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ThesisItem Open Access CLONING, EXPRESSION AND CHARACTERIZATION OF PARAFLAGELLAR ROD GENE OF TRYPANOSOMA EVANSI(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-01) SIVAJOTHI, S; CHENGALVA RAYULU, V(MAJOR); MALA KONDAIAH, P; SREENIVASULU, D; SRILATHA, ChABSTRACT : Present study was undertaken with an objective to isolate, clone, express and characterize the paraflagellar rod gene of Trypanosoma evansi. Local isolate of Trypanosoma evansi collected from naturally infected cow was multiplied in Wistar rats. Total RNA was extracted from DEAE-cellulose column chromatography purified trypanosomes by using Trizol LS reagent. Complementary DNA (cDNA) was synthesized from the RNA of host cell free T. evansi parasites by reverse transcription using oligo dT primers. RT-PCR was standardized to amplify the cDNA by targeting 1800 bp unique for PFR 2 gene of T. evansi. Amplification of cDNA was confirmed on agarose gel electrophoresis. The concentration of PCR amplicon was found to be 40 ng/μl after extracting from the gel. The gel purified PCR product (PFR 2 gene of T. evansi) was cloned into pTZ57R/T vector system. Transformation of competent Escherichia coli DH5α cells with ligated PFR 2 gene T. evansi was successfully carried out in LB agar with X-Gal and IPTG. Developed recombinants were observed as white colonies and non-recombinants as blue colonies. Presence of inserts was confirmed initially by Colony-PCR and then by Plasmid-PCR. Nucleotide sequence of the PFR 2 gene of T. evansi S.V.V.U. isolate (Accession No. KT277497) of the present study revealed 100 % homology with T. evansi China isolate and 99% homology with T. evansi Izatnagar and Bikaner isolates. Variation in nucleotide mutations at 4 positions with T. evansi Izatnagar and 3 positions with T. evansi Bikaner isolates were observed. The amino acid mutations in the PFR 2 gene of T. evansi S.V.V.U. isolate displayed regularity at 4 positions when compared to T. evansi China, Izatnagar and Bikaner isolates. Tree topology based on the Neighbor-joining (NJ) method of phylogenetic analysis has showed a close homology with other Trypanosomatidae species sequences with 100% bootstrap values. Restriction digestion of insert DNA of PFR 2 gene as well as pET 32a vector was carried out with EcoR I and Hind III enzymes and subjected for ligation by using T4 DNA ligase. The recombinant protein was sub-cloned into pET 32a and expressed in the BL21 (DE3) pLysS expression system. A high level of expression of recombinant protein of PFR 2 gene of T. evansi was noted following four hours of induction with 1 mM IPTG. Molecular weight of the Ni-NTA column chromatography purified recombinant protein of PFR 2 gene of T. evansi was found to be approximately 90 kDa after resolving by SDS-PAGE. PFR 2 gene of T. evansi S.V.V.U. isolate was further characterized by determination of its protein profile with SDS-PAGE analysis and western blotting against hyper immune serum. Indirect ELISA was optimized for detection of specific antibody titre against recombinant protein of PFR 2 gene of T. evansi. Based on the ELISA result, it is evident that PFR 2 gene products are eliciting very good immune response. However, further study is required to know the protective effect of the antibodies in laboratory animal models and to explore the PFR 2 gene of T. evansi as potential candidate for diagnostic and vaccine target against surra. Findings of the present study confirmed the existence of PFR 2 gene in Indian cattle isolate of T. evansi. Cloning, expression and characterization of PFR 2 gene of T. evansi of cattle isolate carried out in the present investigation is probably the first report in India.