应用噬菌体随机肽库筛选阴道毛滴虫特异性B细胞表位

发布时间：2019-05-24 09:25

【摘要】：背景与目的阴道毛滴虫(Trichomonas vaginalis, Donne1837),是一种主要寄生于人体泌尿生殖道的鞭毛虫,它所引起的阴道毛滴虫病是常见的性传播疾病之一,据WHO统计,全球每年约有1.8亿人感染阴道毛滴虫,同时阴道毛滴虫的广泛流行还增加了HIV病毒和支原体的感染率。目前对于阴道毛滴虫病的快速诊断方法的灵敏度及特异度有限,阴道毛滴虫疫苗的研发工作也相对滞后。本研究运用分子生物学、免疫学和生物信息学的方法技术,运用噬菌体随机7肽库淘选阴道毛滴虫的特异性B细胞表位,并对淘选结果进行免疫原性,敏感性及特异性检测,旨在为阴道毛滴虫病的快速诊断方法及未来疫苗的研发提供理论基础。材料与方法 1.将采集到的阴道毛滴虫临床分离株(取自于郑州大学第五附属医院检验科)接种于TYM培养基进行连续培养,达到纯培养后将虫体分别制作为全虫抗原和冻存。 2.将全虫抗原经背部皮下多点注射免疫实验用兔(购自于河南省实验动物中心),每隔2w加强免疫2次,末次免疫后ELISA检测抗体效价,达标后采集、分离并纯化免疫血清,并进一步经Western-Blot和间接免疫荧光检测其免疫原性。 3.以纯化后兔抗阴道毛滴虫IgG对噬菌体随机7肽库(购自于美国NEB公司)进行3轮的亲和淘选,ELISA检测淘选结果,并将阳性噬菌体克隆送往生物公司进行测序,将测序结果分别与Genbank上的阴道毛滴虫已知蛋白序列进行比对,并在Protscale在线服务器进行相关生物信息学分析。 4.将选取的阳性噬菌体克隆经背部皮下多点注射免疫BALB/c小鼠(购自于河南省实验动物中心),并同时设立阳性对照、阴性对照及溶剂对照,每隔7d免疫1次,共免疫3次。末次免疫后采集并分离免疫血清,进行ELISA和Western-Blot检测免疫原性,并通过间接免疫荧光试验研究阳性噬菌体展示肽的虫体定位。 5.建立阴道毛滴虫小鼠皮下感染模型,每日观察脓肿形成情况及脓液中活虫的数量。以感染血清通过ELISA的方法检测阳性噬菌体克隆的敏感性及特异性。 6. ELISA检测阳性噬菌体表位肽免疫小鼠血清中细胞因子IL-4和IFN-y的水平。结果 1.阴道毛滴虫在48h左右达到最高生长密度,36～48h为其对数生长期,连续培养3代后即达到纯培养。SDS-PAGE结果表明全虫抗原的蛋白条带分布在10～150kDa之间,其中30-70kDa之间的蛋白条带最为密集。 2. ELISA检测的结果显示阴道毛滴虫全虫抗原经免疫实验用兔后可引起较强的免疫反应,产生的抗体滴度大于1：106。Western-Blot发现共有15条的全虫抗原的蛋白成分可被兔免疫血清识别,比小鼠免疫血清多出3条。免疫荧光染色的结果表明虫体可与全虫抗原的兔免疫血清发生免疫反应,呈现红色荧光。 3.经过3轮的亲和淘选,共得到14个阳性噬菌体克隆,P1～P14。P1～P14与Genbank上已知的阴道毛滴虫蛋白序列无一级结构的相似性。综合噬菌体ELISA及Protscale在线服务器的生物信息学分析结果,P3、P5和P11的免疫原性相对较强。 4. ELISA结果表明P3、P5、P11免疫BALB/c小鼠后都能够引起较强的免疫反应,差异有统计学意义(P0.05)。Western-Blot的结果显示P11噬菌体克隆的免疫血清可以特异性识别全虫抗原在42kDa左右的蛋白条带。免疫荧光染色结果表明P3和P5免疫血清组的虫体呈现出较弱的绿色荧光,而P11免疫血清组则在虫体的膜部呈现出亮绿荧光。 5.皮下接种活虫的小鼠可出现脓肿,在脓液中10d内可检出活虫。ELISA结果表明P3、P5和P11均具有较强的特异性(P0.05),P11的敏感性为1μg/ml,P3的敏感性为2μg/ml,P5的敏感性为3μg/ml。 6. ELISA结果表明P3、P5和P11免疫完成后1w时小鼠血清中IL-4和IFN-γ的水平均高于M13组和PBS组(P0.05),IL-4的升高较为显著,P3、P5和P11这3组之间差异无统计学意义(P0.05)。结论 1.阴道毛滴虫全虫抗原具有良好的免疫原性,其兔免疫血清具有较高的抗体滴度。 2.应用噬菌体随机7肽库淘选出14个阴道毛滴虫模拟B细胞表位。其中P3、P5和P11具有良好的免疫原性,并具有较强的特异性及敏感性,提示这些模拟表位在研制特异性诊断试剂方面可能有潜在应用价值。 3.P3、P5和P11免疫小鼠后可以使小鼠产生体液免疫及细胞免疫,以体液免疫为主。
[Abstract]:Background and Purpose Trichomonas vaginalis, Donne1837, is a kind of trichinella, which is mainly parasitic in the genitourinary tract of the human body. The trichomonas vaginalis is one of the most common sexually transmitted diseases. According to the statistics of the WHO, about 180 million people are infected with vaginal hair drops every year. The widespread popularity of Trichomonas vaginalis also increases the infection of HIV and mycoplasma The sensitivity and specificity of the rapid diagnostic method for Trichomonas vaginalis are limited, and the development of Trichomonas vaginalis vaccine is also relatively slow After using the method of molecular biology, immunology and bioinformatics, the specific B-cell epitopes of Trichomonas vaginalis were selected by using the phage-random 7-peptide library, and the immunogenicity, the sensitivity and the specific test were carried out on the result of the panning. The purpose of this study is to provide a theoretical basis for the rapid diagnosis of trichomonas vaginalis and the development of future vaccines. A. Material The method comprises the following steps of:1, inoculating the collected trichomonas vaginalis clinical isolates (taken from the Fifth Affiliated Hospital of Zhengzhou University) to a TYM culture medium for continuous culture, 2. After the last immunization, the titer of the antibody was detected by ELISA, after the last immunization, the antibody was collected and separated. The serum was purified and the serum was purified by Western-Blot and indirect immunofluorescence. 3. After purification, the rabbit anti-vaginal trichomonas IgG was used for the 3-wheel affinity panning of the phage-random 7-peptide library (available from NEB company in the United States), the panning result was detected by ELISA, and the positive phage clone was sent. sequencing the biological company, comparing the sequencing result with the known protein sequence of the Trichomonas vaginalis in Genbank, and carrying out the sequencing on the Proscale online server, Relevant bioinformatics analysis.4. The selected positive phage clones were immunized with the back subcutaneous multi-point injection of the BALB/ c mice (available from the laboratory animal center in Henan Province), and the positive control, negative control, and solvent control were also established at the same time, every 7 D. The immunosera were collected and isolated after the last immunization, and the immunogenicity was detected by ELISA and Western-Blot, and positive by indirect immunofluorescence test. 5. Establishment of a subcutaneous infection model of Trichomonas vaginalis, and daily observation of the abscess. The number of live insects in the case and in the pus is detected by the method of ELISA in the infected serum. The sensitivity and specificity of phage clone.6. The positive phage epitope peptide was detected by ELISA. factor The results showed that 1. Trichomonas vaginalis reached the highest growth density at about 48 h and 36-48 h. The results of SDS-PAGE show that the protein bands of the whole worm antigen are between 10 and 150 kDa. 2. The results of the ELISA test showed that the whole worm antigen of Trichomonas vaginalis can elicit a strong immune response after being immunized with rabbit, and the antibody titer produced is greater than 1:106. Western-Blot has found a total of 15 protein components of the whole worm antigen. It can be recognized by rabbit immune serum, and is 3 more than that of mouse immune serum. The result of immunofluorescence staining shows that the worm can be combined with the whole worm. The immune response of the rabbit immune serum of the antigen showed red fluorescence.3. After 3 rounds of affinity panning,14 positive phage clones, P1 to P14, P1 to P14 and Genban were obtained. The similarity of the first-order structure of the known Trichomonas vaginalis protein sequence on k. Bioinformatics of the comprehensive phage ELISA and the on-line server of Proscale The results showed that the immunogenicity of P3, P5 and P11 was relatively strong. The results of the immunofluorescent staining show that the insect bodies of the P3 and P5 immune serum groups show a weak green color. Light, and the P11 immune serum group showed bright green fluorescence in the membrane part of the insect body. The results of ELISA showed that P3, P5 and P11 had strong specificity (P0.05), and the sensitivity of P11 was 1. m The sensitivity of P3 was 2.mu. g/ ml, and the sensitivity of P5 was 3.mu. g/ ml. P3 There was no significant difference between the three groups of P5 and P11 (P0.05). The whole worm antigen of Trichomonas vaginalis has good immunogenicity, and the rabbit immune serum has higher antibody 2. The B-cell epitopes of 14 vagintrichomonas vaginalis were selected by using the phage-random 7-peptide library. The P3, P5 and P11 had good immunogenicity and had a strong specificity. And sensitivity, suggesting that these simulated epitopes may have potential application value in the development of specific diagnostic reagents.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.73

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