Background Echinococcosis, coenurosis and cysticercosis are debilitating diseases which prevail in China. Immunological diagnosis of metacestodosis is important in disease control. The 8-kDa glycoproteins from taeniid cestodes have successfully been used for diagnosis of human cysticercosis in immunological assays. The aim of the present study was to investigate genetic variations and phylogenetic relationships of the 8-kDa proteins for evaluating the possibility of utilizing these proteins as diagnostic antigens for other metacestode infections.Methods The genes and complementary DNAs (cDNAs) encoding the 8-kDa proteins from Echinococcus (E.) granulosus, Taenia (T.) multiceps and T. hydatigena were amplified using PCR method. Their amplicons were cloned into the vector pMD18 and the positive clones were sequenced. Sequence data were analyzed with the SeqMan program, and sequence homology searches were performed using the BLAST program. Alignments were conducted using the ClustalX program, and the phylogenetic analyses were performed with the Protein Sequences Program and the Puzzle Program using the Neighbor-joining method.Results Fifteen, 18 and 22 different genomic DNA sequences were identified as members of the 8-kDa protein gene family from E. granulosus, T. multiceps and T. hydatigena, respectively. Eight, four and six different cDNA clones respectively from E. granulosus, T. multiceps and T. hydatigena were characterized. Analysis of these sequences revealed 54 unique 8-kDa protein sequences. Phylogenetic trees demonstrated that the taeniid 8-kDa proteins are clustered into eight clades at least: Ts18, Ts14, TsRS1, TsRS2, T8kDa-1, T8kDa-2, T8kDa-3 and T8kDa-4.Conclusion We found that the gene family encoding for the taeniid 8-kDa antigens is comprised of many members with high diversity, which will provide molecular evidence for cross-reaction or specific reaction among metacestode infections and may contribute to the development of promising immunological methods for diagnosis of metacestodosis. Chin Med J 2011;124(18):2849-2856

【Objective】 This study was conducted to isolate and identify the defense-related proteins in pea albino seedlings in response to GSH treatment, and to clarify the disease-resistant mechanism of these proteins involved in pea disease response. 【Method】 The method of proteomic analysis was used in the study. Four kinds of defense-related proteins closely related to pea disease response were successfully isolated from pea albino seedlings treated with 50 mmol·L-1 reduced glutathione (GSH) after 48 h of treatment. After the identification with MALDI-TOF MS, a pathogenesis-related protein PR10 (PI49), two ABA responsive proteins ABR17 and ABR18, and a glycine-rich RNA binding protein (GRPs) were identified. The possible roles of these four proteins in the disease response of pea seedlings to GSH treatment were discussed. 【Result】 Results showed that ABR17 and PI49 emerged concomitantly, indicating a potential cooperative effects of these two proteins in pea disease response to enhanced disease-resistance of pea. 【Conclusion】 Under GSH treatment, PR and ABA responsive proteins were the main anti-fungal proteins at 48 h after treatment, indicating that these two proteins would play pivotal roles in pea disease response to pathogen attacks.

cDNA-AFLP is a new technique of fingerprinting of mRNA,and its high reproducibility,accuracy and reliability,so that it comprehensively analyse organism transcriptome,this technique is widely applied in gene expression research about analyzing differential expression,genetic marker analysis and identifying genes.With development and apply of this technique,many achievement were obtain in research.The paper summarizes the principle,technical features,prospect and appropriate present situation of the technique.

【Objective】To clone Cysticercus cellulosae AgB gene and express in vitro. 【Method】 The upper and downer fragments of AgB gene were amplified by RT-PCR, respectively. Then, the complete gene of AgB was cloned by splicing overlap extension PCR method using 35 nucleotides overlap between the upper and downer fragments and inserted into the vector pVAX1. This recombinant plasmid pVAX1/B was identified by digestion of endonuclease,PCR and sequencing.Then,it was transfected into BHK-21 cell line mediated by liposome 2000. Specific AgB protein expressed in BHK-21 cell line was detected by SDS-PAGE, Western-blotting and indirect immunofluorescence test. 【Result】 Electrophoresis analysis showed that the specific sequences were amplified and the sizes of products were accord with expectation, respectively. AgB was expressed in BHK-21 cells and was recognized by cysticercosis cellulosae positive serum. 【Conclusion】 Cysticercus cellulosae AgB gene has been cloned using splicing overlap extension PCR method, and expressed in BHK-21 cell line successfully.

This experiment examined the virulence of 6 Eimeria spp.Lanzhou strain by infecting Jiahehuang female broilers at various dosages.Nine trial groups,including negative control group(NC) and 8 challenge infection groups(1-8) were tested.The average relative growth ratio(RGR) of 8 groups was 100 %,77.23 %,77.02 %,76.42 %,72.86 %,63.91 %,67.19 % and 42.06 %,respectively.The RGR of NC group was significantlg higher than other groups(p0.01).The RGRs of the 1st and the 2nd group were significant higher than the 5th and the 6th groups(p0.01) and the 7th group(p0.01).The feed conversion ratio(FCR) in the 7th group was significantlg higher than other groups.The OPG of the 7th group(150.11×104) was significantly higher than the 1st(3.05×104),the 2nd(2.35×104),the 3rd(32.20×104),the 4th(62.23×104) and the 5th group(60.45×104)(p0.01) after challenge.The mortality rate of 9 groups was 0,5.0 %,5.0 %,15 %,25 %,40.0 %,45.0 %,95 % and 100 %,respectively.These results indicated that the Eimeria spp.strains isolated from Lanzhou are highly virulent.Chickens infected with 6×104 oocysts suffered significant suppression in growth and 50 % mortality.The best challenge dosage was 1×104-1×105(each Eimeria spp.).

RNA was extracted from muskmelon fruit(Cucumis melo L.cv.Yindi) by modified Trizol method.The purity,concentration and integrity of the extracted RNA were detected by the agarose gel electrophoresis,ultraviolet ray photometer and RT-PCR.The results showed that extraction of RNA reached optimum in 200 mg sample with 1ml Trizol.A260/A280 of RNA arranged 1.9 to 2.0 by chloroform extractive twice.The room temperature and low temperature sediment had no effects on RNA yield.The electrophoresis bands showed clearly after extracted RNA was reverse-transcripted and amplified gene β-actin fragment.It is suggested that RNA from muskmelon fruit could be extracted by this modified method.

Objective To prepare the recombinant TS-CC18 antigen and to identify its antigenicity.Methods TS-CC18 encoding gene was amplified by PCR from pGEM-TS-CC18 recombinant plasmid and subcloned into the expression vector pET 30a.Then it was used to transform Escherichia coli BL21(DE3)cells to produce TS-CC18 recombinant antigen(rTS-CC18).The His-tagged expressed protein was purified by Ni-FF affinity chromatography.Western blot and ELISA were used to identify the antigencity of the recombinant protein.Results Tricine-SDS-PAGE showed that the 16×103 recombinant protein was successfully expressed in E.coli after induction with 0.5 mmol/L IPTG for 6 h.Western blot analysis showed that swine positive serum against Taenia solium cysticerci reacted well with rTS-CC18 protein.ELISA demonstrated its relative sensitivity and specificity were 84% and 100%,respectively,compared with whole-cyst antigen.Conclusion rTS-CC18 antigen with high sensitivity and specificity was successfully prepared.It might be used as a potential diagnostic antigen for developing immunodiagnostic reagents of cysticercosis.

【Objective】 To investigate the recombinant CE18(Cystic Echinococcosis protein 18 kD) antigen for immunodiagnosis of metacestode diseases in sheep.【Method】 A total of 259 sera samples from sheep infected with larval stage Taenia multiceps(Tm,18),T.hydatigena(Th,15) or Echinococcus granulosus(Eg,19),and sheep from an epidemic area of echinococcosis(194) and normal sheep(control,13) were examined by enzyme-linked immunosorbent assay(ELISA) to detect the antibodies against the rCE18 protein.The significant difference method was used to evaluate the values of rCE18 protein as an antigen for diagnosis of cestode-larva diseases in sheep.Analysis of rCE18 protein from different species was performed using biological softwares.【Result】 Similarities of homologous CE18 protein among different taeniid genera were up to 99% at amino acid level.The sero-positive rate of rCE18-ELISA was 38.89% in Tm,73.33% in Th,100% in Eg,and 76.14% in the epidemic area of echinococcosis,respectively,whereas that of the healthy control was negative.【Conclusion】 CE18 protein may be a useful antigen for the immunodiagnosis of sheep infected with the larva of cestodes,especially for Eg and/or Th.

Based on the published nucleotide sequence of MIC5 gene of Eimeria tenella,a pair of primers was designed.The EnMIC5 gene was amplified by PCR from the genomic DNA of E.necatrix.The amplicons were cloned into pMD18-T simple.Sequence analysis showed that the PCR fragment of EnMIC-5 gene is 1 470 base pairs in length and encodes a partial protein of 490 amino acids.Then the gene was introduced into pET-28a(+) vector,and the recombinant plasmid pET-EnMIC-5 was expressed in the E.coli BL21.The results of SDS-PAGE revealed that the fusion protein with molecular weight about 57.5 ku was over-expressed after induction of IPTG.Western-blot results demonstrated that the expressed recombinant protein was reacted with sera of chickens against E.necatrix infection.A high titer of antibody was detected at two weeks after the injection of the recombinant protein in Kunming mice suggesting that it should have good immunogenicity,and make mice produce high level of specific antibody.

In this study,recombinant expression plasmid pVAX1/TSOL18 was constructed and then transfected into BHK-21 cell line with liposome 2000.The results of SDS-PAGE,Western blotting and fluorescence antibody assay showed that TSOL18 protein was successfully expressed in BHK-21 cell line and could be recognized by positive serum against cysticercosis.Immunization in mice indicated that the expression vectors could efficiently induce the specific cellular and humoral immunoresponse,which laid a solid foundation for the further development of new vaccines of cysticercosis.

To evaluate the mechanism of immune enhancement of β-lentinan(LTN) in chickens,both in vitro and in vivo trials were carried out to study the effect of LTN on NO productive capacity of the splenic and thymus lymphocytes and abdominal macrophage.One-hundred-twenty 21day-old chickens were randomly divided into 4 groups which were injected abdominally with 2 mL of 1 g/L LTN,10 g/L LTN,20 g/L sodium thioglycolate and normal saline solution,respectively.The NO production of the splenic and thymus lymphocytes and abdominal macrophage were measured on day 3 post-injection.And the macrophage activated by sodium thioglycolate were stimulated with LTN at level of 50 mg/L,100 mg/L,200 mg/L and 400 mg/L respectively and the NO production by macrophages were measured.In consequence,1 g/L and 10 g/L LTN stimulated the splenic and thymus lymphocytes and macrophages to produce NO.Different concentrations of LTN were able to stimulate macrophages to increase the production of NO.Moderate stimulation of immunocytes to produce NO may be one of the most important immunoactive mechanisms by LTN in chickens.