应用HRM技术检测MASP2基因D120G突变和MASP2在小儿上呼吸道感染中的意义

发布时间：2018-02-27 13:19

本文关键词： 甘露聚糖结合凝集素相关丝氨酸蛋白酶2 高分辨率熔解曲线分析技术基因分型急性上呼吸道感染　出处：《南方医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：感染性疾病如小儿急性上呼吸道感染(upper respiratory tract infection, URTI,简称上感)、乙型肝炎(hepatitis B,简称乙肝)严重威胁着人类健康与生存质量。上感是小儿内科最为常见的疾病,在春季及冬季较为多发,也是儿科门诊和病房患者所占比例最大的疾病。我国是乙肝的高发流行区,每年约30万人死于因乙肝病毒(hepatitis B virus, HBV)感染所致的重型肝炎、肝硬化和肝癌等疾病。对病原体是否易感取决于机体的抵抗力,而天然免疫是抗御病原体感染的第一道防线。在生物进化史上,补体系统已经存在350万年,是天然免疫防御的重要组成部分,其介导的免疫防御功能包括释放炎症介质、招募炎症细胞、破坏病原体,消除免疫复合物及凋亡细胞等在抵抗各种病原微生物中发挥了重要作用。补体激活的第三条途径—凝集素途径为天然免疫系统中重要组成部分。在凝集素途径中,甘露聚糖结合凝集素(mannan-binding lectin, MBL)和纤维胶原素(ficolins)与甘露聚糖结合凝集素相关丝氨酸蛋白酶1/2 (MBL-associated serine protease 1/2, MASP1/MASP2)等组成类似C1的复合物,MBL通过糖识别域(carbohydrate-recognition domain, CRD)识别并结合病原微生物表面的甘露糖、N-乙酰葡萄糖等相应糖结构后,经其胶原样区(collagen-like region, CLR)活化MASP1和MASP2,裂解补体C4和C2形成C3转化酶C4b2a,由此形成补体激活凝集素途径。MASP2蛋白由686个氨基酸组成,编码基因位于1号染色体p.36.22,全长20717 bp,包含12个外显子和12个内含子。MASP2分子为单肽链,从N端至C端有6个功能区组成,依次为：CUB区(complement subcomponent C1r/C1s-like domain)^ EGF区(epidermal growth factor-like domain)、CUB区、2个CCP区(complement control protein domain)、SP (serine proteases)区。研究表明,MASP蛋白N端及C端的功能相对独立：MASP2N端的3个结构域即2个CUB区和1个EGF区是MASP与MBL-CLR结合的区域,能以Ca2+依赖方式与MBL相互作用,形成MBL-MASPs复合物；而C末端的3个结构域即2个CCP区和1个SP区是丝氨酸蛋白酶的活性中心,激活补体主要依靠SP区发挥丝氨酸蛋白酶的作用,裂解补体C4和C2,产生C3转化酶。所以MASP2为MBL和几种(H、M和L)纤维胶原素激活补体所必需,在凝集素途径中发挥至关重要的作用。在患有多重感染和自身免疫性疾病的患者,首次描述MASP2缺损是由外外显子3所编码的功能区CUB1区产生GAC120GGC点突变,导致编码产物由酸性氨基酸天冬氨酸(Asp, D)变为中性氨基酸甘氨酸(Gly,G),即D120G突变,等电点发生了改变。因此,我们认为MASP2缺损是各种病原微生物反复感染的可能病因之一,而快速进行MASP2基因分型可能预测感染性疾病风险。目前,尚未见血浆MASP2浓度与上感关系的报道,故我们分析了上呼吸道感染儿童血浆MASP2水平并探讨其意义。第一章应用HRM技术检测MASP2基因D120G突变高分辨率熔解曲线(high-resolution melting, HRM)分析技术检测基因点突变具有以下优点：成本低,不需要设计昂贵的探针；在聚合酶链式反应(polymerase chain reaction,PCR)结束后,直接进行熔解曲线分析；快速、高通量,一次可以同时检测96或384个样本；闭管操作,防止样本脱氧核糖核酸(deoxyribonucleic acid,DNA)在PCR扩增后转移过程中污染；经过HRM分析处理后的PCR扩增产物还可以直接进行DNA测序,十分简洁便利。因此HRM技术已广泛应用于生命科学、食品安全、医学、畜牧业等各个领域的研究工作中。基于HRM上述特点,本研究拟应用HRM技术,建立检测分析人MASP2基因D120G突变的方法。材料与方法：1、标本：共收集153例全血标本,采用常规饱和酚/氯仿法提取外周血基因组DNA。2、点突变分析方法：(1)针对MASP2基因D120G变异位点设计PCR扩增体系及优化PCR条件。通过PCR定点诱变构建突变型纯合子基因,本科室构建保存MASP2基因片段经测序验证为野生型纯合子基因从而获得各种基因型样品。(2)建立检测MASP2基因D120G位点突变的HRM分析技术。针对变异位点设计HRM分析的引物,利用经测序已知的基因型样品优化HRM分析条件,从而建立分析MASP2基因的HRM分型方法。(3)根据HRM的分析结果,从各种基因型中选取一定数量的样品进行测序分析,以验证评价该方法的准确性；并且选取每种MASP2基因野生纯合子及突变纯合子各10个样本进行批间及批内重复性实验；对MASP2基因的野生纯合子和突变纯合子样本进行了不同比例混合,得到不同杂合比例质粒模板进行HRM检测突变杂合子灵敏度下限；将质粒DNA模板作10倍梯度稀释进行5个浓度检测,进行HRM检测,分析该方法检测敏感性。3、统计学分析：使用的统计学分析软件为SPSS16.0.结果：建立了稳定可靠的HRM检测体系。此体系中,所选取的扩增目的DNA序列以及所设计的引物的特异性高。MASP2基因的野生纯合子A/A,杂合子A/G,突变纯合子G/G三种基因型能够在HRM上进行明显的区分。用此方法分析广州地区153份人基因组DNA样品(123例为上感患者DNA样本,30例为乙肝患者DNA样本),从每组基因型中随机选出部分样品进行测序对照,结果符合率为100%,证实了该方法的准确性很好；进行批内、批间重复实验,发现10次实验的变异系数(CV)均小于0.1%,证实了该方法的稳定性很好；在10倍稀释试验中,5个浓度均可以准确分型,可以知道1 pg浓度质粒仍然可以用于此检测体系；在不同杂合比例模板HRM分析得知,该体系可以用于检测MASP2基因突变型杂合子比例下限为10%。结论：本课题研究中建立的针对MASP2基因的HRM分析方法能够快速、准确地对MASP2基因进行突变检测,并且重复性和稳定性好、敏感性高、体系可靠。本研究对中国南方人群进行检测,发现所检测的153人中不存在MASP2基因D120G突变型纯合子个体,只有1例D120G突变型型杂合子,推测这种基因型在中国南方地区少见。本研究分析MASP2基因D120G突变建立了一种简单、快速、高通量、经济和灵敏的检测方法,为研究MASP2基因与疾病的关系提供了一项有用的技术。第二章MASP2在小儿上呼吸道感染中的意义已报道MASP2与多种疾病有关,但其与URTI的关系目前不得而知。本章探讨血浆MASP2水平在儿童URTI中的意义。方法：选取2014年10月至12月在南方医院儿科门诊接受诊疗的103例URTI患儿作为研究对象,另选取同期健康体检的正常儿童为对照组,计35例。测定其血浆MASP2和C反应蛋白(C-reaction protein,CRP)浓度并进行白细胞计数(white blood cell,WBC).根据CRP和WBC值、感染不同时期及有无治疗,URTI儿童分为CRP升高组(n=48)与正常组(n=54)、WBC升高组(n=61)与正常组(n=40)、感染早期未用药组(n=68)与感染中后期已用药组(n=35)。根据不同分组分别做统计学分析。应用生物信息学分析MASP2基因与急性期反应蛋白MBL基因、典型急性期反应蛋白CRP基因是否存在共同的应激元件。结果：URTI组血浆MASP2浓度显著高于对照组(P＜0.001),CRP升高组血浆MASP2浓度与CRP值正相关(r=0.310,P0.05),WBC升高组MASP2水平与WBC值显著正相关(r=0.392,P0.01),感染早期未用药组MASP2浓度显著高于感染中后期已用药组(P0.01)。应用生物信息学分析得知,MASP2基因与急性期反应蛋白MBL基因及典型性急性期反应蛋白CRP基因存在共同应激元件一一肝细胞核因子4α(hepatic nuclear factors-4α,HNF-4α)作用区。结论：MASP2蛋白可能为急性期蛋白,血浆MASP2水平可作为诊断小儿上感的一个参考指标。
[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.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R725.6

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