【摘要】：根據(jù)本實(shí)驗(yàn)室首先克隆的mL52基因(GenBank：AY028425)序列設(shè)計(jì)引物，以人胎肝cDNA文庫為模板，通過PCR擴(kuò)增獲得了人類同源新基因，因其富含甘氨酸而命名為—hGlyrichin。在生物信息學(xué)分析提示其為抗菌肽和初步證實(shí)該基因具有抗菌作用的基礎(chǔ)上，為了尋找該蛋白序列的核心功能區(qū)段，進(jìn)一步借助生物信息學(xué)分析結(jié)果，分段合成了系列肽段并對(duì)這些肽段的抗菌活性進(jìn)行了測(cè)試，結(jié)果證實(shí)，從第42到60位氨基酸的19個(gè)氨基酸(pCM-19)為該蛋白的核心功能區(qū)。為此，我們對(duì)pCM-19的抗菌活性、抗菌作用機(jī)理及抗菌肽的普遍副作用進(jìn)行了系統(tǒng)研究。實(shí)驗(yàn)結(jié)果證實(shí)，pCM-19不僅具有抗實(shí)驗(yàn)室工程菌的活性，而且具有抗鼠疫桿菌、傷寒沙門氏菌、綠膿桿菌和腦膜炎球菌的作用。更可喜的是該肽段具有抗攜帶氨芐青霉素抗性基因的實(shí)驗(yàn)室工程菌和抗耐甲氧西林金黃色葡萄球菌的活性，提示pCM-19具有廣譜抗菌作用并為該抗菌肽的應(yīng)用提供了有價(jià)值的實(shí)驗(yàn)數(shù)據(jù)。pCM-19的量效關(guān)系測(cè)定發(fā)現(xiàn)，本實(shí)驗(yàn)室所獲得的曲線與Sunkyu Kim等人報(bào)道的gaegurin 6(GGN 6)的量效關(guān)系曲線類似，即pCM-19需達(dá)到一定濃度才能發(fā)揮抗菌作用。pCM-19抗菌活性的穩(wěn)定性研究發(fā)現(xiàn)，，該肽段經(jīng)100℃加熱30min和60min及反復(fù)凍融3次，其抗菌活性完全不受影響；溶液狀態(tài)在4℃放置80天，抗菌活性仍可保留98％以上；經(jīng)胰蛋白酶處理60min后，抗菌活性保留30％。這些實(shí)驗(yàn)結(jié)果表明，pCM-19具有良好的穩(wěn)定性，也為其發(fā)展成藥提供了重要實(shí)驗(yàn)數(shù)據(jù)。 為了研究該肽段的抗菌作用機(jī)理，采用流式熒光激活細(xì)胞分析(FACScan)和掃描電子顯微鏡分析了pCM-19對(duì)靶細(xì)菌膜完整性的影響。兩種方法所得結(jié)果一致提示，pCM-19可能作用于細(xì)菌的細(xì)胞壁和／或細(xì)胞膜，通過破壞膜的完整性并進(jìn)而導(dǎo)致靶細(xì)菌胞質(zhì)內(nèi)容物外泄而發(fā)揮殺菌作用。這與目前已知陽離子抗菌肽的主要作用機(jī)制類似。 抗菌肽的溶血作用是抗菌肽常見的副作用，也是某些抗菌肽發(fā)展成藥的主要障礙。為此，本文對(duì)pCM-19的溶血作用進(jìn)行了體內(nèi)、外測(cè)試，結(jié)果證實(shí)，pCM-19在體外無溶解人紅細(xì)胞的作用，在體內(nèi)無溶解鼠紅細(xì)胞的作用。
[Abstract]:According to the sequence of mL52 gene (GenBank:AY028425) first cloned in our laboratory, primers were designed and human fetal liver cDNA library was used as template. A new homologous human gene was obtained by PCR amplification, which was named -hGlyrichinin because it was rich in glycine. On the basis of bioinformatics analysis and preliminary confirmation that the gene has antimicrobial effect, in order to find the core functional region of the protein sequence, the results of bioinformatics analysis were further used. A series of peptide segments were synthesized and their antibacterial activities were tested. The results showed that 19 amino acids (pCM-19) from the 42nd to 60th amino acids were the core functional regions of the protein. Therefore, we systematically studied the antimicrobial activity, antibacterial mechanism and common side effects of antimicrobial peptides of pCM-19. The results showed that PCM-19 not only had the activity of anti-engineering bacteria in laboratory, but also had anti-Yersinia pestis, Salmonella typhimurium, Pseudomonas aeruginosa and meningococci. What is more gratifying is that the peptide has the activity of anti-ampicillin resistant genes in laboratory engineering bacteria and methicillin-resistant Staphylococcus aureus. The results indicated that pCM-19 had broad spectrum antibacterial activity and provided valuable experimental data. PCM-19 showed that the curve obtained in our laboratory was similar to that of gaegurin 6 (GGN 6) reported by Sunkyu Kim et al. It was found that the antibacterial activity of the peptide was completely unaffected by heating 30min and 60min at 100 鈩

本文編號(hào)：2251439