I.Isolation And Gene Cloning Of Lysin Protein From Lytic Phage phiNK5II.Effects Of Antibiotics And Metalions On Group A Streptococcus Capsule Increase And Pathogenic Genes Expression

• Main Authors: Li, Tzuching, 李紫晴
• Other Authors: Hung, Chinhsin
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/78836197526828446702

碩士 === 義守大學 === 生物技術與化學工程研究所 === 100 === The Klebsiella pneumonia is a Gram-negative, facultative anaerobic bacterium and always causes the serial nosocomial infections. Recently years, the derivative multiple -drug resistant problems have made the antibiotic treatments on the choke point. The phage therapy is rekindled to combat the multiple-drug resistant bacterium infections. In our laboratory, phage phiNK5 has been isolated and specific to lyse Klebsiella pneumoniae NK5 (abbrev. NK5). In mouse animal model, phage phiNK5 could rescuse the NK5 infected mice and remained 100% survival. The purposes of this stusy will clone the lysin gene of phage phiNK5 and analyzed the activity of the expressed lysine protein. The complete genome sequences of phage phiNK5 has been finished before. Genome sequences was analyzed and blast on NCBI database, the results showed that the putative open reading frame (ORF) ORF45 and ORF46 were with 97% similarities to the endolysin protein of Klebsiella sp. phage KP32 (Accession No.: NC_011043) and K11 (Accession No.: NC_013647). ORF45 was used as the reference to design the primers and use PCR (polymerase chain reaction) to ampify the whole gene of ORF45. The PCR fragment was cloned into the EcoRV site of pBluescript SK+ and sequenced to ensure the correction of insert sequences and gernerate the plasmid pBNK5L. The pBNK5L was digested with NdeI/XhoI and the insert was cloned into pET21b NdeI/XhoI sites to generate the lysin expression plasmid pENK5L. Using IPTG to induce lysin protein expressing and found that the expressed protein was 16 kDa shown on SDS-PAGE, but this protein didn’t have lysis activity after purification. Lysin protein was also crude extracted from the supernatant of phage fNK5 and host coincubated culture medium by using the 20%-40% ammonium sulfate. The crude extracted proteins were separated by SDS-PAGE and the lysis activity protein patterns was determined by bacterium overlay assay. One protein patterns at 120 kDa resulted in clearn zone on the detected plate. This protein pattern was collected from PAGE and analyzed by LC/MS/MS to identified the protein by blast with the genome sequences of phage fNK5. The data showed that the protein was predicted as ORF8 (fusion protein) and ORF24 (DNA polymerase) with 56% and 25% similarity. But both of them were not relative to endolysis, so lysine protein was needed to extract and analyze again later. Group A streptococcus Gram Positive and facultative anaerobes is one of the major clinical pathogenic bacteria in pediatrics. In recent years, the research of Group A streptococcus’ virulence factor, such as hyaluronic acid, M protein, protein F, plasminogen receptor, streptokinase, C5a peptidase, streptokinase O, streptokinase S, streptococcal inhibitor of complement-mediated lysis, streptococcal pyogenic exotoxin B, and IgG-binding proteins. It can discover the bacteria earlier and prevent them start to spreading. Furthermore, it’s helpful to do the research of vaccine by understand the infection of bacteria and the reason of the disease. This research discuss about hyaluronic acid in different penicillin’s concentration, and the excitation of magnesium and Calcium, whether the capsule of bacteria strain getting thick or not, the capsule of streptococcus can cause the disease by this bacteria (Liu et al., 2011).This research use two methods to prove that the penicillin, magnesium and calcium in different concentration, whether their capsule will getting thick or not under excitation. There are two methods could get the conclusion; One is get the hyaluronic acid on Group A streptococcus’s capsule, then analyze it by Enzyme-linked Immunosorbent Assay. Because we can’t survey the standard one, it can’t survey the correct capacity of hyaluronic acid, only can contrast with the control of penicillin, magnesium and calcium’s excitation; so between the different strains, then fifteen kind of clinical strains have seven different kind of situation. The other method is get the mRNA ,then use the Real Time-PCR mRNA quantitative analyze. So far we can discover that the mac and hasB of M72 and M93, two kind of genes’ will get more expression under the excitation, so we can discuss that these two genes are affected by the control of penicillin, and the drug resistance of microbiology.