中国明对虾C 型凝集素基因(Fclectin)的重组表达及活性分析

研究拟通过分析对虾C型凝集素的活性特点,探讨其在对虾先天免疫应答过程中的潜在功能以及在养殖生产实践中的应用。实验利用原核表达系统对中国明对虾C-型凝集素基因的两个串联的糖识别结构域(carbohydrate recognition domain,CRD)进行了重组表达,并通过纯化复性获得了重组目的蛋白(rFclectin-CRD1和rFclectin-CRD2)。活性分析结果显示,重组目的蛋白对多种病原菌有凝集和抑制生长的作用,并且具有Ca2+依赖活性;其凝集活性可被半乳糖、肽聚糖、脂多糖等多种病原相关分子模式所抑制,研究结果证实,Fclectin是一种典型的C-型凝集素,它可能作为中国明对虾先天免疫中重要的模式识别受体,在一定程度上参与了机体应答病原微生物的防御过程。

Recombinant expression and functional characterization of a C-type lectin ( Fclectin ) from the Chinese shrimp ( Fenneropenaeus chinensis )

Chinese shrimp(Fenneropenaeus chinensis)is distributed mainly along Chinese inshore areas,and is one of the most important farmed shrimp in China.The studies on innate immune responses of shrimps,especially on immune defense against the main crustacean pathogens,will provide more knowledge of shrimp immunity to prevent infectious Invertebrates do not possess an adaptive immune system based on highly specific antibodies and antigen receptors.They must rely on efficient immune defenses capable of protecting them against invading microorganisms.The chief issue of crustacean immunity should concern non-self-recognition mechanisms.Proteins that specifically bind to certain carbohydrate components on the surface of microorganisms play an important role in non-self-recognition and cleaning up of the invading microorganisms.Such proteins are known as pattern recognition receptors(PRRs).Lectins exist in almost all living organisms.Due to their ability of binding to terminal sugars on glycoproteins and glycolipids,lectins are primary candidates for pattern recognition receptors in innate immunity.C type Lectin is regarded as a potential molecule involved in immune recognition and phagocytosis through opsonization in crustacean.In the preliminary study,a novel C-type lectin was cloned from hemocytes of Chinese shrimp,(Fenneropenaeus chinensis).It contains two tandem carbohydrate recognition domains(CRDs)/C-type lectin like domains.Both of the CRDs contain a QPD(Gln-Pro-Asp)motif that has a predicted binding specificity for galactose type sugar.In this research,two recombinant target proteins(rFclectin CRD1 and rFclectin CRD2)were expressed by prokaryotic expression system.The result showed that fusion protein was expressed in the form of inclusion bodies.The LC-ESI-MS analysis showed that two peptide fragments of rFclectin-CRD1 and rFclectin-CRD2 were identical with the corresponding sequence of F.chinensis C-type lectin.Recombinant protein was purified by immobilized metal affinity chromatography and Ni-NTA technology.The concentrations of purified target proteins were 0.4 g/L.rFclectin-CRD1 and rFclectin CRD2 had agglutinating and antimicrobial activity against main pathogens in aquaculture in a calcium dependent manner.The agglutinating activity can be inhibited by multiple carbohydrates,such as galactose,peptidoglycan and lipopolysaccharide.These results suggest thatFclectin,as a dependent carbohydrate-recognition protein,is one of the important PRRs.It might play a crucial role in the innate immunity of the shrimp and is expected to be applied in disease control.