Gongying Rukang liniment is a pure and new-type preparation of Chinese herbal medicine, that was developed by Jinhua Animal’s Health Products Limited Company for treating mastitis of cow.This medicine was daubed breast directly and absorbed through skin, using simply and conveniently. Through the compare inhibiting test on the main pathogenic bacteria Staphylococci aureus and Streptococcus agalactiae and that cause mastitis of cow with three kinds of different prescription A , B , C of this preparation and 2-methyl hydrogen quinoid and clindymycin. The results showed that the minimum inhibition consistencies (MIC) of the three different prescriptions to Staphylococci aureus and to Streptococcus agalactiae were respectively 125mg/L, 250mg/mL, 250mg/mL and 250mg/mL, 500mg/mL and 500mg/mL. The MICs of the 2-methyl hydrogen quinoid and clindymycin were 400μg/mL,400μg/mL and 25μg/mL, 50μg/mL. The test indicated that this liniment’s effect was not better than 2-methyl hydrogen quinoid and clindymycin, but prescription A showed better effect, its MIC achieved 125mg/mL and 250mg/mL respectively, while the effect of B and C inhibited the two bacterium similarly.
Expressing condition of gene engineering strain Escherichia coli BL21/pET28a-VP2, which could be used for production of the subunit vaccine against infectious bursal disease, was performed using the method of rocking bottle culture. The result showed that the inducing temperature should be 37℃, the inducing expression time should last 3 to 7 hours, and the inducer could be lactoseor isopropyl-β-D-thiogalactopyranosid(IPTG). The maximum expression capacity could be obtained when the concentration of lactose reached 20mmol/L, or the concentration of IPTG reached 33mmol/L.
Five kinds of recombinant proteins GST-FedA/ab, GST-FedA/ac, GST-FedE GST- FedF/ab and GST-FedF/ac, were extracted by SDS-PAGE method. Five groups of healthy rabbits were immunized with the 5 recombinant proteins respectively and anti-FedA/ab, anti-FedA/ac, anti-FedE anti-FedF/ab and anti-FedF/ac were obtained. The results of the direct agglutination test of the 5 kinds of antisera by using F18ac-positive Escherichia coli 107/86 and F18ac-positive E.coli 8813 respectively showed that all the 5 kinds of antisera could recognize F18ab and F18ac. To investigate the relationship of subunit(s) of F18 fimbrial adhesin with the adhesion effect, adhesive inhibition tests were performed using antibodies specific for the subunits FedA, FedE and FedF. The results showed that anti-FedA and/or anti-FedE (serum) was unable to inhibit adhesion of F18~(+) E.coli to porcine enterocytes, whereas anti-FedF antibody could overtly inhibit the adhesion, which showed the FedF is the adhesive subunit of F18 fimbriae.
Four specificoligonucleotide primers were designedaccording to the gene sequences of Newcastle disease virus(NDV) and the sequence difference at fusion gene cleavage sites between virulent and avirulent strains.A multiplex RT-PCR method was developed to differentiate virulent NDV strains from avirulentNDV strains.Results showed that a specific band of 442 bp and a common band of 671 bp were amplified from the virulent NDV strains,while a specific band of 252 bp and a common band of 671 bp were amplified from the avirulent NDV strains,and as little as 100 pg of NDV RNA can be detected in a few hours,by the developed multiplex RT-PCR method.
The total of 212 swine sera from 6 districts in Jilin Province were examined for antibodies (against) swine hemagglutinating encephalomyelitis virus(HEV) by hemagglutination inhibition test. The antibodies against HEV were prevalent at positive rates of 44.3%. No clinical sign of HEV infection was noticed in any districts. The results showed that pigs had been infected recessively by HEV in these districts.
Ractopamine HCl was activated by glutarate anhydride and was converted into ractopamine-hemiglutarate.Ractopamine-hemiglutarate was linked to carrier protein keyhole limpet hemocyanin(KLH) and bovine serum albumin(BSA) with the mixed anhydride method respectively.The KLH conjugate was used as an immunogen and the BSA conjugate was used as a coating antigen for the development of an ELISA technique.UV scanning spectrum and NMR spectrum proved that the hapten ractopamine was coupled successfully to the carrier protein.The conjugation ratio of ractopamine to KLH was 24∶1 and ractopamine to BSA was 2.5∶1 by UV spectro photometry.The KLH conjugate was used to immunize New Zealand rabbits and polyclonal antibodies against ractopamine were obtained. Titers of the antibodies were over 1∶6000 by the indirect ELISA and IC_(50)was 10ng/mL by the competitive indirect ELISA.Results showed that the artificially-synthesized antigen had immunogenicity.
The maternal antibodies against AEV of chickens were detected dynamically with agar-gel-precipition (AGP) test. The AGP-~positive sate of the progeny chickens from breeder flocks with AGP-~positive 100%-86.7% was 100%-83.3% at one day of age, and decreased to 70%-30% at 14 days of age, most samples converted serological negative at 21 days of age. However, AGP-positive percentage of the progeny chickens from breeder flocks with AGP-positive 76.7%-56.7% was 73.3%-50.0% at one day of age, and decreased to 50%-10% at 7 days of age, most samples converted negative at 14 days of age. The results showed that there was a relationship between the maternal antibody level in the progeny ~chickens and the antibody level of breeder flocks. AE occurred after AEV subcutaneous challenge in some of ~chickens with 50% positive maternal antibody at one-day-age.
A multiplex polymerase chain reaction was developed and optimized to detect simultaneously porcine pseudorabies virus (PRV) and differentiate natural PRV from vaccinal TK~(-)/gI~(-)/gE~(-)PRV. Three pairs of specific primers were designed according tothe conservative sequences of PRV gB,gD and gE genes in GenBank. All samples containing PRV and recombinant PRV were amplified desirably by the multiplex PCR using the three sets of primers and then the multiplex PCR conditions were optimized. Three expected specific bands of PRV gB gene 549bp, gD gene 429bp and gE gene 366bp in length with the templates as little as 756pg of PRV or 106pg of vaccinal TK~(-)/gI~(-)/gE~(-) PRV were obtained by the multiplex PCR.The specific experiment showed that the multiplex PCR is qualified specifically to detect natural PRV and vaccinal TK~(-)/gI~(-)/gE~(-)recombinant PRV.
The specific chicken-egg-yolk antibody(IgY) which was developed from laying hens immuned with K88 and F18 fimbriae,and the mechanism of the antibody against post-weaing Escherichia coli diarrhea(PWECD) were expounded in the light of specificity of the causal agent leading to PWECD with a high rate of incidence.With being characterized by safety,high efficiency,low production cost,high yield,good stability,free antibiotic residues and no induction of antibiotic resistance,IgY was considered as a kind of green bio-preparation and would be applied widely in the future.
Several H10 subtype avian influenza A viruses were isolated from aquatic birds in East China and identified by the specific RT-PCR. The hemagglutinin (HA) gene of one representative virus strain Dk/YZ/502/03 out of the viruses was sequenced and pathogenicity of the strain to SPF chickens and BALB/c mice was determined. The results were as follows. The HA gene's nucleotide sequence of the strain Dk/YZ/502/03 shared high identity with that of A/Mink/Sweden/84(H10N4), and the (sequence) (P-E)-I-M-Q-G-R of cleavage site of the anino acid deduced from the gene was the characteristic sequence of avian influenza viruses with low pathogenicity, which agreed with the low pathogenicity of the strain Dk/YZ/502/03 to SPF chickens and BALB/c mice.
A pair of primers for amplification of σC gene was designed and synthesized according to the published Muscovy duck reovirus (mDRV) S4 gene sequence. The specific RT-PCR product was successfully obtained and cloned from the Muscovy duck reovirus ZJ99 strain. The results showed that the σC gene of mDRV ZJ99 had 99.5% homology with that of mDRV MW9710 isolated in Fujian Province of China, 94.2% and 95.0% homology with that of mDRV 89026 and that of mDRV 89330 reported in France respectively. The homology of the deduced amino acid sequences were 98.9%, 94.1% and ~93.7% , correspondingly. The predicted physicaland chemical characters andstructures of the σC protein of mDRV ZJ99 were similar to that reported by Kuntz-Simon from France.
The purified GST-NcSAG1t was used in an enzyme-linked immunosorbent assay for detection of Neospora caninum antibodies in dairy cows in Geermu County of Qinghai Province.Field serum samples collected from dairy cows in the prefecture were examined for the diagnosis of neosporosis by using the ELISA.Result which 4(11.42%) samples of the detected 35 samples were positive for antibodies to(N.caninum) showed that dairy cows in the prefecture were infected with N.caninum.
A pair of primers was designed and synthesized according to the minor subunit FedE gene of fimbriae F18, and the subgenic fragment of fedE was amplificated from the recombined plasmid TF107E. with appropriate restriction sites. The plasmid vector pGEX-6p-l was used for the expression of the fedE. The 5' terminus of the gene that code for fedE was genetically fused to the 3' terminus of the gene coding for the enzyme glutathione S-transferase , which serves as a carrier in this expression system. The resulting plasmid contained the fedE gene was designed as pPFedE. The pPFedE was transferred into E. coli BL21 and confirmed that it was able to express large quantities of a 43. 632 ku fusion protein (GST-FedE) by SDS-PAGE analysis and Western-blotting.
The recombinant plasmid pAdlox VP2 was obtained after the VP2 gene of infectious bursal disease virus (IBDV) cloned by RT-PCR was sub-cloned into the transfer vector pAdlox. The pAdlox VP2 was transfered into Cre8 cells together with the wild-type adenovirus ψ5 DNA to express the VP2 gene of IBDV. The result demonstrated that the recombinant adenovirus containing the IBDV VP2 gene was (constructed) successfully, and a 37 ku protein was expressed successfuly in the Cre8 cells, which was (confirmed) by Western-blotting method.
The infectious bronchitis virus (IBV) HN strain was successfully isolated from a chicken flock with typical clinical symptom of infectious bronchitis in Henan Province of China. The strain was passaged in SPF chicken embryos, characterized by haemagglutination test and observed by electron microscope. The cDNA fragment including nucleocapsid protein gene, 5a and 5b protein genes of the strain, which is about 1600bp in length, were amplified according to designed specific primers by RT-PCR from viral RNAs. Compared the 5a, 5b genes and N gene respectively with that of the other 11 IBV strains from GenBank on genetic variation, homology of nucleotide and deduced amino acid sequences the IBV HN strain differed from the other strains from GenBank obviously.
阐述了植物雌激素的分类、作用机理及其对动物及人类生殖发育毒性的研究进展,旨在通过探讨植物雌激素对动物体的拟激素作用,证实其对人类生殖健康的影响.
To construct the DNA vaccine against S-1 genefragments containing-CpG motif of SARS coronavirus and investigate immune responses in BALB/c mice, S-1 fragmentsof SARS-CoV S gene, containing different number of CpG motifs introduced by PCR, were inserted into eukaryotic expression vector pVAX1. The recombinant plasmid was injected into quadriceps femoris of BALB/c mice via electroporation and boosting with the same plasmid on week 2 after first immunization. IgG1 and IgG2a were measured weekly. The data indicated that the plasmid can elicit immune system to produce high levels of bothIgG1 and IgG2a significantly (P0.001). The increased ratios of IgG2a to IgG1 indicated that they can elicitan intense Th1 immune response.The constructed eukaryoticexpression plasmid can induceintense immune response, which suggested that DNA vaccination may open up a new avenue for prevention against SARS.
A field strain of infectious bronchitis virus(IBV) was isolated from chicken kidney, trachea and lung of the young layer flocks in Yulin Prefecture of Shaanxi Province. Biological characterizations of the virus were investigated by virus pathogenicity to chicken embryo and healthy chickens, hemagglutination test, hemagglutination inhibition test, dwarfing embryonated chicken eggs, electron microscope and RT-PCR. It was demonstrated that allantoic fluid of each passage virus could hemagglutinate chicken red blood cells by treatment with 10g/L of trypsin. The standard positive serum to IBV could inhibited the hemagglutination. The isolate could make respiratory and kidney diseases in chickens. Gross lesions were observed in chicken embryo after passaging for four times. The spherical virons covered with spike like corona about 100nm in diameter were observed under electron microscope. The RNA was extracted and used to amplify 5′-end of S1 gene of IBV by RT-PCR . The 373bp of fragment was obtained. The nucleotide sequence homologous with that of IBV CQ/01/2004(AY769993) was above 98%. The results showed the isolate is IBV.
In order to analyze sequence similarities of hemagglutinin(HA) gene among avian influenza virus(AIV) subtype H7N2 isolates,1.7kb DNA fragments were amplified with primers specific to HA gene by RT-PCR,and the DNA fragments were sequenced subsequently and compared with GenBank data.A 1664bp nucleotides sequence of HA gene was obtained,which encodes all the 544 amino acids(no signal peptides) of HA protein,including 323 amino acids for HA1 and 221 amino acids for HA2.HA gene of the two isolates shared 99.4% nucleotide sequence identity,and 99.0%-99.4% identity to A/Afri.Star./ Eng-Q/983/79(H7N1) virus,65.0% to A/Chicken/NewYork/13142-5/94(H7N2),and 96.0%-97.0% to other H7N2 viruses isolated from Italy and Israel.Both of these two H7N2 isolates were(situated) in the Euro-Asia branch of the HA phylogenetic tree of H7 subtype.The single basic amino acid at the cleavage site of HA implies the low pathogenic characteristics of the virus isolates.
To study immune activities of nine Chinese herbal ingredients(CHIs),astragalus polysaccharide(APS),propolis polysaccharide(PPS),epimedium polysaccharide(EPS),Chinese angelica polysaccharide(CAPS),isatis root polysaccharide(IRPS),propolis flavone(PF),epimedium(flavone(EF)),ginsenoside(GS) and astragaloside(AS),effects of these CHIs on lymphocytes of mice in vitro were compared by the methods of MTT assay and sandwich ELISA.GS,PF,APS,EF and EPS stimulated lymphocytes significantly,which were induced by LPS and/or ConA,to proliferate and elevated significantly level of antibody IgG produced by splenocytes in vitro.CAPS had the same effects but failed to stimulated lymphocytes to proliferate independently.AS and IRPS stimulated significantly lymphocytes which were inducd by LPS to proliferate.The results suggested that these CHIs have different effects on the functions of lymphocytes of mice in vitro.
