家蚕微孢子虫（Nosema bombycis）ricin B-like蛋白的研究
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摘要
家蚕微孢子虫(Nesoma bombycis)是一种严格的细胞内寄生虫,体外以休眠孢子存在,可通过弹出极丝和细胞吞噬侵入宿主细胞。家蚕微孢子虫宿主范围较广,除家蚕外,柞蚕、果蝇、菜青虫和草地滩夜蛾等多种昆虫都能感染。在家蚕微孢子虫基因组信息分析中,发现一群ricin B-like (RBL)基因存在于家蚕微孢子虫基因组中,与此同时,在另外几种微孢子虫基因组中也发现相应的RBL基因。RBL是一大类与蓖麻毒素ricin蛋白B亚基具有类似结构域的蛋白统称。蓖麻毒素蛋白是一种异源二聚体毒素蛋白,具有A、B两个亚基,分别为RTA和RTB, B亚基能与细胞表面糖基受体结合从而介导和保护A亚基进入细胞内起作用。为阐明家蚕微孢子虫中这些RBL蛋白的功能以及其在家蚕微孢子虫整个生活史中所起作用,我们对家蚕微孢子虫中RBL基因进行了分析鉴定,并克隆表达了其中3个RBL基因,对其功能进行了初步探索研究。其结果如下：
1.家蚕微孢子虫(N. bombycis) RBL基因的鉴定及蛋白结构分析
采用蛋白结构分类(SCOP)在线软件Superfamily1.75分析和SMART在线预测,在家蚕微孢子虫基因组中共鉴定出了21个RBL基因,主要以串联形式分布在基因组数据的第6号Scaffold和第463号Scaffold上,其蛋白归属ricin B-like lectin超家族,由1个蛋白功能域构成。以家蚕微孢子虫RBL序列为基础进行BLSATp分析,结果在兔脑炎微孢子虫(Encephalitozoon cuniculi)、肠道微孢子虫(Encephalitozoon intestinalis)和蜜蜂微孢子虫(Nosema ceranae)各自基因组中分别鉴定出4、2、8个RBL基因,都是以串联形式分布在基因组中。采用RBL蛋白功能域序列进行的多重序列比对分析表明,21个家蚕微孢子虫RBL基因中,有16个基因的蛋白功能域由2个QxW重复基序形成的α、β子域构成,另外5个基因的蛋白功能域仅有1个QxW重复基序,而典型的RBL功能域则由3个QxW重复基序形成的α、β和γ子域构成,家蚕微孢子虫RBL蛋白这种功能域结构的差异可能会导致其蛋白功能的差异。以RBL蛋白功能域构建的系统进化树显示,所有微孢子虫RBL基因聚在一起,家蚕微孢子虫RBL基因又单独聚为一枝,家蚕微孢子虫RBL基因的串联重复是发生在微孢子虫分化之后,其后又发生了一次片段重复事件,导致基因组中RBL基因数量大增。蛋白结构预测分析表明,21个家蚕微孢子虫RBL基因中有9个具有典型的分泌信号,2个具有跨膜域,绝大部分蛋白具有二硫键和糖基化位点。
2.家蚕微孢子虫RBL基因的原核表达及多克隆抗体制备
选择家蚕微孢子虫基因组数据中第463号Scaffold上串联的RBL463-1、RBL463-2、RBL463-4三个RBL基因进行原核表达和抗体制备。以基因组DNA为模板,对RBL463-1、RBL463-2、RBL463-4进行PCR扩增,成功构建了原核表达载体pGEX463-1、pGEX463-2和pGEX463-4,然后转入E.coli BL21中进行诱导表达,所获融合表达蛋白全为包涵体形式,其分子量大小分别在55KD、48kD和48kD左右,与其分子量预测大小相近。融合蛋白经SDS-PAGE电泳后,切取蛋白条带研磨后与弗氏佐剂乳化制备抗原免疫小鼠和家兔,3免后第7天无菌采血分离血清,琼扩法测定这3个融合蛋白制备的多克隆抗体效价都在1：200左右,满足进一步研究需要。
3.RBL基因在家蚕微孢子虫中的转录及表达
为确定RBL463-1,RBL463-2,RBL463-4基因在家蚕微孢子虫中的转录和表达活性,取添食感染家蚕微孢子虫的家蚕中肠组织,从第1d连续采集到第10d,分别提取总RNA,采用RT-PCR方法,对RBL463-1,RBL463-2和RBL463-4基因进行扩增,仅RBL463-1基因在家蚕微孢子虫感染全期都有转录,而RBL463-2和RBL463-4基因直到感染的第3天才检测到明显的转录,在第6天又都未检测到转录,该结果表明,这3个RBL基因在家蚕微孢子虫中的表达时期存在明显差异,在基因组位置上虽串联在一起,但非同步转录。通过对新鲜孢子和4℃保存了3年的孢子总蛋白进行免疫印迹检测发现,仅在新鲜孢子中检测到RBL463-1一种蛋白,而在陈年孢子中3个蛋白都没能检测到；在以成熟孢子和孢子感染细胞为材料进行IFA检测时发现,仅在分裂期的孢子中检测到了3个蛋白的表达,在成熟孢子中没有检测到荧光信号的原因可能在于成熟孢子较厚的孢壁阻止了抗体进入到孢质中与抗原反应或蛋白表达量过少。综合可以得出,这3个RBL基因都具有活性,其中RBL463-1在家蚕微孢子虫整个生活史中都有表达,而RBL463-2和RBL463-4仅在家蚕微孢子虫繁殖的某个阶段才有转录表达。
4.家蚕微孢子虫RBL蛋白性质和功能研究
采用原核和真核表达系统对RBL463-1、RBL463-2和RBL463-4蛋白进行可溶性表达,对其蛋白性质和功能的研究,为RBL蛋白在家蚕微孢子虫中所起作用提供依据。
根据RBL463-1、RBL463-2和RBL463-4蛋白质结构分析,分别设计了扩增全长和去N端疏水部分基因引物,双酶切位点综合考虑了原核和真核表达载体多克隆位点和基因本身酶切位点信息。提取感染家蚕微孢子虫第8天的家蚕中肠组织总RNA,通过RT-PCR.方法,分别扩增出全基因RBL463-1、RBL463-2、RBL463-4和去N端疏水部分基因dRBL463-1、dRBL463-2、dRBL463-4,克隆到原核表达载体pET30α和真核表达载体pPICZα-A上,转入E.coli BL21(DE3)和毕赤酵母X33中,获重组表达菌分别为BL21/pET30-RBL463-1、BL21/pET30-RBL463-2、BL21/pET30-RBL463-4、BL21/pET30-dRBL463-1、BL21/pET30-dRBL463-2、BL21/pET30-dRBL463-4以及X33/pPICZα-RBL463-1、X33/pPICZα-RBL463-2、X33/pPICZα-RBL463-4、X33/pPICZα-dRBL463-1、X33/pPICZα-dRBL463-2、X33/pPICZα-dRBL463-4。
以pET30α为载体的原核表达中,仅去N端疏水部分基因dRBL463-1、dRBL463-2、dRBL463-4获少量可溶表达,可溶蛋白经His亲和层析,超滤除盐浓缩后,分别过乳糖-琼脂糖柱,dRBL463-1和dRBL463-4融合蛋白具有与乳糖结合的能力；分别以可溶表达纯化蛋白注射家蚕和添加sf9细胞,家蚕正常发育并结茧,sf9细胞没有明显病变；以DTSSP交联剂对这3个融合蛋白进行交联处理,结果表明3个蛋白间具有相互作用。在蛋白分离纯化和功能测定的过程中发现,该3个蛋白可溶表达量低并且表达不稳定,易于降解,dRBL463-1部分降解片段具有乳糖结合活性。
在毕赤酵母表达系统中,仅X33/pPICZα-dRBL463-2和X33/pPICZα-RBL463-4有非常低量的融合蛋白表达,采用Western blot方法才能检测得到,所表达的融合蛋白具有不均一性。随着诱导时间的增加,蛋白开始降解,诱导时间越长蛋白降解越严重,当诱导超过120h后基本上检测不到所表达蛋白,以24-48h诱导表达为佳。融合表达蛋白直接经His亲和层析,超滤除盐,过乳糖-琼脂糖柱,没有检测到蛋白与乳糖结合,其原因可能是由于在纯化处理过程中导致的蛋白降解和失活致使其乳糖结合功能的丧失。RBL463-1、RBL463-2、RBL463-4基因在毕赤酵母中不表达或表达量低,表明其融合表达蛋白可能会对酵母细胞的生长代谢产生不利影响。
为了解RBL463-1、RBL463-2和RBL463-4蛋白在细胞中的作用位置,分别把这3个基因克隆到酿酒酵母表达载体pUG35上,获重组表达载体分别为pUG35-RBL463-1、pUG35-RBL463-2和pUG35-RBL463-4,电击转化酿酒酵母CEN.PK2,获重组表达菌分别为CEN/pUG35-RBL463-1、CEN/pUG35-RBL463-2和CEN/pUG35-RBL463-4,经无甲硫氨酸培养基诱导表达,荧光显微镜观测发现,RBL463-1融合蛋白能够结合分布于酵母线粒体和内质网上,RBL463-2融合表达蛋白散在分布于细胞质中,RBL463-4融合表达蛋白结合分布于内质网上。这3个蛋白在酵母细胞中的分布位置差异表明了在家蚕微孢子虫中不同ricin B-like蛋白起作用的位置存在差异。
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. bombycis
Using 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.bombycis
Three 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.bombycis
Here 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.
1.家蚕微孢子虫(N. bombycis) RBL基因的鉴定及蛋白结构分析
采用蛋白结构分类(SCOP)在线软件Superfamily1.75分析和SMART在线预测,在家蚕微孢子虫基因组中共鉴定出了21个RBL基因,主要以串联形式分布在基因组数据的第6号Scaffold和第463号Scaffold上,其蛋白归属ricin B-like lectin超家族,由1个蛋白功能域构成。以家蚕微孢子虫RBL序列为基础进行BLSATp分析,结果在兔脑炎微孢子虫(Encephalitozoon cuniculi)、肠道微孢子虫(Encephalitozoon intestinalis)和蜜蜂微孢子虫(Nosema ceranae)各自基因组中分别鉴定出4、2、8个RBL基因,都是以串联形式分布在基因组中。采用RBL蛋白功能域序列进行的多重序列比对分析表明,21个家蚕微孢子虫RBL基因中,有16个基因的蛋白功能域由2个QxW重复基序形成的α、β子域构成,另外5个基因的蛋白功能域仅有1个QxW重复基序,而典型的RBL功能域则由3个QxW重复基序形成的α、β和γ子域构成,家蚕微孢子虫RBL蛋白这种功能域结构的差异可能会导致其蛋白功能的差异。以RBL蛋白功能域构建的系统进化树显示,所有微孢子虫RBL基因聚在一起,家蚕微孢子虫RBL基因又单独聚为一枝,家蚕微孢子虫RBL基因的串联重复是发生在微孢子虫分化之后,其后又发生了一次片段重复事件,导致基因组中RBL基因数量大增。蛋白结构预测分析表明,21个家蚕微孢子虫RBL基因中有9个具有典型的分泌信号,2个具有跨膜域,绝大部分蛋白具有二硫键和糖基化位点。
2.家蚕微孢子虫RBL基因的原核表达及多克隆抗体制备
选择家蚕微孢子虫基因组数据中第463号Scaffold上串联的RBL463-1、RBL463-2、RBL463-4三个RBL基因进行原核表达和抗体制备。以基因组DNA为模板,对RBL463-1、RBL463-2、RBL463-4进行PCR扩增,成功构建了原核表达载体pGEX463-1、pGEX463-2和pGEX463-4,然后转入E.coli BL21中进行诱导表达,所获融合表达蛋白全为包涵体形式,其分子量大小分别在55KD、48kD和48kD左右,与其分子量预测大小相近。融合蛋白经SDS-PAGE电泳后,切取蛋白条带研磨后与弗氏佐剂乳化制备抗原免疫小鼠和家兔,3免后第7天无菌采血分离血清,琼扩法测定这3个融合蛋白制备的多克隆抗体效价都在1：200左右,满足进一步研究需要。
3.RBL基因在家蚕微孢子虫中的转录及表达
为确定RBL463-1,RBL463-2,RBL463-4基因在家蚕微孢子虫中的转录和表达活性,取添食感染家蚕微孢子虫的家蚕中肠组织,从第1d连续采集到第10d,分别提取总RNA,采用RT-PCR方法,对RBL463-1,RBL463-2和RBL463-4基因进行扩增,仅RBL463-1基因在家蚕微孢子虫感染全期都有转录,而RBL463-2和RBL463-4基因直到感染的第3天才检测到明显的转录,在第6天又都未检测到转录,该结果表明,这3个RBL基因在家蚕微孢子虫中的表达时期存在明显差异,在基因组位置上虽串联在一起,但非同步转录。通过对新鲜孢子和4℃保存了3年的孢子总蛋白进行免疫印迹检测发现,仅在新鲜孢子中检测到RBL463-1一种蛋白,而在陈年孢子中3个蛋白都没能检测到；在以成熟孢子和孢子感染细胞为材料进行IFA检测时发现,仅在分裂期的孢子中检测到了3个蛋白的表达,在成熟孢子中没有检测到荧光信号的原因可能在于成熟孢子较厚的孢壁阻止了抗体进入到孢质中与抗原反应或蛋白表达量过少。综合可以得出,这3个RBL基因都具有活性,其中RBL463-1在家蚕微孢子虫整个生活史中都有表达,而RBL463-2和RBL463-4仅在家蚕微孢子虫繁殖的某个阶段才有转录表达。
4.家蚕微孢子虫RBL蛋白性质和功能研究
采用原核和真核表达系统对RBL463-1、RBL463-2和RBL463-4蛋白进行可溶性表达,对其蛋白性质和功能的研究,为RBL蛋白在家蚕微孢子虫中所起作用提供依据。
根据RBL463-1、RBL463-2和RBL463-4蛋白质结构分析,分别设计了扩增全长和去N端疏水部分基因引物,双酶切位点综合考虑了原核和真核表达载体多克隆位点和基因本身酶切位点信息。提取感染家蚕微孢子虫第8天的家蚕中肠组织总RNA,通过RT-PCR.方法,分别扩增出全基因RBL463-1、RBL463-2、RBL463-4和去N端疏水部分基因dRBL463-1、dRBL463-2、dRBL463-4,克隆到原核表达载体pET30α和真核表达载体pPICZα-A上,转入E.coli BL21(DE3)和毕赤酵母X33中,获重组表达菌分别为BL21/pET30-RBL463-1、BL21/pET30-RBL463-2、BL21/pET30-RBL463-4、BL21/pET30-dRBL463-1、BL21/pET30-dRBL463-2、BL21/pET30-dRBL463-4以及X33/pPICZα-RBL463-1、X33/pPICZα-RBL463-2、X33/pPICZα-RBL463-4、X33/pPICZα-dRBL463-1、X33/pPICZα-dRBL463-2、X33/pPICZα-dRBL463-4。
以pET30α为载体的原核表达中,仅去N端疏水部分基因dRBL463-1、dRBL463-2、dRBL463-4获少量可溶表达,可溶蛋白经His亲和层析,超滤除盐浓缩后,分别过乳糖-琼脂糖柱,dRBL463-1和dRBL463-4融合蛋白具有与乳糖结合的能力；分别以可溶表达纯化蛋白注射家蚕和添加sf9细胞,家蚕正常发育并结茧,sf9细胞没有明显病变；以DTSSP交联剂对这3个融合蛋白进行交联处理,结果表明3个蛋白间具有相互作用。在蛋白分离纯化和功能测定的过程中发现,该3个蛋白可溶表达量低并且表达不稳定,易于降解,dRBL463-1部分降解片段具有乳糖结合活性。
在毕赤酵母表达系统中,仅X33/pPICZα-dRBL463-2和X33/pPICZα-RBL463-4有非常低量的融合蛋白表达,采用Western blot方法才能检测得到,所表达的融合蛋白具有不均一性。随着诱导时间的增加,蛋白开始降解,诱导时间越长蛋白降解越严重,当诱导超过120h后基本上检测不到所表达蛋白,以24-48h诱导表达为佳。融合表达蛋白直接经His亲和层析,超滤除盐,过乳糖-琼脂糖柱,没有检测到蛋白与乳糖结合,其原因可能是由于在纯化处理过程中导致的蛋白降解和失活致使其乳糖结合功能的丧失。RBL463-1、RBL463-2、RBL463-4基因在毕赤酵母中不表达或表达量低,表明其融合表达蛋白可能会对酵母细胞的生长代谢产生不利影响。
为了解RBL463-1、RBL463-2和RBL463-4蛋白在细胞中的作用位置,分别把这3个基因克隆到酿酒酵母表达载体pUG35上,获重组表达载体分别为pUG35-RBL463-1、pUG35-RBL463-2和pUG35-RBL463-4,电击转化酿酒酵母CEN.PK2,获重组表达菌分别为CEN/pUG35-RBL463-1、CEN/pUG35-RBL463-2和CEN/pUG35-RBL463-4,经无甲硫氨酸培养基诱导表达,荧光显微镜观测发现,RBL463-1融合蛋白能够结合分布于酵母线粒体和内质网上,RBL463-2融合表达蛋白散在分布于细胞质中,RBL463-4融合表达蛋白结合分布于内质网上。这3个蛋白在酵母细胞中的分布位置差异表明了在家蚕微孢子虫中不同ricin B-like蛋白起作用的位置存在差异。
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. bombycis
Using 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.bombycis
Three 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.bombycis
Here 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.
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