Objective:To investigate the glycan binding specificity of the P protein in GII.8 and GII.9 noroviruses.Methods:The P particle protein of GII.8 and GII.9 noroviruses was expressed by the Escherichia coli system. The oligosaccharide binding assay and saliva binding assay were carried out to explore the interactions between P particles and the histo-blood group antigens (HBGAs). Sequence alignment and structural superimposition were used to analyze the characteristics of the sequence and structure of the glycan binding sites.Results:The P protein of GII.8 showed no obvious binding to the tested glycans while it bound to some saliva samples of A/B/O types. The P protein of GII.9 recognized H disaccharide and interacted with most of the saliva samples. The P protein of GII.8 possessed the similar potential glycan binding site as that of GII.9 according to the sequence and structural analysis. The site was close to that of prevalent GII.4 strains.Conclusions:The non-dominant strains, GII.8 and GII.9 strains showed different glycan binding ability to different HBGAs, indicating that there may be discrepancy in prevalence. It is necessary to continually monitor the prevalence of non-dominant noroviruses.

Objective:To express and purify VP7 protein of group A rotavirus (RVA) G1P[8]. The VP7 polyclonal antibody was prepared and its function was evaluated.Methods:The G1 VP7 protein was expressed by baculovirus expression system and purified by affinity chromatography. Polyclonal antibody against G1 VP7 was obtained by immunizing rabbits with G1 VP7 protein. The function of the G1 VP7 polyclonal antibody was verified by Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assay.Results:The soluble G1 VP7 protein of human RVA G1P[8] was obtained using the baculovirus expression system and the VP7 protein was mainly in trimer state. The G1 VP7 polyclonal antibody was prepared and displayed relatively high binding titer to G1 VP7 protein by ELISA. The VP7 polyclonal antibodies could recognize multiple G-type RVAs by WB and ELISA. Immunofluorescence assay further demonstrated that G1 VP7 polyclonal antibody can bind to different RVAs, including Wa (genotype G1P[8]), DS-1(genotype G2P[4]), SA11 (genotype G3P[2]), and human G9P[8] RV strains. In addition, double sandwich ELISA showed that VP7 polyclonal antibody could be used to detect rotavirus in clinical samples.Conclusions:The soluble G1 VP7 protein was successfully expressed and VP7 antibody was obtained. The G1 VP7 polyclonal antibody could bind to a variety of G-type rotaviruses, which lays a foundation for the establishment of detection method of different G type rotaviruses.

The widely used rotavirus (RV) vaccine, Rotateq, contained reassortment strains of human and bovine G1/2/3/4P[5] RVs. The functional and structural features of bovine G1P[5] VP8* were investigated. Bovine G1P[5] VP8* was identified to interact with sialic acids and sialic acid-containing glycans. In addition, P[5] VP8* recognized α-Gal histo-blood group antigens (HBGAs). Bovine G1P[5] VP8* did not hemagglutinate the tested red blood cells. The crystal structure of P[5] VP8* was determined at 1.7 Å. Structural superimposition revealed that P[5] VP8* was most close to human P[8] VP8*, while much further to VP8*s of porcine P[7] and rhesus P[3]. Sequence alignment showed that amino acids of the putative glycan binding site in P[5] VP8* were different to those in P[3]/P[7] VP8*s, indicating that P[5] VP8* may interact with glycans using different mechanism. This study provided more understanding of P[5] RV infection and the interactions of RV VP8* and glycans.

Objective:To investigate the glycan binding specificity of VP8* protein of P[15] group A rotavirus.Methods:The VP8* protein of P[15] bovine group A rotavirus was obtained by the Escherichia coli expression system. The binding of P[15] VP8* protein to different glycans was tested by the assays of oligosaccharide binding, biolayer interferometry and hemagglutination.Results:The VP8* protein of P[15] bovine group A rotavirus recognized sialic acids of Neu5Ac and Neu5Gc, and interacted with sialic acid linked to glycans by α2, 3 and α2, 6 linkage. Besides, bovine P[15] VP8* hemagglutinated different human and animal red blood cells.Conclusions:Sialic acid may be the glycan receptor of P[15] bovine group A rotavirus. Similar to rhesus P[3] and porcine P[7] rotaviruses, bovine P[15] VP8* bound to sialic acid and possessed the same potential glycan binding site according to the sequence alignment and structural comparison.

为深入研究大曲中微生物的组成及其产果胶酶特性,本研究对泸州老窖酿酒大曲中微生物进行分离鉴定,得到细菌15株(其中包括6株高温菌株),真菌5株(其中包括3株高温菌株).以桔皮粉为唯一碳源发酵诱导菌株产果胶酶并采用3,5-二硝基水杨酸法(DNS)测定酶活,筛选出了两株酶活力最高的菌株,分别为细菌Q-B2和真菌Q-F5.利用16S rDNA以及ITS鉴定高产果胶酶菌株种属,证明与Q-B2及Q-F5最相近种属分别为鹑鸡肠球菌(Enterococcus gallinarum)和嗜热子囊菌(Thermoascus aurantiacus).本研究为加深对大曲中微生物的组成认识提供理论依据.进一步探索本实验中分离所得微生物产果胶酶的性质,对P-Q-B2 (Q-B2所产果胶酶)的酶学性质进行测定,结果显示,P-Q-B2的最适pH为3,最适温度为50℃,在pH为3以及5～11的区间内,剩余酶活力都在40％以上;该酶在30～50℃具有良好的热稳定性,证明其在实际生产中具有良好的应用潜力.