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A kind of preparation method of porcine elf4 protein egg yolk antibody

A yolk antibody and protein technology, applied in the direction of egg immunoglobulin, anti-animal/human immunoglobulin, etc., can solve the problems of high specificity and high sensitivity, and achieve large antibody yield, low preparation cost, extraction simple method effect

Active Publication Date: 2019-08-16
湖南普菲特动物保健有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0003] At present, the species of animals from which the gene sequence has been cloned are only derived from humans, mice and cattle. There is no relevant report on the prokaryotic expression of the porcine ELF4 gene sequence and its encoded protein, and there are currently no commercial products related to porcine ELF4 protein antibodies In view of the important role of this protein in anti-infection immunity, the present invention is the first time to obtain the ELF4 gene sequence of pigs, and adopts the optimized immunogen preparation scheme to immunize laying hens to prepare corresponding egg yolk antibodies, which can be used to detect pig-derived ELF4 Protein, as a tool, has the characteristics of strong specificity and high sensitivity to carry out in-depth functional research on porcine ELF4

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  • A kind of preparation method of porcine elf4 protein egg yolk antibody
  • A kind of preparation method of porcine elf4 protein egg yolk antibody
  • A kind of preparation method of porcine elf4 protein egg yolk antibody

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preparation example Construction

[0032] A preparation method of porcine ELF4 protein egg yolk antibody, comprising the following steps:

[0033] Step 1. Cloning and sequencing of the porcine transcription regulator ELF4 gene and construction of recombinant expression bacteria:

[0034] (1) Using the total RNA of porcine kidney cells as a template, synthesize the first-strand cDNA, and use primer prime5.0 software to design a pair of primers: pELF4-S: ACT GAATTC ATGGCTATTACCCTGCAGCCCAGT (the underlined sequence is the EcoRI site), pELF4-R: ACT AAGCTT TTATCATATGTCATGGGGCTCCATCTTAATG (the underlined sequence is the HindⅢ site), and the porcine ELF4 gene fragment was amplified by RT-PCR. After the RNA was reverse-transcribed into cDNA with random primers, it was amplified by PCR.

[0035] (2) Utilize the PCR product Eco RI and Hin After dⅢ double enzyme digestion and after Eco RI and Hin The pET28a vector digested with dⅢ was ligated. The ligation product was transformed into Rosetta (DE3) competent ...

Embodiment 1

[0062] This example intends to clone the porcine ELF4 protein gene, construct the prokaryotic expression vector pET28a-pELF4, express and purify the recombinant porcine ELF4 protein, and use it as an immunogen to immunize laying hens to produce anti-porcine ELF4 recombinant protein-specific IgY antibody .

[0063] 1. Primer design and synthesis

[0064] According to the human ELF4 gene sequence, a pair of primers were designed using the software Primer Prime5.0 to amplify the porcine ELF4 gene DNA sequence. pELF4-S:5'-ACT GAATTC ATGGCTATTACCCTGCAGCCCAGT-3' (the underlined sequence is Eco RI site), pELF4-R: 5'-ACT AAGCTT TTATCATATGTCATGGGGCTCCATCTTAATG-3' (the underlined sequence is the Hind III site), and the primers were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.

[0065] 2. PCR amplification of the porcine ELF4 gene, using the RT-PCR method to amplify the porcine ELF4 gene fragment.

[0066] The PCR reaction system is as follows:

[0067] Reagent V...

Embodiment 2

[0173] A preparation method of porcine ELF4 protein egg yolk antibody, comprising the following steps:

[0174] Step 1. Using porcine kidney cells (PK15 cell line) as materials, extracting total RNA as a template, amplifying porcine ELF4 (pELF4) gene fragments by RT-PCR method, combining the amplified porcine ELF4 (pELF4) gene fragments with pET28a The vector is connected and transformed into Rosetta (DE3) competent cells to obtain the recombinant expression strain Rosetta (DE3) / pET28a-pELF4, and the nucleotide sequence of the porcine ELF4 gene is shown in SEQ ID No.1;

[0175] Step 2, using the inducer IPTG to induce the expression of the target protein in the recombinant strain Rosetta(DE3) / pET28a-pELF4, collecting the bacterium, and obtaining the recombinant expression strain Rosetta(DE3) / pET28a-pELF4 expressing the target protein;

[0176] Step 3, separating and purifying the fusion protein from the recombinant expression strain Rosetta(DE3) / pET28a-pELF4 expressing the tar...

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Abstract

The invention provides a preparation method for a swine ELF4 protein yolk antibody. The preparation method comprises the following steps: carrying out amplification at first so as to obtain an ELF4 gene segment and connecting the ELF4 gene segment to a pET28a vector so as to obtain a recombinant expressed bacterial strain Rosetta (DE3) / pET28a-pELF4; inducing the recombinant expressed bacterial strain with an inducer IPTG so as to obtain fusion protein; and adding beta-propiolactone with a final concentration of 0.05% into the purified fusion protein, mixing the treated protein with a vaccine adjuvant ISA71 VG, immunizing a laying hen with the obtained mixture and extracting and collecting a yolk antibody from an egg laid by the laying hen so as to prepare the swine ELF4 protein yolk antibody. The preparation method is simple, high in antibody output and low in preparation cost; and the prepared ELF4 protein yolk antibody for a boar is high in titer and applicable to industrial production.

Description

technical field [0001] The invention relates to an antibody, in particular to a method for preparing the egg yolk antibody of porcine ELF4 protein. Background technique [0002] ETS (E26 transformation specific) family molecules are a class of transcription factors in multicellular organisms, named after the protein encoded by an oncogene first found in the fusion protein expressed by avian myeloerythroblastosis virus E26. The protein structure of this family is highly conserved, and it has a 85aa sequence that can specifically bind to the DNA consensus motif GGAA / T. ELF4 (E74-like factor 4) is a member of the ETS family, which was named myeloid elf-1 (E74-likefactor 1)-like factor (Myeloid elf-1-like factor, MEF) in the early stage, and can constitutively activates the lysozyme gene, upregulates the transcription level of the human β-defensin 2 gene, and is considered a potential tumor suppressor on the X chromosome. At the basal level, ELF4 transcription is regulated by ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C07K16/02
CPCC07K16/02C07K16/18C07K2317/11
Inventor 唐青海阚云超姚伦广王瑾唐存多谷雅静李晓楠李羽焦铸锦李丹丹刘宗才
Owner 湖南普菲特动物保健有限公司