[Screening and analysis of peptides specifically binding to the schistosomulum tegument of Schistosoma japonicum].

OBJECTIVE: To screen and analyze the peptides in 12 phage-display peptide library specifically binding to the schistosomulum, not cercaria, tegument of Schistosoma japonicum. METHODS: A 12 phage-display peptide library was screened with the S. japonicum schistosomula and cercariae as the target cells for biopanning by degrees, 15 positive clones were picked randomly and deduced by DNA sequencing. According the sequencing result, ELISA test, elution recovery test and immunohistochemical staining were performed to determine the specificity of the phages to the tegument. To further examine its binding properties, the positive peptide conjugated to RhB and recombinant pEGFP-C2 plasmid were similarly synthesized. RESULTS: After 3 rounds of biopanning, the phage recovery rate increased from 3.50 x 10(-5)% to 3.20 x 10(-2)%, indicating that the phage library was successfully enriched in the tegument of schistosomula. The analyzed sequences were identical with 3 peptide sequence of ZL6, ZL4 and ZL1. ELISA showed that the P/N value of MppZL4, MppZL6 and MppZL binding the schistosomulum membrane protein was 6.72, 3.65 and 2.22, while 1.58, 5.15 and 1.20 of binding the membrane protein of cercariae, respectively. Elution recovery test showed that the elution recovery rate of MppZL4 [(4.60 +/- 0.27) x 10(-2)%] was much higher than that of MppZL6 [(2.10 +/- 0.23) x 10(-3)%], MppZL1 [(1.20 +/- 0.28) x 10(-3)%] and M13KE [(1.30 +/- 0.60) x 10(-7)%] (P<0.01). Immunohistochemical staining showed that MppZL4 specifically bound to the tegument of schistosomula with a positive rate of 83.0% (83/100). Fluorescent microscopy revealed that the synthesized RhB-ZL4 bound to the tegument of schistosomula. The ZL4/pEGFP-C2 plasmid was introduced into juvenile S. japonicum and expressed in the parasite. CONCLUSION: The peptide of ZL4 specifically binds to the schistosomulum tegument but not to that of cercaria.

[Preparation of agar-paraffin double-embedded longitudinal sections of Schistosoma japonicum].

Schistosoma japonicum adults are pre-embedded in a double-layer agar and made the block, then dehydrated with alcohol, isobutyl alcohol and n-butyl alcohol. Various staining procedures can be conducted after conventional sectioning and dewaxing. Complete longitudinal serial sections of the pre-embedded worms can be obtained, and the desired sections can be easily located accurately.

[An efficient and accurate method for counting target molecules in phage-display peptide library].

The phage titer of samples representing the low, intermediate and high phage number was respectively determined by the double-layer agar plate (DLAP) method and real-time PCR assay. The two methods accurately measured the titer of samples. The plaques from about 1/3 double-agar layer plates could be used to determine the phage titer. The DLAP experiment should repeat 10 times with 10 microl sample each time, while the within-assay coefficient variation (CV) was 4.93%-30.38%. At the same time, the real-time PCR assay only repeated 3 times with 1 microl phage each time, while CV for within-assay ranged from 0.02% to 0.25%. Results indicated that real-time PCR is a simple and quick method for determining bacteriophage titer.

[Detection of Toxoplasma gondii DNA by loop-mediated isothermal amplification].

OBJECTIVE: To detect Toxoplasma gondii DNA by loop-mediated isothermal amplification (LAMP). METHODS: DNA was extracted by phenol-chloroform extraction from T. gondii tachyzoites. Four primers which recognized 6 distinct regions on the B1 gene of T. gondii were designed and used for LAMP assay. To evaluate the specificity of the method, Plasmodium vivax, P. falciparum, Pneumocystis carinii, Schistosoma japonicum, and mouse leucocytes were used gs controls. The parasite extract (T. gondii) was 10-fold serially diluted for evaluating the sensitivity of LAMP, and was amplified by LAMP. LAMP results were read with naked eye and analyzed by electrophoresis. RESULTS: After LAMP reaction, positive amplification was observed with T. gondii, but no positive signal was noted for the negative controls in the study. The sensitivity of LAMP assay reached up to 2-3 T. gondii tachyzoites/ml per reaction. CONCLUSION: LAMP assay shows proper specificity and sensitivity for the detection of T. gondii.