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Riemerella anatipestifer OmpH intercepted recombinant protein, and preparation method and application thereof

A technology of Riemerella anatipestiferus and recombinant protein, which is applied in the field of bioengineering, can solve the problems of low sensitivity and specificity, no research and application research reports on Riemerella anatipestifer, and achieve stable performance and market application The effect of wide prospects and low production cost

Active Publication Date: 2016-05-04
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently established methods for detecting serum antibodies to Riemerella anatipestifer include agar diffusion test, agglutination test, ELISA, etc. These detection methods either have low sensitivity and specificity, or exist due to different antigenic components. Some deficiencies; and the prevention and treatment of Riemerella anatipestifer is mainly to treat with antibiotics or to prevent corresponding serotype infection with bacterium vaccine
OmpH belongs to the porin, and there is no relevant research report on the research and application of OmpH of Riemerella anatipestifer

Method used

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  • Riemerella anatipestifer OmpH intercepted recombinant protein, and preparation method and application thereof
  • Riemerella anatipestifer OmpH intercepted recombinant protein, and preparation method and application thereof
  • Riemerella anatipestifer OmpH intercepted recombinant protein, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The preparation of embodiment 1 Riemerella anatipestifer OmpH truncated recombinant protein

[0045] 1. Main experimental materials

[0046] Plasmid T-VectorpMD19 (simple), MaxDNAPolymerase was purchased from Dalian Bao Biological Engineering Co., Ltd.; prokaryotic expression plasmid pET32a (+), Novagen product; clone host bacteria E.coliDH5α, expression host bacteria E.coliBL21 (DE3) and RA-CH-1 strains, from Sichuan Provided by the Poultry Disease Research Center of Agricultural University.

[0047] 2 Experimental methods

[0048] 2.1 Cloning of RA-CH-1OmpH truncated gene

[0049] 2.1.1 Primer design

[0050] According to the existing RA-CH-1OmpH gene sequence in GenBank, the main antigenic domain (81aa-151aa and named as OmpHM) was selected, and a pair of primers were designed using PrimerPremier5.0 software. Upstream primer as shown in SEQIDNO:3: 5'-CGC GGATCC GAGGCTCAGAGAACTGCT-3' (the underlined part is the BamHI site); the downstream primer shown in SEQ I...

Embodiment 2

[0131] Embodiment 2 Purification of Riemerella anatipestifer OmpH truncated recombinant protein

[0132] After the expression product containing the OmpH truncated recombinant protein of Riemerella anatipestifer prepared in Example 1 was collected from the bacteria, ultrasonically disrupted, and supernatant collected, the recombinant protein was purified by nickel agarose gel affinity chromatography. OmpH truncated recombinant protein. The UV curve of the protein sample after column purification showed three peaks, peak 1 was the breakthrough peak, peak 2 was the elution peak of 50 mmol imidazole, and peak 3 was the elution peak of 100 mmol imidazole. Collect different concentrations of imidazole elution peaks simultaneously, carry out SDS-PAGE electrophoresis, check purity and concentration, the result shows: only contain a large amount of high-purity OmpH truncated recombinant proteins in the 100mmol imidazole elution peak ( Figure 8 ). After the purified OmpH truncated r...

Embodiment 3

[0133] Western-blot (western blot) of embodiment 3 Riemerella anatipestifer OmpH truncated recombinant protein

[0134] 1. Experimental method

[0135] The recombinant protein containing the OmpH truncated fragment of Riemerella anatipestifer obtained in Example 1 was used as a probe for detecting the serum antibody of Riemerella anatipestifer.

[0136] 1SDS-PAGE gel preparation

[0137] 1. Preparation of 112% separating gel

[0138] 1215 μl of deionized water, 950 μl of 1.5MTris-HCl (PH=8.8), 37.5 μl of 10% SDS, 37.5 μl of 10% AP, 1.5 μl of TEMED, and 1.5 ml of 30% acrylamide.

[0139] Preparation of 1.25% Stacking Gel

[0140] 700 μl of deionized water, 125 μl of 1.0MTris-HCl (pH=6.8), 10.0 μl of 10% SDS, 10.0 μl of 10% AP, 1.0 μl of TEMED, and 165 μl of 30% acrylamide.

[0141] 2 Processing of protein samples

[0142] Add an appropriate amount of SDS-PAGE protein loading buffer (0.5MTris-HCl (PH=6.81.2ml), glycerol 1ml, deionized water 4.8ml, 10% SDS2.0ml, 0.1% BPB in ...

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Abstract

The invention discloses a riemerella anatipestifer OmpH intercepted recombinant protein, and a preparation method and application thereof, and belongs to the field of biological engineering. The amino acid sequence of the riemerella anatipestifer OmpH intercepted recombinant protein is shown as SEQ ID NO:1; then, a gene engineering technology is used; on the clone basis, the connection with a prokaryotic expression vector pET-32a (+) is performed; a gene engineering product induced and expressed by a colon bacillus BL21(DE3) expression system is successfully converted; the expression form of the protein is OmpHM / His fusion protein; the molecular weight of the expressed fusion protein is 28kDa; the product is designed into fusion expression, and the effect of being convenient to purify is mainly considered; meanwhile, the expression protein maintains the natural activity of the protein. The product can be applied to the detection of detection antigen of a riemerella anatipestifer antibody by an indirect ELISA (enzyme-linked immuno sorbent assay) method, and can also be used as a gene engineering sigmasubunit vaccine for preventing the riemerella anatipestifer.

Description

technical field [0001] The invention belongs to the field of bioengineering, and particularly relates to a Riemerella anatipestifer OmpH truncated recombinant protein, a preparation method and application thereof. Background technique [0002] Riemerella anatipestifer (RA) is a Gram-negative bacterium belonging to the Flavobacteriaceae family (Flavobacteriaceae), the causative agent of Riemerella anatipestifer disease, which can cause ducks, geese, turkeys and various A chronic or acute septicemia-like contagious disease of poultry, distributed worldwide, with high morbidity and mortality, has become one of the main infectious diseases that seriously endanger the duck industry. The main clinical symptoms of the disease are mainly mental depression, paralysis, neck shrinkage, yellow-green loose stools and other symptoms. The lesion is characterized by fibrinous air sacculitis, meningitis, pericarditis, perihepatitis, and caseous salpingitis in the host, accompanied by sepsis...

Claims

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Application Information

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IPC IPC(8): C07K14/195C12N15/31C12N15/70G01N33/68G01N33/569A61K39/02A61P31/04
CPCA61K39/00C07K14/195G01N33/56911G01N33/68Y02A50/30
Inventor 程安春高群汪铭书朱德康杨乔陈孝跃
Owner SICHUAN AGRI UNIV
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