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Avian chlamydophila psittaci outer membrane protein N-PmpD, preparation method and application

A technology of chlamydia psittaci and outer membrane protein, which is applied in the fields of chlamydia antigen components, botanical equipment and methods, biochemical equipment and methods, etc., to achieve the effect of strong specificity, good application value and market prospect, and good immunogenicity

Inactive Publication Date: 2012-08-22
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, what is the role of the PmpD protein of Chlamydia avulophila in the process of Chlamydia infection in poultry, and whether it has the same immunological value as the MOMP protein of Chlamydia remains to be further studied

Method used

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  • Avian chlamydophila psittaci outer membrane protein N-PmpD, preparation method and application
  • Avian chlamydophila psittaci outer membrane protein N-PmpD, preparation method and application
  • Avian chlamydophila psittaci outer membrane protein N-PmpD, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Cloning and expression of Chlamydia psittaci outer membrane protein N-PmpD gene and preparation of N-PmpD protein

[0046] 1 Main materials and reagents

[0047] 1.1 Strains and plasmids Chlamydia psittacis thermophilic Cal-10 strain, gifted by Professor Patrick, University of Maryland, USA, preserved in our laboratory; Escherichia coli TOP-10, BL21(DH3), purchased from Beijing Quanshijin Biotechnology Co., Ltd. The vector pET-32a(+) is a product of Novagen, which is stored in our laboratory. DNA Ladder 2000 was purchased from Beijing Zhongke Ruitai Biotechnology Co., Ltd. M1: DNA marker KB; M2: λDNA; M3: DNA markerVIII, all purchased from Dalian TAKaLa Biotechnology Co., Ltd.

[0048] 1.2 Reagents PCR-Mix reaction solution (including reaction buffer and dNTP mixture), PBS-T II Vector, restriction enzymes such as Sal I and Not I, T4 DNA ligase, DNALadder1500, and λDNA / HindIII were purchased from Dalian Bao Biology Engineering Co., Ltd.; IPTG (isopropyl-β-D-g...

Embodiment 2

[0072] Example 2 Determination of the activity and reactogenicity of the avian Chlamydia psittaci N-PmpD protein

[0073] 1. Materials and Reagents

[0074] IPTG (isopropyl-β-D2 galactoside), proteinase K, RNase A: purchased from Merk Company (Beijing). Low molecular weight protein marker: Fermentas company, batch number SM0671; urea (analytical grade), Coomassie Brilliant Blue R-250, BPB (bromophenol blue), Lichunhong S: purchased from Beijing Kehaoze Technology Company. HRP-labeled rabbit anti-chicken IgG antibody, HRP-labeled rabbit anti-goat IgG antibody, HRP-labeled rabbit anti-pig IgG antibody, PVDF membrane: purchased from Beijing Dingguo Biotechnology Co., Ltd. 3,3'-diaminobenzidine (DAB), TEMED, ammonium persulfate, acrylamide, N,N'-methylenebisacrylamide: all were purchased from Amresco, USA.

[0075] Chlamydia psittaci standard positive and negative serum: SPF chickens were challenged with the standard strain of Chlamydia psittaci, then the serum of the isolated c...

Embodiment 3

[0080] Embodiment 3 recombinant protein is to the immune protection test of SPF chicken

[0081] 1. Experimental animals

[0082] 36 SPF chickens, 21 days old, male, were purchased from Beijing Meria Weitong Experimental Animal Technology Co., Ltd., and the experimental process was in compliance with the relevant regulations on animal welfare of the Beijing Experimental Animal Management Center.

[0083] 2. Main experimental reagents and consumables

[0084] Thiazolan (MTT), RPMI 1640 medium (batch number 1165062), fetal bovine serum (batch number 506197): all were purchased from Beijing Santai Biotechnology Co., Ltd. HRP-labeled rabbit anti-chicken secondary antibody, HRP-labeled goat anti-rabbit IgG: Beijing Dingguo Biotechnology Co., Ltd.; Costar 96-well cell culture plate, Costar 96-well ELISA plate: Beijing Xinjingke Biotechnology Company. Tetramethylbenzidine (TMB), heparin sodium, concanavalin (ConA), bovine serum albumin: Beijing Kehaoze Biotechnology Co., Ltd.; Chla...

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Abstract

The invention provides an avian chlamydophila psittaci outer membrane protein N-PmpD, a preparation method and the application. The protein N-PmpD refers to: 1) protein composed by an amino acid sequence showed in SEQ ID No.2, or 2) protein derived from 1) and with equal activity by substituting, missing or adding one or several amino acid in the amino acid sequence showed in SEQ ID No.2. The invention further discloses a gene for coding the protein. Experiments show that the protein N-PmpD has good immunogenicity, can serve as an immunogen to enhance resistance of birds to infection of avian chlamydophila psittaci, and is good in immune effect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Chlamydophilapsittaci outer membrane protein N-PmpD, a preparation method and application of the protein. Background technique [0002] Avian Chlamydiosis (Avian Chlamydiosis, AC) is a contact zoonotic disease caused by different syndromes caused by Chlamydophila psittaci infecting poultry. Clinically, it is mainly characterized by pneumonia, pericarditis, Air sacculitis and conjunctivitis are characteristic. Infected poultry flocks can excrete chlamydia for a long time, which seriously threatens human health. The prevalence of this disease has seriously threatened the development of my country's breeding industry and human health. To explore the new protective antigen and immune mechanism of Chlamydia thermophilic psittac. Induction of high levels of humoral and cellular immunity is expected to eliminate the problem of persistent infection with Chlamydia psittaci. [0003] The ...

Claims

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Application Information

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IPC IPC(8): C07K14/295C07K16/12C12N15/34C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A61K39/118A61P31/00G01N33/569
Inventor 何诚凌勇袁吉磊杨君敬
Owner CHINA AGRI UNIV
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