The Function of Lgbp Involved in the Recognition of Seaweed Polysaccharide and Immune Activation in Penaeid Shrimp

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Project title

Code/計畫編號

NSC101-2313-B019-002

Translated Name/計畫中文名

對蝦辨識蛋白LGBP辨識海藻多醣與免疫啟動機制

Project Coordinator/計畫主持人

Jiann-Chu Chen

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Aquaculture

Website

https://www.grb.gov.tw/search/planDetail?id=2643808

Year

2012

Start date/計畫起

01-08-2012

Expected Completion/計畫迄

31-07-2013

Bugetid/研究經費

950千元

ResearchField/研究領域

漁業
生物技術(農)

海藻多醣可提昇蝦類先天性免疫力以及對抗弧菌的侵襲，但有關海藻多醣如何透過辨識，啟動對蝦先天性免疫的研究闕如。LGBP 屬於醣基水解蛋白（glucosyl hydrolase）家族 16 (GH16)的成員，辨識帶β 鍵結的多醣類，對蝦的LGBP 結構上帶有兩個多醣辨識位置 (PsBM，βGRM)，已知可幫助辨識LPS 以及β-glucan，而海藻多醣的結構裡面常帶有β-linked 的醣基鍵結，推測海藻多醣應該可透過LGBP 辨識來達到免疫提昇的作用。本計畫擬表現rLGBP 辨識海藻多醣，並研究其影響對蝦先天性免疫啟動機制。第一年建構pET28-rLGBP、pET28-rPsBM 與pET28-rβGRM 重組蛋白表現質體，表現rLGBP、rPsBM 以及rβGRM，其次研究rLGBP、rPsBM、rβGRM 對於革蘭式陰性菌Escherichia coli、Vibrio alginolyticus，革蘭氏陽性菌Bacillus subtilis、Bacillus thuringiensis 以及酵母菌Pichia pastoris 的辨識與結合能力，證實rLGBP 蛋白質的特性與細菌結合能力。第二年探討rLGBP、rPsBM、 rβGRM 與海藻多醣（如鹿角菜膠、藻酸、褐藻醣膠、龍鬚菜萃取物等）之辨識與結合能力，進一步以rLGBP、rPsBM 或rβGRM 與海藻多醣之結合物注射到對蝦(如白蝦)體內，研究造血組織血淋巴球增生，酚氧化酵素活性，超氧離子生成等免疫因子，細菌吞噬能力與對病原的抵抗。第三年首先以核酸干擾技術（RNAi）進行LGBP 減弱（knock down），探討蝦在 LGBP-silencing 的情況下，對於蝦造血組織血淋巴球增生，免疫因子，細菌吞噬能力與對病原之抵抗的影響，進一步研究當對蝦之LGBP 被knock down (LGBP-silencing)的情況下，注射rLGBP 與海藻多醣結合物，研究其參與對蝦的免疫啟動反應，並以rLGBP-海藻多醣結合物注射對蝦，研究其對於病原之抵抗。 White shrimp Litopenaeus vannamei, tiger shrimp Penaeus monodon, and kuruma shrimp Marsupenaeus japonicus are the important penaeid shrimps currently being cultured worldwide. The world production of white shrimp and tiger shrimp ranked 8th and 13th among the species of fish, crustacean and mollusk in year 2008. It is known that deteriorated environment, weakened host and pathogen aggregation may cause an occurrence of disease outbreak. Previous research indicated that several seaweed polysaccharides including alginate, carrageenan, fucoidan, and seaweed extract like Gracilaria tenuistipitata extract can enhance the innate immunity of shrimp and its resistance against Vibrio alginolyticus. However, little is known on the mechanism involved in the recognition and activation of innate immunity when shrimp have received the seaweed polysaccharide. Lipopolysaccharide and β-glucan binding protein (LGBP) belongs to glucosyl hydrolase 16 family protein (GH16), and is known to recognize polysaccharides having β-linkage glycosyl bond of LGBP, an important pattern recognition proteins (PRPs) has two polysaccharide recognition motifs, PsBM and βGRM that are considered to facilitate the binding of polysaccharides with β-linkage including alginate, carrageenan, β-glucan and LPS, and lead to activation of innate immunity. The purpose of this study is to recombine full length sequence LGBP (rLGBP), partial sequence, PsBM (rPsBM), and βGRM (rβGRM), and examine the binding capability (affinity) of rLGBP, rPsBM, rβGRM with Gram-negative bacteria, Escherichia coli, V. alginolyticus, Gram-positive bacteria Bacillus subtilis, Bacillus. thuringiensis and yeast Pichia pastoris in vitro to confirm the function of LGBP and its binding capability with foreign particles like Gram-positive and Gram-negative bacteria and yeast during the first year. This study is to conduct the binding capability of rLGBP, rPsBM and rβGRM with alginate, carrageenan, fucoidan, and seaweed extract etc., examine the immune parameters like phenoloxidase activity etc., haemocyte proliferation of HPT, and examine phagocytic activity and resistance against V. alginolyticus when shrimp have been injected with the mixture of rLGBP, rPsBM, rβGRM and alginate, carrageenan, fucoidan, and seaweed extract etc. during the second year. This study is to conduct gene knock down of LGBP, produce LGBP-silencing shrimp, and examine the immune parameters and phagocytic activity and resistance against V. alginolyticus of LGBP-silencing shrimp, and then examine the immune parameters, phagocytic activity and resistance against V. alginolyticus when the LGBP-silencing shrimp have received the mixture of rLGBP, rPsBM, rβGRM with alginate, carageenan, seaweed extract etc. during the third year.

Keyword(s)

對蝦
酯多醣與葡聚醣結合蛋白
重組蛋白
藻酸
鹿角菜膠
褐藻醣膠
龍鬚菜萃取物
革蘭氏陰性
革蘭氏陽性
酵母
結合能力
RNAi
免疫因子
免疫啟動
Shrimp
LGBP
recombinant
alginate
carrageenan
fucoidan
Gracilaria tenuistipitata extract
Gram-negative bacteria
Gram-positive bacteria
binging capability
RNAi
immune parameter
immune activation

DSpace CRIS

The Function of Lgbp Involved in the Recognition of Seaweed Polysaccharide and Immune Activation in Penaeid Shrimp

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