Experimental research of miR-132 inhibits proliferation and induces apoptosis of ovarian cancer via Ezrin / 中国免疫学杂志

Objective:To explore the biological function of miR-132 in ovarian cancer and the target. Methods: 22 cases ovarian cancer tissue and non-tumor tissue adjacent were collected,the expression of miR-132 in tumor tissue and non-tumor tissue, normal ovarian epithelial cells and ovarian cancer cell were detected by RT-PCR. The normal ovarian epithelial cells which the expression of miR-132 maximum or minimum were chosen, and they were divided into two groups, respectively with transfection of negative control plasmid ( NC) and miR-132 mimic plasmid. The expression of miR-132 after transfection was detected by RT-PCR,the cell proliferation and cell apoptosis were detected by CCK-8 method and flow cytometry instrument respectively,the expression of Ezrin protein was detected by Western blot. Results:The expression of miR-132 in tumor tissue was significantly lower than the tumor tissue adjacent,the expression of miR-132 in ovarian cancer cell lines was significantly lower than normal ovarian epithelial cells, the differences were statistically significant (P<0. 05). The SKOV3 cell lines was chosed for gene transfection,compared with NC group, transfection with miR-132 mimic plasmid could significantly reduce cell proliferation, increase cell apoptosis, the difference had statistical significance ( P<0. 05 ) . Western blot results showed that up-regulation miR-132 significantly increased the Ezrin protein expression in ovarian cancer SKOV3 cells ( P<0. 05 ) . Conclusion: In ovarian cancer, miR-132;inhibits proliferation and induces apoptosis of ovarian cancer via Ezrin,it may be a tumor suppressor gene.

Potency of a novel multi-epitope vaccine against foot-and-mouth disease type Asia 1 in guinea pigs / 中国人兽共患病学报

The potency of an improved recombinant multi-epitope vaccine against FMDV type Asia1 was evaluated in this study .A multi-epitope gene based on FMDV type Asia1 was designed and a recombinant expression plasmid (pRE-oIgG) was constructed .The proteins ,RE-oIgG and 3D were expressed in E .coli cells and purified with Ni-NTA agarose resin by affinity chromatography .The proteins ,RE-oIgG ,3D and RE-oIgG plus 3D ,were emulsified in an oil adjuvant ISA 206 .Twenty-five female guinea pigs were randomly divided into five groups and intramuscularly vaccinated for with RE-oIgG ,3D ,RE-oIgG plus 3D ,an inactivated FMDV vaccine (type Asia1) ,and PBS .All animals were vaccinated for two times .Anti-FMDV specific an-tibodies ,neutralization antibodies ,protection potency ,and lymphoproliferation assay were detected by ELISA ,virus neutrali-zation assay ,challenge test ,and flow cytometry ,respectively .Results showed that RE-oIgG plus 3D elicited significant high-level anti-FMDV specific antibodies compared to RE-oIgG alone (P<0 .05) .All the vaccinated animals induced higher level lymphoproliferation responses in vitro except PBS .Both 3D alone and PBS produced the negligible neutralizing antibodies and anti-FMDV specific antibodies .RE-oIgG plus FMDV 3D not only elicited high levels of anti-FMDV neutralizing antibodies ,but also induced significant lymphoproliferation responses .More importantly ,RE-oIgG plus 3D conferred complete protection to guinea pigs against challenge with 1 000 GPID50 .Interestingly ,two of five vaccinated animals with 3D alone were full protected against challenge ,and other three animals significantly showed a delay of 2-3 days in the onset of clinical signs .Therefore ,we considered that RE-oIgG plus 3D induces strong humoral and cellular immune responses ,which may be used for control and prevention of FMD in the future .

Generation of Monoclonal Antibodies against Non-structural Protein 3AB of Foot-and-Mouth Disease Virus / 中国病毒学·英文版

To identify linear epitopes on the non-structural protein 3AB of foot-and-mouth disease virus (FMDV),BABL/c mice were immunized with the 3AB protein and splenocytes of BALB/c mice were fused with myeloma Sp2/0 cells.Two hybridoma monoclonal antibodies (mAbs) cell lines against the 3AB protein of foot-and-mouth disease virus (FMDV) were obtained,named C6 and E7 respectively.The microneutralization titer was 1∶1024 for mAb C6,and 1∶512 for E7.Both mAbs contain kappa light chains,and were of subclass IgG2b.In order to define the mAbs binding epitopes,the reactivity of these mAbs against FMDV were examined by indirect ELISA.The results showed that both mAbs can react with FMDV,but had no cross-reactivity with Swine Vesicular Disease (SVD) antigens.The titers in abdomen liquor were 1∶5×106 for C6 and 1∶2×106 for E7.In conclusion,the mAbs obtained from this study are specific for the detection of FMDV,can be used for etiological and immunological researches on FMDV,and have potential use in diagnosis and future vaccine designs.

Expression and utilization of 3AB nonstructural protein of foot-and-mouth disease virus in Escherichia coli / 生物工程学报

To develop a sensitive and specific ELISA for detection of antibodies to the nonstructural protein of FMDV. We cloned and expressed FMDV nonstructural protein 3AB in Escherichia coli expression system. The recombinant protein 3AB was purified with Ni-NTA HisBind Resins and characterized by Western blotting. An indirect ELISA based on purified protein 3AB as a coating antigen was established. The specificity and sensitivity of this assay were evaluated by comparison with a commercial 3ABC-ELISA kit in detecion of serum samples. The results showed that the recombinant protein 3AB was expressed as a formation of inclusion bodies in Escherichia coli. The purified protein could specificially react with FMDV infection antibodies in Western blotting assay, but no reaction with the immune antibodies induced with vaccine. Two assays were no significant differences in specificity and sensitivity for detection of field samples (P>0.05). Therefore, we speculated that the recombinant protein 3AB is a promising molecular marker, which may effectively differentiate FMD-infected from vaccinated animals in a herd.

Develope Monoclonal Antibody against Foot-and-mouth Disease Virus A Type / 中国病毒学·英文版

In order to develop an anti-FMDV A Type monoclonal antibo by (mAb),BABL/c mice were immunized with FMDV A type.Monoclonal antibodies (mAbs) 7B11 and 8H4 against Foot-and-mouth disease virus (FMDV) serotype A were produced by fusing SP2/O myeloma cells with splenocyte from the mouse immunized with A/AV88.The microneutralization titer of the mAbs 7B11 and 8H4 were 1024 and 512,respectively.Both mAbs contain kappa light chains,the mAbs were IgG1.In order to define the mAbs binding epitopes,the reactivity of these mAbs against A Type FMDV,were examined using indirect ELISA,the result showed that both mAbs reacted with A Type FMDV.These mAbs may be used for further vaccine studies,diagnostic methods,prophylaxis,etiological and immunological research on FMDV.Characterization of these ncindicated that prepared anti-FMDV A mAbs had no cross-reactivity with Swine Vesicular Disease (SVD) or FMDV O,Asial and C Type antigens.Their titers in abdomen liquor were 1:5×106 and 1:2×106,respectively.7B11 was found to be of subtype IgG1,8H4 was classified as IgG2b subtype.The mAbs prepared in this study,are specific for detection of FMDV serotype A,and is potentially useful for pen-side diagnosis.

Immune Potential of a Novel Multiple-epitope Vaccine to FMDV Type Asia 1 in Guinea Pigs and Sheep / 中国病毒学·英文版

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.

Fusion expression of Asia I type FMDV neutralizing epitope with heavy chain constant region of sheep IgG and the assessment of its immunogenicity / 生物工程学报

VP1 is a major antigenic protein of foot-and-mouth disease virus(FMDV), which induces the immune response against FMDV infection, and contains several epitopes of the virus. We designed and chemically synthesized a DNA fragment which encoding a tandem repeat protein of 136-160aa and 198-211aa of a strain of type Asia I FMDV, and cloned the gene of heavy chain constant region of sheep IgG. By using the BamH I, EcoR I and Xho I sites, both genes were cloned into pPROExHTb vector in turn to form a recombinant plasmid pPRO-FshIgG A chimeric protein, named FshIgG, was obtained after transforming the pPRO-FshIgG into Escherichia coli BL21 (DE3) host cell and induced by IPTG. Inoculation with 100 microg FsIgG induced strong neutralizing antibody response in guinea pigs, and FshIgG inoculated guinea pigs were also protected against 200 ID50 FMDV challenge. Our study indicated that the heavy chain constant region of sheep IgG can act as the carrier protein for FMDV peptide epitopes, and FshIgG is a potential multiepitope peptide vaccine candidate to prevent FMDV infection.

B Cell Epitopes within VP1 of Type O Foot-and-mouth Disease Virus for Detection of Viral Antibodies / 中国病毒学·英文版

In this study,the coding region of type O FMDV capsid protein VP1 and a series of codon optimized DNA sequences coding for VP1 amino acid residues 141-160(epitopel),tandem repeat 200-213(epitope2(+2))and the combination of two epitopes(epitope1-2)was genetically cloned into the prokaryotic expression vector pPROExHTb and pGEX4T-1,respectively.VP1 and the fused epitopes GST-E1,GST-E2(+2)and GST-E1-2 were successfully solubly expressed in the cytoplasm of Escherichia coli and Western blot analysis demonstrated they retained antigenicity.Indirect VP1-ELISA and epitope ELISAs were subsequently developed to screen a panel of 80 field pig sera using LPB-ELISA as a standard test.For VP1-ELISA and all the epitope ELISAs,there were clear distinctions between the FMDV-positive and the FMDV-negative samples.Cross-reactions with pig sera positive to the viruses of swine vesicular disease virus that produce clinically indistinguishable syndromes in pigs or guinea pig antisera to FMDV strains of type A,C and Asial did not occur.The relative sensitivity and specificity for the GST-E1 ELISA,GST-E2(+2),GST-E1-2 ELISA and VP1-ELISA in comparison with LPB-ELISA were 93.3% and 85.0%,95.0% and 90%,100% and 81.8%,96.6% and 80.9% respectively.This study shows the potential use of the aforementioned epitopes as alternatives to the complex antigens used in current detection for antibody to FMDV structural proteins.

Establishment of colloidal gold-immunochromatography assay strip for detection of type Asia1 foot-and-mouth disease virus / 生物工程学报

To establish a sensitive, rapid and simple gold immunochromatography assay (GICA) for detecting Asia1 type of foot-and-mouth disease virus (FMDV) from the field samples. The purified anti-FMDV type Asia1 monoclonal antibody labeled with colloidal gold and the goat anti-Guinea pig IgG were wrapped onto nitrocellulose membrane as the test line (T line) and the control line (C line), respectively. The strip was then further optimized. A total of 87 field samples were detected. The results indicated a correct rate of 98.8% for detecting FMDV Asia1 type. No cross reaction was found with swine vesicular disease (SVD) and FMDV O, A and C type antigen. The sensitivity of the strip can reach to 10(-4) (TCID50 6.25). It had the same results for positive and negative specimens tested in three times. This strip could be stored at 4 degrees C for three months. In this study, the established gold immunochromatographic strip test kit is simple, rapid, sensitive and specific for detecting FMDV type Asial, and is potentially useful for the for pen-side diagnosis.

Morphological observation of bovine kidney (MDBK) cells effected by foot-and-mouth disease virus L(pro) / 生物工程学报

In order to explore the morphological changes of Bovine Kidney (MDBK) cells induced by foot-and-mouth disease virus (FMDV) L protease, we induced the expression of FMDV L protease in bovine kidney cells (MDBK) artificially. All work is carried out on the basis of a stable MDBK cell line inducibly expresses the Lab gene under the control of tetracycline. We use cell morphology, Hoechst 33258 staining, AO-EB staining, and DNA Ladder abstraction to research the morphological changes of MDBK cells. 24 hours after FMDV L protease were induced and expressed in MDBK cells, cells shown the diminish of cell size, nuclear enrichment and the appearance of transparency circle under the light microscope. Apoptosis characteristics of nuclear condensation, fragmentation, accompanied by apoptotic bodies formation (Hoechst 33258 staining). 36 hours after the expression, nuclear staining of early lesions showed bright green plaque or debris-like dense, and advanced lesions showed Orange and dense plaques (AO-EB staining). 48 hours after the expression, DNA gel electrophoresis showed visible DNA ladder. Results indicate that FMDV L protease can induce apoptosis of MDBK and apoptosis plays an important role in the cytopathogenicity effect of FMDV.

Application of VP1 Protein to Develop Monoclonal Antibody against Foot-and-mouth Disease Virus Asial Type / 中国病毒学·英文版

In order to develop an anti-FMDV Asial type monoclonal antibody (mAb), BABL/c mice were immunized with recombinant FMDV VP1 protein. Three mAbs, 1B8, 5E1 and 5E2, were then further optimized. The result indicated that prepared anti-FMDV Asial mAbs had no cross-reactivity with Swine vesicular disease (SVD) and FMDV O, A and C type antigen. Their titers in abdomen liquor were l:5×106, l:2×106 and l:5×l06, respectively. 1B8 was found to be of IgGi subtype, 5E1 and 5E2 belonged to IgG2b subtype. In this study, the prepared mAbs are specific for detecting FMDV type Asial, and is potentially useful for pen-side diagnosis.

Construction of recombinant retroviral vector carrying Lab gene of foot-and-mouth disease virus and its expression in bovine kidney (MDBK) cells / 生物工程学报

In this study, foot-and-mouth disease virus (FMDV) strain OA/58 RNAs were used as templates for RT-PCR. By the molecular cloning, the Lab gene encoding leader protease called Lpro were cloned in retroviral vector pBPSTR1 to obtain reconstruction retroviral vector termed pBPSTR1-Lab. At different concentrations of puromycin and tetracycline respectively in the cell culture mediums, the growth of bovine kidney cells (MDBK) showed that the optimal puromycin resistant selection concentration was 3 microg/mL and tetracycline regulatory concentration was 1 microg/mL. Pseudotyped retroviral virus particles were produced by transiently co-tansfecting GP2-293 cells with a retroviral vector DNA and VSV-G plasmid. Then MDBK cells were infected by pseudotyped retroviral virus and were continually seeded in the medium at the optimal tetracycline regulatory concentration and puromycin selection concentration for 12 days to obtain puromycin resistant colonies whose genomes contained the Lab gene. After tetracycline removal, synthesis of Lpro induced severe morphological changes in the puromycin resistant MDBK cells. PCR and Western blotting proved that a stable MDBK cell line inducibly expressing the Lab gene under the control of tetracycline was obtained. The experiment might provide a basis for studying that Lpro of FMDV plays an important role in MDBK cell pathogenesis.

Amplification and Characterization of Bull Semen Infected Naturally with Foot-and-mouth Disease Virus Type Asial by RT-PCR / 中国病毒学·英文版

To investigate the security of semen biologically, 15 bull semen samples were collected (of which 5 exhibited clinical signs of Foot-and-mouth disease) and identified by RT-PCR and virus isolation. The results indicated that the semen of the infected bulls were contaminated by Foot-and-mouth disease virus (FMDV), but FMDV was not detected in semen samples from those bulls not showing clinical signs of Foot-and-mouth disease (FMD). This is the first report of the presence of FMDV in bull semen due to natural infection in China. The analysis of the partial sequence of the VP1 gene showed that the virus strain isolated from semen has 97.9% identity with the virus isolated from vesicular liquid of infected bulls showing typical signs of FMD and belonged to the same gene sub-group.