Studies On The Ricin B-like Protein From Nosema Bombycis

As one of microsporidian, Nosema bombycis is an obligate intracellular parasitic eukaryote, forming hypnospore to avoid the disadvantage environment, and invading the host cell by extrusion of the polar tuber and host cell phagocytosis. Many insects such as Bombyx mori, China oak silkworm, Drosophila melanogaster, Pieris rapae, Trichoplusia ni, etc., can be infected by N.bombycis. Based on the genome data of Nosema bombycis, a group of ricin B-like genes were found in the genome of Nosema bombycis. Those relative genes of ricin B-like also were found in other microsporidians such as Encephalitozoon cuniculi, Nosema ceranae and Encephalitozoon intestinalis. Ricin, first was found from castor bean, is a heterodimmer cytotoxin protein consisted of two disulfide-bonded subunits A and B. The A chain (RTA), an RNA-specific N-glycosidase inhibits protein synthesis on eukaryotic ribosomes, and the B chain (RTB), a cell binding lectin could be conjuncting with glycoproteins on the surface of membrane of host cell mediating and protecting RTA penetrating membrane inside the cytoplasm of host cell. For functional analysis, three ricin B-like genes of N. bombycis were chosen for cloning, expression and founctional analysis. The results are as follows:1. Identification of the ricin B-like gene from N. bombycisUsing on-line softwares Superfamily 1.75 and SMART, About 21 ricin B-like genes were identified from N. bombycis. Those ricin B-like genes were members of ricin B-like lectins superfamily and most genes are tandemly distributing on the 6th scaffold and 463th scaffold of N.bombycis genome. Using BLSATp against NCBI protein databases with the amio acid sequences of ricin B-like of N. bombycis, We identified 4.2.8 ricin B-like genes from the E. cuniculi. E. intestinalis and N.ceranae respectively. All of those ricin B-like genes in each of microsporidian are also tandemly distributing on their chromosomes. Mutiple sequence alignment of ricin B-like proteins illuminated that the ricin B-like proteins of N. bombycis have only one functional domain and the domains of 16 of 21 ricin B-like proteins own 2 QxW repeats which forming a and (3 subdomain, and anther 5 of 21 ricin B-like proteins'domain have only one QxW repeat. However, the typical ricin B-like domain has 3 QxW repeats which formingα,βand y subdomain. Phyletic evolutional analysis of ricin B-like genes indicated that the ricin B-like genes of N. bombycis had developed series duplications after the separation of microsporidia, and then the genome fragment duplicates made the dosage of ricin B-like genes increasing in N.bombycis. According to the ricin B-like protein structure prediction,9 of 21 ricin B-like proteins of N. bombycis have typical secretory signal,2 of 21 ricin B-like proteins have one transmembrane domain, and most of 21 ricin B-like proteins have disulfide bonds and glycosylation sites.2. Prokaryotic expression and polyclonal antibodies preparation of ricin B-like genes from N.bombycisThree tandem repeat ricin B-like genes RBL463-1, RBL463-2 and RBL463-4 located on the 463th scaffold of genome of N.bombycis had been chosen for prokaryotic expression and preparation of polyclonal antibodies for the functional analysis of those proteins. The RBL463-1, RBL463-2 and RBL463-4 genes were amplified from genome DNA and then subcloned into the pGEX4T-1 to yield the recombinant plasmid pGEX463-1, pGEX463-2 and pGEX463-4 which transformed into E.coli BL21 respectively, the recombinant E.coli BL21 were obtained through screening with ampicillin. the molecular mass of three recombination proteins expressed in E.coli BL21 induced by IPTG are about 55KD,48kD and 48kD respectively, which were consistent with the calculated molecular mass of recombinant proteins. The expression products of those three recombination genes were existed as cytorrhyctes in the recombinant E.coli BL21. So using the cutting recombinant protein bands of the SDS-PAGE to prepare antigens, which were emulsified with Freund adjuvant to immunize mice and rabbits for the antibodies preparation. At the 7th day after the third immunization.the antiserum were collected and valency of RBL463-1, RBL463-2 and RBL463-4 detected by immune precipitation method were up to 1:200 dilution and were suitable for the next research.3. Transcription pattern and expression of ricin B-like genes in N. bombycis For characterization of the gene activity of RBL463-1, RBL463-2 and RBL463-4, the transcription and expression of those three genes in N.bombycis were investigated. Three pairs of RT-PCR primers were designed to amplify cDNA of those three genes from the total RNA extracted from midgut of Bombyx mori larvae at day 1 to 10 post infection by N.bombycis. The results of RT-PCR showed that RBL463-1 gene were transcribed at whole infection period of N.bombycis, another 2 genes were only detected transcript at day 3 to 5 and day 7 to 10 post infection by N.bombycis. All of the results indicated that the transcription of those three ricin B-like genes in N.bombycis were not co-transcribed though they were tandemly distributed in genome. The expression proteins of those 3 genes in the N.bombycis were investigated by Western blot and IFA assay. By Western blot, only RBL463-1 protein was only detected in the total proteins of new mature spores. By IFA, the expression of three proteins could be detected in unmatured spores but not in mature spores. In conclusion, those 3 genes have transcription activity in N.bombycis and the expression of RBL463-1 gene was at whole life cycle of N. bombycis, however, the expression of RBL463-2 and RBL463-4 genes maybe only at propagation period of N.bombycis.4. Characterization and Functional analysis of ricin B-like proteins from N.bombycisHere we employed the prokaryote expression system and eukaryote expression system to obtain native expression of RBL463-1, RBL463-2 and RBL463-4 genes for characterization and functional analysis.Because of the recombinant expression proteins of those three genes adopting pGEX4T-1 vector were exsisted as cytorrhyctes, then pET30a vector was used for native expression of those three genes. According to the protein structure analysis, PCR primers were designed to amplify the total length genes and truncated genes without hydrophobic N-terminal region of RBL463-1, RBL463-2 and RBL463-4 genes respectively. The total length RBL463-1, RBL463-2, RBL463-4 genes and truncated dRBL463-1, dRBL463-2, dRBL463-4 genes were amplified from the total RNA extracted from midgut of Bombyx mori lavae at day 8 post infection by N.bombycis. The recombinant plasmids were transformed into E.coli BL21(DE3) and Pichia pastoris X33 to obtained recombinant expression E.coli BL21 and Pichia pastoris X33 respectively as follow:BL21/pET30-RBL463-1, BL21/pET30-RBL463-2. BL21/p ET30-RBL463-4. BL21/pET30-dRBL463-1, BL21/pET30-dRBL463-2. BL21/pET30 -dRBL463-4 and X33/pPICZa-RBL463-1. X33/pPICZa-RBL463-2, X33/pPICZa-RBL 463-4, X33/pPICZa-dRBL463-1, X33/pPICZa-dRBL463-2, X33/pPICZa-dRBL463-4. Among the six recombinant expression E.coli clones, only BL21/pET30-dRBL463-1, BL21/pET30-dRBL463-2 and BL21/pET30-dRBL463-4 had a little of native recombinant proteins expressed. The native recombinant proteins were purified by His-tag affinity chromatography and ultrafiltrate desalination, and then loaded on the lactosyl-Sepharose column, washed with 10 mM PBS to remove unbound proteins, eluted bound proteins with 100 mM lactose and 10 mM Tris·cl, only recombinant proteins dRBL463-1 and dRBL463-4 were found that having capability to band with lactose. Using the native expression priteins injected into the Bombyx mori larvaes and incubated with sf9 cell, we found that the development of Bombyx mori larvaes were normal and cocooned consistently with negative control, and the cells of sf9 grew normally. The results of treatment of these three proteins by crosslinking agent DTSSP indicated the interaction among these three proteins. Notably, we found that the native expression of those three proteins were very low and easily degradated, and the degradated peptides of dRBL463-1 protein still had capability to bind with lactose.Among the six recombinant Pichia pastor is clones, only X33/pPICZa-dRBL463-2 and X33/pPICZα-RBL463-4 clones had a little of fusion proteins'expression induced by 1% methanol. Ununiformity of the recombinant proteins expression was the special character among these two proteins, the fusion proteins were easily degradated and the more degradation along with the elongation of culture time. The expression proteins could not be detected when the induction time was beyond 120h, best induction time for the expression of those two proteins was 24h to 48h. The recombinant proteins were purified by His-tag affinity chromatograph and ultrafiltrate desalination, and then incubated with lactose-agarose beads, washing with 10 mM PBS, eluting with 100 mM lactose and 10 mM Tris·cl, both of those two recombinant proteins were not detected in the elution buffer, the possible reason of this phenomenon was that the recombinant proteins inactivation along with the purified processing. And the low expression or non expression of the RBL463-1, BL463-2 and RBL463-4 genes in yeast also indicated that the fusion protein maybe had adverse effects on the host cells.For the functional analysis, the location of the RBL463-1, RBL463-2 and RBL463-4 proteins had been investigated. Those three genes were subcloned into upstream N-terminal gfp gene of Saccharomyces cerevisiae expression plasmid pUG35 to yield recombinant expression plasmids pUG35-RBL463-1. pUG35-RBL463-2 and pUG35-RBL463-4 respectively, which were transformed into Saccharomyces cerevisiae CEN.PK2 through electroporation. The recombinant yeast CEN/pUG35-RBL463-1, CEN/pUG35-RBL463-2 and CEN/pUG35-RBL463-4 were obtained through screening by uracil deficiency medium. After cultured with methionine deficiency medium, visualized by fluorescence microscope. The results of GFP fusion proteins of RBL463-1 localized to mitochondria and endoplasmic reticulum of yeast cells, and RBL463-2 fusion proteins were diffusedly distributed in the cytoplasm of yeast cells, and RBL463-4 fusion proteins localized to endoplasmic reticulum of yeast cells. The special localization of the fusion proteins of those three proteins indicated that the different ricin B-like protein may have different function in N.bombycis.