日本血吸虫含缬酪肽蛋白的克隆表达及其诊断价值

发布时间：2018-05-16 22:26

本文选题：日本血吸虫 + VCP基因　；参考：《江苏省血吸虫病防治研究所》2014年硕士论文

【摘要】：血吸虫病是一种严重危害人类健康的人畜共患病。据世界卫生组织统计，截止到2011年有超过2.43亿的血吸虫病人需要接受治疗，而国家疾病预防控制中心推算，2012年底全国有血吸虫病人240597例。血吸虫病主要影响农业和渔业等容易与疫水接触的人口，生态旅游的兴起以及游客离开常走的路，也使越来越多的游客感染血吸虫病；血吸虫病的传播一方面可由人口移徙至城市地区和难民流动引起，另一方面人口规模的不断扩大引起对水、电相应需求的增加，往往会导致可能助长疾病传播的开发计划和环境改变，引起血吸虫病的传播。对血吸虫病的预防主要以降低接触疫水和污染水源为主，具体措施为保证清洁的饮水、充足的卫生设施和适当的健康教育。我国主要流行日本血吸虫病，经过60多年的努力，我国血吸虫病的防治工作取得了举世瞩目的成就：在有血吸虫病流行的南方各省中，上海、广东、广西、福建、浙江省（市、自治区）阻断了血吸虫病传播，连续5年未发现当地感染血吸虫病例、病畜；四川、云南省丘陵山区和江苏省江滩地区达到了血吸虫病传播控制标准，居民、家畜血吸虫感染率降至1%以下；湖南、湖北、江西、安徽4省流行水平处于1%-5%之间，2010年耕牛血吸虫感染率为1.04%。血吸虫病防治活动的中心环节则是血吸虫病的诊断。诊断具体可用于流行区查病，了解疾病三间分布，在个体或群体水平上确定化疗对象、评价化疗效果以及传播控制或阻断后监测，为防治活动的计划、实施和防治效果评价等各环节提供必要的信息和科学依据。当前我国人群和耕牛血吸虫感染率等疫情指标总体已处于历史最低水平，四川、云南等省的丘陵地区和江苏省的江滩地区分别于2008、2009、2010年达到了血吸虫病传播控制标准，流行较重的湖南、湖北、江西和安徽的部分湖沼地区流行水平也处于1%-5%之间。在低度流行区，传统的病原学检测方法因其灵敏度低、漏诊率高而不适用，寻找一种敏感性高、特异性强、依从性好、快速、经济的理想诊断方法是当前面临的近切需要。研究人员从免疫学和分子生物学角度进行了大量的研究，探索了许多用于血吸虫病诊断的方法，与传统病原学检测方法相比，免疫学和分子生物学如PCR、LAMP等具有更高的灵敏度、特异度，漏诊率低、依从性好，具有较高的早期诊断价值，而免疫学诊断主要依赖抗原抗体反应，流行区病人体内特异性抗体(主要为IgG)因为在体内持续很长时间，即使治愈也不会很快消失，因此用现有的检测抗体的方法无法确定该病人是否治愈，即不能进行疗效考核，从而导致了对流行区人群过度化疗的现象。这不仅会造成吡喹酮的大量浪费，防治成本增大，而且在疫区长期反复服用吡喹酮，将有可能导致目前唯一的抗血吸虫药物——吡喹酮出现抗药性的潜在危险，而现阶段我国急感病人越来越少，发展有疗效考核的诊断方法成为亟待解决的重大问题。含缬酪肽蛋白质（valosin containing protein，VCP）是一种分布很广泛的ATP酶，是与多种细胞活性相关的三磷酸酸腺苷酶超家族中的一员，已被证明参与多种细胞活动，包括有丝分裂，同型膜融合，内质网相关降解机制调控核转录因子，，并参与泛素依赖的蛋白降解途径，有报道VCP与细胞凋亡、癌症的侵袭和转移相关。日本血吸虫VCP是本课题组采用蛋白质谱分析出的日本血吸虫虫卵组分抗原（107-121kDa）中的一种，该组分抗原（107-121kDa）早期被朱荫昌等人证明具有一定的疗效考核价值，而SjVCP是否具有诊断价值或潜在疗效考核价值还不得而知。本研究拟采用分子克隆方法，通过基因克隆和原核表达，对其表达产物进行纯化并评价其免疫诊断价值。研究内容主要包括：一日本血吸虫含缬酪肽蛋白（Sj-VCP）的基因克隆、原核表达及纯化根据Sj-VCP的基因编号，在GenBank中检索获得相关基因序列，运用BioXM、DNAMAN等生物信息学软件对其核酸序列、酶切位点、氨基酸序列、融合蛋白分子量和等电点等进行分析。结果Sj-VCP的DNA序列总长为2409bp，其中A+T占55.09%，含有195个酶切位点；经氨基酸序列分析，SjVCP含有酸性氨基酸179个碱性氨基酸119个；重组日本血吸虫含缬酪肽蛋白（rSjVCP）分子量为90.99KDa，等电点（PI）为6.030。提取日本血吸虫虫卵RNA，逆转录为cDNA，以此为模板PCR扩增日本血吸虫VCP基因，将其亚克隆至原核表达载体pET15b；重组质粒转化入E. coliBL21，在LB培养基中加入异丙基硫代半乳糖苷（IPTG）诱导目的基因表达，并采用包涵体纯化方法获取重组蛋白。因此，通过日本血吸虫含缬酪肽蛋白的基因克隆、原核表达及纯化，成功获得了重组日本血吸虫含缬酪肽蛋白（rSj-VCP）。二荧光定量PCR检测日本血吸虫含缬酪肽蛋白在尾蚴、童虫、成虫(雄虫、雌虫)、虫卵4个生活史阶段的表达情况分别提取日本血吸虫尾蚴、童虫、雌虫、雄虫、虫卵RNA，纯化后逆转录为cDNA第一链；设计目的基因(SjVCP)和内参基因(18s)引物，使两对引物的退火温度和扩增产物大小相近。以cDNA第一链为模板，根据设计的目的基因和内参基因引物荧光定量扩增不同血吸虫生活史期相应基因片段，数据分析采用2-ΔCT法。结果显示，日本血吸虫VCP基因转录的mRNA在尾蚴中水平最高，在童虫、虫卵、雄虫、雌虫中水平较低。因此，日本血吸虫在尾蚴、童虫、成虫(雄虫、雌虫)、虫卵4个生活史阶段均有VCP基因转录的mRNA，而在尾蚴中表达水平最高。三重组日本血吸虫含缬酪肽蛋白（rSj-VCP）的诊断价值研究 11只5-6周龄BALB/c小鼠，按A-K编号，分别感染40条尾蚴，构建小鼠感染日本血吸虫动物模型，于感染前和感染后每周采血并分离血清直至5周。以纯化的日本血吸虫含缬酪肽重组蛋白为抗原，建立ELISA法检测小鼠血清内日本血吸虫VCP特异性抗体，同时以日本血吸虫可溶性虫卵抗原检测小鼠血清，比较二者诊断结果。结果rSjVCP在诊断感染小鼠血清内特异性抗体时，除D号小鼠血清第一周为阳性以外，其余10只均在第二周达阳性，从第三周开始，抗体水平有下降趋势；而虫卵可溶性抗原在诊断感染小鼠血清时，结果于感染后第三周检测现阳性，感染后第四周检测均为阳性，且从第三周开始，血清中IgG抗体水平呈上升趋势。因此，重组日本血吸虫含缬酪肽蛋白具有早期诊断价值，可用于进一步的日本血吸虫早期诊断研究。
[Abstract]:Schistosomiasis is a zoonotic disease that seriously endangers human health. According to the WHO statistics, by the end of 2011, more than 243 million of Schistosoma patients need to be treated, and the National Center for Disease Control and prevention reckon that there are 240597 cases of schistosomiasis in the country at the end of 2012. The population of water, the rise of ecotourism and the way tourists leave often make more and more tourists infected with schistosomiasis; the spread of schistosomiasis can be caused by the migration of the population to the urban areas and the flow of refugees on the one hand. On the other hand, the increasing population size causes the increase of the corresponding demand for water and electricity, which often leads to the increase of the demand for water and electricity. The development plan and environmental changes that can promote the spread of the disease cause the spread of schistosomiasis. The prevention of schistosomiasis is mainly to reduce the contact water and the polluted water. The concrete measures are to ensure clean drinking water, adequate sanitation facilities and appropriate health education.
China's main epidemic of schistosomiasis, after more than 60 years of efforts, has made remarkable achievements in the prevention and control of schistosomiasis in China: in the southern provinces of the epidemic of schistosomiasis, Shanghai, Guangdong, Guangxi, Fujian and Zhejiang (city, autonomous region) have blocked the transmission of schistosomiasis, and have not found the local infection of schistosomiasis for 5 consecutive years, The epidemic level of schistosomiasis in Sichuan, hilly and hilly areas of Yunnan province and Jiangsu province reached the standard of schistosomiasis transmission control. Residents, the infection rate of domestic animal Schistosoma decreased to less than 1%; the prevalence levels of the 4 provinces in Hunan, Hubei, Jiangxi and Anhui were between 1%-5%, and the central link of the infection rate of schistosomiasis in the year of 2010 was the blood of 1.04%. schistosomiasis control. Diagnosis of fluke disease. The diagnosis can be used in epidemic areas to check the disease, understand the distribution of three diseases, determine the target of chemotherapy at the individual or group level, evaluate the effect of chemotherapy and the monitoring of transmission control or after blocking, and provide the necessary information and scientific basis for the plan of prevention and control, the implementation of the prevention and control effect evaluation and so on. The population and the infection rate of Schistosoma japonicum were at the lowest level in history. The hilly areas of Sichuan, Yunnan and Jiangsu province reached the standard of schistosomiasis transmission control in 200820092010 years, and the epidemic levels of some lakes in Hunan, Hubei, Jiangxi and Anhui were also in 1%-5%. In the low epidemic area, the traditional method of etiological detection is not suitable because of its low sensitivity and high rate of missed diagnosis. To find an ideal diagnostic method with high sensitivity, specificity, good compliance, good compliance, rapid and economical is the near cut needs. Researchers have studied a lot of research from the angle of immunology and molecular biology. Many methods for the diagnosis of schistosomiasis, compared with the traditional methods of pathogenic detection, immunology and molecular biology, such as PCR, LAMP, have higher sensitivity, specificity, low leakage rate, good compliance, and high early diagnostic value. Immunological diagnosis mainly depends on antigen antibody reaction, and the specific antibody in the epidemic area patients. It is mainly IgG) because it lasts for a long time, even if the cure does not disappear quickly, so it is impossible to determine whether the patient is cured by the existing method of detecting antibody, that is, it can not be evaluated, which leads to excessive chemotherapy of the population in the epidemic area, which does not only cause a large waste of praziquantel and the cost of prevention and cure is increased. And the long-term use of praziquantel in the epidemic area is likely to lead to the potential risk of resistance to praziquantel, the only anti schistosomiasis drug at present. At the present stage, there are fewer acute patients in China, and the development of the diagnostic method for the evaluation of therapeutic efficacy has become a major problem to be solved.
Valosin containing protein (VCP) is a widely distributed ATP enzyme, a member of the Mei Chao family of adenosine acid adenosine three, which is associated with a variety of cell activities. It has been proved to be involved in a variety of cell activities, including mitosis, Homo membrane fusion, and endoplasmic reticulum related degradation mechanism to regulate nuclear transcription factors and participate in ubiquitin The dependent protein degradation pathway has been reported that VCP is associated with apoptosis, invasion and metastasis of cancer. Schistosoma japonicum VCP is one of the group of Japanese Schistosoma japonicum egg component antigen (107-121kDa) analyzed by protein mass spectrometry (107-121kDa). The component antigen (107-121kDa) was proved to be of a certain therapeutic value by Zhu Yinchang et al. It is not known whether SjVCP has diagnostic value or potential efficacy assessment. This study intends to use molecular cloning and cloning and prokaryotic expression to purify the expression products and evaluate its diagnostic value. The main contents of this study include:
Cloning, prokaryotic expression and purification of valerib protein (Sj-VCP) from Schistosoma japonicum
According to the gene number of Sj-VCP, the sequences of related genes were retrieved in GenBank, and the nucleic acid sequence, the enzyme cutting site, the amino acid sequence, the molecular weight of the fusion protein and the isoelectric point were analyzed by using BioXM, DNAMAN and other bioinformatics software. The results showed that the total length of DNA sequence of Sj-VCP was 2409bp, in which A+T accounted for 55.09% and contains 195 enzyme cutting sites. After analysis of amino acid sequence, SjVCP contains 119 acidic amino acids and 179 basic amino acids, and the recombinant Japanese Schistosoma japonicum (rSjVCP) has a molecular weight of 90.99KDa, and the isoelectric point (PI) is 6.030. extracted from the egg RNA of Schistosoma japonicum, and reverse transcriptase cDNA, which is used as a template PCR to amplify the VCP gene of Schistosoma japonicum, and subcloned to the prokaryotic expression. Vector pET15b, recombinant plasmid was transformed into E. coliBL21, and isopropyl thiosulfate galactoside (IPTG) was added to the LB medium to induce the expression of the target gene, and the recombinant protein was obtained by the inclusion body purification method. Therefore, the recombinant Japanese Schistosoma japonicum was successfully obtained by cloning and prokaryotic expression and purification of valerein protein in Schistosoma japonicum. Containing valerein protein (rSj-VCP).
Two fluorescent quantitative PCR was used to detect the expression of Valin protein in Schistosoma japonicum, cercariae, larvae, adults (males, females) and eggs in 4 life cycle stages.
Extraction of Schistosoma japonicum cercariae, children, females, males, eggs RNA, purified and retroviral to the first chain of cDNA, and designed the target gene (SjVCP) and the internal reference gene (18S) primers to make the annealing temperature of the two pairs of primers similar to the size of the amplified products. The first chain of cDNA was used as the mold plate, and the fluorescence quantitative primers were primed according to the designed target gene and the internal reference gene. 2- Delta CT was used to amplify the corresponding gene fragments in the life history of different Schistosoma Schistosoma. The results showed that the mRNA of the VCP gene of Schistosoma japonicum was the highest in the cercariae, and the level was low in the larvae, eggs, males and females. Therefore, the 4 life history stages of Schistosoma japonicum were VCP based in the life history of the cercariae, the children, the adult (male, female) and the eggs. The transcription level of mRNA was the highest in cercariae.
Three diagnostic value of recombinant Schistosoma japonicum containing valibutycasin protein (rSj-VCP)
11 5-6 week old BALB/c mice infected with 40 cercariae were infected by A-K, and the mice infected with Schistosoma japonicum were constructed. The blood was collected and separated from the serum to 5 weeks before and after infection. The purified Japanese Schistosoma japonicum containing the recombinant protein of valerein as antigen was used to establish a ELISA method to detect the specific resistance of VCP in the sera of mice. Meanwhile, the serum levels of Schistosoma japonicum soluble egg antigen were detected, and the diagnostic results of the two groups were compared.
Results in the diagnosis of serum specific antibodies in infected mice, rSjVCP was positive except for the first week of D mice serum, and the other 10 were positive in the second week. From third weeks, the antibody level had a downward trend, and the soluble antigen of the eggs was detected in the infected mice serum, and the results were detected at third weeks after infection and after infection. All four weeks of detection were positive, and the level of IgG antibody in serum was rising from third weeks. Therefore, the recombinant protein of recombinant Japanese Schistosoma japonicum has early diagnostic value and can be used for further diagnosis of Schistosoma japonicum.
【学位授予单位】：江苏省血吸虫病防治研究所
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R532.21

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