Objective To clone and express a membrane protein(Tetraspanin 2) gene of Schistosoma japonicum (SjTsp2). Methods A pair of primers was designed to amplify the SjTsp2 gene which was subcloned into prokaryotic plasmid pET28a(+). The recombinant plasmid was transformed into E. coli BL21(D3) and followed by expression of the protein induced by IPTG. The protein was purified by affinity chromatography and used to immunize BALB/c mice. Dilution of antibody against SjTsp2 was determined by ELISA. The protein was also identified by Western blotting. Results Big loop of SjTsp2-A, 228 bp, was amplified in vitro by PCR. Its deduced amino sequence shared 52% similarity with SmTsp2. The soluble recombinant SjTsp2-A was expressed in the experiment and high dilution antibody against the recombinant (1 ∶ 32 000 in maximum) was produced in immunized mice. SjTsp2-A reacted positively with sera of acute and chronic schistosomiasis patients but not with sera from healthy persons by Western blotting. Conclusion SjTsp2 has been expressed and shows certain antigenicity.

Objective To obtain genes encoding the novel molecules for diagnosis of schistosomiasis. Methods The cDNA library of Schistosoma japonicum (Sj) schistosomula of day 15 post-infection was screened with positive patients sera,and the inserts of positive clones were sequenced. Then the sequences were compared with all sequences in GenBank database by Internet. The positive clones were analyzed by the software in bioinformatics. Results After three rounds of screening,15 positive clones were obtained,and among them 11 clones had the meaningful sequences,in which 5 genes coding for Sj mitochondrion,1 gene coding for Sj myosin and the others were SjCHGC05315,SjCHGC01371,SjCHGC04782,SjCHGC05166,SjCHGC09769,respectively. Conclusion Immunological screening by using positive patients sera can be used to discover Sj antigen genes with a potential value for diagnosis.

Objective To obtain genes encoding the novel molecules for diagnosis of schistosomiasis.Methods Juvenile S.japonicum cDNA library was immunoscreened to obtain positive clones.By DNA sequencing and sequence analysis,the target gene was amplified by PCR and subcloned into prokaryotic plasmid pET28a.The recombinant plasmid was transformed into E.coli BL21 followed by expression of the protein induced by IPTG.The protein was identified by Western blotting.Results 34 positive clones were obtained,24 of which were chosen to be sequenced,13 of which were Sj22600 gene.The protein were recognized with sera of infected rabbits and patients with acute and chronic schistosomiasis by Western blotting.Conclusion The gene coding for Sj22600 membrane protein was screened with high frequency in the cDNA library.The E.coli BL21 transformed with the recombinant plasmid can express the fusion protein,which shows immunoactivity.

Tetraspanin 2-A(SjTsp2-A) gene was amplified by PCR.pcDNA3.1(+)/SjTsp2-A recombinant plasmids were constructed and transformed into E.coli DH5?.Twenty four BALB/c mice were randomly divided into pcDNA3.1(+) /SjTsp2-A group(A),pcDNA3.1(+)/SjGST group(B) and pcDNA3.1(+) group(C).Each mouse was injected through musculus quadriceps femoris by three times(two weeks interval) respectively with 100 ?g pcDNA3.1(+)/SjTsp2-A,pcDNA3.1(+)/SjGST,or pcDNA3.1(+).At two weeks after the final inoculation,mice were each challenged by 40?2 cercariae of S.japonicum.Forty-five days after infection,all mice were sacrificed,the number of worms collected and eggs in liver tisssue was counted.Anti-pcDNA3.1(+)/SjTsp2-A antibody was detected by ELISA and protein expression in quadriceps muscle by immunohistochemical staining.The worm reduction rate(44.4%) and egg reduction rate(28.4%) of group A was higher than those of group B and C(P

Objective To screen a new schistosome vaccine candidate. \ Methods\ Schistosoma japonicum adult cDNA library was screened using sera from immune rabbits vaccinated with irradiated cercariae and monoclonal antibodies against membrane antigen of S.japonicum schistosomula. Three different fragments of S.japonicum cDNA genes were cloned into pGEM-T vector. The sequences of the inserts were determined using an automatic DNA sequencer and were analysed using Blast program. One of the unknown genes (B8) was selected and its ORF sequence (291 bp) was subcloned into eukaryotic expression vector. The recombinant plasmids were identified by restrictive enzymes and PCR amplification. The positive recombinant plasmids (pBK/SjB8) were transformed into host bacteria XL1-blue, and were then induced by IPTG for expression. SDS-PAGE and Western blotting analysis of total cellular protein from the bacteria were performed to detect the gene products. Results The results demonstrated that ORF of SjB8 gene was subcloned into the plasmid pBK-CMV and could express as fusion protein in XL1-blue. The results of SDS-PAGE and Western-blot also showed that the molecular weight of the fusion protein with 3 kDa ?-galactosidase was approximately 13\^6 kDa and the actual molecular weights of the SjB8 was 10\^6 kDa. The expressed fusion product of pBK/Sj-B8 could be recognized by immune serum and McAb. Conclusion A new gene of S.japonicum vaccine candidate (SjB8) was cloned into eukaryotic expression vector pBK-CMV and could express 10\^6 kDa schistosome protein. The results provide foundation for further study of the protein for its posibility as candidate vaccine.

Objective To investigate the morphological features of reproductive system of adult Schistosoma japonicum under an optical microscope.Methods Adult schistosomes were obtained from infected mice with cercariae shedding from Oncomelania snails.The adult worms fixed with 10% formalin,dehydrated,imbedded in paraffin,cut at 3 ?m thick,stained by HE staining and then observed under an optical microscope.Results The reproductive organs of adult Schistosoma japonicum such as testicle,ovary,fallopian tube,vitellarium,yolk duct and hystera were displayed distinctly and typically.Conclusions The morphological features of reproductive system of adult Schistosoma japonicum are distinct and typical by using routine pathological techniques preparing and HE staining,which establishes a morphological foundation for the morphological teaching of schistosomes and reproductive biology research.

OBJECTIVETo prepare sodium alginate-nanohydroxyapatite composite material and to explore its feasibility as a bone repair material.

METHODSSodium alginate-nanohydroxyapatite composite material was prepared using chemical cross-linking and freeze-drying technology. The composite was characterized by X-ray diffraction (XRD) and scanning electron microscope (SEM) and its porosity was measured by liquid displacement method. The fifth passage of bone marrow stromal stem cells (BMSCs) were incubated on the composite material and then growth was observed by inverted microscope and SEM. BMSCs were cultured with liquid extracts of the material, methyl thiazolyl tetrazolium (MTT) assay was used to calculate the relative growth rate (RGR) on 1, 3, 5 d and to evaluate the cytotoxicity. Fresh dog blood was added into the liquid extracts to conduct hemolysis test, the spectrophotometer was used to determine the optical density (OD) and to calculate the hemolysis rate.

RESULTSSodium alginate-nanohydroxyapatite composite material displayed porosity, the porous pore rate was (88.6 +/- 4.5)%. BMSCs showed full stretching and vigorous growth under inverted microscope and SEM. BMSCs cultured with liquid extracts of the material had good activities. The toxicity of composite material was graded as 1. Hemolysis test results showed that the hemolysis rate of the composite material was 1.28%, thus meeting the requirement of medical biomaterials.

CONCLUSIONThe composite material fabricated in this study has high porosity and good biocompatibility.

Alginates ; Biocompatible Materials ; Cells, Cultured ; Glucuronic Acid ; Hexuronic Acids ; Humans ; Mesenchymal Stromal Cells ; Porosity ; Tissue Engineering ; Tissue Scaffolds

Objective To investigate physiological changes in peri extracorporeal circulation period of patients who underwent cardiac valve replacement surgeries with crystalloid solution mixed with colloidal solutions and pure crystal solution as extracorporeal circulation priming solution, and explore the clinical value and practicability of crystalloid solution as the sole extracorporeal circulation priming solution.Methods A retrospective analysis was performed in 130 patients who underwent cardiac valve replacement surgeries.Pure lactated Ringer's solution liquid and Lactated Ringer's solution mixed with Voluven as the extracorporeal circulation priming solution were used.We respectively compared hematocrit at different time points, postoperative blood routine, liver and kidney function, blood coagulation index, duration of intensive care and trachea cannula in two groups.Results There were no significant differences in ages, preoperative blood routine, kidney function, blood coagulation function, duration of operation, clamping time, bypass time, intensive care, postoperative blood routine, kidney function, blood coagulation function and hematocrit at different time points in two groups (P ＞0.05).However, the hospital day of group which used crystalloid solution as extracorporeal circulation priming solution was significant shorter compared to group which used lactated Ringer's solution mixed with Voluven (P ＜ 0.05).Alanine aminotransferase of group which used crystalloid solution as extracorporeal circulation priming solution was significant higher compared to group which used lactated Ringer's solution mixed with Voluven (P ＜0.01).Conclusions Crystalloid solution as extracorporeal circulation priming solution is safe and economy in cardiopulmonary bypass.Pure crystalloid solution as the sole extracorporeal circulation priming solution can be safely used on patients (New York Heart Association class Ⅱ-Ⅲ) who have normal liver and kidney function before the operation of adult heart valve replacement with cardiopulmonary bypass.

Objective To screen the mimic epitopes of Rh(D)blood group antigens and identify their immunity from phage display peptide library.Methods A twelve mer phage peptide library was biopanned with anti-Rh(D)monoclonal antibody immobilized on plastic surface.After three round panning,thirty-five clones were randomly selected and positive clones were identified by ELISA and cross-reaction,followed by antibody competition inhibition assay and DNA sequencing to obtain the mimic epitopes of Rh (D)blood type antigens.The target phage clones were characterized and the antigenicity was analyzed by Western blot.Results After the third round screening,phages were enriched,and eleven positive clones were obtained.According to sequencing and competition inhibition analysis,the same"-WP-Q-"structure existed in seven of the eleven clones,and they had more than 40%inhibition ratio.The other clones had no same characteristics with low inhibition ratio possibly due to non-specific binding.Western blot analysis indicated that these phage clones could be specifically recognized by the anti-Rh(D)serum and they shared the same antigenicity of Rh(D)protein.Conclusions Rh(D)mimotope of"-WP-Q-"structure is successfully obtained by phage peptide library screening with anti-Rh(D)monoelonal antibody.The results lay the foundation for further exploration of pathogenesis and vaccine development of Rh(D)hemolytic diseases of newborn.

Objective To obtain novel vaccine candidate antigens against Schistosoma japonicum. Methods S. japonicum schistosomula cDNA library was screened by using sera of Microtus fortis that was naturally resistant to schistosomiasis. The positive clones were transformated into Escherichia coli BM25.8, E. coli clones containing the plasmid cultured in LB, and then selected for plasmid extraction, the plasmid DNA was digested by EcoRⅠand Hind Ⅲ, and analysed by agarose gel electrophoresis. The positive clones were also sequenced and the data were analysed through the internet Nucleotide BLAST software of NCBI and Expert Protein Analysis system of GeneRunner and HNN. Results Twelve positive clones were obtained after repeatedly immunoscreening the library and their sizes ranged from 300 bp to 1100 bp. Two novel genes (named as Sj-sMf1 and Sj-sMf2) with complete ORF were obtained. The deduced protein of Sj-sMf1 consisted of 93 amino acids while Sj-sMf2 consisted of 61 amino acids. Sj-sMf1 protein predicted containing one cAMP phosphorylation site and Casein kinase C phosphorylation site, respectively. Sj-sMf1 protein predicted containing one Casein kinase C phosphorylation site and two Protein kinase C phosphorylation sites. Conclusion Two novel genes predictably encoding unknown proteins are obtained from immunoscreening of Schistosoma japonicum schistosomula cDNA library by M. fortis sera.