| Serine proteases are enzymes that hydrolyze proteins, named from the Ser at the active site which show nucleophilic attack on substrate.As an important member of the protease family, their role is to break the peptide bonds of protein molecules,and make them become small proteins. The catalysis is achieved by the conformational change of amino acid residues at the active center.There must be a serine at the active center of protease, so we call it serine protease. In mammals, serine proteases play very important roles, many proteins are well-known in the serine proteases family, including digestive enzymes, clotting factors and members of the complement system. Because that serine proteases play important roles in embryonic development, tissue remodeling, cell differentiation, angiogenesis and defense against pathogen, there are lots of research reports on animal serine protease. However, studies on serine proteases of insect are relatively little. Ross et al.(2003) identified the serine protease gene family of the drosophila based on the genome data. A total of119genes were identified, being divided into SP and SPH categories which contain86and33genes, respectively. They evolutionary analysis revealed that gene duplication and mutation play important roles in the formation of this gene family.Silkworm has as many serine protease genes.A variety of serine protease have been found in different tissues of Bombyx mori. By referring to the serine protease sequences in other species, Ping Zhao et al.(2010) comprehensively analyzed the serine protease gene of B.mori base on the genome data. A total of143serine protease gene were identified from silkworm genome,24genes more than that in Drosophila. These genes were constructed scientific names and recorded as:BmSP1-143.The analysis on amino acid sequence showed that there are two types of serine proteases in B.mori, one is the SP class with full three active sites, including51serine protease genes, the other is the SPH class with incomplete active sites. The isoelectric point and molecular weight of B.mori serine protease are widely distributed. Twenty B.mori serine proteases have been reported, including ovarian protease, cocoonase, yolk protein degrading protease and midgut alkaline protease, Therefore, silkworm serine proteases were involved in digestion, embryonic development and egg formation, and even the formation of silk protein. Presumably serine proteases plays indispensable roles in maintaining normal physiological activities of silkworm, such as the growth and development.There is a class of serine proteases containing the CUB domain, which were involved in important biological processes. CUB domain is an extracellular domain containing110amino acids, named for initially identification in three types of proteins, including Complement subcomponents Clr/Cls, Uegf, and Bone morphogenic protein-1. CUB domain-containing proteins have multiple functions, mostly associated with growth and development. CUB domains are usually couple with other domain, such as the serine protease domain. Many CUB domain-containing proteins contain a composite CUB domain and a compound protease domain. They play important physiological roles in cell differentiation, growth factor activation, degradation of peptides, extracellular growth and developmental process.Silkworm is an economic insect, could produce high amout of silk, and thus have important economic value. Previous study of our group have analyzed the silkworm genome microarray data, and found BmSP82showing specific high-level expression in the anterior and middle silk gland. Functional research of serine protease BmSP82is helpful for us to clarigy the specific function of serine protease in the silk gland inquiry, improve our understanding to the secretory mechanism of silk gland, and help to improve the quality and quantity of silk, and make contribute to the silk production.Given all that, we choose BmSP82as the research object. Firstly, we carried on the information analysis and sequence alignment, and then used RT-PCR to analyzed its spatial and temporal expression level, and prepared the polyclonal antibody using prokaryotic expressed protein, and then detected the BmSP82protein in the silkworm body use the polyclonal antibody, and revealed its tissue localization using CUB domain antibody of BmSP82. Finally, we explored the activity of BmSP82from prokaryotic expression and eukaryotic yeast expression system. The main results are as follows:1. Bioinformatics and expression characteristics analysis of silkworm serine protease BmSP82;The gene number of BmSP82on SilkDB website is BGIBMGA004445. This gene located on the twentieth chromosome with position:nscaf2795:1108881-1115723(+strand).Microarray datas and RT-PCR results showed that BmSP82is mainly expressed in the Malpighian tubules and the anterior and middle silk gland of B. mori.Bioinformation analysis indicated that BmSP82contain a CUB domain and a trypsin domain. The first17amino acids of BmSP82constitute its signal peptide, the following N-terminus is a CUB domain consists of114amino acids containing8cysteines. Its C-terminus has a trypsin-like domain, which contains the catalytic triad:His, Asp and Ser. The trypsin-like domain also contains eight cysteines. A sequence of18amino acid residues connects the two domains, which contains two cysteines. The Zymogen activation sites of BmSP82is isoleucine(I).2. The prokaryotic expression and polyclonal antibody preparation of serine protease BmSP82in Bombyx mori;By sequence analysis of BmSP82, we designed expression primers, use the silk gland cDNA on day3of the fifth instar as the template for PCR. The fragment was subcloned in p28(pET28a transformation) vector, then transformed into the expression strain Transetta (DE3) after sequencing successfully. Expression detection showed that BmSP82expressed in the form of inclusion bodies. The recombinant protein was purified by gel slices and then injected into Kunming mice. After30days of feeding,the titer of the polyclonal antibody was1:10000.3. Spatial and temporal expression analysis of B.mori serine protease BmSP82;We found that BmSP82expressed in the silk gland and Malpighian tubule, further testing found that its expression in the anterior silk gland and middle silk gland, but not in the posterior silk gland by using the polyclonal antibody. We also reveal the tissue localization of serine protease BmSP82in the anterior areas of middle silk gland of silkworm, the result show that BmSP82main distribution in the silk gland cell layer and the chitin layer between the intima of silk gland and silk gland cavity of the middle silk gland of silkworm, indicate their possible involvement in the formation of the extracellular matrix in lumen of the anterior area of middle silk gland, in order to protect the silk gland cells from the pressure of silk proteins to the inner cells of silk gland cavity and shear damage in the spinning process.4. Eukaryotic expression and activity inquiry of the serine protease BmSP82in silkworm.We obtained BmSP82mature body with GST tag by prokaryotic expression, have an affinity chromatography using GST tag, got high purity recombinant protein GST-BmSP82. After using the TEV enzyme excision the GST tag of the BmSP82recombinant protein and then incubation with casein, found that the prokaryotic expressed BmSP82protein has no activity. Then conducted a Pichia pastoris eukaryotic expression. Construct the mature body fragment of serine protease gene BmSP82to the eukaryotic expression vector pPIC9K, transferred into Pichia pastoris GS115by electroporation and then cultured. Finally, use the BMGY and BMMY liquid medium system for inducing expression. Yeast transformants significant expression was detected after induction24h. And make an ammonium sulfate gradient precipitation, obtain a more pure mature protein BmSP82, laid the foundation for further study of function and protease activity detection. |
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