• 한국축산식품학회지
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• 제36권6호
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• pp.769-778
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• 2016

In this study, we improved the eggshell-membrane separation process by separating the shell and membrane with EDTA solution, evaluating effects of three different extraction solutions (acetic acid, EDTA, and phosphate solution), and co-purifying multiple eggshell proteins with two successive ion-exchange chromatography procedures (CM Sepharose Fast Flow and DEAE Sepharose Fast Flow). The recovery and residual rates of eggshell and membrane separated by the modified method with added EDTA solution were 93.88%, 91.15% and 1.01%, 2.87%, respectively. Ovocleidin-116 (OC-116) and ovocalyxin-36 (OCX-36) were obtained by loading 50 mM Na-Hepes, pH 7.5, 2 mM DTT and 350 mM NaCl buffer onto the DEAE-FF column at a flow rate of 1 mL/min, ovocleidin-17 (OC-17) was obtained by loading 100 mM NaCl, 50 mM Tris, pH 8.0 on the CM-FF column at a flow rate of 0.5 mL/min. The purities of OCX-36, OC-17 and OC-116 were 96.82%, 80.15% and 73.22%, and the recovery rates were 55.27%, 53.38% and 36.34%, respectively. Antibacterial activity test suggested that phosphate solution extract exhibited significantly higher activity against the tested bacterial strains than the acetic acid or EDTA extract, probably due to more types of proteins in the extract. These results demonstrate that this separation method is feasible and efficient.

• Asian-Australasian Journal of Animal Sciences
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• 제26권5호
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• pp.609-624
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• 2013

The objective of this study was to get a comprehensive understanding of how genes in chicken shell gland modulate eggshell strength at the early stage of active calcification. Four 32-week old of purebred Xianju hens with consistent high or low shell breakage strength were grouped into two pairs. Using Affymetrix Chicken Array, a whole-transcriptome analysis was performed on hen's shell gland at 9 h post oviposition. Gene ontology enrichment analysis for differentially expressed (DE) transcripts was performed using the web-based GOEAST, and the validation of DE-transcripts was tested by qRT-PCR. 1,195 DE-transcripts, corresponding to 941 unique genes were identified in hens with strong eggshell compared to weak shell hens. According to gene ontology annotations, there are 77 DE-transcripts encoding ion transporters and secreted extracellular matrix proteins, and at least 26 DE-transcripts related to carbohydrate metabolism or post-translation glycosylation modification; furthermore, there are 88 signaling DE-transcripts. GO term enrichment analysis suggests that some DE-transcripts mediate reproductive hormones or neurotransmitters to affect eggshell quality through a complex suite of biophysical processes. These results reveal some candidate genes involved with eggshell strength at the early stage of active calcification which may facilitate our understanding of regulating mechanisms of eggshell quality.

• KSBB Journal
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• 제30권6호
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• pp.296-301
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• 2015

Eggshell-based biocomposites have become attractive due to their exquisite nanostructure and biological properties, which are mainly composed of highly organized calcium carbonate crystals controlled by organic macromolecules such as proteins and polysaccharides. Here, we designed the recombinant fusion protein of a putative eggshell matrix protein named as GG1234 and a fluorescent reporter protein of DsRed. The protein was successfully over-expressed in E. coli and purified by Ni-NTA affinity chromatography. In vitro calcium carbonate crystallization was conducted in the presence of the fusion protein, and morphological change was investigated. The protein inhibited the calcite growth in vitro, and spherical calcium carbonate micro-particles with the diameter of about $20-30{\mu}m$ were obtained. We expect that this study would be helpful for better understanding of eggshell-based biomineralization.

• 한국가금학회지
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• 제44권1호
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• pp.19-28
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• 2017

계란의 난각은 닭의 자궁에서 형성되는 복잡하고 고도화된 다층 석회화 구조이다. 본 연구는 닭의 산란연령에 따른 자궁내막조직의 변화 양상과 난각 형성에 관여하는 주요 유전자들의 발현 양상을 고찰하였다. 난각 형성과 관련하여 난각 단백질과 관련한 OCX-32, OCX-36, OC-17, OC-116 유전자와 난각 이온과 관련한 CALB1, SPP1, SCNN1G, ATP2A2, CA2, CALM1 유전자를 분석대상으로 하였다. 자궁내막조직의 현미경적 관찰 결과, 연령이 증가함에 따라, 자궁 섬모상피세포의 탈락 및 세포의 위축과 섬유화 진행양상이 심화되었다. 그러나 연령 증가에 따른 혈중 이온성분의 농도차이는 없었다. 자궁내막 상피세포의 텔로미어 함량 분석 결과, 산란연령이 증가함에 따라 텔로미어 함유율이 감소됨을 확인하였다. 대부분의 난각 형성 관련 유전자의 발현량은 산란연령에 따라 유의한 변화를 나타내었다. 일부 난각 단백질 관련 유전자의 발현량은 산란연령이 증가함에 따라 증가양상을 보였으나, 이들 유전자들 간의 상호 관련성은 유의한 관계를 보이지 않았다. 난각 형성 이온 관련 유전자인 ATP2A2, SCNN1G, CA2, CALM1 유전자 간에는 상호 밀접한 관련이 있는 것으로 나타났다. 또한 난각 단백질 관련 OCX-32 및 OCX-36 유전자는 대부분의 이온 관련 유전자들과 높은 정의 상관을 보였다. 따라서 OCX-32, OCX-36, CALB-1, ATP2A2, SCNN1G, CA2 및 CALM1유전자들이 난각 형성에 상호 협력하거나 또는 독립적으로 작용한다. 결론적으로 산란연령이 증가함에 따라 자궁내막세포의 손상이 일어나지만, 그럼에도 불구하고 정상적인 난각을 형성할 수 있는 이유는 난각 단백질 및 난각 형성 이온 관련 일부 유전자들이 발현을 조절하여 난각 형성의 항상성 유지하기 때문인 것으로 사료된다.