Expression And Localization Of Serpin Gene NBO₃9gi001 And Functional Analysis Of Serpin In Nosema Bombycis

Microsporidia are a group of unicellular eukaryotic parasites infecting almost all invetibrates and vetibrate, including human. Especially, microsporidia are important pathogens to the economic species such as economic insect, fish, fur animals and so on. Approximately 150 generas and over 1400 species have been reported to date. Nosema bombycis is the first identified microsporidia species, which is obligately parasitize Bombyx mori causing destructive pebrine disease and leading to serious economic losses for their vertical transmission. So N. bombycis is set to be an officinally medical inspection object. China is the biggest sericultural country, however, the pebrine disease cause up to million losses in sericulture. So, preventing the pebrine is an important and difficult issue for sericulture, and how to detect the disease in early stage and take effective measures are very urgently.The whole genome sequence project of N. bombycis has been finished,which provided the basis for studied on N. bombycis.We identified twelve serpin-like serine protease inhibitors.None of serpins was found in other microsporidias except Nosema ceranae.Serpins have been shown to possess diverse function in many processes.Pathogens have evolved various strategies to escape the host immune system.It is reported parasitoid serpins can destroy the host immune system,so we presume that serpins of N.bombycis possibly used as a virulence factor that disrupt the host B. mori immune reaction by inbibiting serine protease.In our work, we have identified the serpins of N.bombycis with bioinformatics and RT-PCR, then choose one of them for prokaryotic expression and ultrastructural localization analysis, and other serpin for further functional analysis, which could provide light on interaction between N. bombycis and the host B. mori. The main results are as follows:1. Sequences and RT-PCR analysis of N. bombycis serpinsWe indentified 12 serpin-like genes and compared them with serpins from other insects. The amino acid sequence of N. bombycis serpins have low homology to other indentified serpins. Multiple sequence alignment of RCL hinge region revealed that non-conservative substitutions within P16-P9 sites of NBO₃4g0030, NBO₄2g0004, NBO₁8g0021, NBO₁8g0004, NBO₃72g0002, NBO₃4g0036 and NBO₁9g0009, indicated those genes may not display inhibitory behavior.We have predicted of their proteolytic cleavage site P1. Serpin NBO₁24g0001 NBO₁569g0001,NBO₁9g0007,NBO₁570g000,NBO₁9g0009,NBO₃72g0002(with Asp located at the PI site), NBO₁8g0004,NBO₃4g0036(with Glu located at the P1 site) may inhibit unknowed enzymes. Serpin NBO₃4g0030,NBO₄2g0004(with Phe located at the P1 site), and NBO₃9gi001(with Tyr located at the P1 site) may inhibit chymotrypsin-like enzymes. NBO₁8g0021(with Lys located at the P1 site) may inhibit trypsin-like enzymes.To analysis the transcription level of serpins, silkworm were infected with N. bombycis spores at 4th instar, then the midgut were obtained at post-infection(p.i.)1st day to 7th day. RT-PCR were performed and the results indicated serpin NBO₁570g0002 were transcribed from the 1st to 7th day after infection. NBO₁8g0004 and NBO₃9gi001 were transcripted on p.i.4th-7th,p.i.5th-7th day,respectively; NBO₃72g0002 mRNA detected on p.i.7th and NBO₃4g0030 on 5th day, the transcription pattern have not regularity. These results suggested that secretory-serpin NBO₁570g0002, NBO₃9gi001 may participate in the interaction between N. bombycis and the host B. mori.2. Molecular cloning and prokaryotic expression of serpin NBO₃9gi001Based on the analysis of the bioinformation and combined with the results of RT-PCR, serpin NBO₃9gi001 was cloned into the pGEX4T-1 vector. The GST-NBO₃9gi001 fusion protein were expressed in BL21 E. coli. Polyclonal antibodies were raised against GST-NBO₃9gi001 with mouse. The results of western blotting show a specific band which was recognized by anti-GST-NBO₃9gi001 serum immuboltting with total proteins of N. bombycis, but it is not coincidence with the predicted band 42KD. Serpin may comprise complex formation with its target enzymes then result this phenomenon.3. Ultrastructural localization and fuctional of serpinsImmunoelectron microscopy (IEM) was used to determine the cellular location of NBO₃9gi001. the results revealed that the labelld gold particles are present in sporoplasm. It indicated NBO₃9gi001 is expressed in mature spores. Due to the serpin GST-NBO₃72g002. GST-NBO₃9gi001 expression in the forms of inclusion body, we choose GST-NBO₁570g0002 for further functional analysis. The results showed GST-NBO₁570g0002 has no inhibitory effect on PO cascade in B. mori, as well as Subtilisin and protease K.The reasons for this as following: Firstly, GST-NBO₁570g0002 has no activity because of no protein modification in prokaryotic expression system; Secondly, serpins have evolved to regulate various processes, NBO₁570g0002 possibly don't regulate PO cascades;Thirdly, Subtilisin and protease K were not the targets of NBO₁570g0002.