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Korean J Parasitol > Volume 35(3):1997 > Article

Original Article
Korean J Parasitol. 1997 Sep;35(3):203-210. English.
Published online Sep 20, 1997.  http://dx.doi.org/10.3347/kjp.1997.35.3.203
Copyright © 1997 by The Korean Society for Parasitology
cDNAs encoding the antigenic proteins in pathogenic strain of Entamoeba histolytica
K I Im,*J T Choi,Y P Hong,T E Kim and M S La
Department of Parasitology and Institute of Tropical Medicine, Yonsei University College of Medicine, Seoul 120-752, Korea.
Received June 20, 1997; Accepted September 01, 1997.


The differential display reverse transcription polymerase chain reaction (DDRT-PCR) analysis was performed to identify the pathogenic strain specific amplicons. mRNAs were purified from the trophozoites of the pathogenic strain YS-27 and the non-pathogenic strain S 16, respectively. Three kinds of first stranded cDNAs were reverse transcribed from the mRNAs by one base anchored oligo-dT11M (M: A, C, or G) primers. Each cDNA template was used for DDRT-PCR analysis. A total of 144 pathogenic strain specific amplicons was observed in DDRT-PCR analysis using primer combinations of the 11 arbitrary primers and the 3 one base anchored oligo-dT11M primers. Of these, 31 amplicons were verified as the amplicons amplified only from the mRNAs of the pathogenic strain by DNA slot blot hybridization. Further characterization of the 31 pathogenic strain specific amplicons by DNA slot blot hybridization analysis using biotin labeled probes of the PCR amplified DNA of cysteine proteinase genes revealed that 21 of them were amplified from the mRNAs of the cysteine proteinase genes. Four randomly selected amplicons out of the rest 10 amplicons were used for screening of cDNA library followed by immunoscreening and all of them were turned out to be amplified from the mRNA.


Fig. 1
An example of differential display using one base anchored oligo-dT primers and one arbitrary 10-mer primer (#228). P denotes pathogenic Entamoeba histolytica YS-27 and NP denotes non-pathogenic E. dispar S 16. Arrows denote the bands specific for pathogenic E. histolytica YS-27. S lane denotes standard size marker of 100 bp DNA ladder.

Fig. 2
Identification of DDRT-PCR amplicons of differentially expressed genes by DNA slot blot analysis using the pooled DDRT-PCR products as hybridization probes. Duplicated sets of the reamplified PCR products of the selected 31 YS-27 specific amplicons were immobilized onto nylon membranes. Each membrane was hybridized with the pooled DDRT-PCR products from Entamoeba histolytica YS-27 (left) and from E. dispar S 16 (right), respectively. Amplicons representing differentially expressed mRNA are indicated by arrow-heads.

Fig. 3
Identification of DDRT-PCR amplicons of Entamoeba histolytica cysteine proteinase genes by DNA slot blot analysis. PCR reamplified products of the selected 31 unique amplicons for the YS-27 were immobilized onto nylon membrane and hybridized with the PCR amplified sequences of the E. histolytica cysteine proteinase genes. Twenty-one amplicons representing differentially expressed cysteine proteinase mRNA are indicated by arrows.

Fig. 4
Result of the immunoscreening. Dots on each blot denote the positive clones detected by anti-Entamoeba histolytica human serum. (A) clone #232-G, (B)clone #250-G, (C)clone #282-G, (D)clone #282-C.


Table 1
Number of differential amplified fragment of the combination of 11 arbitrary 10-mer and one base anchored oligo-dT11 primers

Table 2
DDRT-PCR products hybridized only with the probe synthesized from the pooled DDRT-PCR products fo Entamoeba histolytica YS-27

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