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تاثير انزيم الكلوكوسيل ترانسفيريز المنقى من العزلة المحلية Streptococcus mutans النمط C في انتاج الاضداد (IgY) من صفار بيض طيور الدجاج == The Effect of Glucosyltransferase Purified From Local Isolate Streptococcus Mutans (Serotype C) On Egg Yolk Antibodies (IgY) Generation In Layer Hens
Author name:
هاشم محمد زهراو الصبيحاوي
Supervisor name:
عصام فاضل علوان الجمیلي | فارس عبد الكريم
General topic:
Biology
Specific topic:
Biotechnologies
Degree:
Doctorate
University:
University of Baghdad
Language:
English
University location:
Baghdad
First pages:
24T2659 - p.pdf
Abstract:
استهدفت الدراسة الحالية عزل وتشخيص بكتيريا Streptococcus mutans المسؤولة عن تنخر الاسنان البشري والتكلسات (plaque) واستخراج اضداد لها من صفار بيض الدجاج Yolk Immunoglobullin (IgY) لغرض استخدامها مستقبلا كمثبطات لنمو هذه البكتيريا الخطيرة ويمكن مزجها مع مع | The presented study aimed to isolate the main agent for dental caries and teeth plaque, Streptococcus mutans bacteria, and then production of specific antibodies against these harmful bacteria by the use of chicken egg yolk immunoglobulin (IgY). S. mutans had been proposed as the main etiological agents of dental caries and high levels of mutans streptococci in the plaque is correlated with a higher risk for dental caries. Seventy five plaque samples were collected from human teeth. Forty two samples were considered to be positive bacterial isolates using MS - agar (Mitist Salivares agar). Thirty five isolates were considered belonging to the group Streptococci; among these isolates 29 isolates were expected to be belonging to mutans streptococci group according to ability of producing special kind of exopolysaccharides. Ten isolates were considered as S. mutans with a percentage of 41% depending on staining with triphenyltetrazolium chloride and tolerance with NaCl 4%, 6 isolates were classified as serotype C by using Lancefield grouping identification. These isolates were tested for production of extracellular Glucosyltransferase (GTF) through determination of their enzyme specific activity. All isolates were able to produce the enzyme; Streptococci isolate (H5) identified as Streptococcus mutans serotype C was selected as the best producible isolate for GTF with a specific activity of 2.6 U/mg. It was found that GTF of the chosen isolate (H5) was produced during the middle stationary phase (18 - 35 hr.) and its maximal productivity was reached at 22 hr. Purification of S. mutans serotype (C) H5 GTF were done by ammonium sulfate, ion - exchange chromatography (DEAE - Sephacel column), and gel - filtration chromatography using Sepharose 6B column. The best percent saturation use for precipitating GTF by ammonium sulfate was 20 - 40% with specific activity 3.4 U/mg. Two purified GTF enzymes (GTF - I and GTF - II) were detected with specific activity 8.3 U/mg, 35.5 U/mg after 22.6, and 96.1 fold of purification respectively with yield 17.2%. Purification S. mutans CA - GTF (H5) were done by 8M urea, ammonium sulfate, DEAE - Sephacel column and gel - filtration (sepharose 6B) column chromatography. The purified CA - GTF was detected with specific activity 18.1 U/mg after 24.5 fold of purification with yield 20.2%. Determination of purified GTF (GTF - I, GTF - II) and CA - GTF molecular weight was done by using gel - filtration chromatography (sepharose 6B) column with presence of standards proteins. It was found that the molecular weight of GTF - I, GTF - II and CA - GTF was 125.819, 112.201 and 84.139 dalton, respectively. The ability of GTF, CA - GTF and whole cell of S. mutans to stimulate the immune system of avian hens was tested. The intramuscular rout injection of three purified antigens (GTF, CA - GTF and whole cell) in the chest of experimental hens was done. IgG from egg yolk hens (IgY) was purified through the post immunization period (9 weeks) by using polyethylene glycol (PEG) precipitation and protein content of IgY antibodies was estimated from egg yolk and serum. Each one milliliter of purified IgY egg yolk samples GTF, CA - GTF and Whole cell, protein contained 7.06, 6.97, 3.9 mg/ml, respectively while in serum protein content about2.6, 3.1 and 3.25 mg/ml, respectively. The Sodium dodecyle sulfate polyacrylamide gel electrophoresis (SDS - PAGE) of anti - GTF (IgY) indicated that purified IgY gave two bands; 47.863 and 34.673dalton which were considered to be IgY heavy and light chains respectively. the IgY - CA - GTF sample is the best in terms IgY specificity 34.07% while the two samples (GTF, Whole cell) performed 30.5% and 29.3% respectively, Igy - GTF the best in terms purity 49% followed IgY - CA - GTF 47% and IgY - whole cell 46.3%. The immunological specificity of the three IgY samples preparations was assessed by ELISA test and the best sample that produced high titration was IgY - GTF with concentration 3.5 mg/ml, followed by the IgY - CA - GTF and IgY - whole cell with concentration 3.28 and 3.1 mg/ml respectively. The IgY - GTF inhibited approximate 75% of the specific activity GTF, while IgY - CA - GTF inhibited 50% of the specific activity CA - GTF. A double immunodiffusion test for detection of the immune response between anti - GTF IgY and purified GTF, CA - GTF and Whole cell antigens were recorded. The immunological response of anti - GTF and anti - CA - GTF was indicated by the appearance of precipitation lines on the surface gel between anti - GTF and two antigens GTF and CA - GTF while in the anti - CA - GTF and anti whole cell only with homologues antigen. The effect of different concentration of inhibitor (Amoxicillin) and anti - GTF, anti - CA - GTF and anti - whole cell on the growth of S. mutans bacteria were tested using broth dilution method and diffusion method on solid medium. Anti - GTF and anti - CA - GTF had no effect on the growth of S. mutans(H5) serotype C, while anti - whole - cell and Amoxicillin were capable to inhibit the growth of bacteria at concentration 20µg/ml and 15µg/ml respectively. The minimal inhibitory concentrations in which these concentrations were noticed at 35µg/ml and 30 µg/ml respectively. The highest zone of inhibition (40 mm) was recognized with Amoxicillin at concentration of 50 µg/ml, followed by anti - whole cell with a zone of inhibition of 34 mm at concentration of 70 µg/ml.
