To clone and construct a prokaryotic expression system containing the galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium, the p30 gene was amplified from genomic DNA of Cryptosporidium parvum by PCR and cloned into vector pMD18-T directly. The positive clones were identified by EcoR I, Xho I digestion and sequenced. The gene structure and its possible function were analyzed and predicted by using related bioinformatics softwares. The P30 gene was recombined with plamid pET-28 a (+) to construct the prokaryotic expression vector and was expressed in E. Coli with IPTG induction. Ni-NTA affinity chromatography was used to extract P30 protein and the expression effect and purification of P30 protein were determined by SDS-PAGE and Western blotting. It was demonstrated that the galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium parvum was specifically amplified, and its sequence homology of nucleotide and the deduced amino acid sequence of P30 gene with relevant sequences in GenBank were 98%-100% and 99%-100% respectively. Its theoretical iso-electric point and molecular weight were found to be 6.4854 and 31842 dalton.It was predicted to contain 9 potential epitopes. The expressed plasmid was identified by EcoR I/ Xho I digestion and sequenced and the recombinant P30 protein could be identified by SDS-PAGE and Western blotting assay. It is evident that the prokaryotic expression system for galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium parvum has been constructed successfully.

Objective To clone and express prokaryotic recombinant plasmid of nucleoside triphosphate hydrolase (NTPase) gene of Toxoplasma gondii, and analyze its antigenicity. Method NTPase gene was amplified by PCR from RH strain of T. gondii and cloned into pGEM-T Easy vector. Positive clones were screened and identified by BglⅡ, HindⅢ digestion and sequenced. The target gene was then subcloned into prokaryotic expression vector pBAD-HisB and transformed into E. coli BL21(DE3). The expressed recombinant protein was purified with Ni-NTA agarose and further analyzed by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blotting. Results NTPase-Ⅱ gene was specifically amplified, and the homology of DNA sequence was 100% to that in the GenBank. SDS-PAGE showed that the recombinant NTPase protein with correct molecular weight was expressed highly in E.coli BL21(DE3). Western blotting testified that the purified recombinant protein could be specifically recognized by mouse serum immunized with T. gondii and mouse anti-recombinant protein serum. Conclusion The NTPase-Ⅱ gene has been cloned and expressed in E.coli BL21(DE3), and the purified protein of NTPase-Ⅱ gene displays a specific antigenicity.

Objective To develop and identify monoclonal antibodies (McAbs) against adult worm of Angiostrongylus cantonensis and observe its applicability. Methods BALB/c mice were immunized with soluble antigen of adult worms of A.cantonensis. The spleen cells of immunized mice were fused with myeloma cell, and the hybridoma secreting high titer of McAbs with high specificity was screened. By using the McAbs, serum of angiostrongyliasis patient and sera of the rats infected with A.cantonensis were detected by Western blotting and double antibody sandwich ELISA respectively. Results Three McAbs were established (2A2,3F1,4H2), which all showed no cross reaction with antigens of Schistosoma japonicum, Paragonimus westermani, Cysticercus cellulosae and Trichinella spiralis. Western blotting analysis demonstrated that the three McAbs recognized a Mr 15 000 soluble antigen of adult worm of A.cantonensis and recognized the Mr 24 000 and Mr 15 000 circulating antigens from the serum of angiostrongyliasis patient. The double antibody sandwich ELISA detection showed a positive rate of 76^5%. Conclusion Three hybridoma cell lines against adult worm of A.cantonensis have been established which secret high titer of McAbs with high specificity and seem promising in detecting the circulating antigen of the angiostrongyliasis patient.

Objective To prepare and characterize the monoclonal antibodies (McAbs) against recombinant adhesion protein 33 (AP33) of Trichomonas vaginalis. Methods The purified recombinant fusion protein AP33 was used as antigen to immunize BALB/c mice. Sp2/0 myeloma cells were fused with the splenocytes from immunized BALB/c mice. After ELISA screening and 4 times of limited dilution, 5 positive hybridoma cell lines were obtained, and the biological properties of the McAbs were identified by Western blotting. Indirect immunofluorescent antibody test (IFAT) was performed and the inhibition effect of McAbs on the cytoadherence of T.richomonas vaginalis to HeLa cell was assayed. Results Western blotting demonstrated that 5 McAbs, designated as 4A2, 4F11, 4F8, 4E7 and 4H11, specifically combined with the recombinant AP33 of T.vaginalis. The McAbs were IgG1 isotypes. Four of them (4F11, 4F8, 4E7 and 4H11) showed parasite recognition by IFAT. Parasite cytoadherence to a monolayer of HeLa cells was inhibited in vitro with a inhibition rate of 50.08%, 65.03%, 50.70% and 49.08% by the 4 McAbs under a concentration of 200, 200, 400 and 200?g/ml, respectively. Conclusions The prepared McAbs against the recombinant AP33 show a protective inhibition on cytoadherence of Trichomonas vaginalis in vitro.

Due to the effect of structural characteristics and service environment of esophageal stent, fatigue damage of esophageal stent is developed easily, which may lead to serious complications. At present, the researches on fatigue performance of esophageal stent involve load spectrum, stress-strain relationship, fatigue crack and fatigue life prediction, and there are three main research method: theoretical analysis, numerical simulation and experimental research. In this paper, various analysis methods and limitations for measuring fatigue performance of esophageal stent are elaborated and summarized in detail, and the future research of esophageal stent is prospected.

Objective To investigate the influence of different end shapes of esophageal stents on the mechanical behavior of the stent-esophagus system. Methods Through finite element simulation, the mechanical behavior of the coupling system between braided esophageal stents with different end shapes (straight-tube-shaped, cup-spherical-shaped, double-trumpet-shaped) and the esophageal cavity was analyzed. The influences of bare stents and covered stents with three different end shapes on stress distributions in the inner wall of the esophagus and on dilatation of the esophageal stenosis zone were compared. Results The reduction in stenosis rate caused by the bare stent was larger than that of the covered stent. Moreover, the equivalent stress and the contact stress caused by the bare stent were much larger than those of the covered stent. Different end shapes had a significant influence on the stress occurring in the healthy esophageal zone. Stress concentration occurred in the zones where the esophagus contacted the central part of the cup-shaped end and the edge of the double-trumpet-shaped end. The braided esophageal stents with three different end shapes all had good apposition.Conclusions Different end shapes of stents cause different stress states in the esophagus. A larger stress involves a higher probability of occurrence of esophageal tissue hyperplasia, but a smaller possibility of stent migration. Therefore, understanding the effect of the end shapes on stent performance can provide important theoretical references for optimization design of the braided stent and its clinical selection.