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Murine IL-27 recombinant protein eukaryotic expression vector and construction method
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A eukaryotic expression vector, IL-27 technology, applied in the fields of immunology and molecular biology, can solve the problems of difficulty in constructing protein expression vectors, and achieve the effect of high protein expression efficiency
Inactive Publication Date: 2012-12-12
NANKAI UNIV
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However, since IL-27 is composed of EBI3 and p28 subunits to form a heterodimer, it is relatively difficult to construct the expression vector of this protein
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[0033] Construction method of murine IL-27 recombinant protein eukaryotic expression vector
[0034] 1. Primer design and amplification of key fragment pSZ12-insert
[0035] Primer PRM-149, as shown in the sequence table SEQ ID No.1, includes a part of the KpnI restriction site and the 5' end coding sequence of EBI3;
[0036] Primer PMR-150, as shown in the sequence table SEQ ID No.2, includes (GGGS) 4 strand coding sequence, part of the 3' coding sequence of EBI3 and part of the 5' coding sequence of p28;
[0037] Primer PMR-151, as shown in the sequence listing SEQ ID No.3, includes a part of the 5' end coding sequence of p28;
[0038] Primer PMR-152, as shown in the sequence table SEQ ID No.4, contains a part of the 3' end coding sequence of p28, 6×histidine coding sequence, stop codon and XhoIrestriction site;
[0039] First extract the total RNA of mouse dendritic cells stimulated by IFN-γ+CpG, reverse...
Embodiment 2
[0043] Embodiment 2 (attached image 3 )
[0044]Use mouse IL-27 recombinant protein eukaryotic expression vector pSZ12 to express IL-27 recombinant protein in vitro. 1.293T cells were cultured in a 10cm cell culture dish, and 10ml of DMEM fresh medium containing 10% calf serum was added to a growth density of about 80%. .
[0045] 2. Add 10 μg of pSZ12 plasmid and control plasmid to 500 μL of Opti-MEM medium, mix well, then add 15 μL FuGENE HD transfectionreagent, mix well. Let stand at room temperature for 15 minutes.
[0046] 3. Add the transfectionreagent mixture dropwise to the cell culture plate, shake gently, and continue to culture in the incubator.
[0047] 4. Cell supernatants were collected 24 hours and 48 hours after transfection, and IL-27 recombinant protein expressed in the supernatants was detected with an IL-27 ELISA kit. The expression of IL-27 recombinant protein could not be detected in the supernatant of 293T cells transfected with control control pl...
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Abstract
The invention discloses a murine IL-27 recombinant protein eukaryotic expression vector and a construction method. The construction process comprises the following steps that primers are designed to amplify the cDNA (complementary deoxyribonucleic acid) sequence of encoding mouse EBI3 amino acids 1-228 and the cDNA sequence of encoding mouse p28 amino acids 29-234; and then an integrated DNA fragment is obtained by an SOEPCR (gene splicing by overlap extension polymerasechain reaction) method. The fragment is connected with a pcDNA3.1 + vector to obtain a murine IL-27 recombinant protein eukaryotic expression vector pSZ12. EBI3 and p28 are connected by the pre-designed amino acid encoding sequence. The vector constructed by the invention can not only effectively express a heterodimer IL-27 formed by EBI3 and p28, but also contain 6 * histidine encoding sequences to greatly facilitate the purification of recombinant protein. The key fragment can be cloned to other vectors so as to facilitate the application of protein expression in different cases, and an effective tool is provided for the study of the IL-27 function and the gene therapy method.
Description
technical field [0001] The invention belongs to the technical field of immunology and molecular biology. Background technique [0002] IL-27 (Interleukin-27) is a new interleukin discovered in recent years. This cytokine plays an extensive and important role in inflammation and tumors in the immune system. IL-27 recombinant protein is of great significance in the research of this field. However, since IL-27 consists of EBI3 and p28 subunits to form a heterodimer, it is relatively difficult to construct the expression vector of this protein. Contents of the invention [0003] The purpose of the present invention is to solve the problem of constructing the expression vector of IL-27 recombinant protein, provide a eukaryotic expression vector for highly expressing IL-27, and a construction method with simple method and convenient operation. [0004] The present invention firstly provides a mouse IL-27 recombinant protein eukaryotic expression vector pSZ12, and related prime...
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