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Plecoglossus altivelis CD46 gene, recombinant engineering bacterium and preparation method of polyclonal antibody of CD46 gene

A technology of genetically engineered bacteria and ayu, applied in the field of genetic engineering, can solve problems such as the research on CD46 polyclonal antibody without and without ayu

Active Publication Date: 2020-04-14
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is also no report on the expression of sweetfish CD46 in Pichia pastoris in vitro, let alone the research on sweetfish CD46 polyclonal antibody

Method used

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  • Plecoglossus altivelis CD46 gene, recombinant engineering bacterium and preparation method of polyclonal antibody of CD46 gene
  • Plecoglossus altivelis CD46 gene, recombinant engineering bacterium and preparation method of polyclonal antibody of CD46 gene
  • Plecoglossus altivelis CD46 gene, recombinant engineering bacterium and preparation method of polyclonal antibody of CD46 gene

Examples

Experimental program
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Effect test

specific Embodiment 1

[0033] Analysis of Cloning and Sequence Analysis of CD46 Gene of Sweetfish

[0034]RNAiso Plus was used to extract total RNA from peripheral blood mononuclear cells (PBMC) of sweetfish. After being treated with DNase I, the first-strand cDNA was synthesized with AMV reverse transcriptase using Oligo(dT)-adaptor as a primer. Two isoforms of sweetfish CD46 were obtained from the monocyte-macrophage transcriptome derived from sweetfish head kidney, and the open reading frame sequences were used to design amplification primers PaCD46t-F (5'-ATGCAGTCCCTTGATACATCTCGTT-3') and PaCD46t-R (5'-CTATAGTGAATCAACAGGAGGACTGC-3'), using the above cDNA as a template, PCR amplification and sequencing verification. The obtained sweetfish CD46 isoform sequence was analyzed by BLAST search for homologous sequence alignment; the molecular weight and isoelectric point were predicted by p I / Mw calculation tool; SignalP 4.1 software was used for signal peptide prediction; SMART online software was ...

specific Embodiment 2

[0041] Construction method of recombinant sweetfish CD46 genetically engineered bacteria

[0042] 1. Construction of Pichia pastoris expression plasmid for the extracellular region of recombinant sweetfish CD46 protein

[0043] According to the nucleotide sequence of sweetfish CD46-1 gene SEQID NO.1, primers rPaCD46sex-F and rPaCD46sex-R, which can amplify the extracellular region containing 4 SCR and STP parts, were designed, and the PCR reaction conditions were carried out according to conventional settings , using the above-mentioned PBMC cDNA as a template to amplify to obtain the nucleotide sequence of the extracellular region. The PCR product containing the extracellular region of the recombinant sweetfish CD46 protein was recovered with a gel cutting recovery kit, and the recovered product was recovered with a restriction endonuclease Eco RI and Kpn I double enzyme digestion, ligate the digested PCR product with the same digested plasmid pPICZαA with T4 DNA ligase,...

specific Embodiment 3

[0054] 1. Preparation of polyclonal antibody against sweetfish CD46 protein

[0055] The recombinant protein purified in Example 2 was emulsified with Freund's adjuvant, and then immunized into male New Zealand rabbits. Immunization injections are given every 10 days for a total of four injections. For the first immunization, the recombinant protein was emulsified with complete Freund's adjuvant (1:1), and 1 mg of the purified recombinant protein was subcutaneously injected into the back of each rabbit. After three immunizations, the recombinant protein was emulsified with Freund's incomplete adjuvant (1:1), and 500 μg of the recombinant fusion protein was injected in the same way on the 10th, 20th and 30th day. 10 days after the fourth immunization, food was fasted for 1 day, and then heart blood was collected. Stand overnight in the refrigerator at 4°C, centrifuge at 15,000g for 10 minutes in a low-temperature centrifuge at 4°C, and gently absorb the supernatant with a pip...

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Abstract

The invention discloses a plecoglossus altivelis CD46 gene, a recombinant engineering bacterium and a preparation method of a polyclonal antibody of the CD46 gene. The plecoglossus altivelis CD46 geneis characterized in that the plecoglossus altivelis CD46 gene comprises plecoglossus altivelis CD46-1, CD46-2 and CD46-3 genes, and the cDNA sequences are shown as SEQID NO.1, SEQID NO.3 and SEQID NO.5 correspondingly. The preparation method of the recombinant plecoglossus altivelis CD46 gene engineering bacterium comprises the following steps: constructing a recombinant plecoglossus altivelis CD46 protein extracellular region pichia pastoris expression plasmid; screening a high-expression recombinant engineering strain; culturing the high-expression yeast strain in a fermentation tank; purifying the recombinant plecoglossus altivelis CD46 extracellular region protein to obtain a recombinant plecoglossus altivelis CD46 gene engineering bacterium; and preparing a rabbit anti-plecoglossus altivelis CD46 protein polyclonal antibody through a rabbit immunization method. The advantage is that a plecoglossus altivelis CD46 protein isomer can be specifically recognized.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for preparing sweetfish CD46 gene, recombinant engineering bacteria and polyclonal antibody thereof. Background technique [0002] Membrane cofactor protein (MCP or CD46) is one of the members of membrane-bound complement regulatory protein (mRCPs), which acts as a cofactor of complement factor I to mediate the cleavage of C3b and C4b, thereby inhibiting the complement level Cascade reaction to prevent autologous cell damage caused by overactivation of complement. In addition, CD46 is a co-stimulatory protein of T cells and a key molecule that regulates the immune function of T cells. The above effects reveal that CD46 plays an important role in connecting innate and adaptive immunity. Recently, studies have found that CD46 is involved in chromatin assembly and gene expression regulation; it can reduce the pro-inflammatory ability of monocyte-derived dendri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/81C12N15/66C12N1/19C07K14/46C07K14/705C07K16/28C07K16/06C07K1/18C07K1/16C12R1/84
CPCC07K14/461C07K14/70596C07K16/2896C07K16/065C12N15/815C12N15/66C07K2317/20
Inventor 陈炯史雨红马文静
Owner NINGBO UNIV
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