Conglutinin, a liver synthesized versatile innate immune marker consisting C-type lectin domain belongs to collectin superfamily of proteins. The protein, first detected in bovine serum as soluble pattern recognition receptor (PRR) has wide range of antimicrobial activities. In the present study, open reading frame (ORF) encoding neck and carbohydrate recognition domain (NCRD) of goat conglutinin gene ligated to the vector pRSET-A was expressed in E. coli BL-21(pLys) cells. The 27 kDa recombinant protein (rGCGN) purified by single step Ni+2 -NTA affinity chromatography was found to cross-react with recombinant anti-buffalo conglutinin antibody raised in poultry. Further, it displayed calcium-dependant sugar binding activity towards yeast mannan and calcium-independent binding activity towards LPS. The mannan binding activity of rGCGN was inhibited in the presence of N-acetyl-glucosamine because of higher affinity towards this sugar. The recombinant protein was found to stimulate production of superoxide ions and hydrogen peroxide in goat neutrophils, which are instrumental in stimulating phagocytic activity of cells. When used as antigen in Sandwich ELISA, straight line (Y = 0.299x + 0.067, R2 = 0.997) was observed within the concentration range of 200-1000 ng/100 μl of rGCGN. Using this equation, the native conglutinin concentration in goat sera was estimated to be 0.5-7.5 μg/ml. The results indicated that prokaryotically expressed functionally active rGCGN can be used as antigen to assess native serum conglutinin levels in Sandwich ELISA and as immunomodulator in therapeutic applications to sequester unwanted immune complexes from the circulation.

中文翻译：

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部分重组山羊凝集素的功能表征：其作为先天免疫标记物的作用，并在三明治ELISA中用作抗原。

凝集素是肝脏合成的由C型凝集素结构域组成的通用先天免疫标记物，属于蛋白质的collectin超家族。该蛋白质首先在牛血清中被检测为可溶性模式识别受体（PRR），具有广泛的抗菌活性。在本研究中，在大肠杆菌BL-21（pLys）细胞中表达了编码与载体pRSET-A连接的山羊凝集素基因的颈部和糖基识别结构域（NCRD）的开放阅读框（ORF）。发现通过一步Ni + 2-NTA亲和层析纯化的27 kDa重组蛋白（rGCGN）与家禽中产生的重组抗水牛凝集素抗体发生交叉反应。此外，它表现出对酵母甘露聚糖的钙依赖性糖结合活性和对LPS的钙非依赖性结合活性。在N-乙酰基-葡萄糖胺存在下，rGCGN的甘露聚糖结合活性受到抑制，因为对这种糖的亲和力更高。发现重组蛋白可刺激山羊嗜中性粒细胞中超氧离子和过氧化氢的产生，这在刺激细胞的吞噬活性中起重要作用。在夹心ELISA中用作抗原时，在200-1000 ng / 100μlrGCGN的浓度范围内观察到直线（Y = 0.299x + 0.067，R2 = 0.997）。使用该方程式，山羊血清中的天然凝集素浓度估计为0.5-7.5μg/ ml。