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Home > Expression, Purification of Toxoplasma Rop 18 Recombinant Protein and its Antigenic and Immunogenic Trials in Mice

Expression, Purification of Toxoplasma Rop 18 Recombinant Protein and its Antigenic and Immunogenic Trials in Mice

Thesis Info

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Author

Habibun Nabi

Program

PhD

Institute

University of Veterinary and Animal Sciences

City

Lahore

Province

Punjab

Country

Pakistan

Thesis Completing Year

2016

Thesis Completion Status

Completed

Subject

Parasitology

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/10059/1/thesis%20Habibun%20Nabi%20-%2026-12-2016%20final%20PhD%20thesis.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726174480

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Toxoplasma gondii is an obligate intracellular, apicomplexan parasite that infects all warm-blooded vertebrates, including mammals and birds. Human beings can be infected by ingestion of oocysts from cat feces or through the consumption of meat containing Toxoplasma gondii cysts. There are potential vaccines candidates among which ROP18 has its major role in host gene expression along with the modulatory effect on key regulators of the host immune system. Therefore in this study, ROP18 sequence was amplified from local T. gondii strain, recombinant ROP18 was expressed through recombinant DNA technology and this recombinant protein was then tested for its antigenicity and immunogenicity in a mouse model. Approximately 200 fecal samples were collected from domestic, wild and stray cats in and around city of Lahore, Pakistan. Oocysts of T. gondii from cat feces were identified by using light microscopy and flotation technique. The oocysts were measured by micrometry having diameter of 8-10 μm. Out of 200 fecal samples, only three were suspected for T. gondii through direct microscopic examination and flotation technique. From 3 fecal samples, genomic DNA was extracted using a stool DNA extraction kit. After DNA extraction, the 3 samples were confirmed and characterized by PCR and nested PCR by using B1 gene and SAG2 primer sets. Reference DNAs (RH) of toxoplasma were kindly provided by Dr. Henrik Vedel Nielsen (Statens Serum Institut, Denmark) and Dr. Jorge Enrique Gomez Marin (COLOMBIA, South America). For detection of the B1 gene of T. gondii, the diagnostic method was optimized to amplify a 529 base pair (bp) repetitive sequence by PCR using DNA extracted from cat feces. Then a nested PCR was employed using internal primers to amplify a 102 bp from 391 bp product. The SAG2 gene was targeted at 5 different regions to amplify 5 amplicons. Genotype analysis was done using SAG2 sequence by Dr. Jorge Enrique Gomez Marin using 10 different markers. For amplification of ROP18, 54 sequences of the ROP18 gene retrieved from Genbank (National Center for Biotechnology Information (NCBI)) We used Geneious R8.1.6 software for sequence alignment and creating consensus sequence from all 54 ROP18 sequences. Primers were designed manually from the consensus sequence of ROP18. Primer pair namely ROP18-F 5‟ATCTAGAATGTTTTCGGTACAGCGG3‟ and ROP18-R Reverse 5‟TTCGAATTCTGTGTGGAGATGTTCC3‟ were designed to have restriction sites XbaI and HindIII respectively. The rop18 sequence was first cloned in pGMT easy vector system and then subcloned in pET28. BL21 competent cells were transformed with pET28-ROP18 and rROP18 was expression using IPTG for induction. The rROP18 was quantified through protein quantification kit (BCA). The rROP18 was formulated into nanospheres using PLGA as coating material. The Swiss-Webster mice were inoculated either intranasal or subcutaneous with rROP18 with or without montanide as adjuvant 3 times with 2 week interval. The blood was collected 2 weeks after each immunization. The control groups were inoculated with PLGA I/n or montanide s/c. For western blotting, ROP18 protein was electrophoresed on SDS-PAGE and blots were immune-blotted with the sera of immunized or infected mice. Bound antibodies were detected through Goat anti-mouse IgG–alkaline phosphatase conjugated. For evaluation of humoral response, ELISA plate was coated overnight at 4°C with rROP18 protein at 5μg/ml in 50mM sodium carbonate buffer (pH 9.6) @ 100 μl/ well. The absorbance of each sample was measured at OD 405 nm using ELISA (Bio-Tek, E-800, USA). Comparisons of quantitative values in the different groups were performed using ANOVA test, after checking the homogeneity of variances. Comparisons between groups for the antibody titre were performed by Dunn multiple range tests test. Comparisons were considered significant when a probability of equality was less than 5% (P<0.05). It was observed that rROP18 in nanospheres administered intranasal elicited elevated responses of specific intestinal IgA and IgG2a as compared to other groups inoculated intranasally rROP18 alone or injected subcutaneously rROP18 adjuvanted in montanide. It was concluded that nanospheres of ROP18 would be a non-invasive approach to develop vaccination against toxoplasmosis. Further experiments are needed to conclude the cellular response of these nanospheres in a chronic mouse model.
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قلم نے آپ ؐ کی توصیف جب لکھی آقاؐ


قلم نے آپؐ کی توصیف جب لکھی آقاؐ!
ورق ورق پہ اُتر آئی روشنی آقا!ؐ

مکانِ چشم کے ویران دو دریچوں کو
عطا ہو روضۂ انور کی دلکشی آقاؐ!

جہاں میں صرف مقدّر کا اِک سکندر ہے
غلام آپؐ کے قسمت کے سب دھنی آقاؐ!

حضوریوں کے سفر کی یہی تو منزل ہے
عطا ہو روضۂ انور پہ حاضری آقاؐ!

یہی ہے خواہشِ عرفاںؔ یہی ہے فخرِ سخن
ثنا میں آپؐ کی کٹ جائے زندگی آقاؐ!

Physical activity in prevention of cancer and better prognosis

Cancer is a multifactorial disease with genetic and environmental risk factors. Environmental factors may also be termed as modifiable risk factors and these contribute towards 35% of cancer related mortalities as reported by World Health Organization. Obesity is the leading risk factor in this regard, causing not only deaths due to cancer but also to many other diseases. Among different factors causing obesity, a major contributor is lack of physical activity. In this era of modern technology and digitalization, sedentary mode of life has become a part of life and is mostly unavoidable. At the same time, there is a rise in the incidence of cancer. In the olden times, people used to do all manual work, a lot of walk, exercise and had healthy life style. Such healthy life style may have prevented them from various diseases.             Physical activity as a therapy on daily basis, is associated with a reduction in incidence of various carcinomas. It may improve overall wellbeing of healthy people as well as diseases persons from various ailments. As it is a common proverb that, “prevention is better than cure”, physical activity serves as a preventive measure for various diseases and also for fitness of normal healthy people. Although it is a known fact, yet planned population studies are required to provide evidence. Instead of unorganized physical activities, a structured physical activity may help in improvement of condition of cancer patients, prevention of cancer, cancer related deaths as well we quality of life. Healthcare providers should guide the patients in this regard.             There is lack of awareness among physicians and mostly they don’t refer them to physical therapists. There is also lack of information regarding the implementation of the programs and regimens of physical exercises for different diseases and cancers. Physical therapists may guide the patients in terms of frequency, intensity, duration of exercises which may serve as a betterment of their condition.

Pharmacognosy of Skimmia Laureola Dc. Siebold. & Zucc. Ex Walp. and Zanthoxylum Armatum Dc. , Family Rutaceae

Present study is about Skimmia laureola and Zanthoxylum armatum belonging to Family Rutaceae, comprises phytosociology, ethnobotany, pharmacognostic study, physicochemical and pharmacological activities of these plants. Phytosociological attributes of S. laureola were studied in six different localities of khyber Pukhtunkhwa, Pakistan showing that this plant grows gregariously in laomy and clay loamy soil at high altitude ranging from 2400- 3400 meters toward North facing slopes. S. laureola was found dominent in five out of six localities in association with a total of 44 plants with a density of 312 to 4437.5 hectare-1. Various other ecological, ethnobotanical and commercial aspects of the plants are also worked out. Similar studies of Z. armatum showed that the plants grows in association with 51 species on the North and North West slopes in the foothills of khyber Pukhtunkhwa at an elevation of 850- 1600 meters with a density ranging from 560 to 1020 hectare-1. Pharmacognostic study included leaf and bark of S. laureola and leaf, bark and fruit of Z. armatum. Leaf of S. laureola is punctate with glabrous surfaces. Transverse section of the leaf through the midrib region showed usual bifacial structure with prominent oil cavities in the midrib regions. Other leaf features such as palisade ratio (7.8±0.21), vein islets number (15.4±0.63 per mm2), vein termination number (19.1±0.43 per mm2), stomatal number (196.1±3.07 per mm2) and stomatal index (12.96±0.14) were worked out. Eight different stomatal types were detected, in which actinostephanocytic was the most frequently occurring one . Stomatal cluster was also observed. Morphology and anatomy of Z. armatum was also carried out. Leaf of this plant lacks any type of trichome, where as in the midrib region, prominent oil cavities were observed. Palisade ratio (8.2±0.32), vein islets number (16.8±0.64 per mm2), vein termination number (11.3±0.47 per mm2), stomatal number (122.1±4.32 per mm2) and stomatal index (12.32± 0.26) were cworked out. Anatomy of Z. armatum fruit showed two portion i.e. fruit wall and seed, the later being non endospermic and contained small elongated embryo. Powder drug microscopy of the parts was carried out. Ash analysis for both plants were carried out. Results of qualitative and quantitative preliminary phytochemical screenings of selected parts of S. laureola and Z. armatum are given, showing the presence of carbohydrates, proteins, alkaloids, phytosterols, triterpenoids, phenols, flavonoids, tannins, anthocyanins, saponins, glycosides, fixed and volatile oils. Similarly results of extractive values and fluorescence analysis are also given. Elemental analysis showed presence of Zn, Mn and Cr in fairly good amounts and may contribute to hypoglycemic effect of these plants. Correlations among various elements were also determined. Proximate analysis of both plants showed carbohydrate, proteins, fibers, fats and moisture contents in fairly large amounts. Results of essential oils obtained from the leaves of S. laureola (SVO) and Z. armatum (ZVO) and fixed oils (ZHO and ZEO) from the fruit of Z. armatum, extracted with different solvents were evaluated for physicochemical characteristics including color, odor, % yield, density, optical activity, refractive index, specific gravity, carbon residue, absolute viscosity, kinematic viscosity, total acid number, iodine number and saponification value are presented. A total 31 different components in SVO and 34 in ZVO, 14 in ZHO and 14 in ZEO were identified through GC-MC analysis and their percent concentration is given. Toxicological studies showed that both plants are safe for human use. Antipyretic effect was found dose dependant. SLE showed maximum antipyretic action of 72.31%, ZLE showed 85.42 percent pyrexia inhibition of whereas ZFE showed a maximum antipyretic action of 83.84%. Hypoglycemic effect of SLE was found to be dose dependent and like the standard allopathic drug and it induced reduction of blood glucose level after 2 hour of dose administration. All the doses showed significant reduction (p < 0.05) in glucose level at 6th hour post administration, but 300 mg/Kg body weight dose showed maximum reduction of blood glucose level at 6 hour (80.54±0.04). Antispasmodic effects of ethanolic and n-hexane extracts of leaf (SLE, SLH), bark (SBE, SBH) and leaf essential oil (SVO) of S. laureola and ethanolic and n-hexane extracts of leaf (ZLE, ZLH), bark (ZBE, ZBH), fruit (ZBE, ZBH) and leaf essential oil of Z. armatum on the isolated rabbit jejunum for both spontaneous and KCl induced contractions showed that SVO and ZVO were the most efficient one, causing 100 % relaxation of the smooth muscles at very low concentrations, thus providing a scientific proof for its ethnopharmacological use as an antispasmodic drug. The two plants were also evaluated for cytotoxic, phytotoxic, antibacterial and anti fungal activities. SLE, SLH and SVO of S. laurola and ZBE, ZBH and ZVO of Z. armatum showed outstanding cytotoxic results with LD50 values of 5.34, 7.44, 11.01, 16.79 17.06 and 15.90 μg/ml. respectively. Lemna minor phytotoxicity asssay of SBE showed 100% inhibition at 1000 μg/ml followed by SBH, SLE and SLH with FI50 of 25, 1.38, 4.54 and 8.67 μg/ml respectively. ZBE, ZFE and ZLH also showed excellent inhibitions with FI50 values of 7.98, 9.24 and 19.13 μg/ml respectively. Antibacterial bioassays showed that all the samples were effective against various bacterial strains (Micrococcus leutus, Escherichia coli, Staphylococcus aureus, Pasteurella multocida, Pseudomonas aeruginosa, Bacillus subtilis, and Streptococcus viridanes). SVO and ZVO being the highly effective against all the test strains. Results of various extracts of both plants against various bacterial strains are presented in this dissertation. Dose dependent antifungal activities against test species (Trichophyton longifusis, Candida albicans, Fusarium solani, Microsporum canis, Aspergillus flavus and Candida glabrata) were found for all the samples but SVO and ZVO inhabited all the test strains and C. albicans, A. flauus, T. longifusis and F. solani being the most susceptible species. The present study has revealed the immense and diverse medicinal properties of S. laureola and Z. armatum, both can be exploited for therapeutic preparations on commercial scale.