ABSTRACT
A major issue in many gene expression studies utilizing small amount of biological materials is the limited quantity of RNApurified from clinical samples, which is often used for RT-PCR or standard Northern blot analysis. The SMART cDNA synthesis method and subsequent SMART-cDNA-PCR technique was used to analyse 3 genes in macroschizonts of Theileria annulata in small lymph node biopsy material. The SMART-cDNA of TaSp gene was cloned in pTZ57R/T-vector and sequenced. We focused on genes encoding surface proteins TaSp, TaD and HSP70. Our results showed that SMART cDNA dependably reproduces the expression profile found in messenger RNA. The RT-SMART-PCR showed the amplification of the processed mRNAs. The sequencing analysis showed that the amplified cDNA was coded for TaSp protein in Theileria annulata. It was concluded that the SMART PCR technique is practical for amplification of complete sequence of mRNAs in the form of cDNAs, and therefore for gene expression studies if only small amounts of starting material are available
ABSTRACT
Because of the strong immunologic responses of surface protein TaSp in Theileria annulata infected host, we tried to characterize this protein in a T. annulata isolate from Iran. The RNA prepared from T. annulata infected cells was used to produce SMART-DS-cDNA. The Double strand cDNA was then amplified with primers derived from TaSp mRNA sequences. The PCR product was cloned in pTZ57R/T vector, sequenced and registered under accession no. JQ003240 in GenBank. The sequence analysis showed 90%-94% nucleotide sequence identity and 68%-94% amino acid homology to the corresponding sequences of TaSp gene by T. annulata, T. sp. china I, T. sp. china and T. lestoquardi and three T. annulata reported from Iran respectively. Interestingly, the sequence analysis also showed small nucleotide sequence region near the 5` end in which the presented TaSp protein differed very strongly from the other known TaSp sequences. For the preparation of the recombinant protein, the cDNA was cloned in pQE-32 vector, the recombinant protein was prepared and assayed by Theileria infected bovine serum. The polymorphism in TaSp gene could be detected in intra- as well as inter species. The different characterized TaSp proteins had a common identic region, which may be helpful for development of broad band vaccine based on the recombinant proteins. The polymorphism in this gene, make this protein also interesting for the diagnostic purposes
Subject(s)
Cloning, Molecular , Protozoan Proteins , Theileria , Polymorphism, GeneticABSTRACT
We used the PCR technique based on the abovementioned primer pair and sequencing to demonstrate the Theileria infection in the sheep samples collected from Sultanate of Oman. According to the frame work of "integrated control of ticks and tick borne diseases in globalized world managed by EU-ICTTD-3 project, the samples from blood, liver, spleen, lymph node and lung were sent to the laboratory of Iranian Research Center for Ticks and Tick-borne Diseases [IRCTTD]. Samples from blood smear and impression smears from liver, spleen, lymph node, and lung were analyzed by Geimsa staining. The DNA was extracted from the abovementioned samples and analyzed by PCR technique using specific primers derived from the nucleotide sequences of 18S rRNA gene of T. lestoquardi, which can amplify the common region in other Theileria and Babesia spp. Subsequently the amplified DNA was sequenced. The analysis of blood smears of the sheep was negative for piroplasmosis performed through the Giemsa staining. The impression smears prepared from liver, spleen, lymph node, and lung showed suspicious structures mimicking Theileria schizonts in some cells. The results showed an expected PCR product of 428 bp in length, which is specific for Theileria spp. The PCR products were subsequently sequenced. The corresponding nucleotide sequence is registered under accession number JF309152 in GenBank. The sequence alignment in Gen Bank showed that the PCR products had 99% homology to the known T. lestoquardi registered under accession number AF081135 in the GenBank. Oman sheep are highly susceptible for Theileria infection and the infected sheep mostly die before the microschizonts or erythrocytic form of Theileria appears in the nucleated or erytrocytic cells respectively