本文關(guān)鍵詞： 甘露聚糖結(jié)合凝集素相關(guān)絲氨酸蛋白酶2 高分辨率熔解曲線分析技術(shù) 基因分型 急性上呼吸道感染　出處：《南方醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：感染性疾病如小兒急性上呼吸道感染(upper respiratory tract infection, URTI,簡稱上感)、乙型肝炎(hepatitis B,簡稱乙肝)嚴重威脅著人類健康與生存質(zhì)量。上感是小兒內(nèi)科最為常見的疾病,在春季及冬季較為多發(fā),也是兒科門診和病房患者所占比例最大的疾病。我國是乙肝的高發(fā)流行區(qū),每年約30萬人死于因乙肝病毒(hepatitis B virus, HBV)感染所致的重型肝炎、肝硬化和肝癌等疾病。對病原體是否易感取決于機體的抵抗力,而天然免疫是抗御病原體感染的第一道防線。在生物進化史上,補體系統(tǒng)已經(jīng)存在350萬年,是天然免疫防御的重要組成部分,其介導(dǎo)的免疫防御功能包括釋放炎癥介質(zhì)、招募炎癥細胞、破壞病原體,消除免疫復(fù)合物及凋亡細胞等在抵抗各種病原微生物中發(fā)揮了重要作用。補體激活的第三條途徑—凝集素途徑為天然免疫系統(tǒng)中重要組成部分。在凝集素途徑中,甘露聚糖結(jié)合凝集素(mannan-binding lectin, MBL)和纖維膠原素(ficolins)與甘露聚糖結(jié)合凝集素相關(guān)絲氨酸蛋白酶1/2 (MBL-associated serine protease 1/2, MASP1/MASP2)等組成類似C1的復(fù)合物,MBL通過糖識別域(carbohydrate-recognition domain, CRD)識別并結(jié)合病原微生物表面的甘露糖、N-乙酰葡萄糖等相應(yīng)糖結(jié)構(gòu)后,經(jīng)其膠原樣區(qū)(collagen-like region, CLR)活化MASP1和MASP2,裂解補體C4和C2形成C3轉(zhuǎn)化酶C4b2a,由此形成補體激活凝集素途徑。MASP2蛋白由686個氨基酸組成,編碼基因位于1號染色體p.36.22,全長20717 bp,包含12個外顯子和12個內(nèi)含子。MASP2分子為單肽鏈,從N端至C端有6個功能區(qū)組成,依次為：CUB區(qū)(complement subcomponent C1r/C1s-like domain)^ EGF區(qū)(epidermal growth factor-like domain)、CUB區(qū)、2個CCP區(qū)(complement control protein domain)、SP (serine proteases)區(qū)。研究表明,MASP蛋白N端及C端的功能相對獨立：MASP2N端的3個結(jié)構(gòu)域即2個CUB區(qū)和1個EGF區(qū)是MASP與MBL-CLR結(jié)合的區(qū)域,能以Ca2+依賴方式與MBL相互作用,形成MBL-MASPs復(fù)合物；而C末端的3個結(jié)構(gòu)域即2個CCP區(qū)和1個SP區(qū)是絲氨酸蛋白酶的活性中心,激活補體主要依靠SP區(qū)發(fā)揮絲氨酸蛋白酶的作用,裂解補體C4和C2,產(chǎn)生C3轉(zhuǎn)化酶。所以MASP2為MBL和幾種(H、M和L)纖維膠原素激活補體所必需,在凝集素途徑中發(fā)揮至關(guān)重要的作用。在患有多重感染和自身免疫性疾病的患者,首次描述MASP2缺損是由外外顯子3所編碼的功能區(qū)CUB1區(qū)產(chǎn)生GAC120GGC點突變,導(dǎo)致編碼產(chǎn)物由酸性氨基酸天冬氨酸(Asp, D)變?yōu)橹行园被岣拾彼?Gly,G),即D120G突變,等電點發(fā)生了改變。因此,我們認為MASP2缺損是各種病原微生物反復(fù)感染的可能病因之一,而快速進行MASP2基因分型可能預(yù)測感染性疾病風(fēng)險。目前,尚未見血漿MASP2濃度與上感關(guān)系的報道,故我們分析了上呼吸道感染兒童血漿MASP2水平并探討其意義。第一章應(yīng)用HRM技術(shù)檢測MASP2基因D120G突變高分辨率熔解曲線(high-resolution melting, HRM)分析技術(shù)檢測基因點突變具有以下優(yōu)點：成本低,不需要設(shè)計昂貴的探針；在聚合酶鏈?zhǔn)椒磻?yīng)(polymerase chain reaction,PCR)結(jié)束后,直接進行熔解曲線分析；快速、高通量,一次可以同時檢測96或384個樣本；閉管操作,防止樣本脫氧核糖核酸(deoxyribonucleic acid,DNA)在PCR擴增后轉(zhuǎn)移過程中污染；經(jīng)過HRM分析處理后的PCR擴增產(chǎn)物還可以直接進行DNA測序,十分簡潔便利。因此HRM技術(shù)已廣泛應(yīng)用于生命科學(xué)、食品安全、醫(yī)學(xué)、畜牧業(yè)等各個領(lǐng)域的研究工作中�；贖RM上述特點,本研究擬應(yīng)用HRM技術(shù),建立檢測分析人MASP2基因D120G突變的方法。材料與方法：1、標(biāo)本：共收集153例全血標(biāo)本,采用常規(guī)飽和酚/氯仿法提取外周血基因組DNA。2、點突變分析方法：(1)針對MASP2基因D120G變異位點設(shè)計PCR擴增體系及優(yōu)化PCR條件。通過PCR定點誘變構(gòu)建突變型純合子基因,本科室構(gòu)建保存MASP2基因片段經(jīng)測序驗證為野生型純合子基因從而獲得各種基因型樣品。(2)建立檢測MASP2基因D120G位點突變的HRM分析技術(shù)。針對變異位點設(shè)計HRM分析的引物,利用經(jīng)測序已知的基因型樣品優(yōu)化HRM分析條件,從而建立分析MASP2基因的HRM分型方法。(3)根據(jù)HRM的分析結(jié)果,從各種基因型中選取一定數(shù)量的樣品進行測序分析,以驗證評價該方法的準(zhǔn)確性；并且選取每種MASP2基因野生純合子及突變純合子各10個樣本進行批間及批內(nèi)重復(fù)性實驗；對MASP2基因的野生純合子和突變純合子樣本進行了不同比例混合,得到不同雜合比例質(zhì)粒模板進行HRM檢測突變雜合子靈敏度下限；將質(zhì)粒DNA模板作10倍梯度稀釋進行5個濃度檢測,進行HRM檢測,分析該方法檢測敏感性。3、統(tǒng)計學(xué)分析：使用的統(tǒng)計學(xué)分析軟件為SPSS16.0.結(jié)果：建立了穩(wěn)定可靠的HRM檢測體系。此體系中,所選取的擴增目的DNA序列以及所設(shè)計的引物的特異性高。MASP2基因的野生純合子A/A,雜合子A/G,突變純合子G/G三種基因型能夠在HRM上進行明顯的區(qū)分。用此方法分析廣州地區(qū)153份人基因組DNA樣品(123例為上感患者DNA樣本,30例為乙肝患者DNA樣本),從每組基因型中隨機選出部分樣品進行測序?qū)φ?結(jié)果符合率為100%,證實了該方法的準(zhǔn)確性很好；進行批內(nèi)、批間重復(fù)實驗,發(fā)現(xiàn)10次實驗的變異系數(shù)(CV)均小于0.1%,證實了該方法的穩(wěn)定性很好；在10倍稀釋試驗中,5個濃度均可以準(zhǔn)確分型,可以知道1 pg濃度質(zhì)粒仍然可以用于此檢測體系；在不同雜合比例模板HRM分析得知,該體系可以用于檢測MASP2基因突變型雜合子比例下限為10%。結(jié)論：本課題研究中建立的針對MASP2基因的HRM分析方法能夠快速、準(zhǔn)確地對MASP2基因進行突變檢測,并且重復(fù)性和穩(wěn)定性好、敏感性高、體系可靠。本研究對中國南方人群進行檢測,發(fā)現(xiàn)所檢測的153人中不存在MASP2基因D120G突變型純合子個體,只有1例D120G突變型型雜合子,推測這種基因型在中國南方地區(qū)少見。本研究分析MASP2基因D120G突變建立了一種簡單、快速、高通量、經(jīng)濟和靈敏的檢測方法,為研究MASP2基因與疾病的關(guān)系提供了一項有用的技術(shù)。第二章MASP2在小兒上呼吸道感染中的意義已報道MASP2與多種疾病有關(guān),但其與URTI的關(guān)系目前不得而知。本章探討血漿MASP2水平在兒童URTI中的意義。方法：選取2014年10月至12月在南方醫(yī)院兒科門診接受診療的103例URTI患兒作為研究對象,另選取同期健康體檢的正常兒童為對照組,計35例。測定其血漿MASP2和C反應(yīng)蛋白(C-reaction protein,CRP)濃度并進行白細胞計數(shù)(white blood cell,WBC).根據(jù)CRP和WBC值、感染不同時期及有無治療,URTI兒童分為CRP升高組(n=48)與正常組(n=54)、WBC升高組(n=61)與正常組(n=40)、感染早期未用藥組(n=68)與感染中后期已用藥組(n=35)。根據(jù)不同分組分別做統(tǒng)計學(xué)分析。應(yīng)用生物信息學(xué)分析MASP2基因與急性期反應(yīng)蛋白MBL基因、典型急性期反應(yīng)蛋白CRP基因是否存在共同的應(yīng)激元件。結(jié)果：URTI組血漿MASP2濃度顯著高于對照組(P＜0.001),CRP升高組血漿MASP2濃度與CRP值正相關(guān)(r=0.310,P0.05),WBC升高組MASP2水平與WBC值顯著正相關(guān)(r=0.392,P0.01),感染早期未用藥組MASP2濃度顯著高于感染中后期已用藥組(P0.01)。應(yīng)用生物信息學(xué)分析得知,MASP2基因與急性期反應(yīng)蛋白MBL基因及典型性急性期反應(yīng)蛋白CRP基因存在共同應(yīng)激元件一一肝細胞核因子4α(hepatic nuclear factors-4α,HNF-4α)作用區(qū)。結(jié)論：MASP2蛋白可能為急性期蛋白,血漿MASP2水平可作為診斷小兒上感的一個參考指標(biāo)。
[Abstract]:Infectious diseases such as infantile acute upper respiratory tract infection (upper respiratory tract infection URTI, referred to as URI (hepatitis B), hepatitis B, hepatitis B) is a serious threat to the health and quality of life of human beings. Infection is the most common disease in pediatric medicine, spring and winter are more common is the outpatient department of Pediatrics and ward patients accounted for the largest proportion of the disease. Our country is a high incidence of hepatitis B epidemic area, about 300 thousand people die each year due to hepatitis B virus (hepatitis B, virus, HBV) caused by infection of severe hepatitis, cirrhosis and liver cancer and other diseases. The pathogen is susceptible depends on the resistance of the organism, and natural immunity is the first line of defense against pathogen infection in the history of biological evolution, the complement system has existed for 3 million 500 thousand years, is an important part of the innate immune defense and immune defense function mediated release of inflammatory mediators including recruitment, inflammation Cells, destroy pathogens, eliminate immune complexes and apoptosis play an important role in resistance to various pathogenic microorganisms. In third ways: the lectin pathway of complement activation is an important component of the innate immune system. The lectin pathway, mannan binding lectin (mannan-binding lectin, MBL) and collagen fiber (ficolins) lectin associated serine protease 1/2 combined with mannan (MBL-associated serine protease 1/2, MASP1/MASP2) compound composition similar to C1, MBL through the carbohydrate recognition domain (carbohydrate-recognition domain, CRD) and identification of mannose binding surface of pathogenic microorganisms, N- acetyl glucose and corresponding sugar structure, the collagen like region (collagen-like region, CLR the activation of MASP1 and MASP2), C4 and C2 formed C3 cleavage complement convertase C4b2a, lectin pathway of complement activation of.MASP2 formed by the The protein consists of 686 amino acids, encoding gene is located on chromosome 1 p.36.22 and 20717 BP in length, including 12 exons and 12 introns of.MASP2 molecule as a single polypeptide, from N end to end C is composed of 6 functional areas as follows: CUB (complement subcomponent C1r/C1s-like EGF (domain) ^ region epidermal growth factor-like domain), CUB, 2 CCP (complement control protein domain), SP (serine proteases) district. The results showed that MASP protein N and C end function independently: 3 domains of MASP2N end is 2 CUB and 1 EGF area is MASP combined with MBL-CLR the area, in a Ca2+ dependent manner and MBL interaction, the formation of the MBL-MASPs complex; and the 3 domain of C at the end of the 2 CCP and 1 SP area is the active center of the serine protease, complement activation mainly depends on the SP region play serine protein enzyme function, complement C4 and cracking C2, C3. So MASP2 MBL invertase and several (H, M and L) collagen fiber activation necessary complement, play a crucial role in the lectin pathway. In patients with multiple infections and autoimmune disease, first described the MASP2 defect by means of exon 3 encoding function zone CUB1 GAC120GGC point mutation, resulting in encoding product from aspartic acid (Asp, D) into neutral amino acid glycine (Gly, G), D120G mutation, changed the isoelectric point. Therefore, we believe that MASP2 is one of the causes of various defects may be repeated infection of pathogenic microorganism, and fast MASP2 genotyping may predict infectious disease risk. At present, no relationship between the concentration of plasma MASP2 and on the report, so we analyze and evaluate the significance of plasma MASP2 levels in children with upper respiratory tract infection. In the first chapter, the application of HRM technology in detection of MAS The P2 gene mutation of D120G high resolution melting curve (high-resolution melting HRM) gene point mutation detection analysis technology has the following advantages: low cost, does not need expensive probe; polymerase chain reaction (polymerase chain reaction, PCR) after the end of direct melting curve analysis; fast, high throughput, time can be 96 or 384 samples at the same time; closed tube operation, to prevent sample DNA (deoxyribonucleic acid, DNA) after PCR amplification in the transfer process of pollution; after HRM analysis of PCR amplification products can be used for DNA sequencing, very simple and convenient. Therefore, HRM technology has been widely applied in life science, food safety. The research work in various fields of medicine, animal husbandry in HRM. Based on the characteristics, this study intends to use HRM technology, establish the detection methods of human MASP2 gene D120G mutation material. Methods: 1 specimens, a total of 153 patients were collected blood samples, using conventional phenol chloroform method to extract genomic DNA.2 from peripheral blood, point mutation analysis method: (1) for MASP2 gene D120G variant design of PCR amplification system and optimization of PCR conditions. By PCR mutagenesis to construct mutant homozygote gene, the Department of save construction of MASP2 gene fragment by sequencing homozygous for the wild-type gene to obtain various genotype samples. (2) the establishment of detection of MASP2 gene mutation of D120G HRM analysis technique. According to the analysis of mutation of HRM primer design, the optimized analysis conditions of HRM genotype were known, so as to establish the analysis of the MASP2 gene of HRM typing method. (3) according to the results of the HRM, select a certain number of samples from each genotype were sequenced and analyzed, to verify the accuracy of the method is evaluated and selected for each MASP; Nobu Juriko and the 2 gene mutation in the homozygous 10 samples between batch and batch repetitive experiments; the MASP2 gene homozygous wild and mutant homozygote samples with different mixing ratio, different proportion of heterozygous plasmid template for HRM mutation detection of heterozygous sensitivity limit; plasmid DNA template for 10 fold dilution of 5 concentration detection, HRM detection,.3 detection sensitivity analysis, the method of statistical analysis: statistical analysis software used for SPSS16.0. results: the establishment of the HRM detection system is stable and reliable. In this system, the selected target DNA sequence and the high specificity of.MASP2 gene primers designed by wild homozygous A/A, heterozygous homozygous A/G mutant homozygote G/G three genotypes can be clearly distinguished in HRM. Analysis of Guangzhou area of 153 human genomic DNA samples by this method (123 cases On patients with DNA samples, 30 cases of patients with hepatitis B DNA samples from each genotype), randomly selected samples were sequenced the control results, the coincidence rate is 100%, confirmed the accuracy of this method is very good; the batch, repeat the experiment, found that the variation of 10 experiments (CV) were less than the number of 0.1%, confirm the stability of this method is very good; in 10 times dilution test, 5 concentration can be accurately classified, can know the concentration of 1 pg plasmid can still be used for the detection system; in different proportion of heterozygous HRM template analysis showed that this system can be used to detect MASP2 gene mutation heterozygote proportion limit conclusion: 10%. method was established for MASP2 gene HRM analysis in this study was able to quickly and accurately to detect the mutation of MASP2 gene, and good repeatability and stability, high sensitivity, reliable system. The research on the South Chinese Group testing, found that the MASP2 gene of D120G homozygous mutant individuals do not exist in the detected 153 people, only 1 cases of D120G mutant heterozygote, rare speculated that this genotype in this area in the south of Chinese. Research and analysis of the MASP2 gene D120G mutation to establish a simple, rapid, high-throughput, economical and sensitive detection methods that provides a useful technique for the study of relationship between MASP2 gene and disease. In the second chapter, MASP2 in children with respiratory tract infection in significance has been reported that MASP2 is associated with many diseases, but its relationship with URTI. This chapter discusses the current can make nothing of it in the level of plasma MASP2 in children with URTI. Methods: October 2014 to December in Nanfang Hospital Outpatient Department of Pediatrics for treatment of 103 cases of URTI patients as the research object, the normal children were selected as healthy control group of 35 cases. The determination of the plasma levels of MASP2 and C. White (C-reaction protein, CRP) concentration and white blood cell count (white blood, cell, WBC). According to CRP and WBC, and there is no infection in different periods of treatment, URTI children were divided into CRP group (n=48) and normal group (n=54), WBC group (n=61) and normal group (n=40) the early stage of infection, untreated group (n=68) and infection in late treatment group (n=35). According to the different groups were statistically analyzed. Analysis of MASP2 gene and acute phase protein MBL gene by bioinformatics, C-reactive protein CRP gene in acute stage of the existence of typical stress components in common. Results: in group URTI, the plasma concentration of MASP2 significantly higher than the control group (P < 0.001), CRP group increased the concentration of plasma MASP2 and CRP were positively correlated (r=0.310, P0.05), WBC group increased MASP2 levels and WBC values were positively correlated (r=0.392, P0.01), the early stage of infection were significantly higher than untreated group MASP2 infection in late treatment group (P0.01). Application of bioinformatics analysis showed that the reactive protein MBL gene and typical acute phase protein CRP gene MASP2 gene and acute stress one common element of hepatocyte nuclear factor 4 alpha (hepatic nuclear factors-4 HNF-4 alpha, alpha) zone. Conclusion: MASP2 protein may be an acute phase protein, plasma MASP2 levels can be used as a the reference index for the diagnosis of infantile upper respiratory tract infection.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R725.6

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1 熊思敏;應(yīng)用HRM技術(shù)檢測MASP2基因D120G突變和MASP2在小兒上呼吸道感染中的意義[D];南方醫(yī)科大學(xué);2015年

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