用重組的克雷伯氏肺炎菌OmpK35及OmpK36來製備抗體以檢測具有carbapenem 抗藥性的克雷伯氏肺炎分離菌株

Preparation of polyclonal antibodies against recombinant OmpK35 and OmpK36 from Klebsiella pneumoniae and application in detecting carbapenem-resistant isolates

洪唯力(Wei-Li Hung)

指導教授 ： 曾銘仁

國立中正大學/理學院/生命科學系分子生物研究所/碩士(2015年)

摘要

克雷伯氏肺炎菌屬於革蘭氏陰性菌，也是院內感染常見的病原菌。雖然碳青黴烯類抗生素(carbapenem)仍能有效的治療這些菌種的感染，但由於克雷伯氏肺炎菌產生-lactamases 以及外膜蛋白的缺失，使得細菌對carbapenem產生抗藥性。 OmpK35及OmpK36是克雷伯氏肺炎菌兩個主要的外膜蛋白，為三聚體的親水性通道，使得親水性的小分子，像營養物質、抗生素，能穿越外膜。絕大多數的抗生素都是透過這兩個膜蛋白穿越克雷伯氏肺炎菌的細胞壁。在台灣的加護病房感染，發現超過95%的carbapenem-resistant K. pneumoniae (CRKP)有OmpK35 或/和 OmpK36 porin的表達缺失。本研究的目標在建立一個與克雷伯氏肺炎菌Ompk35/36 porin表達缺失有關的碳氫黴烯類抗生素抗藥性的抗體檢測平台，可作為抗生素投藥的參考。
先利用菌落PCR由對carbapenem具敏感性和不具敏感性的菌株分別得到全長的ompk35和ompk36的DNA片段，接合到pQE30後轉殖到E.coli M15以進行蛋白表現。利用IPTG誘導表現的OmpK35和OmpK36蛋白質均為不可溶的inclusion bodies，經由含Urea的緩衝液溶解後，以Ni-column純化。濃縮後的蛋白質以SDS-PAGE分離，再將其從SDS-PAGE萃取，用以免疫兔子制備抗體。
利用OmpK36抗體檢測純化後的OmpK36以及對碳氫黴烯類抗生素敏感的克雷伯氏肺炎菌粗蛋白質萃取液，發現此抗體的效價很高。但OmpK35抗體以相同的方法檢測，發現其效價不如預期，無法進行西方墨點法的檢測。50株臨床CRKP懸浮在SDS buffer中，以直接加熱破菌萃取粗蛋白，利用西方墨點法檢測是否有OmpK36的表現。同時以菌落PCR得到這些菌的全長ompk36 genes 並選殖到yT&A質體進行DNA定序，分析每株CRKP的ORF是否有無意義突變、缺失突變或嵌入突變。以西方墨點法分析檢測其
OmpK36的訊號為陰性(無OmpK36的表達)的菌株，除了7株分離菌以外，他們的對等ompk36 gene 的轉譯過程也提早中斷，形成較短的 OmpK36 蛋白。
ompk35 genes 也以菌落PCR的方式對臨床抗碳氫黴烯類的克雷伯氏肺炎菌得到全長的ompk35 genes，選殖後也進行DNA定序，分析每株CRKP的ORF是否有無意義突變、缺失突變或嵌入突變。 本研究的結果發現這50株臨床CRKP菌株大多數都會有Ompk35 或/和 Ompk36膜蛋白的缺失。

關鍵字

克雷伯氏肺炎菌

並列摘要

Klebsiella pneumoniae is a Gram-negative opportunistic pathogen and a common cause of nosocomial infections. Carbapenems are still effective drugs for these strains; however, resistance to carbapenems could result from the production of -lactamase and additional loss of outer membrane porins. OmpK35 and OmpK36 porins exist as trimers and act as water-filled protein channels. They provide as a channel that allows a wide range of antibiotics to penetrate the K. pneumoniae cell wall. More than 95% of carbapenem-resistant K. pneumoniae (CRKP) isolates exhibited either OmpK35 or OmpK36 porin loss or both in intensive care units in Taiwan. The aim of this study is to establish an antibody-based detecting platform for carbapenem-resistant related loss of OmpK35/36 porin in K. pneumonia. The outcome can serve as a reference for antibiotic administration.
The ompk35 and ompk36 genes from a carbapenem-sensitive and a carbapenem-resistant isolates, respectively, were obtained by colony PCR and subcloned into pQE30 E. coli expression vector and overproduced both OmpK proteins as inclusion bodies in E.coli M15 upon IPTG induction. The inclusion bodies were solubilized with urea containing buffer and purified with Ni-column. The concentrated purified proteins were further resolved by SDS-PAGE to exclude trace contaminates and used to immunize rabbits for antisera production.
The OmpK36 antisera showed very high specificity on purified OmpK36 and crude lysate of a carbapenem-sensitive K. pneumonia isolate by Western blot. However, the OmpK35 antisera failed to give satisfied results with purified OmpK35 and bacterial lysates. The crude lysates of 50 clinical CRKP
isolates were prepared by direct boiling in SDS buffer. The presence/absence of OmpK36 in each CRKP lysate was screened by Western blot with OmpK36 antisera. The ompk36 gene was also obtained by colony PCR, cloned into yT&A vector and sequencing for detecting the presence of nonsense mutation, deletion or insertion in the open reading frame for each CRKP isolate. Except 7 isolates, others all showed negative signal in Western blot, i.e., not OmpK36 protein, their ompk36 genes also showed translational disruptions, i.e., truncated OmpK36 proteins. Our data suggested that the OmpK36 antisera can specifically and efficiently identified CRKP with intact OmpK36 protein.
The ompk35 gene was also obtained by colony PCR, cloned into yT&A vector and sequencing for detecting the presence of nonsense mutation or insertion sequence in the open reading frame for each CRKP isolate. The final finding showed there are either OmpK35 or OmpK36 porin loss or both in these 50 c linical CRKP isolates.

並列關鍵字

carbapenem-resistant ； OmpK36 ； OmpK35 ； Klebsiella pneumoniae

參考文獻

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