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K99-987P-F41 recombinant protein and application thereof

A recombinant protein, F41 technology, applied in the field of animal molecular biology and genetic engineering, can solve the problems of loss of important antigens, incomplete immunity, and high production cost of inactivated vaccines, achieve elimination of side reactions, high specificity, and overcome the problem of whole bacteria. Effects of inactivated vaccines

Inactive Publication Date: 2013-09-04
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the immune effect of self-made vaccine is relatively good, it can only be applied in a small area
In addition, inactivated vaccines are expensive to produce and require adjuvants to enhance the body's immune response
In addition, during inactivation, important antigens are lost and immunity is incomplete

Method used

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  • K99-987P-F41 recombinant protein and application thereof
  • K99-987P-F41 recombinant protein and application thereof
  • K99-987P-F41 recombinant protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Primers were designed according to the complete sequences of enterotoxigenic Escherichia coli adhesin K99, 987P, and F41 genes published in GenBank (accession numbers M35282.1, M35257.1, and M21788.1, respectively). The genome of Bacillus C83919 was used as a template, and target fragments were amplified by PCR respectively; among them, Escherichia coli C83912, Escherichia coli C83916, and Escherichia coli C83919 contained adhesin K99, adhesin 987P, and adhesin F41 respectively, and the sequences of the primers used were as follows:

[0046] P1: K99(F): CGG GGTACC ATGACTATTAACTTCAATGGCAAA (KpnI) (SEQ ID No. 3);

[0047] P2: K99(R): CGC GGATCC TGAAGTAGTAAATACGCCAGC (BamHI) (SEQ ID No. 4);

[0048] P3: 987P(F): CGC GGATCC GGTGGTGGTGGTTCTGCTGAAAACAACACCAGCCAG (BamHI) (SEQ ID No. 5);

[0049] P4: 987P (R): C GAGCTC ACTAGCAGTGACAGTACCGGC (SacI) (SEQ ID No. 6);

[0050] P5: F41(F): C GAGCTC GGCGGTGGCGGCAGCGTATCTGGTTCAGTGATGGCT (SacI) (SEQ ID No. 7);

[0051] P6: F...

Embodiment 2

[0108] Using the same method as in Example 1, 987P was amplified using P3 / P4 and using the Escherichia coli C83916 genome as a template. The gel was recovered, connected to pMD-18T, and transformed into DH5α competent cells. After identification by PCR and double enzyme digestion, the positive plasmids were named pMD-18T-987P-DH5α respectively. pMD-18T-987P-DH5α and pET30a-K99 were digested with BamHI and SacI, the target fragments were recovered by gel cutting, and then ligated to construct pET30a-K99-987P, which was identified by PCR and double digestion. F41 was amplified using P5 / P6 and using the Escherichia coli C83919 genome as a template. The gel was recovered, connected to pMD-18T, and transformed into DH5α competent cells. After identification by PCR and double enzyme digestion, the positive plasmids were named pMD-18T-F41-DH5α respectively. pMD-18T-F41-DH5α and pET30a-K99-987P were digested with SacI and NotI, the target fragments were recovered by gel cutting, and ...

Embodiment 3

[0110] This example illustrates the induced expression of recombinant proteins.

[0111] The positive recombinant expression plasmid pET30a-K99-987P-F41-BL21(DE3) was transformed into Escherichia coli BL21(DE3), and a single colony was picked and cultured with shaking in LB liquid medium containing Kana at 37°C. When the OD600 value reached 0.6, IPTG was added to a final concentration of 0.5 mmol / L for induction. At the same time, the expression vector control group was set, and the bacterial liquid was taken 5 hours after induction of expression, centrifuged at 12000r / min for 1min, and the precipitate was preserved. PBS resuspended bacteria pellet, after ultrasonic lysis, centrifuged at 10000r / min for 20min, collected supernatant and precipitate for SDS-PAGE electrophoresis, the results are as follows Figure 4 shown.

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Abstract

The invention relates to a K99-987P-F41 recombinant protein, wherein the amino acid sequence thereof is shown by SEQ ID No.2. The invention also provides a coding gene of the recombinant protein, a preparation method and an application in preparing an enterotoxigenic escherichia coli (ETEC) vaccine. The three-section target genes ETEC adhesins K99, 987P and F41 are serially connected and sub-cloned to an escherichia coli prokaryotic expression vector pET30a(+), and can be used for preparing an escherichia coli K99-987-F41 series connection expression subunit vaccine; multiple antibodies can be obtained by immunizing once, and the vaccine is convenient to use and overcomes the shortcomings of a monovalent vaccine; meanwhile, since a few surface proteins are contained, many unrelated antigenic determinants of bacteria and side reaction caused by crude extraction or semi-purification preparation are eliminated; and the K99-987P-F41 recombinant protein has the characteristics of good safety and good stability, can overcome the shortcomings of whole-cell inactivated vaccines, and has the advantage of high specificity of subunit vaccines.

Description

technical field [0001] The invention belongs to the fields of animal molecular biology and genetic engineering, and relates to a K99-987P-F41 recombinant protein and its application. Background technique [0002] Enterotoxigenic Escherichia coli (ETEC) is the most common cause of calf diarrhea, usually causing acute diarrhea in cattle, pigs and other animals. ETEC mainly acts on the jejunum and ileum, especially the ileum. The virulence factors of ETEC include adhesins (also known as pili) and enterotoxins. The pathogenic mechanism of ETEC is that the specific pili bind to the specific receptors of the host small intestinal mucosal epithelial cells, and then colonize the small intestinal mucosa to prevent its loss due to intestinal peristalsis and intestinal content flow. ETEC proliferates in large numbers in the intestinal tract. secrete enterotoxin. Enterotoxin can lead to enhanced secretory function and decreased absorption function of small intestinal mucosal cells, c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K39/116A61K39/108A61P31/04
Inventor 倪宏波郎秀艳张洋龙邢思疑蒋慧婷刘娇
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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