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Preparation method and application of chlamydia psittaci polytype outer membrane protein PmpG protein

A technology of outer membrane protein and chlamydia, applied in the field of agricultural biology, can solve the problem of unclear effect, achieve good immune efficacy, reduce immune stress and high synergistic effect

Active Publication Date: 2021-07-09
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the role of C. psittaci PmpG protein in diagnosis and immune protection is still unclear

Method used

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  • Preparation method and application of chlamydia psittaci polytype outer membrane protein PmpG protein
  • Preparation method and application of chlamydia psittaci polytype outer membrane protein PmpG protein
  • Preparation method and application of chlamydia psittaci polytype outer membrane protein PmpG protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Cloning and expression of Chlamydia psittaci polytype outer membrane protein Pmp17G, 19G, 20G, 21G gene and preparation of recombinant protein

[0041] 1 Experimental materials

[0042] 1.1 Bacterial strains, plasmid vectors, cell lines and serum

[0043] The 6BC strain of Chlamydia psittaci was donated by Professor Wu Yimou of Nanhua University and kept in the Laboratory of Veterinary Medicine, China Agricultural University. Saccharomyces cerevisiae CEN.PK 2-1C strain, Escherichia coli Rosseta GM-48 strain: donated by Heinrich Heine University, Germany. Escherichia coli RosettaGM48 was purchased from Nonages (USA). Hela229 cells and Chlamydia psittaci 6BC positive serum were preserved in the School of Veterinary Medicine, China Agricultural University. pKM32 vector: based on pAC2, integrates CEN6, ARS, URA3, and fuses His tag at the N-terminus of the target protein, donated by Heinrich Heine University, Germany.

[0044] 1.2 Main reagents and kits

[004...

Embodiment 2

[0141] Embodiment 2 Establishment of Chlamydia psittaci PmpG-ELISA method

[0142] 1 Main reagents and kits

[0143] IPTG (isopropyl-β-D2 galactoside), proteinase K, RNase A: purchased from Merk Company (Beijing). Low molecular weight protein marker: Fermentas company, lot 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 purchased from Amresco, USA. MOMP recombinant protein: prepared and identified by the School of Veterinary Medicine, China Agricultural University.

[0144] 1.1 Experimental animals and serum

[0145] 28-day-old SPF chickens were purchased from Beij...

Embodiment 3

[0233] Example 3 Chlamydia psittaci Pmp17G, Pmp19G, Pmp20G, Pmp21G protein vaccine to the immune protection test of SPF chicken

[0234] 1 test animal

[0235]SPF chickens, 50, 21-day-old, male, were purchased from Beijing Meria Weitong Experimental Animal Technology Co., Ltd., and the experimental process complied with the relevant regulations on animal welfare of the Beijing Experimental Animal Management Center.

[0236] 2 Main experimental reagents and consumables

[0237] Chicken IL-10, IL-2 and IFN-γ cytokine ELISA kits and ELISA Accessory Pack were purchased from Abcam, USA. Concanavalin protein ConA (C8110), BCA protein concentration assay kit (50T), 4% tissue cell fixative and chicken peripheral blood lymphocyte separation kit were purchased from Beijing Soleibao Company; BrdU cell proliferationELISA Kit (colorimetric) Purchased from Abcam, USA. Mouse anti-chicken CD3 / SPRD marker (8200-13), CD8a / PE marker (8220-09) and CD4 / FITC marker (8210-02) were purchased from ...

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Abstract

The invention provides a preparation method of an avian chlamydia psittaci polymorphic outer membrane protein (PmpG) protein and an application thereof. The method includes: 1) chlamydia psittaci PmpG protein which comprises primers, vectors, expression conditions and a protein renaturation technology for 17G, 19G, 20G and 21G protein expression are provided; 2) the prepared Pmp20G protein is used as a coating antigen to prepare an ELISA diagnostic kit, the kit has good specificity, sensitivity and repeatability, the coincidence rate of the kit reaches 98.1% compared with a foreign commercial kit, and the kit has no cross reaction with other related respiratory pathogens; 3) the four protein compositions of chlamydia psittaci Pmp17G, 19G, 20G and 21G are used as antigens, and the prepared vaccine has the characteristics of small dosage, high synergistic effect, high immune protection efficacy and the like. and 4) a composition of four proteins of chlamydia psittaci Pmp17G, 19G, 20G and 21G is taken as an antigen, chitosan gel is taken as an adjuvant, and good respiratory mucosa immunity can be generated through an aerial fog immune way, so that infection of the chlamydia psittaci is prevented and treated, and spreading of the chlamydia psittaci from livestock and poultry to people is blocked.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a preparation method and application of a polytype outer membrane protein PmpG protein of Chlamydia psittaci. Background technique [0002] Avian Chlamydiosis (Avian Chlamydiosis) is a contact zoonotic disease caused by different syndromes caused by Chlamydia psittaci (C.psittaci) infecting poultry. , Conjunctivitis is a feature. Infected poultry flocks can excrete chlamydia for a long time, which seriously threatens human health. Due to the lack of reliable commercial diagnostic kits and vaccines, it is difficult to effectively prevent and control the spread of Chlamydia psittaci. The prevalence of the disease has seriously threatened the development of the breeding industry and human health. Therefore, it is urgent to explore the reactivity and immunity of Chlamydia psittaci. Antigens for the development of new detection kits and vaccines to prevent and c...

Claims

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Application Information

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IPC IPC(8): C07K14/295C12N15/31C12N15/81A61K39/118A61K39/39A61P31/04G01N33/569G01N33/68C12R1/865
CPCC07K14/295C12N15/81A61K39/118A61K39/39A61P31/04G01N33/56927G01N33/6854A61K2039/55583A61K2039/552A61K2039/544G01N2469/20Y02A50/30
Inventor 何诚李强隋卓君王艺晖李小慧
Owner CHINA AGRI UNIV
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