基于噬菌體展示技術(shù)篩選內(nèi)毒素結(jié)合肽并建立內(nèi)毒素檢測新方法
發(fā)布時(shí)間：2018-09-19 11:43
【摘要】：內(nèi)毒素又被稱作為脂多糖是革蘭氏陰性細(xì)菌的細(xì)胞壁中的一種重要的組成部分。內(nèi)毒素進(jìn)入血液中,將會出現(xiàn)發(fā)熱、血壓降低、彌散性血管內(nèi)凝血、內(nèi)毒素?cái)⊙Y等一系列臨床反應(yīng),嚴(yán)重時(shí)會導(dǎo)致休克、甚至是死亡。所以一些食品、藥品、醫(yī)療器械在出廠前都要進(jìn)行內(nèi)毒素的檢測,以避免內(nèi)毒素對人體造成的危害。中國藥典中已經(jīng)收錄了光度法和凝膠法這兩種用于測定細(xì)菌內(nèi)毒素含量的的檢查方法,這兩種方法都是在利用鱟血提取物的基礎(chǔ)上對內(nèi)毒素的含量進(jìn)行的定性甚至定量的測定。鱟試劑是從鱟血中提取的,原材料稀缺、價(jià)格昂貴、不穩(wěn)定。本文利用噬菌體展示技術(shù)從構(gòu)建好的十二肽庫中經(jīng)過了三輪篩選得到能夠與內(nèi)毒素結(jié)合的噬菌體克隆,再提取噬菌體ssDNA,運(yùn)用DNASTAR和BLAST軟件對序列進(jìn)行分析,最后用固相合成法合成十二肽序列,利用分子互作技術(shù)鑒定十二肽與內(nèi)毒素的結(jié)合能力,并建立增強(qiáng)比濁法檢測內(nèi)毒素。利用噬菌體展示技術(shù),以內(nèi)毒素做為目標(biāo)靶分子并將其包被在聚丙烯平板上,然后將噬菌體展示出的肽庫與已經(jīng)預(yù)先包被好靶分子的平板共溫育,經(jīng)過兩輪非特異性洗脫篩選及擴(kuò)增和一輪特異性洗脫篩選,最后成功篩選出能夠與內(nèi)毒素高親和性結(jié)合的噬菌體克隆。將篩選得到的結(jié)合噬菌體克隆進(jìn)行培養(yǎng),提取噬菌體ssDNA,其大小為6400bp,經(jīng)測序及軟件分析得到十二肽序列:QVTPQVPRSTQM和QVNGLGERSQ QM。運(yùn)用固相合成法合成十二肽各1mg,純度達(dá)95%。運(yùn)用分子互作技術(shù)對合成的十二肽進(jìn)行親和性分析,其解離平衡常數(shù)(KD)分別達(dá)到3.52×10-9和8.012×10-9,Full^R2分別達(dá)到0.9979和0.9969,表明合成的十二肽與內(nèi)毒素具有良好的親和性。利用所合成的十二肽建立內(nèi)毒素檢測-增強(qiáng)比濁法,獲得增強(qiáng)比濁檢測內(nèi)毒素標(biāo)準(zhǔn)曲線其線性范圍是0.03~0.48EU/mL,R2達(dá)到0.9925。該方法能夠簡便、準(zhǔn)確的檢測內(nèi)毒素,為臨床快速檢測內(nèi)毒素奠定基礎(chǔ)。
[Abstract]:Endotoxin, also known as lipopolysaccharide, is an important component of the cell wall of Gram-negative bacteria. A series of clinical reactions such as fever, decreased blood pressure, disseminated intravascular coagulation, endotoxemia and so on will occur when endotoxin enters the blood, which can lead to shock or even death. Therefore, some foods, drugs and medical devices should be tested for endotoxin before they leave the factory to avoid the harm of endotoxin to human body. Photometric and gel methods have been included in the Chinese pharmacopoeia for the determination of bacterial endotoxin. These two methods are qualitative and even quantitative determination of endotoxin on the basis of Limulus blood extract. Limulus reagent is extracted from Limulus blood, raw materials are scarce, expensive, unstable. In this paper, phage display technology was used to obtain phage clones which could bind with endotoxin from the constructed dodecapeptide library. The phage ssDNA, was extracted and sequenced by DNASTAR and BLAST software. Finally, the dodeceptide sequence was synthesized by solid state synthesis, the binding ability of dodecapeptide to endotoxin was identified by molecular interaction technique, and an enhanced turbidimetric method was established for the detection of endotoxin. Using phage display technology, endotoxin was used as target molecule and coated on polypropylene plate, then the peptide library was incubated with the plate which had been precoated with target molecule. After two rounds of non-specific elution screening and amplification and one specific elution screening, the phage clones which could bind to endotoxin with high affinity were successfully screened. The phage ssDNA, was extracted from the screened phage clone. The size of phage ssDNA, was 6400bp. the sequence of dodecapeptide was obtained by sequencing and software analysis. The sequence of dodecapeptide: QVTPQVPRSTQM and QVNGLGERSQ QM. were obtained. The dodecapeptide was synthesized by solid state synthesis with a purity of 95 mg each. The molecular interaction technique was used to analyze the affinity of the synthesized dodeceptide. The dissociation equilibrium constant (KD) reached 3.52 脳 10 ~ (-9) and 8.012 脳 10 ~ (-9) full ^ R2, respectively, 0.9979 and 0.9969, respectively, which indicated that the synthesized dodeceptide had good affinity with endotoxin. The standard curve of enhanced turbidity for endotoxin detection was obtained by using the synthesized dodeceptide to establish endotoxin-enhanced turbidimetry. The linear range was 0.03 ~ 0.48 EUU / mL ~ (2) ~ (2) up to 0.9925. This method is simple and accurate for the detection of endotoxin, and lays a foundation for rapid detection of endotoxin in clinic.
【學(xué)位授予單位】：長春理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R446.5;O652
[Abstract]:Endotoxin, also known as lipopolysaccharide, is an important component of the cell wall of Gram-negative bacteria. A series of clinical reactions such as fever, decreased blood pressure, disseminated intravascular coagulation, endotoxemia and so on will occur when endotoxin enters the blood, which can lead to shock or even death. Therefore, some foods, drugs and medical devices should be tested for endotoxin before they leave the factory to avoid the harm of endotoxin to human body. Photometric and gel methods have been included in the Chinese pharmacopoeia for the determination of bacterial endotoxin. These two methods are qualitative and even quantitative determination of endotoxin on the basis of Limulus blood extract. Limulus reagent is extracted from Limulus blood, raw materials are scarce, expensive, unstable. In this paper, phage display technology was used to obtain phage clones which could bind with endotoxin from the constructed dodecapeptide library. The phage ssDNA, was extracted and sequenced by DNASTAR and BLAST software. Finally, the dodeceptide sequence was synthesized by solid state synthesis, the binding ability of dodecapeptide to endotoxin was identified by molecular interaction technique, and an enhanced turbidimetric method was established for the detection of endotoxin. Using phage display technology, endotoxin was used as target molecule and coated on polypropylene plate, then the peptide library was incubated with the plate which had been precoated with target molecule. After two rounds of non-specific elution screening and amplification and one specific elution screening, the phage clones which could bind to endotoxin with high affinity were successfully screened. The phage ssDNA, was extracted from the screened phage clone. The size of phage ssDNA, was 6400bp. the sequence of dodecapeptide was obtained by sequencing and software analysis. The sequence of dodecapeptide: QVTPQVPRSTQM and QVNGLGERSQ QM. were obtained. The dodecapeptide was synthesized by solid state synthesis with a purity of 95 mg each. The molecular interaction technique was used to analyze the affinity of the synthesized dodeceptide. The dissociation equilibrium constant (KD) reached 3.52 脳 10 ~ (-9) and 8.012 脳 10 ~ (-9) full ^ R2, respectively, 0.9979 and 0.9969, respectively, which indicated that the synthesized dodeceptide had good affinity with endotoxin. The standard curve of enhanced turbidity for endotoxin detection was obtained by using the synthesized dodeceptide to establish endotoxin-enhanced turbidimetry. The linear range was 0.03 ~ 0.48 EUU / mL ~ (2) ~ (2) up to 0.9925. This method is simple and accurate for the detection of endotoxin, and lays a foundation for rapid detection of endotoxin in clinic.
【學(xué)位授予單位】：長春理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R446.5;O652
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