本文選題：日本血吸蟲 + VCP基因��； 參考：《江蘇省血吸蟲病防治研究所》2014年碩士論文

【摘要】：血吸蟲病是一種嚴(yán)重危害人類健康的人畜共患病。據(jù)世界衛(wèi)生組織統(tǒng)計(jì)，截止到2011年有超過2.43億的血吸蟲病人需要接受治療，而國家疾病預(yù)防控制中心推算，2012年底全國有血吸蟲病人240597例。血吸蟲病主要影響農(nóng)業(yè)和漁業(yè)等容易與疫水接觸的人口，生態(tài)旅游的興起以及游客離開常走的路，也使越來越多的游客感染血吸蟲��；血吸蟲病的傳播一方面可由人口移徙至城市地區(qū)和難民流動(dòng)引起，另一方面人口規(guī)模的不斷擴(kuò)大引起對水、電相應(yīng)需求的增加，往往會(huì)導(dǎo)致可能助長疾病傳播的開發(fā)計(jì)劃和環(huán)境改變，引起血吸蟲病的傳播。對血吸蟲病的預(yù)防主要以降低接觸疫水和污染水源為主，具體措施為保證清潔的飲水、充足的衛(wèi)生設(shè)施和適當(dāng)?shù)慕】到逃?我國主要流行日本血吸蟲病，經(jīng)過60多年的努力，我國血吸蟲病的防治工作取得了舉世矚目的成就：在有血吸蟲病流行的南方各省中，上海、廣東、廣西、福建、浙江�。ㄊ�、自治區(qū)）阻斷了血吸蟲病傳播，連續(xù)5年未發(fā)現(xiàn)當(dāng)?shù)馗腥狙x病例、病畜；四川、云南省丘陵山區(qū)和江蘇省江灘地區(qū)達(dá)到了血吸蟲病傳播控制標(biāo)準(zhǔn)，居民、家畜血吸蟲感染率降至1%以下；湖南、湖北、江西、安徽4省流行水平處于1%-5%之間，2010年耕牛血吸蟲感染率為1.04%。血吸蟲病防治活動(dòng)的中心環(huán)節(jié)則是血吸蟲病的診斷。診斷具體可用于流行區(qū)查病，了解疾病三間分布，在個(gè)體或群體水平上確定化療對象、評價(jià)化療效果以及傳播控制或阻斷后監(jiān)測，為防治活動(dòng)的計(jì)劃、實(shí)施和防治效果評價(jià)等各環(huán)節(jié)提供必要的信息和科學(xué)依據(jù)。當(dāng)前我國人群和耕牛血吸蟲感染率等疫情指標(biāo)總體已處于歷史最低水平，四川、云南等省的丘陵地區(qū)和江蘇省的江灘地區(qū)分別于2008、2009、2010年達(dá)到了血吸蟲病傳播控制標(biāo)準(zhǔn)，流行較重的湖南、湖北、江西和安徽的部分湖沼地區(qū)流行水平也處于1%-5%之間。在低度流行區(qū)，傳統(tǒng)的病原學(xué)檢測方法因其靈敏度低、漏診率高而不適用，尋找一種敏感性高、特異性強(qiáng)、依從性好、快速、經(jīng)濟(jì)的理想診斷方法是當(dāng)前面臨的近切需要。研究人員從免疫學(xué)和分子生物學(xué)角度進(jìn)行了大量的研究，探索了許多用于血吸蟲病診斷的方法，與傳統(tǒng)病原學(xué)檢測方法相比，免疫學(xué)和分子生物學(xué)如PCR、LAMP等具有更高的靈敏度、特異度，漏診率低、依從性好，具有較高的早期診斷價(jià)值，而免疫學(xué)診斷主要依賴抗原抗體反應(yīng)，流行區(qū)病人體內(nèi)特異性抗體(主要為IgG)因?yàn)樵隗w內(nèi)持續(xù)很長時(shí)間，即使治愈也不會(huì)很快消失，因此用現(xiàn)有的檢測抗體的方法無法確定該病人是否治愈，即不能進(jìn)行療效考核，從而導(dǎo)致了對流行區(qū)人群過度化療的現(xiàn)象。這不僅會(huì)造成吡喹酮的大量浪費(fèi)，防治成本增大，而且在疫區(qū)長期反復(fù)服用吡喹酮，將有可能導(dǎo)致目前唯一的抗血吸蟲藥物——吡喹酮出現(xiàn)抗藥性的潛在危險(xiǎn)，而現(xiàn)階段我國急感病人越來越少，發(fā)展有療效考核的診斷方法成為亟待解決的重大問題。 含纈酪肽蛋白質(zhì)（valosin containing protein，VCP）是一種分布很廣泛的ATP酶，是與多種細(xì)胞活性相關(guān)的三磷酸酸腺苷酶超家族中的一員，已被證明參與多種細(xì)胞活動(dòng)，包括有絲分裂，同型膜融合，內(nèi)質(zhì)網(wǎng)相關(guān)降解機(jī)制調(diào)控核轉(zhuǎn)錄因子，，并參與泛素依賴的蛋白降解途徑，有報(bào)道VCP與細(xì)胞凋亡、癌癥的侵襲和轉(zhuǎn)移相關(guān)。日本血吸蟲VCP是本課題組采用蛋白質(zhì)譜分析出的日本血吸蟲蟲卵組分抗原（107-121kDa）中的一種，該組分抗原（107-121kDa）早期被朱蔭昌等人證明具有一定的療效考核價(jià)值，而SjVCP是否具有診斷價(jià)值或潛在療效考核價(jià)值還不得而知。本研究擬采用分子克隆方法，通過基因克隆和原核表達(dá)，對其表達(dá)產(chǎn)物進(jìn)行純化并評價(jià)其免疫診斷價(jià)值。研究內(nèi)容主要包括： 一日本血吸蟲含纈酪肽蛋白（Sj-VCP）的基因克隆、原核表達(dá)及純化 根據(jù)Sj-VCP的基因編號(hào)，在GenBank中檢索獲得相關(guān)基因序列，運(yùn)用BioXM、DNAMAN等生物信息學(xué)軟件對其核酸序列、酶切位點(diǎn)、氨基酸序列、融合蛋白分子量和等電點(diǎn)等進(jìn)行分析。結(jié)果Sj-VCP的DNA序列總長為2409bp，其中A+T占55.09%，含有195個(gè)酶切位點(diǎn)；經(jīng)氨基酸序列分析，SjVCP含有酸性氨基酸179個(gè)堿性氨基酸119個(gè)；重組日本血吸蟲含纈酪肽蛋白（rSjVCP）分子量為90.99KDa，等電點(diǎn)（PI）為6.030。提取日本血吸蟲蟲卵RNA，逆轉(zhuǎn)錄為cDNA，以此為模板PCR擴(kuò)增日本血吸蟲VCP基因，將其亞克隆至原核表達(dá)載體pET15b；重組質(zhì)粒轉(zhuǎn)化入E. coliBL21，在LB培養(yǎng)基中加入異丙基硫代半乳糖苷（IPTG）誘導(dǎo)目的基因表達(dá)，并采用包涵體純化方法獲取重組蛋白。因此，通過日本血吸蟲含纈酪肽蛋白的基因克隆、原核表達(dá)及純化，成功獲得了重組日本血吸蟲含纈酪肽蛋白（rSj-VCP）。 二熒光定量PCR檢測日本血吸蟲含纈酪肽蛋白在尾蚴、童蟲、成蟲(雄蟲、雌蟲)、蟲卵4個(gè)生活史階段的表達(dá)情況 分別提取日本血吸蟲尾蚴、童蟲、雌蟲、雄蟲、蟲卵RNA，純化后逆轉(zhuǎn)錄為cDNA第一鏈；設(shè)計(jì)目的基因(SjVCP)和內(nèi)參基因(18s)引物，使兩對引物的退火溫度和擴(kuò)增產(chǎn)物大小相近。以cDNA第一鏈為模板，根據(jù)設(shè)計(jì)的目的基因和內(nèi)參基因引物熒光定量擴(kuò)增不同血吸蟲生活史期相應(yīng)基因片段，數(shù)據(jù)分析采用2-ΔCT法。結(jié)果顯示，日本血吸蟲VCP基因轉(zhuǎn)錄的mRNA在尾蚴中水平最高，在童蟲、蟲卵、雄蟲、雌蟲中水平較低。因此，日本血吸蟲在尾蚴、童蟲、成蟲(雄蟲、雌蟲)、蟲卵4個(gè)生活史階段均有VCP基因轉(zhuǎn)錄的mRNA，而在尾蚴中表達(dá)水平最高。 三重組日本血吸蟲含纈酪肽蛋白（rSj-VCP）的診斷價(jià)值研究 11只5-6周齡BALB/c小鼠，按A-K編號(hào)，分別感染40條尾蚴，構(gòu)建小鼠感染日本血吸蟲動(dòng)物模型，于感染前和感染后每周采血并分離血清直至5周。以純化的日本血吸蟲含纈酪肽重組蛋白為抗原，建立ELISA法檢測小鼠血清內(nèi)日本血吸蟲VCP特異性抗體，同時(shí)以日本血吸蟲可溶性蟲卵抗原檢測小鼠血清，比較二者診斷結(jié)果。 結(jié)果rSjVCP在診斷感染小鼠血清內(nèi)特異性抗體時(shí)，除D號(hào)小鼠血清第一周為陽性以外，其余10只均在第二周達(dá)陽性，從第三周開始，抗體水平有下降趨勢；而蟲卵可溶性抗原在診斷感染小鼠血清時(shí)，結(jié)果于感染后第三周檢測現(xiàn)陽性，感染后第四周檢測均為陽性，且從第三周開始，血清中IgG抗體水平呈上升趨勢。因此，重組日本血吸蟲含纈酪肽蛋白具有早期診斷價(jià)值，可用于進(jìn)一步的日本血吸蟲早期診斷研究。
[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.
【學(xué)位授予單位】：江蘇省血吸蟲病防治研究所
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R532.21

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10 張e

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