] The reaction system is 50 μL, and the PCR amplification refers to pfx DNA polymeras...

Embodiment 2

[0053] Example 2 Construction of pLVX-pCD163-PGK-Puro and pCDNA3.1-Siglec-10 recombinant plasmids

[0054] The PCR product of the pCD163 gene and the pLVX-PGK-Puro vector ( figure 1 ), the pCD163 gene and vector gene fragments recovered from the gel were ligated with T4 DNA ligase, T4 DNA Ligase 1 μL, 5×Ligase Reaction Buffer 2 μL, pCD163 recovered product 6 μL, pLVX-PGK-Puro vector plasmid 3 μL, ddH 2 O 8 μL, the above mixture was ligated at 22°C for 2 h, and then the recombinant plasmid was transformed into E. coli DH-5α competent cells. At the same time, use BamHI / Kpn I to double-enzyme digest the PCR product of Siglec-10 gene and pCDNA3.1 vector respectively, recover the gel to obtain Siglec-10 gene and vector gene fragments and connect them with T4 DNA ligase, T4 DNA Ligase 1μL, 5× LigaseReaction Buffer 2 μL, pSiglec-10 recovery product 6 μL, pCDNA3.1 vector plasmid 3 μL, ddH 2 O 8 μL, the above mixture was ligated at 22°C for 2 h, and then the recombinant plasmid was ...

Embodiment 3

[0055] Example 3 lentiviral packaging

[0056] The recombinant plasmid pLVX-pCD163-PGK-Puro carrying the target gene, pSPAX2 plasmid, and pMD2.G plasmid (Huayueyang Biological (Beijing) Technology Co., Ltd.) were introduced into 293T cells together to produce a high-titer lentivirus carrying the target gene ( Hereinafter referred to as rLV-pCD163); at the same time, the recombinant plasmid pLVX-PGK-Puro was used as a control virus (abbreviated as rLV-PP). The specific operation was carried out according to the instructions of the lentiviral packaging kit (Huayueyang Biological (Beijing) Technology Co., Ltd.).

[0057] 24h morphological observation of 293T cells transfected with pLVX-PGK-Puro see figure 2 , 293T cells transfected with pLVX-pCD163-PGK-Puro 48h morphological observation picture image 3 .

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Abstract

The invention discloses a recombinant MARC-145 cell, and a construction method and application thereof, and belongs to the technical field of genetic engineering. The recombinant MARC-145 cell disclosed by the invention is a MARC-145<pCD163-pSiglec10> cell line capable of simultaneously expressing swine source CD163 and a sialic acid binding Ig-like lectin 10 gene in the MARC-145 cell. The MARC-145<pCD163-pSiglec10> cell line constructed by the invention can quickly carry out proliferation on PRRSV, and a foundation is laid for developing and doing relevant research on PRRSV vaccines.

Description

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Claims

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Application Information

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Owner LONGYAN UNIV
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