Characteristics And Functional Analysis Of C-type Lectin 11 (BmCTL11) Of Silkworm, Bombyx Mori

Insect is a big animal group on the earth with more than one million species, they play important roles in ecological system. As invertebrates, insects lack the adaptive immune system, but they have formed a highly effective defense system to resist the invasion and parasitization of microbes during the process of evolution. The recognition of pathogen-associated molecule patterns (PAMPs) by pattern recognition receptors (PRRs) triggers the first step of defense.Silkworm is an important economic insect, they are also a typical model system for Lepidoptera with abundant PRRs, such as C-type lectin. Based on Genome-wide transcriptional response of silkworm induced by Nosema bombycis, we focused on BmCTL11 which can be constantly and abundantly expressed in the middle-late stages of silkworm induced by Nosema bombycis. However, the function of BmCTL11 is not clear in innate immune system of silkworm. In this study, we analysed the function of BmCTLll in innate immune system of silkworm. Firstly, we analysed the sequence feature of BmCTL11. Subsequently, heterologous expression and preparation of polyclonal antibodies were carried out. In addition, the analysis of characteristics and fuction of BmCTL11 was implemented. The main results obtained are listed as follows:1. The sequence analysis of BmCTL11Bioinformatic analysis indicated that BmCTL11 located in the 10th chromosome and it is composed of 307 amino acids with a molecular weight of 34.51KDa and an isoelectric point of 4.69. It also has two carbohydrate recognition domains(CRDs) called CRD1(47 to 148 amino acid) and CRD2(184 to303 amino acids). The length of the coding-sequence of BmCTL11 is 924bp without intron. BmCTL11 has one signal peptide region (1 to 21 amino acids) and two N-glycosylation sites (N214 and N253). The secondary structure of protein encoded by BmCTL11 was predicted,which showed this protein is composed of 4 α-helixes and 18 β-sheets and 23 random coils, and there also has one mannose binding site in C terminal of this protein. According to microarray data, the expression of BmCTL11 was in a high level in many tissues, including fat body,integument, ovary, head and testis of silkworm, the highest expression level of BmCTLll was detected in fat body,these results suggested the BmCTLll may play an important role in the innate immune system. In addition, we found that the expression level of BmCTL11 was higher in ovary than that in testis and the BmCTL11 could be up-regulated by the induction of N. bombycis.2. The BmCTLll expression profileRT-PCR analysis of the expression of BmCTL11 showed that it was detected in fat body, malpighian tube, testis and ovary. RT-PCR assay showed there was transcription of BmCTL11 in the period of larva instead of in late stage of pupae. In addition, the analysis of qRT-PCR showed the expression of BmCTL11 could be up-regulated by N. bombycis, B. bassiana, P. pastoris, B. bombyseptieus, S. marcescens and BmNPV, the change was obvious within 48 hours. These results suggested in the larva period BmCTL11 may play an important role in the early stage of pathogen infection.3. Cloning and expression of BmCTL11 and preparation of rBmCTL11 polyclonal antibodyBmCTL11 sequence was amplified using specific primers and the amplified fragment was inserted into the heterologous expression vector pET32 and over-expressed in Transetta(DE3) under 16℃ with induction by 0.1mmol/L IPTG. The fusion protein of BmCTL11 was expressed in form of soluble expression and inclusion body expression. Then the purified rBmCTL11 was injected into mice to prepare polyclonal antibody (PAb). The titer of Anti-BmCTL11 was more than 1:102400 determined by Enzyme-Linked Immuno Sorbent Assay (ELISA). The tissue protein of silkworm were extracted and analyzed with anti-rBmCTL11 by Western blotting, the results presented a specific band with an approximated size of 60KDa which is larger than the predicted theoretical molecular weight of rBmCTL11. The possible reasons caused this phenomenon may be BmCTL11 forming complex with other proteins of silkworm or forming heterodimer between BmCTL11 and the other CTLs of silkworm. The results also indicated the BmCTL11 can express in all tissues except head and integument of silkworm.4. Function analysis of BmCTL11The binding experiments of rBmCTL11 with carbohydrate or microorganism were carried out after the soluble protein obtained.The results showed rBmCTL11 could bind to mannose, lipopolysaccharide and galactose except peptidoglycan. Besides, the binding of rBmCTL11 to LPS is depended on calcium ion, suggesting calcium ion participated in the binding process of BmCTL11 to E. coli. In addition, rBmCTL11 could agglutinate N. bombycis, P. pastoris, B. bombyseptieus and S. marcescens obviously detected by Western blotting. The results suggested rBmCTL11 could recognize microorganisms and trigger innate immunity. To testify this point, rBmCTL11, hemocytes and saccharides were used in encapsulation and melanization, the results showed nickel agarose beads coated with rBmCTL11 enhanced encapsulation and melanization of haemocytes, and the phenomenon was more evident with existence of rBmCTL11 and mannose, lipoteichoic acid, lipopolysaccharide or trehalose. A combination of rBmCTL11 with saccharides activated phenol oxidase more rapidly and to a higher level than immulectin alone, whereas lipopolysaccharide by itself had little effect on phenol oxidase activation and the combination between rBmCTLll and lipoteichoic acid was the most valid. The study of BmCTL11 will provide a new view to recognize the innate immune system of silkworm and its resistance on infection.