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Home > Establishment of Stable Cell Lines Expressing Proteins of Hcv Genotype 3A Pakistani Isolate and Their Molecular Characterization

Establishment of Stable Cell Lines Expressing Proteins of Hcv Genotype 3A Pakistani Isolate and Their Molecular Characterization

Thesis Info

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Author

Butt, Sadia

Program

PhD

Institute

University of the Punjab

City

Lahore

Province

Punjab

Country

Pakistan

Thesis Completing Year

2013

Thesis Completion Status

Completed

Subject

Molecular Biology

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/9765/1/Sadia_Butt_Molecular_Biology_Univ_of_the%20Punjab_2013_HEC_Scholar_Received%20date%2028.01.2016.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726090219

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Hepatitis C virus (HCV) infection is the leading cause of chronic hepatitis which progresses to hepatocellular carcinoma (HCC) afflicting > 170 million people worldwide. HCV 3a is the most common genotype (about 70% of all genotypes) circulating in Pakistan. Expression of HCV individual gene of 3a would facilitate therapeutic and vaccines strategies against chronic HCV and liver Cirrhosis. The aim of the present study was the establishment of stable Huh-7 cell lines expressing structural and non structural proteins of HCV Genotype 3a Pakistani isolate obtained from chronic HCV patients. Blood samples were obtained from chronic HCV-3a positive patients. HCV individual genes were amplified using PCR with gene specific primers having restriction sites and kozak sequence. These gene amplicons were cloned in TA and mammalian expression vector PcDNA3.1+. Restriction digestion analysis and sequencing results confirmed the successful cloning of HCV genes in PcDNA3.1 vector. Huh-7 cell lines were transfected with these constructed plasmids having structural or non-structural HCV genes in confluent cells with lipofectamine. Positive clones were selected with G418 and then confirmed by genome PCR. Subsequently, transcription and expression of the integrated genes were demonstrated by RT-PCR, sequencing and Western blot analysis. We successfully cloned and expressed seven HCV-3a genes in PcDNA3.1 mammalian expression vector. Results of western blot and sequencing PCR confirmed the stable expression of these seven genes. Various cytokines like Tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF- β), leptin and Angiotensin were used as indicator of disease progression in huh-7 cells stably expressing HCV structural and nonstructural proteins in vitro. Collectively, these observations suggest a significant involvement of HCV huh-7/Core, huh-7/E2 and huh-7/NS5A in the up-regulation of gene expression proinflammatory cytokine TNF-α and profibrotic TGF-β. No significant change in the expression of Angiotensin-II in vitro was observed. Up-regulation of leptin was observed in huh-7/E2 cell line only. The stable cell-lines expressing HCV-3a individual genes would be a useful tool to investigate the role of various HCV proteins on HCV disease outcome and testing of new therapeutic strategies against HCV.
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سلطان کھاروی دیاںکافیاں

سلطان کھاروی دیاںکافیاں

کجھ و دواناں دے وچار نیں کہ کافی عربی زبان دا لفظ اے جیدے معنی نیں مکمل ، پورا یا کافی ہونا۔ کیوں جے قرآن مجید وچ ربی فرمان اے۔

’’ کفیٰ باللہ شہیدا                               اللہ دی گواہی کافی اے ‘‘ (۱)

’’ کفیٰ باللہ وکیلا                              اللہ کارساز کافی اے ‘‘(۲)

’’قرآن حکیم وچ ایہہ لفظ کافی تے مکمل دے معنی وچ استعمال ہویا اے۔ ایس لئی شاعری دی اجہی صنف نوں کافی آکھیا گیا ہے۔ جہدے وچ اک مکمل مضمون اک مکمل خیال یاں اک مکمل جذبے دا اظہار کیتا گیا سی۔ شاعری دی ایس صنف نوں پڑھ کے یاںسن کے ہور کسے صنف یاں شاعری دے مطالعے یاں سنن دی لوڑ نہیں رہندی ۔ کیوں جے ایہدے راہیں بیان ہون والا مضمون یاں خیال ، ادبی ذوق ، روحانی لوڑاں تھوڑاں تے وجدان دی مکمل حد تک تسکین کر دیندا اے۔ ایسے پاروں شاعری دی ایس صنف نوں کافی آکھیا گیا اے۔‘‘(۳)

کافی تے سنگیت دا آپسی گوہڑا سمبندھ ہے ۔ کافی وچ سنگیت پکھ اگھڑواں ہوندا اے۔ ایس لئی سنگیت توں بنا ں کافی مکمل نہیں ۔جے ایہہ آکھیا جاوے کہ شاہ حسین نے کافی تے سنگیت نوں لازم و ملزوم بنا دتا اے تاں کجھغلط  وی نہ ہووے گا۔ اونہاں اپنیاں کافیاں بارے واضح شبداں وچ لکھ دتا، پئی ایہہ کافی فلاں راگ یاں راگنی وچ گائی جاوے مثال دے طور تے اوہناں نے اپنی ایس کافی نوں راگ جے جے ونتی وچ گاون دی ہدایت کیتی اے۔

 

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Sufism and Personality Development

Personality development is the process of improvement in behaviors and attitudes to make an individual a unique person. Sufism brings about positive changes in the behaviors of individuals. The positive changes are brought through moral development within   inductive environment griped by building of character. Moral development guides through “Zuhud” and “Taqwa” for minimization of material luxuries, enhancement of repentance and self-purification. The purpose of moral development is achieved with an institution articulated with teacher-student (Murshid-Murid) relationships; this environment ensures the provision of guidance and scholarships for the engaged students. The engagement of students revolves around knowledge, awareness and behavior. In the dimension of knowledge students are helped with raising moral and spiritual awarenessto enhance firmness in faith and encouragement towards good deeds. In addition, behavioral (amal) dimension is covered with remembrance of Allah, optional prayers, fasting services, alms giving along with obligatory ones. All these behavioral deeds are performed with devotion for the sake of closeness “Taqarrub” to Allah. Finally, ma’rifat (Recognition) is applied to secure soundness in building of character.

Production of Bacterial Pectinase Using Submerged Culture for Various Applications

Pectinases are pectin degrading enzymes and are naturally produced by plants, animals and microorganisms. Their major source of production at industrial scale is from microorganisms especially Bacillus sp, Aspergillus sp. and yeast sp. which are generally regarded as safe. Aspergillus sp. generally produce acidic pectinases which are used in the food and beverage industry for the extraction and clarification of fruit juices and maceration of vegetables for production of purees and pastes. Bacillus sp. are usually capable of producing alkaline pectinases which have diverse functions and are in use in many industrial processes, successfully substituting the use of harsh chemicals which not only causes the deterioration of product quality but also the deterioration of environment. Pectinases from Bacillus sp. are generally active at broad ranges of pH and temperature and due to this reason, they are multi-functional enzymes. Pectinases account for more than 10% of the industrial enzymes market and they constitute 25% of the global food enzymes market. The present study was concerned with the search for a novel bacterial isolate for the lab scale production of pectinase (Polygalacturonase). Keeping in view the increasing demand of pectinase, specially its need in Faisalabad, a textile city of Pakistan, isolation of new hyper producer bacterial strains locally is an easy and cheap way of getting the desirable products at low cost. Therefore, isolation of new strains for industrial enzyme production has been, and will be, a part of research. This method alone can also provide raw material for further research such as enzyme engineering or molecular directed evolution. Pectinase positive cultures were isolated using PSAM, the medium that is able to grow and differentiate pectin consuming bacteria from others. The pectinase producing bacteria form clear halos around their colonies while others do not form any clear zones. For the identification of hyper producer strains, colony PCR was done for 16S rRNA analysis. The reason to use the 16S rRNA gene for identification purposes is that there is a large database of DNA sequences available for the gene from the widest range of microbial species as compared with any other genetic target. The selected bacterial isolate NS1 (source of pectinase enzyme) was identified based on PCR amplification of 16S rRNA and for this purpose the amplified product was electrophoresed in agarose gel against a known species of Bacillus licheniformis. The 16S rRNA sequencing confirmed the Bacillus status of the strain NS1 and the nucleotide sequence BLAST results showed 98% similarity of strain NS1 having Accession No. KX765286 with few species of Bacillus licheniformis. The growth conditions of the newly isolated Bacillus licheniformis strain were investigated using submerged fermentation to understand the fermentation behaviour of the microorganism and the pattern of pectinase production by it. The growth of the organism and enzyme production by it was investigated using some local agrowastes such as wheat bran, gram bran, citrus peel, apple pomace, carrot pomace and peanut shells as carbon sources. Among these agrowastes citrus peel powder at 2.5% concentration proved as best substrate for pectinase production followed by wheat bran. Among various nitrogen sources investigated for their role in pectinase production, organic sources such as tryptone and yeast extract gave better results than inorganic nitrogen sources. Among inorganic sources Diammonium hydrogen phosphate gave more pectinase units than other inorganic nitrogen sources. Physical parameters like pH, temperature, inocula size and incubation period for high yield of pectinase in submerged fermentation were optimized by using Response Surface Methodology. Which is an efficient tool for increasing product yield many folds in short time due to limited number of experiments and lab work. In the present study, the yield of pectinase was increased 5.6 fold that optimization produced 219U/mL as compared to one variable at a time method which produced only 38.86U/mL. Several purification methods were evaluated to observe that which one is more advantageous and cost effective in the present study for pectinase purification. Three methods of protein purification (aqueous two phase purification system, macro-affinity ligand facilitated three phase partitioning and gel filtration chromatography) were used in the present study and macro-affinity ligand facilitated three phase partitioning were found to give high purification of pectinase with purification fold of 13.05. The pectinase from newly isolated Bacillus licheniformis showed some novelty in characteristics as compared to most of the pectinases produced by other species. Although its optimum activity was achieved at a temperature of 70ºC in glycine buffer pH 8 but it also showed considerable activity (26.75U/mL) even at 100ºC in phosphate buffer pH 7. Addition of 15mM CaCO3 to the enzyme assay mixture increased the pectinase activity by 3.1 fold and addition of chloroform to enzyme assay mixture increased the pectinase activity by 7.45 fold. Surfactants (CTAB, SDS and Triton X-100) increased the pectinase activity many fold as compared to control. Among various sugars investigated for their effect on pectinase activity, sorbitol was found as a stimulator of pectinase activity by increasing its activity by 1.8 fold while glucose, lactose and sucrose inhibited its activity. The pectinase produced in this study was investigated for applications such as oil extraction from sunflower seeds, apple juice extraction and clarification and starch extraction from potatoes. In all of the above applications, the locally produced pectinase enhanced the yield of apple juice, oil and starch several fold as compared to control without the application of pectinase enzymes. Apple juice yield was increased by 2.06 fold due to pectinase treatment while the juice clarification was increased by 1.62 fold. Pectinase application also increased the oil yield some 3.15 fold as compared to oil extraction by water without the addition of pectinase. Its effect on potato starch extraction was also appreciable and 3.95 fold increase in starch yield was observed due to pectinase treatment of potato slurry. All these investigations showed that the low cost pectinase produced by locally isolated microorganisms using low cost agrowastes as nutrient supplements are able to compete with costly commercial enzymes and can bring a revolution in product quality and yield if used by local industries.