大片形吸虫ES与宿主互作抗原的筛

发布时间：2018-05-02 23:51

本文选题：大片形吸虫 + ES　；参考：《黑龙江八一农垦大学》2017年硕士论文

【摘要】：片形吸虫不仅感染牛羊等反刍动物,而且也可感染人体,严重威胁着人和动物的健康。因此,建立高效准确的诊断方法是防控该病的研究重点。片形吸虫包括肝片形吸虫、大片形吸虫及其中间型,目前对于大片形吸虫及其中间型的相关研究相对较少。因此,本研究选择大片形吸虫为研究对象,通过对大片形吸虫排泄分泌(ES)抗原与宿主的互作,旨在筛选、鉴定及分析出具有诊断潜力的蛋白,为建立大片形吸虫的诊断方法奠定基础。本研究分为三部分。第一部分,对大片形吸虫的鉴定。首先,将采自广西南宁的144条片形吸虫虫体进行形态学鉴定和分子生物学鉴定。形态学鉴定这144条虫体均为片形吸虫。对这144条虫体的核糖体第二内转录间隔区(ITS-2)序列进行了扩增、测序,结果表明144个PCR样品的测序结果区别于肝片形吸虫及中间型,而均与大片形吸虫ITS-2序列(GenBank accession AJ557569)匹配率达99.7%,从而判定分离得到的144条片形吸虫均为大片形吸虫,不含中间型。第二部分,大片形吸虫ES抗原的制备及互作抗原的筛选。首先,利用体外培养方法收集大片形吸虫虫卵,对虫卵进行体外培养孵出毛蚴感染淡水螺,使其在淡水螺体内生长发育,直至尾蚴逸出螺体脱尾形成囊蚴,收集形成的囊蚴(感染虫卵后35 d),并进一步用囊蚴经口感染水牛(约500个囊蚴/头),在水牛感染后的14 d、42 d、70 d、98 d,分别采血制备血清。其次,利用体外培养方法分别收集大片形吸虫ES抗原,将收集的ES抗原免疫家兔制备阳性血清,用间接ELISA方法检测其抗体效价,结果表明ES抗原制备的阳性血清效价可达到1:1 000,说明大片形吸虫ES抗原的免疫原性较好,可用于后续的试验。然后,将大片形吸虫ES抗原分别与水牛各时间段的血清互作,通过免疫共沉淀和LC-MS/MS(质谱)试验,共获得了465个非冗余蛋白。进一步用BlastP同源性比对、UniProt鉴定,找到57个已知蛋白,分别在42 d、70 d和98 d的蛋白有13(22.8%)个、5(8.8%)个和7(12.3%)个,同时存在于42 d和70 d、70 d和98 d、42 d和98 d的可被鉴定的蛋白分别有8(14%)个、5(8.8%)、1(1.8%)个,同时存在于42 d、70 d和98 d可被鉴定的蛋白有18(31.6%)个。进一步用生物信息学分析,预测α-微管蛋白和亮氨酸氨肽酶等蛋白具有诊断价值。第三部分,对筛选出的具有代表性的α-微管蛋白和亮氨酸氨肽酶进行克隆、原核表达,并探索其作为片形吸虫病诊断抗原的可行性。结果表明,成功克隆了片段长为1 356 bp、能编码452个氨基酸、理论分子量为48.99 kDa的α-微管蛋白基因和片段长为1 572 bp、能编码523个氨基酸、理论分子量为56.38 kDa的亮氨酸氨肽酶基因。这两个蛋白的二级结构预测都以无规卷曲为主,抗原表位在整个序列均有分布。三级结构的主要功能区域都在N端。将α-微管蛋白基因插入载体pGEX-6P-1(+),亮氨酸氨肽酶基因插入载体pET-30a分别进行原核表达。发现经IPTG诱导表达的重组蛋白pGEX-6P-1(+)-FgAT分子量约为76 kDa(含载体携带的26 kDa的GST标签),且以融合蛋白形式表达,经Glutathione-Sepharose beads纯化后,获得了较高纯度的重组蛋白pGEX-6P-1(+)-FgAT;经IPTG诱导表达的重组蛋白pET-30a-FgLAP分子量约为56 kDa,以包涵体形式表达,经Ni-NTA His bind Resin纯化后获得较高纯度的重组蛋白pET-30a-FgLAP。这两个重组蛋白分别与大片吸虫ES抗原免疫家兔后制备的阳性血清、水牛感染大片吸虫不同期的阳性血清反应,经Western Blot分析证实均具有免疫反应性。本研究通过以上三个部分的试验,成功鉴定出大片形吸虫ES抗原中与宿主互作的蛋白,包括465个非冗余蛋白。利用生物信息学分析,筛选出57个已知蛋白,并进一步鉴定出具有代表性的α-微管蛋白基因和亮氨酸氨肽酶基因。通过对这两个基因的克隆、表达及免疫反应性分析,推断出它们在大片形吸虫病诊断中具有很大的潜力。
[Abstract]:Lamellar flukes not only infect ruminants such as cattle and sheep, but also can infect human beings, which seriously threaten the health of people and animals. Therefore, the establishment of an efficient and accurate diagnosis method is the key point of prevention and control of the disease. The study is relatively small. Therefore, in this study, large tracts are selected as the research object. Through the interaction between ES antigen and the host, this study aims at screening, identifying and analyzing the diagnostic potential proteins to establish a diagnostic method for large tracts. This study is divided into three parts. Identification of insects. First, 144 flake bugs collected from Nanning, Guangxi, were identified by morphological identification and molecular biological identification. Morphological identification of these 144 worms were lamellar bugs. The sequence of the ribosome second internal transcriptional interval (ITS-2) sequence of the 144 worm bodies was amplified and sequenced, and the results showed that the sequence of 144 PCR samples was sequenced. Different from the liver Fasciola and the intermediate type, and the matching rate of the ITS-2 sequence (GenBank accession AJ557569) with the large tracts of Fasciola (accession AJ557569) is up to 99.7%. Thus, it is found that all of the 144 flake like flukes are large tracts, without intermediate type. Second, the preparation of ES antigen of large Fasciola and the screening of mutual antigen. First, the culture method is used in vitro The eggs were collected and cultured in vitro, and the eggs were incubated in vitro to infect freshwater snails, to grow and develop in fresh water spire, until the cercariae escaped from the spire to form the cycariae, and the cycariae were collected (35 d after the infection of the eggs), and the buffalo (about 500 cysts / heads) infected with the cysts, 14 d, 42 d, 7 after buffalo infection. 0 d and 98 D were collected to prepare blood serum respectively. Secondly, using the in vitro culture method to collect the large tracer ES antigen, the collected ES antigen was immunized to prepare the positive serum, and the antibody titer was detected by the indirect ELISA method. The results showed that the positive serum titer of the ES antigen preparation could reach 1:1 000, indicating the immunogen of the large Fasciola ES antigen. 465 non redundant proteins were obtained by immunoprecipitation and LC-MS/MS (mass spectrometry) test, and 57 known proteins were found in 42 d, 70 D and 98 D proteins, respectively, with BlastP homology comparison and UniProt test. There are 13 (22.8%), 5 (8.8%) and 7 (12.3%), which exist at 42 d and 70 D, 70 D and 98 D, 42 d and 98 D, respectively. The protein has diagnostic value. In the third part, we clone the representative alpha microtubulin and leucine aminopeptidase, prokaryotic expression, and explore its feasibility as a diagnostic antigen for flake like worm disease. The results showed that the fragment was successfully cloned to 1356 BP and could encode 452 amino acids, and the theoretical molecular weight was 48.99 kDa. The alpha microtubulin gene and fragment length are 1572 BP, which can encode 523 amino acids and a leucine aminopeptidase gene with a theoretical molecular weight of 56.38 kDa. The two structure prediction of these two proteins is dominated by random curling, and the epitope of the antigen is distributed throughout the sequence. The main functional areas of the three stage structure are at the N end. PGEX-6P-1 (+) and leucine aminopeptidase gene inserted into the vector pET-30a to express the prokaryotic expression. The molecular weight of the recombinant protein pGEX-6P-1 (+) -FgAT induced by IPTG was about 76 kDa (including the GST tag carried by the carrier), and expressed in the form of the fusion protein. The purity of the recombinant protein was purified by Glutathione-Sepharose beads, and the high purity was obtained. The recombinant protein pGEX-6P-1 (+) -FgAT, the molecular weight of recombinant protein pET-30a-FgLAP induced by IPTG was about 56 kDa, expressed in the form of inclusion body, and purified by Ni-NTA His bind to obtain a high purity recombinant protein pET-30a-FgLAP., the two recombinant proteins, which were immunized with the positive sera of the large Fasciola ES antigen to immunize rabbits respectively. Cattle infected with positive sera from different stages of Fasciola were immunoreactive by Western Blot analysis. This study successfully identified the proteins that interacted with the host, including 465 non redundant proteins, in the three parts of the test, and 57 known proteins were screened by bioinformatics analysis. Further identification of the representative alpha microtubulin gene and leucine aminopeptidase gene is further identified. By cloning, expressing and immunoreactive analysis of these two genes, it is concluded that they have great potential in the diagnosis of large tracepimiasis.

【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.7

【参考文献】