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2020 - 20222
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Subject: Animal Biotechnology × End date: 2022 × Start date: 2020 × Type: Thesis × Thesis Type: M.V.Sc. ×
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    ThesisItemOpen Access
    DNA POLYMORPHISM IN MITOCHONDRIAL GENES ENCODING ND1, CO1 AND CYTB IN CANINE MALIGNANT TUMOURS
    (College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2020-01) REHMAN, SHAKEEL-UL; Borah, Probodh
    Malignant tumours in dogs are frequently reported. The types of malignancies commonly reported in canines include female breast cancers, lymphomas, adenomas and carcinomas of mast cells. Specific mutations and polymorphism in mitochondrial genes have been shown to be associated with different types of human malignancies. However, similar studies in respect to malignant tumours in dogs are very limited. Hence in the present study, an attempt was made to identify frequency of occurrence of mutation and polymorphism in gene sequences encoding NADH dehydrogenase subunit 1 (ND1), cytochrome b (CYTB) fragments of mtDNA and cytochrome c oxidase subunit 1 (CO1) in dogs, and to define the association of DNA polymorphic mutations with different tumour types. Based on histopathology, out of 10 tumours examined 5 (50%) were found to be of epithelial and the rest 5 (50%) of mesenchymal origin. Two of the five epithelial tumors were recognized as adenonocarcinoma and three as squamous cell carcinoma. Of the five mesenchymal tumors, four were identified as fibrosarcoma and one as liposarcoma. Of the 10 cases, 8 (80%) were recorded in local and 2 (20%) in crossbred dogs. While 7 (70%) cases were recorded in male, 3 (30%) were observed in females. Location-wise, two each of the tumours were observed in skin and mammary gland, while one each was observed in mouth, left flank, abdominal region, testicle, right elbow and left forelimb. The dogs suffering from the neoplastic growth in different parts of the body were within the range of 5 - 13 years of age. Analysis of three mtDNA gene fragments established a relatively low level of molecular genetic variation between the tissues (tumour tissue, normal tissue and blood) of the individuals examined. Majority of the mutational changes in the ND1, COI and CYTB gene fragments in the analyzed tissues in most of the dogs with tumours were insertions and deletions. Only a few polymorphisms were noted in the partial gene fragments of the analyzed tissues when compared to reference successions. Multiple substitutions and insertions have been noted in ND1 gene fragment; these included four substitutions (C218T, T455C, G498A and C666T) and three insertions (341InsC, 355InsC and 718InsT). However, no mutations were recorded in ND1 gene fragment from any of the three types of tissues examined in case of a dog affected with squamous cell carcinoma. Changes in CYTB gene fragment included two substitutions (C322T and T799C) and one insertion (303InsG) mutation. Polymorphism C322T in the CYTB fragment was noted in 40% of the samples analysed. No mutation was, however, detected in this gene fragment in one case of fibrosarcoma. In the COI gene fragment, A735G polymorphic mutation was recorded in all (100%) the 10 cases of malignant tumours investigated in the present study. In this gene fragment, instances of mutations recorded were comparatively lesser. Except for C218T mutation observed in ND1 gene fragment seven cases of canine malignant tumour that induced S (Serine) to Y (Tyrosine) variation in the amino acid sequence of the coded protein at position 72, no other substitution mutation recorded in this gene fragment could cause a variation at the level of amino acid sequence. On the other hand, none of the mutations detected in CYTB gene fragment could induce any change in the level of amino acid sequence of the coded protein. Similarly, the only substitution mutation in the CO1 gene fragment that induced a change at the amino acid level was A813T mutation observed in a case of fibrosarcoma, which caused a G (Glycine) to A (Alanine) variation at 71 position. Results of the present study showed the effect of two alleles (ND1: 218, CO1: 813) on the amino acid sequence of the coded proteins which suggested consequently their potential role in carcinogenesis. However, the sample size in the present study was too small to infer conclusively about the association of the mutations and polymorphisms identified in the present study with specific malignant tumours in dog.
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    ThesisItemOpen Access
    EXPRESSION OF CAP PROTEIN OF PORCINE CIRCOVIRUS TYPE 2 (PCV2) AND EVALUATION OF ITS IMMUNOGENICITY IN MICE
    (College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2022-03) BORAH, DEBARUN; Hazarika, Girin
    The present study was conducted with an aim to express the Cap protein of Porcine Circovirus Type 2 (PCV2) in a prokaryotic host and to assess its immunogenicity in mice. The recombinant pET32a/CT-His plasmid containing a codon-optimised PCV2 cap gene was transformed into E. coli BL21(DE3) cells for expression of the Cap protein after 8 hours of IPTG induction at 37°C. Subsequent SDS-PAGE analysis revealed an intense band of 46 kDa. Following successful purification by affinity chromatography using Ni-NTA column under denaturing conditions, the recombinant protein was refolded by dialysis. In western blot analysis of the purified protein, a 46.0 kDa-sized intense band was detected in the PVDF membrane. In four groups of Swiss albino mice consisting of six mice in each group, the immunological response to the purified recombinant Cap protein was assessed and compared to that of a commercial vaccine. Group 1 served as the control group receiving PBS, while Group 2 received a commercial vaccine, Group 3 received the recombinant Cap protein, and Group 4 received the Cap protein combined with Freund's adjuvant. On the 14th day post-primary immunization, each group of mice received a booster dose of the corresponding inoculum. Serum was collected from all the experimental animals on 0, 7th, 14th, 21st, 28th, 35th, and 42nd days after immunization. As detected by indirect ELISA, the mean antibody level against PCV2 was significantly higher in Group 4 getting Cap protein with Freund's adjuvant, than in Group 2 receiving the commercial vaccine, and Group 3 receiving the Cap protein alone. The recombinant Cap protein was found to be immunogenic in mice and was capable of inducing a specific IgG response, as detected by indirect ELISA. Based on the results of the present study, it may be concluded that the recombinant Cap protein expressed in E. coli is a promising immunogen and may be further explored as a vaccine candidate against PCV2. Use of the recombinant protein as a potential diagnostic antigen may also be explored in future.