宫颈癌患者核糖核酸酶抑制因子基因突变分析和常见食源性致病菌快速检测技术体系的建立

【作者】 卢行安；

【导师】 崔秀云；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 目的：核糖核酸酶（RNase）是降解RNA的酶类，核糖核酸酶抑制因子（ribonuclease inhibitor，RI）能和碱性RNase（AKR）结合而抑制其活性，从而减少RNA的降解。核糖核酸酶抑制因子（RI）是一种存在于细胞浆中的50KD的酸性蛋白质，广泛分布与哺乳动物的各种组织器官中，其中在人的胎盘中含量最为丰富。血管生成因子（angiogenin，Ang）是一种单链的碱性蛋白质，分子量为14KD，属于核糖核酸酶（RNase）超家族的一员，其氨基酸序列与核糖核酸酶A有35％的一致性。血管生成因子主要催化18S和28S rRNA的有限水解，但它重要的生物学功能是具有诱导新血管网形成的作用。肿瘤生长依赖于其周围新血管的生成，以便给其提供生长所需的必要物质。RI是由7个亮氨酸重复序列排列折叠而成，每个重复单位含有一个短的β片层和一个长的α螺旋，β片层排列在马蹄形的内表面形成平行的β片层，而α螺旋装饰外表面，7个亮氨酸重复单位有规律的环状排列是N末端C末端在空间上较为接近。RI可与RNase A以1：1化学计量比紧密结合，对其产生竞争性抑制作用，以极低的Ki值(4×10^-14 mol／L)控制胞内RNA水平。血管生成因子的氨基酸残基组成与核糖核酸酶A（RNase A）具有35％的同源性，它们有着极其相似的空间结构都可以作为配体与RI结合。而RI-Ang复合体的解离常数要比RI-RNase A复合体低约60倍。RI抑制肿瘤的机制目前认为包括两个方面：①RI能强烈抑制血管生成因子的血管生成活性。②RI能阻断肿瘤细胞对于血管生成因子的黏附，防止瘤细胞又黏附到血管生成因子上被带到新生成的血管上。方法：核糖核酸酶抑制因子基因（RNH）位于11号染色体短臂，即11P¹⁵。此部位分布着许多重要的基因，如ras基因。RNH基因包括11个外显子，10个内含子，为了进一步研究RNH基因与肿瘤发生的关系，RI基因是否是抑癌基因，本实验设计21对引物，利用PCR-SSCP方法，研究核糖核酸酶抑制因子全基因在肿瘤细胞中有无突变，采用正常人的全血标本与宫颈癌患者全血标本。为了增强PCR的检测效果，阳性内参采用β-actin。对于宫颈癌患者的核糖核酸酶抑制因子全基因的突变研究尚没有成型SSCP分析方法的条件，所以在实验中对于各种条件进行了初步的摸索和优化。结果：PCR实验结果表明，从普通琼脂糖凝胶电泳可以看出正常人和宫颈癌患者都扩增出了相应的21个片段，电泳图谱未显示有差别。SSCP技术在不含变性剂的中性聚丙烯酰胺凝胶中电泳时，DNA单链的迁移率除与DNA链的长短有关外，更主要的是取决于DNA单链所形成的构象。此次聚丙烯酰胺凝胶电泳尽量避免其影响因素，采用4％的聚丙烯酰胺凝胶以提高检出率。实验中扩增的片段最小231bp，最大970bp。结论：SSCP分析的结果显示正常人与宫颈癌患者的核糖核酸酶抑制因子基因未发现其在遗传学上的差异，但尚不能肯定其他肿瘤没有发生突变。创新点：1．首次对宫颈癌患者核糖核酸酶抑制因子基因突变进行了分析；2．设计了全基因序列引物，共21对引物；3．建立了SSCP分析核糖核酸酶抑制因子基因突变的最适条件。目的：据WHO估计，全世界每年发生食源性疾病数十亿人，每年几乎有二百万儿童死于腹泻，而大部分腹泻是由被微生物污染的食品和水引起，其中66％以上是由细菌性致病菌所致。常见的食源性致病菌主要有沙门氏菌、空肠弯曲菌、单核细胞增生李斯特氏菌、大肠杆菌O₁₅₇：H₇、志贺氏菌、金黄色葡萄球菌、副溶血弧菌、霍乱弧菌等。对上述食源性致病菌的检测在一定程度上可以有助于其得到有效的控制；传统的检测程序复杂，所用试剂繁多，费时费力，灵敏度低，假阴性比较严重，已经不能适应现代检食品安全的要求。PCR方法、实时荧光PCR方法、全自动免疫荧光方法等快速检测方法是近年了发展起来的全新检测技术，具有快速、简便、灵敏度高、特异性强、自动化程度高等优点。本研究着重于常见致病菌快速检测方法体系的建立，并应用于样品的检测；同时对于检出的沙门氏菌进行流行病学调查，针对传统的检测方法鉴定到血清型后，依赖详细评价阳性案例和一批适当的对照，确定调查与特殊疾病的关联因素，不能区分同一血清型不同菌株同源性的特点，本研究用脉冲凝胶电泳方法对其进行分子分型，为研究其流行病学特征和同源性提供分子生物学技术依据。方法：①本研究采用PCR和实时荧光PCR技术，根据尽量避免假阴性（漏检）的基本原则进行靶基因的选择。再依据尽量从理论上避免检测的假阳性及假阴性并提高检测灵敏度的原则来设计引物和探针。从基因序列库中下载所有已经公开发表的靶基因序列，运用DNAStar软件中的SeqMan和MegAlign模块进行排序比较。利用ABI Primer Express 2.0，DNAStar PrimerSelect等寡核甘酸设计软件在保守区段设计多套引物和探针，确保引物之间、引物与探针之间不会形成稳定的二聚体，然后在GenBank上做BLAST分析，确认所设计的引物和探针与其它生物物种的基因序列不存在互补配对现象，然后进行引物、探针筛选，针对8种食源性致病菌分别建立普通PCR和实时荧光PCR检测体系并进行优化。②采用平板菌落记数的做为对照试验，以确定各个检测方法的灵敏度。在独立进行的灵敏度的验证实验中，每个稀释梯度均与国标法和行标法进行了对照。③同时采用了对照菌株进行了检测方法的特异性实验。④运用以上建立的方法，对鸡胴体淋洗液样品进行沙门氏菌检测；即样品经过前增菌和选择性增菌后，分别采用4种不同的方法进行检测，即普通PCR方法、实时荧光PCR方法、免疫学方法（VIDAS）和传统的微生物检验方法。⑤PFGE方法对分离到的7株鼠伤寒沙门氏菌进行增菌，制成一定浓度的菌悬液后，制备琼脂糖菌体包埋胶块，用溶菌酶、蛋白酶K和TE缓冲液依次处理提取菌体DNA后，用限制性内切酶XbaI消化胶块，再用脉冲凝胶电泳仪进行DNA分子分型；同时使用全球统一参考菌株沙门菌Braenderup血清型H9812，XbaI酶切，作为分子量标准（Marker）；对电泳后的图谱，采用BioNumerics软件对其同源性进行聚类分析比较，菌株的同源性用相似性系数（矩阵）和百分数表示。结果：①建立了沙门氏菌、空肠弯曲菌、单核细胞增生李斯特氏菌、大肠杆菌O₁₅₇：H₇、志贺氏菌、金黄色葡萄球菌、副溶血弧菌以及霍乱弧菌8种常见食源性致病菌的普通PCR和实时荧光PCR快速检测的标准操作程序。其核心内容包括靶基因的确定、特异性扩增引物和探针的设计和筛选以及扩增反应体系的优化。②对所研制的8种常见食源性致病菌的普通PCR和实时荧光PCR快速检测方法最优反应体系的灵敏度进行了测试，结果显示：单核细胞增生李斯特氏菌核酸提取法优于直接煮沸法，其他的细菌没有显著性差异；实时荧光定量PCR检出限数小于普通PCR，前者检测体系的灵敏度可达到了10²cfu／mL左右：8种常见食源性致病菌中单核细胞增生李斯特氏菌、大肠杆菌O₁₅₇：H₇和志贺氏菌的检出限比较低；上述8种常见致病菌的检测方法的灵敏度均优于国标法。③采用对照菌株测试了各个检测体系的特异性，结果表明各个检测体系对所有对照菌株都没有阳性检测信号产生，显示其特异性极高，没有出现一例非特异性的检测结果。④应用上述建立的方法，共检测了56份鸡胴体淋洗液样品中的沙门氏菌，普通PCR检出阳性样品34份，实时荧光PCR阳性样品36份，VIDAS阳性样品28份；PCR和实时荧光定量PCR均无假阳性和假阴性结果。结果显示该3种检测方法均可以用于鸡胴体中沙门氏菌的快速检测。⑤针对以上检出的鼠伤寒沙门氏菌，进行菌体DNA经酶切、脉冲凝胶电泳后，沙门氏菌参考菌株和每个待测菌株均出现了15条以上的条带，图谱经软件分析后结果显示：FJ003号和FJ004号两个菌株的相似度约为96.2％，FJ001号和FJ002号相似度为95.8％，FJ007号和FJ001、FJ002号的相似度为83.8％，7个菌株彼此的相似度范围为61.0％～96.2％。按照85％相似度的评定标准，同一血清型的7株鼠伤寒沙门氏菌可以分成5个型。PFGE具有分辨力高，是研究鼠伤寒沙门菌分子流行病学较好的基因分型方法。结论：本课题成功建立了8种常见食源性致病菌一整套普通PCR和实时荧光PCR检测方法，并且把实时荧光PCR反应条件一致化，使之更具有可操作性；在靶基因选择方面的有创新，设计了有自主知识产权的引物和探针，具有灵敏度高、特异性强等特点；建立的快速检测方法适用于多种国外食物微生物权威方法的前增菌液，同时即可用于筛选，又可用于确认；特别适用于检验检疫、疾病控制、临床诊断、产品质量检验等对上述致病菌的检测。本次试验采用Xba I酶作为鼠伤寒沙门菌染色体DNA的限制性内切酶，产生的电泳条带较理想，每株约产生16～25条电泳带，说明本次试验所选用的限制性内切酶和电泳条件适合鼠伤寒沙门菌的分型；按照85％相似度的评定标准，将7株菌株分成5个型。本实验方法可以帮助确定同一血清型菌株之间的亲缘关系。创新点：1．成功建立了常见食源性致病菌的普通PCR和实时荧光PCR检测方法，方法具有技术先进、特异性好、灵敏度高、快速准确等特点，检出限可达10²cfu／mL，与传统检测方法相比，灵敏度提高了3个数量级；2．新建立的方法已经通过了行业标准的审定；3．方法中有自主知识产权的引物和探针序列；4．方法中含8种常见的致病菌的检测，致病菌的数目比较多；5．成功应用PFGE对检出的鼠伤寒沙门氏菌进行了分子分型，分型精确，确定了同一血清型菌株之间的亲缘关系。更多还原

【Abstract】 Objective: Ribonuclease Inhibitor （RI） is a 50KD acidic protein existing in cytoplasm, which lies in all tissues and organs of the mammals extensively, especially affluent in the placenta of humans. RI can decrease the RNA degradation by RNase A, because RI can inhibit the activity of RNase A for the combination with alkaline RNase. Angiogenin （Ang） is a single strand 14 KD alkaline protein, belonging to RNase A superfamily, and which can induce the formation of newly formed vascular net. The growth of tumor depends on the formation of the newly formed circumambient vascular, they can supply the necessary nourishment for the growth of tumor. There is 35% homology between the amino acid residues of the Ang and RNase A and their similar spatial structure can both combine with RI as ligand. The dissociation constant of the RI-RNase complex was higher than the RI-Ang complex by 60 times, and RI can inhibit the formation of the newly formed vascular effectively. The objective of the paper was to establish an effective method for analysis of gene mutation of Ribonuclease Inhibitor （RNH） in the blood cells of patients with cervical carcinoma, what’s more, the RNH of 30 normal persons and 18 patients with cervical carcinoma were detected by means of the method established in the experiment.Method: The RNH lies in the galianconism of the No. 11 chromosomes (11P^15.5), the whole length of the gene is 12310bp, including in 11 extron and 10 intron. The target gene fragments must contain the whole sequence of extron when primers were designed, and several target gene fragments contained two adjacent extron and parts of the adjoining introns, and therewere 11 pairs of such primers. But the longer intron were divided into several fragments eligibly for amplification, and all the target gene fragments were less than 1000 bp, and there were 10 pairs of such primers, so there were 21 pairs of primers in the experiment, and most of the target gene fragments were between 300-700bp. The conditions of Polymerase Chain Reaction （PCR） were optimized by using the genome DNA extracted from the normal persons’ blood cells, then the target gene fragments were amplified using the DNA extracted from the normal persons and the patients with cervical carcinoma as mould respectively, the 21 primers were designed as above. β-actin were adopted as the internal standard to verify the results of PCR. Single strand conformation polymorphism （SSCP） was used to the mutation analysis. The amplification products of the normal persons’ DNA were used for the optimization of the experiment. The gene mutations were judged by the difference of the electrophorestic mobility shift of the degenerative single strand DNA on the polyacrylamide gel. All the target gene fragments were amplified used the 21 designed primers after optimization.Results: The PCR products were undertaken the electrophoresis on the agarose gel, then the electrophorestic mobility shift of each product（s） of the 21 primers were compared, but there is no significant differences. The most optimized conditions of the SSCP in the experiments were as follows: The ratio of the acrylamide and Methylene-bis-Acrylamide was 49:1, the concentration was 4%, and glycerin was not contained, the thickness of the gel was 1 mm, the voltage was 220v, the temperature was 4℃, buffer solution was 0.5×TBE, the volume of the sample was 5 μ l. The groups of SSCP analysis were same as the agarose gel electrophoresis, the electrophorestic mobility shifts were obtained according to equation of M=Ld/Vt, the fine repeatabilities were checked by the General Linear Model. There were not significant differences in the results of the single strand DNA electrophorestic mobility shift in polyacrylamide gel electrophoresis. The results showed that there was not significant genetic difference between the gene of RI of the normal persons and patients with cervical carcinoma.Objective The aim of our study was to establish the techniques protocols for the rapid detecton of 8 species foodborn pathogen, and we compare the efficiency of different methods for rapid Salmonella detection in whole chicken rinses samples and to optimize the most appropriated method of detection. For the Salmonella typhimurium from the above samples, we also study the relationship among different strains, a molecular epidemiological analysis method — pulsed-field gel electrophoresis （PFGE） for the analysis of DNA restriction fragment length polymorphism （RFLP） in S. tyhpimurium was used.Methods Accroding to the pathogen’s high reserve sequence, designing the prime and probe, and the specificity and sensitivity of the rapid protococls were done to evaluate the new methods suitabiltiy. The microbiological culture, the broth culture-PCR, the real-time PCR and VIDAS methods were used for the detection of Salmonella spp. in Whole Chicken Rinses. Each Salmonella typhimurium strain isolated from Whole Chicken Rinses was enriched, the Bacteria Cell Suspension Buffer （CSB） were mixed with melted 1% SeaKem Gold:1% SDS agarose, then dispense part of mixture into appropriate well（s） of reusable plug mold. The total genome DNA in agarose plugs was extracted in turn with lysozyme solution, proteinase K solution and TE buffer. The agarose pulgs were digested with restriction enzyme Xba I, followed by pulsed-field gel electrophoresis （PFGE）. Salmonella ser. Braenderup H9812 standards was used. The bands were analyzed with statistics software.use BioNumerics software for database maintenance, tiff image normalization, and analysis and patterncomparisons.Results The rapid detection methods are more sensitive , specific and efficient, the detecting limite for PCR was about 10⁴cfu/mL, 10²cfu/mL for real-time PCR, suitable for Salmonella, Staphylococcus aureus, Shigella, Campylobacter jejuni, O₁₅₇.H₇, Listeria monocytogenes, Vibrio paraheamolyticus and Vibrio cholera. The processing is rapid and simple, will be a routine and practical protocols for detecting and identifying pathogenic microorganisms. Using the above methods for Salmonella, total 56 Whole Chicken Rinses samples were detected, Salmonella were founded in 34 samples by the PCR, 36 positive by Real-time PCR, and 28 by VIDAS. Conclusion The above three detecting method were suitable for the rapid detection of Salmonella in Whole Chicken Rinses. 7 S. tyhpimurium isolated from Whole Chicken Rinses were subtyed using PFGE, the lanes with the Salmonella ser. Braenderup H9812 and 7 S. tyhpimurium had above 15 bands, the similarity between of FJ001 and FJ002 is 95.2%, 96.2% similarity is found between FJ003 and FJ004, the similarity range among total 7 S. tyhpimurium is from 61.0% to 96.2%. Taking 85% similitude as criterion, 7 strains of S. tyhpimurium were divided into 5 PFGE types. The typeability, the high discriminatory power and the stability of PFGE-RFLP make this a valuable method to be used in conjunction with serotyping.更多还原