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A polyclonal antibody against Saccharomyces spp. and its preparation method

A polyclonal antibody, the technology of buckling the capsule and the membrane, which is applied in the field of animal genetic engineering to achieve the effect of high purity, high titer and good specificity

Inactive Publication Date: 2017-10-10
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many types of β-glucosidase antibodies currently on the market, but there is no β-glucosidase antibody that specifically targets the yeast Saccharomyces spp.

Method used

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  • A polyclonal antibody against Saccharomyces spp. and its preparation method
  • A polyclonal antibody against Saccharomyces spp. and its preparation method
  • A polyclonal antibody against Saccharomyces spp. and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Construction and identification of Escherichia coli recombinant expression vector pET--21a(+)--BGL1 and recombinant bacteria

[0049] Antigen epitope analysis and prediction were carried out on the sequence of the CB protein of Saccharomyces fumigatus (GeneBank accession number: HQ891006.1). ) encodes and expresses 859 amino acids (as shown in SEQ ID NO: 2 in the sequence table), and finally obtains a protein fragment containing more antigenic epitopes after prediction analysis, encoding 242 amino acids (as shown in SEQ ID NO: 4 in the sequence table ), expressed by the 726bp nucleotide sequence shown in SEQ ID NO:3.

[0050] 1.1 Cloning of part of the BGL1 gene

[0051] 1.1.1 Primer design:

[0052] Using the Primer5.0 software, refer to the BGL1 gene sequence (GeneBank accession number: HQ891006.1) to synthesize primers from Treasure Bioengineering Dalian Co., Ltd. An EcoRI restriction site and a CCG protection base were added to the 5' end of the upstre...

Embodiment 2

[0064] Example 2: Expression of recombinant expression vector pET--21a(+)--BGL1 in Escherichia coli BL21

[0065] 2.1 Extraction and identification of recombinant plasmids

[0066] Bacteria to be extracted from the plasmid and LB medium (containing Amp 50ug / ml) were expanded at a volume ratio of 1:1000, 37°C, 220r / min shaking culture for 12-15h. Refer to the plasmid mini-extraction kit from TIANGEN Company for the plasmid extraction of the bacteria solution with correct sequencing. For the specific method, refer to the kit instruction manual. After the extracted plasmid is digested with EcoRI and XhoI for 1 hour, it is identified by 1% agarose gel electrophoresis. (See image 3 ). The results of electrophoresis showed that the cut out bands were consistent with the expected results.

[0067] 2.2 Induced expression of recombinant plasmid pET-21a(+)-BGL1

[0068] Transform the recombinant expression vector with correct sequencing into BL21 competent cells, pick a single clon...

Embodiment 3

[0074] Example 3: Large-scale expression and purification of CB protein of Saccharomyces spp.

[0075] 3.1 Massive expression of CB protein, the specific operation steps are as follows:

[0076] (1) Transform BL21 competent cells with the correctly sequenced plasmid, pick a single clone and inoculate it into 3ml of P-0.5G medium, and culture at 37°C for 8h at 250rpm;

[0077] (2) Transfer the culture solution to 1000ml ZYP-5052 medium at a volume ratio of 1:1000, and culture overnight at 37°C and 250rpm;

[0078] (3) Centrifuge at 6000rpm for 5min, discard the supernatant, and collect the bacteria;

[0079] (4) Add 30-50ml of lysate to resuspend the bacteria, and ultrasonically break the resuspended bacteria solution on ice (600w, run for 5s with an interval of 10s, about 25 times) until translucent;

[0080] (5) Centrifuge at 13,000rpm at 4°C for 15min, collect the supernatant and precipitate (inclusion body), dissolve the inclusion body with bindingbuffer containing 8M ure...

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Abstract

The invention belongs to the field of animal gene engineering, and particularly relates to a saccharomycopsis fibuligera polyclonal antibody and a preparation method thereof. A nucleotide sequence of a BGL1 gene of separated saccharomycopsis fibuligera CB protein provided by the invention is as shown in SEQ ID NO:1; an amino acid sequence encoded by the gene is as shown in SEQ ID NO:2; the method comprises the following steps: predicting and analyzing saccharomycopsis fibuligera CB protein epitope to obtain a segment containing more epitopes; building an escherichia coli recombinant expression carrier, and transforming echerichia coli with the expression carrier to obtain a recombinant strain of expressing the CB protein; and carrying out inducible expression on the recombinant strain, greatly expressing and purifying the CB protein, and adopting the purified CB protein as an immunogen immune animal of the saccharomycopsis fibuligera polyclonal antibody, so as to obtain the polyclonal antibody. The saccharomycopsis fibuligera polyclonal antibody is good in multi-resistant specificity, high in purity and valence and long in storage life, and can be applied to immunoblotting, immunohistochemical analysis and the like in genetic engineering.

Description

technical field [0001] The invention belongs to the technical field of animal genetic engineering, and in particular relates to a polyclonal antibody of the yeast Saccharomyces spp. and a preparation method thereof. Background technique [0002] Saccharomycopsis fibuligera, also known as Endomycopsis fibuligera, is a kind of fungus that can express raw starch liquefaction enzyme and glucoamylase, Saccharomycopsis fibuligera is considered It is one of the best ascomycete for producing amylolytic enzymes. [0003] The β-glucosidase (β-D-Glucosidase, CB for short) in the present invention is a kind of cellulase produced by the expression of the BGL1 gene derived from Saccharomyces fornicatus. Cellulase is a complex enzyme system with multi-enzyme components. It exists widely in nature. Bacteria, fungi, and animals can produce cellulase, but the cellulase generally used for production comes from fungi. Cellulase is mainly composed of three enzymes: endoglucosidase, exoglucosid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C07K16/40
CPCC07K16/40C12N9/2445C12N15/70C12Y302/01021
Inventor 蒋思文李玉娇彭健柴进李凤娥
Owner HUAZHONG AGRI UNIV
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