發(fā)布時(shí)間：2018-03-11 18:10

  本文選題：嵌合蛋白　切入點(diǎn)：抗菌肽　出處：《四川大學(xué)》2005年博士論文　論文類型：學(xué)位論文

【摘要】：細(xì)菌及病毒感染是困擾人類很久的問(wèn)題,尤其是近年來(lái)由于抗生素不規(guī)范使用及細(xì)菌突變等多方面原因,耐藥菌的感染成為日益嚴(yán)重而急需解決的一個(gè)難題。內(nèi)源性抗菌肽是一類陽(yáng)離子短肽,廣泛分布于人體多個(gè)部位,具有廣譜抗微生物活性,在不久的將來(lái)可能作為新一代抗感染藥物代替?zhèn)鹘y(tǒng)抗生素。防御素是人體中一類重要抗菌肽,新近的研究發(fā)現(xiàn)該類分子還是一種多功能免疫活性肽,廣泛地參與了機(jī)體的免疫應(yīng)答。本研究應(yīng)用重組嵌合蛋白技術(shù)試圖改造抗菌肽以提高抗菌活性和穩(wěn)定性,構(gòu)建抗菌肽嵌合核酸疫苗以增強(qiáng)抗腫瘤免疫。 本文分三個(gè)部分。 第一部分是對(duì)本實(shí)驗(yàn)室從人子宮頸粘液中分離鑒定出的1個(gè)新抗菌肽進(jìn)行N-端氨基酸序列測(cè)定和cDNA克隆,獲得其全長(zhǎng)編碼基因,以便用作嵌合肽的構(gòu)建。測(cè)序結(jié)果顯示氮端氨基酸序列從第一至第十位依次為脯氨酸(Pro,P),賴氨酸(Lys,K),精氨酸(Arg,R),賴氨酸(Lys,K),丙氨酸(Ala,A),天冬氨酸(Asp,D),甘氨酸(Gly,G),谷氨酸(Glu,E),丙氨酸(Ala,A),賴氨酸(Lys,K)。根據(jù)其氮端氨基酸序列,設(shè)計(jì)簡(jiǎn)并引物,3’-RACE-PCR技術(shù)擴(kuò)增出全長(zhǎng)cDNA,通過(guò)BLAST程序檢索對(duì)比分析,確定其為高遷移率蛋白N2(High mobility group chromosomal protein N2, HMG N2),用蛋白質(zhì)二結(jié)構(gòu)軟件分析發(fā)現(xiàn)該分子含1個(gè)跨膜α-螺旋結(jié)構(gòu)域,位于該分子氨基酸序列的第17位至42位之間�；瘜W(xué)合成該分子N-端、α-螺旋和C-端肽片段,抗菌活性檢測(cè)顯示僅α-螺旋肽片段具有抗菌活性,證明α-螺旋是HMG N2抗菌功能結(jié)構(gòu)域。 第二部分是構(gòu)建HMG N2 α-螺旋結(jié)構(gòu)域與富組蛋白-5嵌合抗菌多肽。采用PCR拼接技術(shù)將二者連接起來(lái),構(gòu)建出融合肽原核表達(dá)質(zhì)粒
[Abstract]:Bacterial and viral infections have been a problem for a long time, especially due to the irregular use of antibiotics and bacterial mutations in recent years. The infection of drug-resistant bacteria has become an increasingly serious and urgent problem. Endogenous antimicrobial peptides are a kind of cationic short peptides widely distributed in many parts of human body and have broad spectrum antimicrobial activity. In the near future, defensins may replace traditional antibiotics as a new generation of anti-infective drugs. Defensins are an important class of antimicrobial peptides in the human body, and recent studies have found that these molecules are also a multifunctional immunoreactive peptide. In this study, recombinant chimeric protein technology was used to modify antimicrobial peptides to improve their antibacterial activity and stability, and to construct an antimicrobial peptide chimeric nucleic acid vaccine to enhance anti-tumor immunity. This paper is divided into three parts. In the first part, a new antimicrobial peptide isolated from human cervical mucus in our laboratory was sequenced by N-terminal amino acid and cloned into cDNA, and its full-length encoding gene was obtained. Sequence analysis showed that the N-terminal amino acid sequences from the first to the 10th were proline, lysine, arginine, Alaanine, aspartic acid, glycine, glutamate, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamate, glutamate, glutamate, glutamate, glutamic acid, glutamate, glutamic acid, glutamate, glutamate, glutamic acid, glutamate, glutamate, glutamic acid, glutamic acid, and alanine. The amino acid sequence of the nitrogen terminal of Alaa, Lyschon, and its amino acid sequence was determined by analyzing the amino acid sequence of the amino acid terminal of the amino acid. The full-length cDNA was amplified by degenerate primer 3Case -RACE-PCR, and was identified as high mobility protein N2 high mobility group chromosomal protein N2, HMG N2 by BLAST program search and contrast analysis. A transmembrane 偽 -helix domain was found by protein two-structure software analysis. The N-terminal, 偽 -helix and C-terminal peptide fragments were chemically synthesized from the 17th to the 42nd position of the amino acid sequence of the molecule. The antibacterial activity of 偽 -helix peptide fragment was only found to have antibacterial activity, which proved that 偽 -helix was an antibacterial functional domain of HMG N2. The second part is the construction of HMG N2 偽 -helix domain and rich histone 5 chimeric antibacterial polypeptide. The fusion peptide prokaryotic expression plasmid was constructed by PCR splicing technique.
【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392

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本文編號(hào)：1599312