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Recombinant vector for expressing swine-derived sialoadhesin (Sn) receptor gene, recombinant cell as well as construction methods and application thereof

A technology for recombining vectors and receptor genes, applied in the field of molecular biology, can solve problems such as low immunity and lack of immunity

Pending Publication Date: 2019-12-20
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The study confirmed that the root cause of the low immunity of PRRS vaccine produced by Marc145 cells is the lack of highly efficient PRRSV infection receptors on the cell surface

Method used

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  • Recombinant vector for expressing swine-derived sialoadhesin (Sn) receptor gene, recombinant cell as well as construction methods and application thereof
  • Recombinant vector for expressing swine-derived sialoadhesin (Sn) receptor gene, recombinant cell as well as construction methods and application thereof
  • Recombinant vector for expressing swine-derived sialoadhesin (Sn) receptor gene, recombinant cell as well as construction methods and application thereof

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preparation example Construction

[0032] In the present invention, the preparation method of the recombinant vector preferably comprises the following steps: 1) artificially synthesizing the gene Sn, and connecting a Hind III restriction enzyme site and a BamH I restriction enzyme site at both ends of the gene Sn Obtain the gene Sn fragment of band restriction site; 2) carry out the double gene Sn fragment of band restriction site and pEGFP-N1 carrier described in step 1) respectively with Hind III restriction enzyme and BamH I restriction enzyme After enzyme digestion, the enzyme digestion product was ligated to obtain a recombinant vector.

[0033] In the present invention, the gene Sn is artificially synthesized, and the artificial synthesis is preferably entrusted to Suzhou Jinweizhi Biotechnology Co., Ltd. In the present invention, after obtaining the artificially synthesized gene Sn, a Hind III restriction site and a BamH I restriction site are connected at both ends of the gene Sn to obtain a gene Sn fr...

Embodiment 1

[0042] The nucleotide sequence of the full-length gene of the porcine sialoadhesin receptor gene (Sn) is shown in SEQ.ID.NO.1.

[0043] Construction of recombinant vectors:

[0044] Synthetic pig-derived salivaridhesin receptor gene (Sn) (Suzhou Jinweizhi Biotechnology Co., Ltd.). Use oligo7.0 software to design corresponding specific primers, and add Hind III and BamH I restriction enzyme sites at both ends of the porcine sialoadhesin receptor gene (Sn): Sn-Hind III: 5`-GAT AAGCTT GCCACCATGGACTTCCTGCTCCT-3`; Sn-BamH I:5`-TCAGACTGTGCTTTTCACAGACTG GGATCC GCG-3`;

[0045] Use TAKARA’s Premix PrimeSTAR HS for amplification: PCR reaction system: Sn-Hind III primer 0.3 μM, Sn-BamH I primer 0.3 μM, Premix PrimeSTAR HS: 25 μL, template DNA: 100ng, sterilized distilled water: add to 50 μL ;PCR amplification system is: 95°C pre-denaturation for 5min; (95°C denaturation for 45s, 62°C renaturation for 40s, 72°C extension for 3min), a total of 35 cycles; finally 72°C extension for 10...

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Abstract

The invention provides a recombinant vector for expressing a swine-derived sialoadhesin (Sn) receptor gene, a recombinant cell as well as construction methods and application thereof and belongs to the technical field of molecular biology. The recombinant vector comprises an initial vector and a gene Sn; and a host cell of the recombinant cell is a Marc145 cell. The preparation method of the recombinant cell comprises the steps as follows: artificially synthesizing the gene Sn, and linking enzyme digestion sites at two ends of the gene Sn; after performing double enzyme digestion on gene Sn fragments with the enzyme digestion sites and a pEGFP-N1 vector by Hind III restriction enzyme and BamH I restriction enzyme respectively, and linking enzyme digestion products to obtain the recombinantvector; transfecting the recombinant vector with the Marc145 cell to obtain the recombinant cell. The recombinant cell can efficiently and stably express the gene Sn and can be applied to preparationof vaccines.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a recombinant vector expressing a porcine sialoadhesin receptor gene, a recombinant cell, a construction method and application thereof. Background technique [0002] Sialoadhesin (Sn), also known as CD169 or siglec-1, is a restriction lectin of macrophages and can bind to sialic acid. In 1985, Sn was first recognized as a binding receptor for sheep erythrocytes. Sn, a member of the immunoglobulin superfamily, is a type I transmembrane glycoprotein consisting of 17 repeating extracellular Ig-like domains and a short cytoplasmic tail. Since the cytoplasmic tail of Sn is poorly conserved and lacks tyrosine motifs, it is speculated that it mainly interacts between cells through receptor binding to sialic acid, rather than through cell signal transduction. At present, studies have shown that the expression of Sn in macrophages can promote the interaction of pa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/12C12N15/65C12N15/66C12N5/10A61K39/00A61P31/14
CPCA61K39/00A61P31/14C07K14/70503C12N15/65C12N15/66C12N15/85
Inventor 常艳燕邵军军常惠芸李扬帆张永光
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI