Cloning And Analysis Of The Gene BmNPC1 In Silkworm,Bombyx Mori

Niemann-Pick C(NPC)disease is a rare,panethnic,autosomal recessive disorder,leading to progressive neurodegeneration,which is caused by mutations either NPC1(95%)or NPC2(5%).The gene NPC1 acts as an intracellular cholesterol transporter in mammals,which encodes a 13-transmembrane protein locating in endosome and lysosome membrane and possessing a sterol-sensing domain.Cells with mutation of NPC1 reveal an accumulation of large amounts of unesterified cholesterol in the endosomal/lysosomal(E/L)compartment.Different functions of NPC1 have been observed,such as: NPC1 was involved in the transport of cholesterol;transmembrane molecular pump activity of NPC1 protein was proved;recent work on EBOV demonstrated that NPC1 protein is an essential entry receptor for Ebola virus.Take drosophila as an insect model,NPC1 was found participate in cholesterol trafficking,molting process and permatogenesis.There are two NPC1 orthologs,NPC1 a and NPC1 b,in the drosophila genome.However,none was reported about Bm NPC1 in Bombyx mori.To investigate the function of Bm NPC1 in Bombyx mori,we cloned Bm NPC1,and analyzed the expression of Bm NPC1.Then we prepared the Bm NPC1 antibody.Finally we explored the function of Bm NPC1 in Bombyx mori by ds RNA interference technology.The main research results are as following:1.c DNA clone and structural analysis of Bm NPC1Taking advantage of the genomics of silkworm and NCBI datebase,analysis the basic information of Bm NPC1,then designed the primers.Bm NPC1 was cloned by the rapid amplification of c DNA ends(RACE).The genomic DNA was 4591 bp long with its ORF 3702 bp long,and the gene located in the chromosome 3.Bm NPC1 coded 1233 amino acid,and encoded a protein 137.44 k D,the protein predicted to have a sterol-sensing domain and 13 transmembrane domain.Choosing NPC1 protein sequences of many spices to build an NJ phylogenetic tree,and the result showed that the genetic relationship of Bm NPC1 was nearest with drosophila.2.The expression analysis of Bm NPC1 in Bombyx mori B.mori was killed to obtain all tissues on day 3 of 5th instar larvae and the hemocytes from the molting period of the 4th instar larvae to the wandering stage.The c DNA was prepared taking advantage of the tissues,then we test the expression of Bm NPC1 through q RT-PCR.Expression analysis showed that Bm NPC1 was expressed in all tissues of silkworm,and highly expressed in testis,ovary,hemocytes and malpighian tubule.Bm NPC1 was highly expressed at molting stages in hemoccytes.3.Preparation of Bm NPC1 antibody and its specific detectionPolyclonal antibody of Bm NPC1 was prepared by four steps: construction of recombinant prokaryotic expression plasmid p ET32,a large amount of protein expressed that induced by IPTG,then the purification of protein expressed in inclusion body,afterwards,5 times of mice injection.Finally we gained the polyclonal antibody of Bm NPC1.We used ALISA to test antibody titer.Western blot domenstrated that the polyclonal anti-Bm NPC1 antibody can specifically detect Bm NPC1,and the weight of Bm NPC1 was about 130 k D as predicted.Immunofluorescence assay showed that Bm NPC1 was expressed in cytoplasm in hemocytes and germ lines(SID-1)of silkworm as the same as in mammal cells(NPC1 protein located on the membrane of endosome and lysosome).Polyclonal antibody of Bm NPC1 was successfully prepared,which laid the foundation for further study of Bm NPC1 in B.mori.4.Taking advantage of ds RNA interference technique to explore the function of Bm NPC1Design and synthesis the ds RNA segments.Using the ds RNA segments in SID-1 cells at the cellular level,stained by Filipin III,it was observed that there is an accumulation of cholesterol in cytoplasm,indicating a participation of Bm NPC1 in cholesterol trafficking;Using the ds RNA segments to inject silkworm at the individual level,the molting of some silkworm was delayed,some silkworm was unable to molt to next instar and finally died,indicating a participation of Bm NPC1 in molting.