日本血吸虫铁蛋白基因的克隆及其在油菜中的表达和活性检测

发布时间：2018-01-25 22:32

本文关键词： 日本血吸虫铁蛋白基因转基因植物疫苗油菜　出处：《湖南农业大学》2007年硕士论文　论文类型：学位论文

【摘要】： 血吸虫病是一种历史悠久、分布面广、对人类健康危害严重的人兽共患寄生虫病。目前流行于亚洲、非洲和美洲的74个国家，受威胁人口达6亿，2亿人受感染。血吸虫疫苗是控制血吸虫病的重要补充途径，因而是血吸虫病防治研究的热点。粘膜是机体免疫的第一道防线。与非粘膜免疫比较，粘膜免疫(如经口、滴鼻等)除可引起机体产生全身免疫应答(包括细胞免疫和体液免疫)外，还可诱导产生粘膜免疫应答(sIgA)，从而有效地提高机体的免疫保护力。研究业已表明粘膜免疫参与了血吸虫的保护性免疫应答，植物基因工程疫苗具有通过食用(口服)接种诱导粘膜免疫应答等优点。本研究以日本血吸虫铁蛋白基因为目的基因，探讨油菜作为日本血吸虫口服疫苗的载体的可能，为血吸虫疫苗和口服疫苗载体的研究提供新的思路。 1．日本血吸虫铁蛋白基因(SjFer)的克隆、表达和抗血清的制备：雌性昆明鼠10只，30g，，经腹部贴壁感染40条日本血吸虫尾蚴，42天后处死门静脉冲虫取成虫，经液氮粉碎，提取RNA，制备cDNA文库。根据GenBank中SjFer序列设计特异性引物一对，以日本血吸虫成虫cDNA文库为模板PCR扩增SjFer，将其插入到pMD18-T Simple vector中，经测序比较，发现该序列与GenBank中日本血吸虫铁蛋白基因序列(AF040385)具有100％同源性。将SjFer定向克隆到pET32α(+)中，构建重组质粒SjFer／pET32α(+)，将重组质粒转化大肠杆菌BL21，提取质粒经PCR、双酶切和测序，鉴定插入片段正确后，经终浓度为1mmg／L的IPTG诱导，提取重组蛋白rSjFer，SDS-PAGE电泳分析显示表达的融合蛋白为42kD，与预期结果一致。western-blotting鉴定呈阳性，说明该蛋白具有免疫活性。用8M尿素纯化该融合蛋白并复性，与弗氏完全佐剂(第二开始使用弗氏不完全佐剂)等体积混匀，按0-2-4周皮下免疫小鼠。第三次免疫后15天ELISA方法检测抗体效价。得到效价为1：6400的抗血清。 2．日本血吸虫铁蛋白基因(SjFer)在油菜中表达：根据SjFer序列设计一对特异性引物，用PCR方法从cDNA文库中扩增铁蛋白基因，然后克隆到pMD18-T Simple vector载体中，从T载体中切下目的片段后，定向克隆到pBI121中，重组质粒经PCR、双酶切和测序鉴定以检测插入片段是否正确。利用冻融法直接将重组质粒转入根癌农杆菌LAB4404，挑取阳性克隆进行PCR分析确定后，阳性根癌农杆菌介导转化油菜子叶，经过不同浓度的筛选培养基(含卡那霉素)的筛选和生根培养基中的诱导生根，开瓶锻炼3～5天后移入花盆中，即获得转化植株。提取转化植株的总DNA和RNA，PCR分析显示在约520bp处出现一亮带，说明SjFer已成功转入油菜中；Northern-blot杂交呈阳性，说明SjFer已在转录水平上得到正确的表达。提取转化植株的总蛋白，以日本血吸虫铁蛋白抗血清作为一抗，进行western-blotting活性检测，结果呈阳性，且定位于22kD处，与预期结果一致。可见SjFer已在油菜中正确表达，且具有良好的免疫原性。本研究在构建cDNA文库的基础上，成功地扩增了日本血吸虫铁蛋白基因，并在油菜中实现了表达，且具有免疫活性，该研究为进一步研究日本血吸虫基因工程疫苗奠定基础。
[Abstract]:Schistosomiasis is a long history, wide distribution, serious zoonotic parasitic diseases harmful to human health. The current epidemic in 74 countries in Asia, Africa and the Americas, threatened a population of 600 million, 200 million people infected. Schistosomiasis vaccine is an important complementary way to control schistosomiasis, which is the research focus of schistosomiasis control. Mucosal immunity is the first line of defense. Compared with the non mucosal immunity, mucosal immunity (such as oral, nasal etc.) in addition can cause the body produce immune response (including cellular immunity and humoral immunity), also can induce mucosal immune responses (sIgA), so as to effectively improve the protective immunity of organism the research has shown that mucosal immunity in protective immunity of Schistosoma japonicum, plant genetic engineering vaccine is by eating (oral) vaccination to induce mucosal immune response and other advantages. In this study, the Japanese blood The target gene of fluke protein is the target gene, and the possibility of rapeseed as an oral vaccine of Schistosoma japonicum is discussed. It will provide new ideas for the research of schistosomiasis vaccine and oral vaccine carrier.
1. Schistosoma japonicum ferritin gene (SjFer) cloning, expression and preparation of antiserum: female Kunming 10 rats, 30g wall, infected with 40 cercariae by the belly, after 42 days of portal vein from adult worm punch, by liquid nitrogen crushing, extracting RNA, cDNA library preparation, according to GenBank. SjFer sequence specific primers were designed by a pair of Schistosoma japonicum adult worm cDNA library SjFer template for amplification of PCR and insert it into the pMD18-T Simple vector, after sequencing, found Schistosoma japonicum ferritin gene sequence with the sequence of GenBank (AF040385) with 100% homology. SjFer was cloned into pET32 (+ alpha), the recombinant plasmid SjFer / pET32 alpha (+), the recombinant plasmid was transformed into Escherichia coli BL21, plasmid was extracted by PCR, double enzyme digestion and sequencing, identification of the inserted fragments correctly, with the final concentration of 1mmg / L induced by IPTG, extraction of recombinant protein rSjFer, SDS-PAGE Electrophoretic analysis showed that the expression of the fusion protein was 42kD, consistent with the expected results of.Western-blotting identification was positive, indicating that the protein had immune activity. 8M urea was used to purify the fusion protein and renaturation, with complete Freund's adjuvant (second began using incomplete Freund's adjuvant) such as volume mixing, according to 0-2-4 weeks of immunized mice 15 days after the third immunization. ELISA method for detection of antibody titer. The antiserum titer was 1:6400.
2. Schistosoma japonicum ferritin gene (SjFer) expression in rape: the primers designed according to the sequence of SjFer amplified ferritin gene from the cDNA library by PCR method, then cloned into pMD18-T Simple vector vector, cut fragment from T vector, cloned into pBI121, recombinant plasmid PCR, double enzyme digestion and sequencing to detect the insertion fragment is correct. Using freeze-thaw method directly to the recombinant plasmid was transformed into Agrobacterium LAB4404, positive clones were analyzed by PCR to determine the positive after Agrobacterium mediated transformation of rapeseed leaf, after screening culture medium (containing different concentrations of kanamycin) screening and rooting culture induced rooting medium, open a bottle of exercise 3 ~ 5 days into the pot, to obtain transgenic plants. The transgenic plants extract total DNA and RNA, PCR showed a bright band at around 520bp, indicating that SjFer has The success of transgenic rapeseed; Northern-blot hybridization was positive, indicating that SjFer has been correctly expressed at the transcription level. To extract the total protein of transgenic plants, with Schistosoma japonicum ferritin antiserum as the first antibody, Western-blotting activity detection, the result was positive, and is located at 22kD, consistent with the expected results. The visible SjFer has the correct expression in the rape, and has a good immunogenicity.
Based on the construction of cDNA library, we successfully amplified Schistosoma japonicum ferritin gene and expressed it in rapeseed, and it has immunological activity. This research lays the foundation for further research of Schistosoma japonicum genetic engineering vaccine.

【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346;S852.5;Q943.2

【引证文献】