I= CLONING OF SOMATOTROPIN FROM INDIGENOUS BUFFALO BREEDS, OVER EXPRESSION, PURIFICATION AND CHARACTERIZATION
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Title of Thesis
CLONING OF SOMATOTROPIN FROM INDIGENOUS BUFFALO BREEDS, OVER EXPRESSION, PURIFICATION AND CHARACTERIZATION

Author(s)
SUMBUL KHALID
Institute/University/Department Details
Department of Biochemistry/ Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi
Session
2008
Subject
Biochemistry
Number of Pages
139
Keywords (Extracted from title, table of contents and abstract of thesis)
somatotropin, buffalo breeds, milk yield, nili-ravi, growth hormone

Abstract
The economy of Pakistan is basically dependent on agriculture and livestock is the second important sector of agriculture. While the share of agriculture in GDP declined, the share of livestock in agriculture increased. Pakistan is rich in its livestock wealth and there is significant increase in milk production mostly due to increase in the number of buffalo, cattle and milk breeds of goats and sheep. But at the same time the import of milk and milk byproducts is also increasing to meet the demand. The reason is that milk is a very important food item in Pakistan like wheat and it provides two third of the daily calories to majority population. Therefore the question is how milk production can be increased to meet the increasing demand. In 1930s it was discovered that pituitary contains growth hormone (GH) or somatotropin (ST) which is responsible to enhance milk yield in lactating rats and goats and reduce carcass fat in growing rats. Breakthroughs in biotechnology in the early 1980s enabled ST to be produced by recombinant DNA technology. Since then ST gene has been cloned from a number of species in different vectors and are expressed under different conditions. It is generally accepted that genetically, Nili-ravi, an indigenous to Pakistan, buffalo breed has the maximum potential to yield more milk. Therefore a study was planned to develop an indigenous technology by exploiting the ST cDNA through recombinant technology and over express it to be used for enhancing the milk production in Nili-ravi. Therefore buffalo ST cDNA (buST) was cloned using RT - PCR in T/A cloning vector. The nucleotide sequence was determined and cDNA was sub cloned in pET Acceptor vector for expression studies. The best conditions for expression of ST were determined by using various concentrations of IPTG and different time intervals. The results of SDS- PAGE showed the expected size protein i.e. 22 kDa was expressed. The expression was further characterized by Western blotting. Finally protein was purified using immuno affinity column chromatography and electrophoresis on SDS-PAGE to check the homogeneity. The buST cDNA sequence was further subjected to various modeling softwares to obtain a validated model of protein. Structure prediction studies of bust revealed that the bust structure was very similar to the reported 3D structure of bovine ST (1bst. pdb) and showed no changes but buST show structural differences with human GH (1hGH.pdb).

Download Full Thesis
2096.15 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
176.67 KB
2 1 Introduction 1
95.57 KB
3 2 Review Of Literature 10
381.15 KB
  2.1 Livestock of Pakistan 10
  2.2 Buffalo 14
  2.3 Nili-Ravi 17
  2.4 Milk 19
  2.5 Growth Hormone 22
  2.6 Structure of Growth Hormone 26
  2.7 Bioinformatics 27
  2.8 Comparative Modeling and 3D Structure of buGH 32
4 3 Materials and Methods 35
224.44 KB
  3.1 Sample Collection 35
  3.2 RNA Isolation 35
  3.3 Primer Designing 36
  3.4 RT €“ PCR 37
  3.5 Nested PCR 38
  3.6 Agarose Gel Electrophoresis 39
  3.7 Purification of PCR Amplified Product 40
  3.8 Restriction Analysis of PCR Amplified Product 41
  3.9 Ligation 42
  3.10 Preparation of Competent Cells by Trituration Buffer 42
  3.11 Transformation 43
  3.12 Colony PCR to Select the Positive Clones 44
  3.13 Plasmid Extraction from Positive Clones 44
  3.14 Restriction Analysis to Confirm the Presence of Insert in Vector 45
  3.15 Restriction Analysis for Orientation Confirmation of the Insert 46
  3.16 Sequencing of the buST Gene 46
  3.17 Protein Expression Studies 47
  3.18 Antibody Production against ST 51
  3.19 Dot Blot 52
  3.20 Western Blot Analysis 52
  3.21 Immuno -Affinity Column Preparation 53
  3.22 Purification of buST 54
5 4 Results and Discussion 56
1173.16 KB
  4.1 RNA Isolation, RT-PCR and Nested PCR 56
  4.2 Gel Purification of PCR Amplified Product 56
  4.3 Restriction Analysis of PCR Amplified Purified Product 59
  4.4 Cloning in T/A Vector and Restriction Analysis 62
  4.5 Sequencing of buST Gene 62
  4.6 Nested PCR for Expression Vector 66
  4.7 Cloning in pET AccepTor TM Vector 68
  4.8 Expression Studies 72
  4.9 Production of Antisera in Rabbit 76
  4.10 Protein Purification Through Immuno -Affinity Column 81
  4.11 Modeling Studies 84
6 5 Summary 114
225.35 KB
  5.1 Literature Cited 118
  5.2 Appendices 130