【作者】 余招锋；

【导师】 于三科； 才学鹏； 张志芳；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2004， 硕士

【摘要】 猪带绦虫 ４５Ｗ 基因家族是由一群结构和功能相似的基因组成的。４５Ｗ 基因在反式剪接作用下产生 Ａ、Ｂ 和 Ｃ 三型转录本，其中 Ａ 型转录本是由外显子Ⅰ、Ⅱ、Ⅲ和Ⅳ组成；Ｂ 型转录本由Ⅰ、Ⅲ和Ⅳ组成；Ｃ 型转录本由Ⅰ和Ⅳ组成，在不同的转录本中的同一外显子有高度的同源性，也存在变异。　 ４５Ｗ 基因已被认为是预防猪囊尾蚴病的基因工程疫苗侯选基因之一。本试验成功地从六钩蚴中克隆到 ４Ｂ 基因。通过对 ４Ｂ 基因序列的比较，发现 ４Ｂ 基因是 ４５Ｗ 基因中较为特殊的一个基因，该基因与 ４５Ｗ 其他相关基因之间变异范围为 １７．８％－２０．６％，但该基因在不同虫株间和不同克隆间高度保守。为了研究 ４Ｂ 蛋白的结构和功能，并为研制和开发有效的猪带绦虫基因工程疫苗打下基础，本研究将 ４Ｂ 基因及其 ３′－端的剪切片段克隆到 ｐＥＴ－２８ａ 载体中，并在大肠杆菌 ＢＬ２１ 中成功地表达，与预期分子大小一致，为 １８　ｋＤａ，经 Ｗｅｓｔｅｒｎ　ｂｌｏｔ 检测，表达的重组蛋白能被猪囊尾蚴病人血清识别，为开发高效猪囊尾蚴基因工程疫苗打下了基础。　 ４５Ｗ 基因的这独特的交替剪接形式引起了寄生虫学家们的广泛兴趣。为了研究４Ｂ 蛋白在猪囊尾蚴组织中的结构和功能，试验中利用大肠杆菌表达系统表达的 ４Ｂ 重组蛋白，分子量约为 １８　ｋＤａ，腹腔注射免疫小白鼠，经两次免疫后，采血，并用该血清与猪囊尾蚴组织匀浆蛋白做免疫印迹，发现在 ５０　ｋＤａ 处有一明显的印迹条带，而 １８　ｋＤａ 处没有。表明在猪囊尾蚴组织蛋白中 ４５Ｗ 蛋白各型蛋白可能以同型或异型寡聚体形式存在。用计算机软件分析发现 ４Ｂ 蛋白的抗原性很好，用蛋白质结构数据库　（Ｐｒｏｔｅｉｎ　Ｄａｔａ　Ｂａｓｅ，　ＰＤＢ）比较，发现 ４Ｂ 蛋白（包括 ４５Ｗ 其他型）存在纤连蛋白（Ｆｉｂｒｏｎｅｃｔｉｎ，　ＦＮ）结构域。４５Ｗ 蛋白寡聚体形式组成与纤链蛋白的组织构成十分相似，推测 ４５Ｗ 蛋白与猪囊尾蚴囊壁的形成有关。　 家蚕表达系统通过将外源目的基因插入家蚕核多角体病毒（ＢｍＮＰＶ）多角体启动子（ｐｏｌｙｈｅｄｒｉｎ　ｐｒｏｍｏｔｅｒ）的下游，外源基因利用多角体启动子进行转录表达，并分泌到细胞外，从而在细胞上清获得重组目的蛋白。家蚕表达系统（ｓｉｌｋｗｏｒｍ　ｂａｃｕｌｏｖｉｒｕｓ　ｅｘｐｒｅｓｓｉｏｎ　ｖｅｃｔｏｒ　ｓｙｓｔｅｍ，　ＢＥＶＳ）表达的目的蛋白能够进行正确的加工、修饰，使目的蛋白更接近于天然蛋白。同时家蚕细胞能识别哺乳动物的分泌蛋白的信号肽序列。４Ｂ 基因克隆到家蚕杆状病毒转移载体 ｐＶＬ１３９３ 中，通过与家蚕杆状病毒（Ｂｏｍｂｙｘ　ｍｏｒｉ　ｎｕｃｌｅａｒ　ｐｏｌｙｈｅｄｒｏｓｉｓ　ｖｉｒｕｓ，　ＢｍＮＰＶ）在家蚕 Ｂｍ 细胞中共转染，重组到 ＢｍＮＰＶ 中，通过筛选纯化，获得重组病毒。重组病毒感染家蚕细胞和 ５龄家蚕，表达 ４Ｂ 蛋白，分子量约为 １６　ｋＤａ，　与预测的大小相吻合。并用 Ｗｅｓｔｅｒｎ　ｂｌｏｔ<WP=6>鉴定该重组蛋白能识别猪囊虫病人（猪）阳性血清。４Ｂ 基因在家蚕细胞中的表达对进一步研究 ４５Ｗ 基因的结构功能，并开发出更可行、方便的基因工程疫苗打下基础。更多还原

【Abstract】 45W gene family of Taenia solium constitutes of a group of genes that have familiarstructure and function. Three types of 45W genes of T. solium, A type, B type and C typewere produced through alternative splicing. A transcript is composed of the four exonsⅠ, Ⅱ, Ⅲ and Ⅳ, B transcript is lack of exon Ⅱ, and C transcript is lack of exons Ⅱand Ⅲ. The same exon between different genes is highly homologous, but shows somevariations. 45W genes of T. solium were identified to be a talent candidate gene for generecombinant vaccine. In this study, 4B full-length and 3′truncated gene were successfullycloned into pET-28a vector, and expressed in BL21. The products of expression weredetected by Western blot with serum of human infected with Cysticercus Cellulosae, andshowed its antigenicity. The form of alternative splicing of 45W gene family interested many parasitologists.In these transcripts, 4B gene is a special one with the most variation contrast with theother genes of 45W. In this study, 4B protein was isolated and used to vaccinateintraperitoneally the Kunming line mice for twice, then the anti-serum of the mice waspooled for the following Western blot with the tissue protein of Cysticercus cellulosae.The Western blot appears the band in the 50 kDa, which shows that 4B protein fused with6 HIS have antigenicity, and can stimulate mice to produce antigen-specific antibody. Onthe other hand, it can be ascertained that 4B protein is presented in the tissue ofcysticercus cellulosae with the polymer. 4B gene and protein sequence is analyzed usingthe DNAstar software, and blasted using Protein Data base (PDB), showed that 4B proteinhave potential antigenicity, and in this protein there is a Fibronectin (FN) domain. It ispredicted that the 45W protein is related to forming of cysticercus.　 Expression system of BmNPV-Bm is high effective for producing recombinantprotein approach to its nature structure by the correct post-transcript process andmodification. 45W-4B gene of T. solium was cloned into baculovirus transfer vector ofpVL1393 and cotransfected into Bombyx mori nuclear polyhedrosis virus (BmNPV). Therecombinant BmNPV was screened, and used to infect Bm-N cells and silkworm. 4Bprotein was detected in the cell supernatant and haemolyph by Western blot using serumof human (and swine) infected with Cysticercus Cellulosae. Successful expression of 4B<WP=8>recombinant protein in BmNPV-Bm will be significant for study the structure and functionof 4B protein, and development of the effective recombinant vaccine against T. solium andCysticercus Cellulosae.更多还原