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Hepatit B yüzey antijenleri polipeptitlerinin kimyasal yolla sentezi ve karakterizasyonu

Başlık çevirisi mevcut değil.

  1. Tez No: 75472
  2. Yazar: DİLGİMEN SALMAN
  3. Danışmanlar: PROF. DR. MUHAMMED MUSTAFAEV
  4. Tez Türü: Yüksek Lisans
  5. Konular: Kimya, Chemistry
  6. Anahtar Kelimeler: Belirtilmemiş.
  7. Yıl: 1998
  8. Dil: Türkçe
  9. Üniversite: İstanbul Teknik Üniversitesi
  10. Enstitü: Fen Bilimleri Enstitüsü
  11. Ana Bilim Dalı: Kimya Ana Bilim Dalı
  12. Bilim Dalı: Belirtilmemiş.
  13. Sayfa Sayısı: 130

Özet

ÖZET Bu çalışmada, Hepatit B virüsünün yüzey antijenleri, Merrifield Peptit sentezi yöntemi ile, Katı Faz Peptit Sentezi tekniği kullanılarak kimyasal yolla sentezlenmiş ve bu sentezlenen peptitlerin çeşitli fizikokimyasal yöntemlerle incelenmesi yapılmıştır. Hepetit B virüsü tüm dünya için ve de ülkemizde oldukça yaygın olan ve günümüzde korunması iyi yapılmazsa öldürücü olabilecek bir hastalıktır. Bu hastalığa karşı geliştirilebilinecek yeni tipli aşıların başarılı olması halinde bu çalışmaların diğer başka virütik hastalıklarada uygulanabileceği bilinmektedir. Bu amaç doğrultusunda diagnostik tankitlerinde ve yeni tipli aşı oluşumunda kullanılabilecek karakterde virüsün yüzey antijen epitoplan olan polipeptitleri seçilmiş ve bunlar kimyasal olarak, sentezlenmiştir. Katı Faz Peptit Sentezi sırasında Fmoc yöntemi ve kimyasalları kullanılmıştır. Bu yöntem, laboratuarımızda, sentez için kullandığımız peptit sentezleyicisinin içine, bilgisayar programına yüklenmiş olan bir program sayesinde otomatik olarak işlemiştir. Bu yolla sentezlenmiş ürünler daha sonra çeşitli spektroskopik yöntemlerle analiz edilmiştir. Bu analizler aynı zamanda ürünlerin karakterizasyonunu da sağlamıştır. Ele geçen sonuçlan değerlendirdiğimizde ise sentezin istenilen aminoasit dizilerinde elde edildiğini ve bu sentezlenen yapıların saflığının yeterli oranlarda olduğunu görürüz. Bu çalışmanın hepatit hastalığına karşı geliştirilmesi düşünülen aşı sistemleri için ve peptit sentezi üzerinde yapılan çalışmalar için olumlu bir adım olmasını dilerim. XI

Özet (Çeviri)

SUMMARY CHEMICAL SYNTHESIS OF HEPATISIS B SURFACE ANTIGENIC POLYPEPTIDES AND THEIR CHARACTERIZATION In this study, Hepatosis B surface antigenic polypeptides had been synthesized chemically and analized with physico-chemical concepts. Hepatisis B Virus is one of the most common problem and potential danger of all over the world and also in our country. This concept drive us to create new protection shemes against Hepatisis B Virus. Taking all these into account, as creating a new basis for the novel vaccines, in this study, I had synthesized 4 epitopes of antigenic parts of surface antigens of Hepatisis B Virus. Hepatisis B is a virus that couses the liver to be inflamed. The most people fight off the infection themselves. However, approximately 5-10% of those people who are infected with the virus will become carriers, an astimated 5-10% of those people infected each year will progress to chronic liver disease, cirrhosis and possible liver cancer. This disease is more infectious than AIDS and is transmitted through infected blood and other body fluids. However, in approximately 30-40% of cases the method of transmission is unknown. The Hepatisis Virus B (HBV) is a 42 nm double-shelled round particle. It is the prototype of a virus family (hepatisis DNA viruses or Hepadnaviridae) composed of two genera, mammalian and avian viruses, which infect man, non-human primates, woodchucks, ducks and grey herons. HBV-DNA contains 4 major open readingframes or genes on the same DNA strand, namely pol, s, pre c/c, andx. The s gene resides within the pol gene and is devided into its three fractions, pre- si, pre-s2 and s. The HBsAg is devided into three hydrophobic and two hydrophilic areas in the molecule. The largest and most hydrophobic region spans approximately positions 80-100. The domain is flanked by two hydrophilic domains encompassing positions 45-80 and 110-150. The other two hydrophobic domains are found at the NH2 and COOH termini. In our study we had synthesized the regions 2-16, 22-35, 95-109 and 115-129 of the s gene. The amino acid sequences are as fallows: xu1.2-16 AH2 Glu-Asn-De-Thr-Ser-Gly-Phe-Leu-Gly-Pro-Leu-Leu- Val-Leu-Gln-Cys 2. 22-35 AH3 Leu-Thr-Arg-De-Leu-Thr-Ile-Pro-Gln-Ser-Leu-Asp-Ser-Trp-Cys 3. 95-109 AH4 Leu-Val-Leu-Leu-Asp-Tyr-Gln-Gly-Met-Leu-Pro-Val-Cys-Pro-Leu 4. 115-129 AHİ Thr-Thr-Ser-Thr-Gly-Pro-Cys-Lys-Thr-Cys-Thr-ne-Pro-Ala-Gln The development of synthetic peptide vaccines started with the hope that it would be possible to tigger the immune system specifically with certain isolated determinants or epitopes. This idea lead us to produce the specified antigenic epitopes that would enable us to lead further studies. Primarily, synthetic peptides are used to identify key antigenic epitope regions of proteins. This identification is achieved by initiating the immune response against the native protein. One of the most commonly used techinque of synthesizing peptides is the Merrifield technique. This Merrifield technique contains different paths leading to the targetted product. In this study, Solid Phase Peptit Synthesis (SPPS) path had bee used. The two major chemical methods for solid phase peptide synthesis employ either t-BOC or FMOC protection of a- amino groups. This T-BOC and FMOC also reffers to the techniques names. In this study, FMOC technique is used involving stepwise syntheses whereby a single amino acid is added at each step starting from the C- terminus of the peptide, and both use very similar chemistries in the formation of peptide bonds. The reaction order that was fallowed during the synthesis is N- Deprotection, Neutralization, Coupling, N- Deprotection and Neutralization, Side Chain Deprotection and Cleavage as in order. These steps are all performed by the Millipore's peptide Synthesizer that is beeing used in our laboratory. During the synthesis, print outs of coupling persentages gives a proof that the coupling was performed. After all amino acids were coupled, the product was cleaveged from the polymer support with TFA coctails. These coctails are performed according to the amino acid residues and the protection shemes. In general TFA, 1,2- Etandithiol, Anisole, Water solvents are used in different ratios. The excess solvents then evaporated in low temperatures in order not to couse any bond defections. After evaporation the products were decanted and lyophlization was applied. The four synthesized antigenic epitopes were white in color and they were powder like substances. After the synthesis, the characterization and purification steps were performed. These steps include chramotografic, spectroscopic, fluorometric analysis, and also gel electrophoresis techniques. The most commonly used analytical techniques are HPLC systems, electrophoresis and some types of spectroscopy applications. Mass spectroscopy is also a very good character analyser but it is very expensive to be a rutine analyser. In general we can say that, Reverse Phase HPLC assesses level of heterogenity and useful for purification. All besides these, it is not possible to yield any structural informations. Electrophoresis ( gel electrophoresis is used) is usefull to form an idea about the peptides heterogeneity and gives an idea of Mw approximately. Ultraviolet Spectroscopy directly enableing the purification monitoring. Also spectroscopic applications of some analytic methods like TNBS- method and Lowry-method enables us to determine the concentration of peptides. TNBS method is also practiced in this work. xuiBesides all these methods, Millipore's Automated Peptide Synthesizer gives us chance to detect if the coupling reactions are going in proper order and at needed % persantages. We also have chance to stop the synthesis if the coupling % persantages are lower then the required ones. The synthesizer automatically stops the synthesis if the coupling and deprotection parameters lower then the specified. This system is a kind of self detection. The system that was used in the synthesizer as said, a kind of self detection system. Here also it means, if you find the coupling % low you have the choice : One can eliminate the synthesis and start the other. One can stop the synthesis and refill the amino acid vial and restart the synthesis where it had stoped. This characteristic helped us to save the reactives. The synthesized four antigenic peptides are all monitored during the synthesis to eliminate the low quality products. Returning back to analytical techniques, it would be more clear if we give a brief explanation about how they were used in this study. The HPLC system is very important for peptides for it is used for both purification and characterizations. So it would be good if we first talk about this system. In HPLC systems gel permeation column chromatograph is used. The graphs are as in below. * Chromatogram mAbs ?* Filename:AHl-UV.C01 0 10 Figure 1 HPLC for AHİ min XIV.** Chromatogram *** Filename :AH2-UV.C01 mAbs 0 10 Figure 2 HPLC for AH2 rain * Chromatogram *** Filename :AH3-UV.C01 mAbs Figure 3 HPLC for AH3 XV*** Chromatogram **. Filename:AH4-4UV.c01 mAbs 10 20 30 40 min Figure 4 HPLC for AH4 From the figures it can be drived that oligomeric structures with low molecular weights are just similar to that synthesized. Before the HPLC analysis, also Normal chramotograpy had been used for the four products. This was a need of pre-column treatment. Bio-Rad Chromatography instrument was used and its collector was connected to enable to have the fractions. Different column materials were used and the fractions had been collected. Figure 5 Sephadex G-200 for AHİ xviQ O 1.4 1.2 H 0.8 H 0.6 0.4 0.2 Figure 6 Sephacryl S-300 for AHİ O Tüp No. Figure 7 Sephacryl S-300 for AH2 XV13Q O Figure 8 Sephaciyl S-300 for AH3 C Tüp No. Figure 9 Sephaciyl S-300 for AH4 xvillSAMPLE WAVENUMBERS Nicolet Instrument Corporation Figure 10 IR for AHİ SUNS: 3? nrs: 4.0 HHF: oq/iB/ ir:0B:

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