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