In order to develop a double antibody sandwich assay (DAS-ELISA) for detecting bovine respiratory syncytial virus (BRSV),New Zealand white rabbits and BALB/c mice were immunized with the purified G protein as an antigen to prepare anti-G protein polyclonal and monoclonal antibodies.The antibody concentration and reaction conditions of DAS-ELISA were optimized by square titration,and its sensitivity,specificity,and coincidence rate were validated.Five hybridoma were stably secreting Mab which subclass belonged to IgG1κ.Western blot and IFA test showed that PcAb and Mab could react specifically with G protein and BRSV.The PcAb and Mab as the capture antibody and detection antibody respectively,and their optimal working concentrations were determined to be 2.5 μg/mL and 10μg/mL,the critical value 0.22 and the detection limit of 1.43 μg/mL,batch,inter-assay coefficient of variation less than 10 ％.The DAS-ELISA had no cross-reaction with several pathogens which often caused bovine respiratory disease.When 45 nasal swabs of clinical samples were simultaneously detected by the DAS-ELISA and RT-PCR,the sensitivity,specificity and coincidence rate were 92.0 ％,100 ％,95.6 ％,respectively.It' s indicated that the established DAS-ELISA detection method can be used to detect a large number of clinical samples.It was the foundation of monitoring and quick diagnosis for BRSV.

In order to determine immunogenicity and protective effect in chickens, we used the IBDV (Infectious bursal disease virus)-Vp2/Lactobacillus casei as antigen transfer system. First, the immunized and control chickens were challenged by IBDV/DQ at lethal dose to determine the protective ratio. Second, chickens were orallyand intranasally vaccinated twice with 10(9) CFU/mL pLA-VP2/L. casei, pLA/L. casei and PBS as negativecontrol and commercial vaccine as positive control. The bursa injury and the lesion score wererecorded post challenge. The level of specific IgG and sIgA in pLA-VP2/L. casei and positive control groups was significantly higher than that in negativecontrol groups. The protection efficacy in pLA-VP2/L. casei oral group was higher than that inintranasal group. The SI. of pLA-VP2/L. casei oral group was significant higher than other groups. The lesion score indicated the pLA-VP2/L. casei was safer than commercial vaccine for bursa. Collectively, the pLA-VP2/L. casei could be a vaccine candidate for IBDV.
Animals ; Antibodies, Viral ; blood ; Antibody Formation ; Birnaviridae Infections ; prevention & control ; veterinary ; Chickens ; Infectious bursal disease virus ; Lactobacillus casei ; Poultry Diseases ; prevention & control ; Recombinant Proteins ; immunology ; Viral Structural Proteins ; immunology ; Viral Vaccines ; immunology

To obtain and analyze the sequence of the nucleocapsid gene from bovine coronavirus, and to produce the fusion protein of the N gene in E.coli in order to use this recombinant protein for the study of bovine coronavirus. The N gene of BCV-DQ strain was amplified by RT-PCR, in which the primers were designed on the basis of N gene sequence of BCV-Mebus strain. The PCR products of 1 347 bp in length were cloned and sequenced, and then inserted into the prokaryotic vector pET30a. The recombinant plasmids were then transformed into Escherichia coli BL21 and identified by SDS-PAGE and Western blot assay. ELISA assay was optimized of N protein as the coating antigen to detect the viruses in the clinical samples. In comparison with 6 BCV strains in GenBank, the sequence identity was proved to be more than 98.3%. Result in SDS-PAGE showed that the fusion protein had a molecular weight of 60 ku, and could be specifically recognized by mouse serum against BCV. The indirect ELISA was used to test 256 serum samples collected from Heilongjiang province and 65.23% samples were positive. On testing field samples, an overall agreement of 95.31% was generated between the the neutralization test of viruses (VN) and indirect ELISA. It is apparent that the N gene was highly conservative and is expressed in E. coli in high level,also the prokaryotic expression products of this gene show a fine reactiongenicity in immune responses. It was also suggested that the N protein may be a useful antigen for sero-diagnosis and epidemiological investigation of BCV.

OBJECTIVETo prepare pulsed-release tablet (PTS) according to the rhythm of coronary heart disease based on efficacy material and the mechanism of compound Danshen.

METHODPTS were achieved by coating the core which contains drugs, CMS-Na, lactose, succinic acid and MCC with separation layer (Eudragit RL), swelling layer (HPMC E5), and controlled-release membrane (Eudragit RS-RL-EC).

RESULTThe results of in vitro experiments showed that no difference was observed among the profiles of Danshensu, protocatechuic aldehyde, ginsenoside Rg1, Rb1, notoginsenoside R1 release from the two-step release system. And it indicated that swelling was the basis and prerequisite for drug release from PTS, and the diffusion, organic acid-induced, and osmotic pumping mechanism were involved in drug release, but the latter they were the dominant factors.

CONCLUSIONSuccessfully obtained the PTS of a certain lag-time behind the rapid release which indicate that after bed time administration of such device, the drug plasma concentration-time curve CAN meet the requirements of chronotherapy of cardiovascular disease.

Benzaldehydes ; metabolism ; Catechols ; metabolism ; Chromatography, High Pressure Liquid ; Coronary Disease ; drug therapy ; Diffusion ; Drug Compounding ; methods ; Drug Delivery Systems ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; metabolism ; therapeutic use ; Ginsenosides ; metabolism ; Osmosis ; Salvia miltiorrhiza ; chemistry ; Tablets ; Time Factors

K88ac-LT(B) gene derived from pQE30-K88ac-LT(B) was cloned into the expression vector pLA and then the recombinant vector was transformed into the competent cells Lactobacillus casei 525. The recombinant bacteria were grown at 37 degrees C, in MRS broth. Western blotting analysis with rabbit-anti-K88ac-LT(B) polyclonal serum indicated that the recombinant protein reacted with the specific antibodies. The results showed that the molecular weight of the recombinant protein was about 71.2 kD. The K88ac-LT(B) fusion protein on the cell surface was confirmed by immunofluorescence mciroscopy and flow cytometric analysis. In addition, the survival of recombinant Lactobacillus casei 525 was studied in imitative gastrointestinal environments such as artificial gastro fluid (pH 1.5-5.5), artificial intestinal fluid, bile(0.3-3.0 g/L). The results indicated that the recombinant strain survived well in artificial gastric fluids at pH 2.5-4.5 in 5 h. The recombinant Lactobacillus casei 525 could slowly grow in the artificial intestinal fluid for different time, and could survive in 0.3% bile.
Antigens, Bacterial ; genetics ; Bacterial Toxins ; genetics ; metabolism ; Enterotoxigenic Escherichia coli ; genetics ; metabolism ; Enterotoxins ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; metabolism ; Fimbriae Proteins ; genetics ; Gastric Juice ; Lactobacillus casei ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Recombination, Genetic

TrkA is essential components of the high-affinity NGF receptor necessary to mediate biological effects of the neurotrophins NGF. Here we report on the expression of trkA in the cerebral cortex and diencephalon of mongolian gerbils during postnatal development. The expression of trkA was identified by immunohistochemical method. In parietal cortex and piriform cortex, higher levels of trkA-IR (immunoreactivity) were detected at 3 days postnatal (P3) and at P9. Although trkA was not expressed till P3 in the parietal cortex, it was detectable at birth in the piriform cortex. Several regions, such as Layers I, IV & VI, did not show much expression. Layer I showed especially weak labeling. In the hippocampus, thalamus, and hypothalamus, higher levels of trkA-IR were detected at P6 and P12 than earlier days. But trkA was not expressed at birth in the hippocampus, at P3 in the reticular thalamic nucleus (Rt), or neonatally in the dorsomedial hypothalamic nucleus (DM). This data shows that expression of trkA is developmentally regulated and suggests that high affinity neurotrophin-receptors mediate a transient response to neurotrophines in the cerebral cortex and diencephalon during mongolian gerbil brain ontogeny.
Animals ; Animals, Newborn ; Cerebral Cortex/*metabolism ; Diencephalon/*metabolism ; Gerbillinae/*metabolism ; Immunohistochemistry/veterinary ; Nerve Growth Factor/metabolism ; Receptor, trkA/*metabolism

Background: The genetic basis for hyperglycaemia in pregnancy remain unclear. This study aimed to uncover the genetic determinants of gestational diabetes mellitus (GDM) and investigate their applications. Methods: We performed a meta-analysis of genome-wide association studies (GWAS) for GDM in Chinese women (464 cases and 1,217 controls), followed by de novo replications in an independent Chinese cohort (564 cases and 572 controls) and in silico replication in European (12,332 cases and 131,109 controls) and multi-ethnic populations (5,485 cases and 347,856 controls). A polygenic risk score (PRS) was derived based on the identified variants. Results: Using the genome-wide scan and candidate gene approaches, we identified four susceptibility loci for GDM. These included three previously reported loci for GDM and type 2 diabetes mellitus (T2DM) at MTNR1B (rs7945617, odds ratio [OR], 1.64; 95% confidence interval [CI],1.38 to 1.96]), CDKAL1 (rs7754840, OR, 1.33; 95% CI, 1.13 to 1.58), and INS-IGF2-KCNQ1 (rs2237897, OR, 1.48; 95% CI, 1.23 to 1.79), as well as a novel genome-wide significant locus near TBR1-SLC4A10 (rs117781972, OR, 2.05; 95% CI, 1.61 to 2.62; Pmeta=7.6×10-9), which has not been previously reported in GWAS for T2DM or glycaemic traits. Moreover, we found that women with a high PRS (top quintile) had over threefold (95% CI, 2.30 to 4.09; Pmeta=3.1×10-14) and 71% (95% CI, 1.08 to 2.71; P=0.0220) higher risk for GDM and abnormal glucose tolerance post-pregnancy, respectively, compared to other individuals. Conclusion Our results indicate that the genetic architecture of glucose metabolism exhibits both similarities and differences between the pregnant and non-pregnant states. Integrating genetic information can facilitate identification of pregnant women at a higher risk of developing GDM or later diabetes.

Background: The genetic basis for hyperglycaemia in pregnancy remain unclear. This study aimed to uncover the genetic determinants of gestational diabetes mellitus (GDM) and investigate their applications. Methods: We performed a meta-analysis of genome-wide association studies (GWAS) for GDM in Chinese women (464 cases and 1,217 controls), followed by de novo replications in an independent Chinese cohort (564 cases and 572 controls) and in silico replication in European (12,332 cases and 131,109 controls) and multi-ethnic populations (5,485 cases and 347,856 controls). A polygenic risk score (PRS) was derived based on the identified variants. Results: Using the genome-wide scan and candidate gene approaches, we identified four susceptibility loci for GDM. These included three previously reported loci for GDM and type 2 diabetes mellitus (T2DM) at MTNR1B (rs7945617, odds ratio [OR], 1.64; 95% confidence interval [CI],1.38 to 1.96]), CDKAL1 (rs7754840, OR, 1.33; 95% CI, 1.13 to 1.58), and INS-IGF2-KCNQ1 (rs2237897, OR, 1.48; 95% CI, 1.23 to 1.79), as well as a novel genome-wide significant locus near TBR1-SLC4A10 (rs117781972, OR, 2.05; 95% CI, 1.61 to 2.62; Pmeta=7.6×10-9), which has not been previously reported in GWAS for T2DM or glycaemic traits. Moreover, we found that women with a high PRS (top quintile) had over threefold (95% CI, 2.30 to 4.09; Pmeta=3.1×10-14) and 71% (95% CI, 1.08 to 2.71; P=0.0220) higher risk for GDM and abnormal glucose tolerance post-pregnancy, respectively, compared to other individuals. Conclusion Our results indicate that the genetic architecture of glucose metabolism exhibits both similarities and differences between the pregnant and non-pregnant states. Integrating genetic information can facilitate identification of pregnant women at a higher risk of developing GDM or later diabetes.

Background: The genetic basis for hyperglycaemia in pregnancy remain unclear. This study aimed to uncover the genetic determinants of gestational diabetes mellitus (GDM) and investigate their applications. Methods: We performed a meta-analysis of genome-wide association studies (GWAS) for GDM in Chinese women (464 cases and 1,217 controls), followed by de novo replications in an independent Chinese cohort (564 cases and 572 controls) and in silico replication in European (12,332 cases and 131,109 controls) and multi-ethnic populations (5,485 cases and 347,856 controls). A polygenic risk score (PRS) was derived based on the identified variants. Results: Using the genome-wide scan and candidate gene approaches, we identified four susceptibility loci for GDM. These included three previously reported loci for GDM and type 2 diabetes mellitus (T2DM) at MTNR1B (rs7945617, odds ratio [OR], 1.64; 95% confidence interval [CI],1.38 to 1.96]), CDKAL1 (rs7754840, OR, 1.33; 95% CI, 1.13 to 1.58), and INS-IGF2-KCNQ1 (rs2237897, OR, 1.48; 95% CI, 1.23 to 1.79), as well as a novel genome-wide significant locus near TBR1-SLC4A10 (rs117781972, OR, 2.05; 95% CI, 1.61 to 2.62; Pmeta=7.6×10-9), which has not been previously reported in GWAS for T2DM or glycaemic traits. Moreover, we found that women with a high PRS (top quintile) had over threefold (95% CI, 2.30 to 4.09; Pmeta=3.1×10-14) and 71% (95% CI, 1.08 to 2.71; P=0.0220) higher risk for GDM and abnormal glucose tolerance post-pregnancy, respectively, compared to other individuals. Conclusion Our results indicate that the genetic architecture of glucose metabolism exhibits both similarities and differences between the pregnant and non-pregnant states. Integrating genetic information can facilitate identification of pregnant women at a higher risk of developing GDM or later diabetes.

Background: The genetic basis for hyperglycaemia in pregnancy remain unclear. This study aimed to uncover the genetic determinants of gestational diabetes mellitus (GDM) and investigate their applications. Methods: We performed a meta-analysis of genome-wide association studies (GWAS) for GDM in Chinese women (464 cases and 1,217 controls), followed by de novo replications in an independent Chinese cohort (564 cases and 572 controls) and in silico replication in European (12,332 cases and 131,109 controls) and multi-ethnic populations (5,485 cases and 347,856 controls). A polygenic risk score (PRS) was derived based on the identified variants. Results: Using the genome-wide scan and candidate gene approaches, we identified four susceptibility loci for GDM. These included three previously reported loci for GDM and type 2 diabetes mellitus (T2DM) at MTNR1B (rs7945617, odds ratio [OR], 1.64; 95% confidence interval [CI],1.38 to 1.96]), CDKAL1 (rs7754840, OR, 1.33; 95% CI, 1.13 to 1.58), and INS-IGF2-KCNQ1 (rs2237897, OR, 1.48; 95% CI, 1.23 to 1.79), as well as a novel genome-wide significant locus near TBR1-SLC4A10 (rs117781972, OR, 2.05; 95% CI, 1.61 to 2.62; Pmeta=7.6×10-9), which has not been previously reported in GWAS for T2DM or glycaemic traits. Moreover, we found that women with a high PRS (top quintile) had over threefold (95% CI, 2.30 to 4.09; Pmeta=3.1×10-14) and 71% (95% CI, 1.08 to 2.71; P=0.0220) higher risk for GDM and abnormal glucose tolerance post-pregnancy, respectively, compared to other individuals. Conclusion Our results indicate that the genetic architecture of glucose metabolism exhibits both similarities and differences between the pregnant and non-pregnant states. Integrating genetic information can facilitate identification of pregnant women at a higher risk of developing GDM or later diabetes.