OBJECTIVE: To study the effect of samples having different pH values in the bacterial endotoxins test (BET). METHODS: The BET was conducted as an assay of the concentration of endotoxins by using kinetic-turbidimetric technique. Samples having different pH values but containing same concentration reference standard endotoxin (RSE) were tested by TAL/LAL reagents. The average result on the standard curve was calculated and the mean recovery observed. RESULTS: The reagents had three highly sensitivities in pH 5.20, pH 7.83/pH 6.23 and pH 10.55. After adjustment to pH 6 to 8, the mean recovery was 76.5%～115.9% or 85.8%～99.8%. When the sample pH value was less than 3 or more than 12, the test was unsuitable for the inhibition test, and the mean recovery was less than 56.8%. CONCLUSION: It is necessary to adjust the pH of the solution to be tested in the BET, adjust the sample to pH (6.5～7.5) when using TAL reagents, and adjust to pH (6.2±0.1) or pH(6.7～7.8) when using LAL reagents.

Objective To study the effect of Danggui-beimu-kushen Pill on benign prostatic hyperplasia (BPH) and its possible mechanism. Methods BPH model mouse was produced by intraperitoneal injection of testosterone propionate. The high, medium and low dose treatment group was fed high, middle and low dose of Danggui-beimu-kushen Pill respectively, while the positive control group was fed Qianliekang liquid, 600 mg/kg. All mice were executed on the 21 day. Such values were observed as the prostate index changes, pathological changes of prostate by HE staining light microscopy and the expression changes of PCNA,bcl-2 and bax by immunohistochemistry. Results The prostate index of the treatment group was lower than the model group; the PCNA and bcl-2 expression were lower than the model group, while the bax expression was higher than the model group. Meanwhile; there is no significant difference among the treatment groups.Moreover, the difference between the treatment groups and the positive control group has no statistic meaning.Conclusion Danggui-beimu-kushen Pill can suppress BPH in mice. The mechanism may be related to the reduction expression of cell-proliferation protein PCNA and apoptosis profilin bcl-2, and the increase of bax expression.

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.

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.

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.

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.

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.
Animals ; Antibodies, Monoclonal ; Chromatography ; methods ; Foot-and-Mouth Disease ; diagnosis ; Foot-and-Mouth Disease Virus ; classification ; immunology ; isolation & purification ; Gold Colloid ; Immunoassay ; methods ; Immunoglobulin G ; immunology ; Reagent Strips ; Sensitivity and Specificity

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.
Animals ; Antibodies, Viral ; analysis ; Antigens, Viral ; biosynthesis ; genetics ; immunology ; Cattle ; Cattle Diseases ; diagnosis ; immunology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Foot-and-Mouth Disease ; diagnosis ; immunology ; Foot-and-Mouth Disease Virus ; chemistry ; genetics ; isolation & purification ; Genetic Vectors ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; immunology

Objective: To investigate the expression level and clinical significance of a long non-coding RNA (LncRNA), BC200 in non-small cell lung cancer (NSCLC) and analyze its correlation with the epithelial-mesenchymal transition (EMT)-related proteins, E-cad-herin, N-cadherin, and Snail. Methods: Sixty NSCLC and sixty paired adjacent tissue samples were collected to detect the BC200 levels. Furthermore, 40 samples of NSCLC and 40 samples of normal lung tissues were collected to quantify the messenger RNA (mRNA) lev-els of E-cadherin, N-cadherin, and Snail using quantitative polymerase chain reaction. The relationship between the BC200 level and mRNA levels of E-cadherin, N-cadherin, and Snail was explored in NSCLC tissues. The correlation between BC200 and clinical pathologi-cal parameters (gender, age, TNM stage, tumor size, lymph node metastasis, and pathologic type) was also analyzed. Receiver operat-ing characteristic curve (ROC) was constructed to evaluate the diagnostic efficiency of BC200. Immunohistochemical staining was per-formed to determine the expression levels of E-cadherin, N-cadherin, and Snail in 40 specimens of NSCLC and 20 specimens of normal lung tissue and their correlation to the expression levels of BC200 was evaluated. Results: 1) The expression of BC200, N-cadherin mRNA, and Snail mRNA was significantly upregulated in the tumor tissues when compared to that in normal lung tissues (P<0.05). The expression of E-cadherin mRNA was significantly lower in tumor tissues than in the normal lung tissues (P<0.05). 2) The positive rate of E-cadherin, N-cadherin, and Snail in NSCLC was 40% (16/40) , 57.5% (23/40), and 57.5% (23/40), respectively, while that of normal lung tissues was 95% (19/20), 5% (1/20), and 10% (2/20), respectively. There was a significant difference between these two data sets (P<0.05). 3) BC200 is highly expressed in the NSCLC tissues. The high expression of BC200 in lung cancer was correlated with lymph node metastasis, clinical stage, and positive rate of E-cadherin, N-cadherin, and Snail. The expression of BC200 in NSCLC tissues was negatively correlated with the expression of E-cadherin mRNA (r=-0.31, P<0.05) and positively correlated with the expression of Snail and N-cadherin mRNA (r=0.305, r=0.257, P<0.05). 4) ROC analysis of BC200 indicated a potential diagnostic value of BC200 levels in NSCLC . Conclusions: BC200 is highly expressed in NSCLC tissues, which was correlated with lymph node metastasis, clinical stage, E-cadherin, N-cadherin, and Snail positive rate. The expression of BC200 in NSCLC tissues was negatively correlated with E-cadherin and positively correlated with Snail and N-cadherin. BC200 may regulate the invasion and migration ability of NSCLC by EMT. BC200 may be a potential tumor marker for NSCLC diagnosis.

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.
Animals ; Cattle ; Cell Line ; Cloning, Molecular ; Endopeptidases ; biosynthesis ; genetics ; Foot-and-Mouth Disease Virus ; genetics ; Genetic Vectors ; genetics ; Puromycin ; pharmacology ; Recombinant Proteins ; biosynthesis ; genetics ; Retroviridae ; genetics ; metabolism ; Tetracycline ; pharmacology ; Transfection