To develop a safe and efficient recombinant subunit vaccine to foot-and-mouth disease virus (FMDV) type Asia 1 in sheep, a tandem repeated multiple-epitope gene consisting of residues 137-160 and 197-211 of the VP1 ge...To develop a safe and efficient recombinant subunit vaccine to foot-and-mouth disease virus (FMDV) type Asia 1 in sheep, a tandem repeated multiple-epitope gene consisting of residues 137-160 and 197-211 of the VP1 gene of FMDV was designed and artificially synthesized. The biologically functional molecule, the ovine IgG heavy constant region (oIgG) as a protein carrier was introduced for design of the multiple-epitope recombinant vaccine and recombinant expression plasmids pET-30a-RE and pET-30a-RE-oIgG were successfully constructed. The recombinant proteins, RE and RE-oIgG, were expressed as a formation of inclusion bodies in E. coli. The immune potential of this vaccine regime in guinea pigs and sheep was evaluated. The results showed that IgG could significantly enhance the immune potential of antigenic epitopes. The recombinant protein RE-oIgG could not only elicit the high levels of neutralizing antibodies and lymphocytes proliferation responses in the vaccinated guinea pigs, but confer complete protection in guinea pigs against virus challenge. Although the recombinant protein RE could not confer protection in the vaccinated animals, it could delay the appearance of the clinical signs and reduce the severity of disease. Inspiringly, the titers of anti-FMDV neutralizing antibodies elicited in sheep vaccinated with RE-oIgG was significantly higher than that for the RE vaccination. Therefore, we speculated that this vaccine formulation may be a promising strategy for designing a novel vaccine against FMDV in the future.展开更多
Objective:To explore the expression,biological function and possible mechanism of action of microRNA molecular-196a(miR-196a) in epithelial ovarian cancer.Methods:RT-PCR was used to detect the expression quantities of...Objective:To explore the expression,biological function and possible mechanism of action of microRNA molecular-196a(miR-196a) in epithelial ovarian cancer.Methods:RT-PCR was used to detect the expression quantities of epithelial ovarian tissue,benign ovarian tissue,normal ovary epithelial tissue,ovarian cancer cell lines and miR-196 a in normal ovarian epithelial cells to analyze the relationship between the expression of miR-196 a and the clinical pathologic parameters of ovarian cancer.Among those cell lines,the cell line of which miR-196 a expressed the most or least was selected and transfected the ovarian cancer cell line by using negative control plasma and miR-196 a inhibitor.After transfection,RT-PCR was used to test the expression quantity of miR-196 a,Transwell chamber method was applied to determine the migration and invasion abilities of ovarian carcinoma cells and Western blot was employed to detect the expression of HOXA10 protein.Results:The relative expression quantities of miR-196 a in ovarian cancer tissue and benign ovarian tissue were significantly higher than that in normal ovarian epithelial tissue,and the expression quantity of miR-196 a in ovarian cancer tissue was distinctively higher than that in benign ovarian tissue(P < 0.05).Among 78 cases of epithelial ovarian cancer,the expression quantities of miR-196 a in patients with low differentiation were all significantly higher than those in patients with high differentiation(P< 0.05).The expression of miR-196 a showed no significant relation with age,clinical stage and whether CA125 was positive or not in patients(P > 0.05).Compared with normal ovarian epithelial cell line IOSE80,the expression quantities of miR-196 a of all ovarian cancer cell lines increased obviously and differences were statistically significant(P < 0.05).Among them,the expression of miR-196 a of ovarian cancer cell line SKOV3 was the highest,while it decreased significantly(4.678 ± 0.785 vs.2.131 ± 0.345,t = 2.938,P < 0.05) after the ovarian cancer cell line SKOV3 was transfected by miR-196 a inhibitor.The results of Transwell chamber method showed that the migration and invasion abilities of ovarian cancer cells SKOV3 were declined significantly after the expression of miR-196 a was down-regulated and the difference showed statistical significance(P < 0.05).The results of Western blot revealed that the relative expression of HOXA10 decreased distinctly after the expression of miR-196 a was down-regulated and also the difference showed statistical significance(P < 0.05).Conclusions:The miR-196 a might serve as a cancer-promoting gene to promote the migration and invasion of epithelial ovarian cancer by downstream target gene HOXA10.展开更多
Monoclonal antibodies (McAbs) 1A9 and 9F12 against Foot-and-mouth disease virus (FMDV) serotype O were produced by fusing SP2/0 myeloma cells with splenocyte from the mouse immunized with O/China99. Both McAbs reacted...Monoclonal antibodies (McAbs) 1A9 and 9F12 against Foot-and-mouth disease virus (FMDV) serotype O were produced by fusing SP2/0 myeloma cells with splenocyte from the mouse immunized with O/China99. Both McAbs reacted with O/China99 but not with Asia 1, as determined by immunohistochemistry assay. The microneutralization titer of the McAbs 1A9 and 9F12 were 640 and 1 280, respectively. Both McAbs contain kappa light chains, but the McAbs 1A9 and 9F12 were IgG1 and IgM, respectively. In order to define the McAbs binding epitopes, the reactivity of these McAbs against VP1, P20 and P14 were examined using indirect ELISA, the result showed that both McAbs reacted with VP1 and P20. McAbs may be used for further studies of vaccine, diagnostic methods, prophylaxis, etiological and immunological researches on FMDV.展开更多
基金National Science and Technlogy Pillar Program(2006DAD06A03)National High Technology Research and Development Program of China(2006AA10A204)Key Project of Science and Technology of Gansu Province(092NKDA030)
文摘To develop a safe and efficient recombinant subunit vaccine to foot-and-mouth disease virus (FMDV) type Asia 1 in sheep, a tandem repeated multiple-epitope gene consisting of residues 137-160 and 197-211 of the VP1 gene of FMDV was designed and artificially synthesized. The biologically functional molecule, the ovine IgG heavy constant region (oIgG) as a protein carrier was introduced for design of the multiple-epitope recombinant vaccine and recombinant expression plasmids pET-30a-RE and pET-30a-RE-oIgG were successfully constructed. The recombinant proteins, RE and RE-oIgG, were expressed as a formation of inclusion bodies in E. coli. The immune potential of this vaccine regime in guinea pigs and sheep was evaluated. The results showed that IgG could significantly enhance the immune potential of antigenic epitopes. The recombinant protein RE-oIgG could not only elicit the high levels of neutralizing antibodies and lymphocytes proliferation responses in the vaccinated guinea pigs, but confer complete protection in guinea pigs against virus challenge. Although the recombinant protein RE could not confer protection in the vaccinated animals, it could delay the appearance of the clinical signs and reduce the severity of disease. Inspiringly, the titers of anti-FMDV neutralizing antibodies elicited in sheep vaccinated with RE-oIgG was significantly higher than that for the RE vaccination. Therefore, we speculated that this vaccine formulation may be a promising strategy for designing a novel vaccine against FMDV in the future.
基金supported by the General Project of Provincial Natural Science Research of the Anhui Higher Education Institutions(Grant No. KJ2015B096by)
文摘Objective:To explore the expression,biological function and possible mechanism of action of microRNA molecular-196a(miR-196a) in epithelial ovarian cancer.Methods:RT-PCR was used to detect the expression quantities of epithelial ovarian tissue,benign ovarian tissue,normal ovary epithelial tissue,ovarian cancer cell lines and miR-196 a in normal ovarian epithelial cells to analyze the relationship between the expression of miR-196 a and the clinical pathologic parameters of ovarian cancer.Among those cell lines,the cell line of which miR-196 a expressed the most or least was selected and transfected the ovarian cancer cell line by using negative control plasma and miR-196 a inhibitor.After transfection,RT-PCR was used to test the expression quantity of miR-196 a,Transwell chamber method was applied to determine the migration and invasion abilities of ovarian carcinoma cells and Western blot was employed to detect the expression of HOXA10 protein.Results:The relative expression quantities of miR-196 a in ovarian cancer tissue and benign ovarian tissue were significantly higher than that in normal ovarian epithelial tissue,and the expression quantity of miR-196 a in ovarian cancer tissue was distinctively higher than that in benign ovarian tissue(P < 0.05).Among 78 cases of epithelial ovarian cancer,the expression quantities of miR-196 a in patients with low differentiation were all significantly higher than those in patients with high differentiation(P< 0.05).The expression of miR-196 a showed no significant relation with age,clinical stage and whether CA125 was positive or not in patients(P > 0.05).Compared with normal ovarian epithelial cell line IOSE80,the expression quantities of miR-196 a of all ovarian cancer cell lines increased obviously and differences were statistically significant(P < 0.05).Among them,the expression of miR-196 a of ovarian cancer cell line SKOV3 was the highest,while it decreased significantly(4.678 ± 0.785 vs.2.131 ± 0.345,t = 2.938,P < 0.05) after the ovarian cancer cell line SKOV3 was transfected by miR-196 a inhibitor.The results of Transwell chamber method showed that the migration and invasion abilities of ovarian cancer cells SKOV3 were declined significantly after the expression of miR-196 a was down-regulated and the difference showed statistical significance(P < 0.05).The results of Western blot revealed that the relative expression of HOXA10 decreased distinctly after the expression of miR-196 a was down-regulated and also the difference showed statistical significance(P < 0.05).Conclusions:The miR-196 a might serve as a cancer-promoting gene to promote the migration and invasion of epithelial ovarian cancer by downstream target gene HOXA10.
基金The national high technology research and development program of China 863 (2006AA10A204)The national science and technology pillar program (2006BAD06A17)
文摘Monoclonal antibodies (McAbs) 1A9 and 9F12 against Foot-and-mouth disease virus (FMDV) serotype O were produced by fusing SP2/0 myeloma cells with splenocyte from the mouse immunized with O/China99. Both McAbs reacted with O/China99 but not with Asia 1, as determined by immunohistochemistry assay. The microneutralization titer of the McAbs 1A9 and 9F12 were 640 and 1 280, respectively. Both McAbs contain kappa light chains, but the McAbs 1A9 and 9F12 were IgG1 and IgM, respectively. In order to define the McAbs binding epitopes, the reactivity of these McAbs against VP1, P20 and P14 were examined using indirect ELISA, the result showed that both McAbs reacted with VP1 and P20. McAbs may be used for further studies of vaccine, diagnostic methods, prophylaxis, etiological and immunological researches on FMDV.
