發(fā)布時(shí)間：2018-01-25 22:32

  本文關(guān)鍵詞： 日本血吸蟲 鐵蛋白基因 轉(zhuǎn)基因植物 疫苗 油菜　出處：《湖南農(nóng)業(yè)大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 血吸蟲病是一種歷史悠久、分布面廣、對(duì)人類健康危害嚴(yán)重的人獸共患寄生蟲病。目前流行于亞洲、非洲和美洲的74個(gè)國(guó)家，受威脅人口達(dá)6億，2億人受感染。血吸蟲疫苗是控制血吸蟲病的重要補(bǔ)充途徑，因而是血吸蟲病防治研究的熱點(diǎn)。粘膜是機(jī)體免疫的第一道防線。與非粘膜免疫比較，粘膜免疫(如經(jīng)口、滴鼻等)除可引起機(jī)體產(chǎn)生全身免疫應(yīng)答(包括細(xì)胞免疫和體液免疫)外，還可誘導(dǎo)產(chǎn)生粘膜免疫應(yīng)答(sIgA)，從而有效地提高機(jī)體的免疫保護(hù)力。研究業(yè)已表明粘膜免疫參與了血吸蟲的保護(hù)性免疫應(yīng)答，植物基因工程疫苗具有通過食用(口服)接種誘導(dǎo)粘膜免疫應(yīng)答等優(yōu)點(diǎn)。本研究以日本血吸蟲鐵蛋白基因?yàn)槟康幕�，探討油菜作為日本血吸蟲口服疫苗的載體的可能，為血吸蟲疫苗和口服疫苗載體的研究提供新的思路。 1．日本血吸蟲鐵蛋白基因(SjFer)的克隆、表達(dá)和抗血清的制備：雌性昆明鼠10只，30g，，經(jīng)腹部貼壁感染40條日本血吸蟲尾蚴，42天后處死門靜脈沖蟲取成蟲，經(jīng)液氮粉碎，提取RNA，制備cDNA文庫。根據(jù)GenBank中SjFer序列設(shè)計(jì)特異性引物一對(duì)，以日本血吸蟲成蟲cDNA文庫為模板PCR擴(kuò)增SjFer，將其插入到pMD18-T Simple vector中，經(jīng)測(cè)序比較，發(fā)現(xiàn)該序列與GenBank中日本血吸蟲鐵蛋白基因序列(AF040385)具有100％同源性。將SjFer定向克隆到pET32α(+)中，構(gòu)建重組質(zhì)粒SjFer／pET32α(+)，將重組質(zhì)粒轉(zhuǎn)化大腸桿菌BL21，提取質(zhì)粒經(jīng)PCR、雙酶切和測(cè)序，鑒定插入片段正確后，經(jīng)終濃度為1mmg／L的IPTG誘導(dǎo)，提取重組蛋白rSjFer，SDS-PAGE電泳分析顯示表達(dá)的融合蛋白為42kD，與預(yù)期結(jié)果一致。western-blotting鑒定呈陽性，說明該蛋白具有免疫活性。用8M尿素純化該融合蛋白并復(fù)性，與弗氏完全佐劑(第二開始使用弗氏不完全佐劑)等體積混勻，按0-2-4周皮下免疫小鼠。第三次免疫后15天ELISA方法檢測(cè)抗體效價(jià)。得到效價(jià)為1：6400的抗血清。 2．日本血吸蟲鐵蛋白基因(SjFer)在油菜中表達(dá)：根據(jù)SjFer序列設(shè)計(jì)一對(duì)特異性引物，用PCR方法從cDNA文庫中擴(kuò)增鐵蛋白基因，然后克隆到pMD18-T Simple vector載體中，從T載體中切下目的片段后，定向克隆到pBI121中，重組質(zhì)粒經(jīng)PCR、雙酶切和測(cè)序鑒定以檢測(cè)插入片段是否正確。利用凍融法直接將重組質(zhì)粒轉(zhuǎn)入根癌農(nóng)桿菌LAB4404，挑取陽性克隆進(jìn)行PCR分析確定后，陽性根癌農(nóng)桿菌介導(dǎo)轉(zhuǎn)化油菜子葉，經(jīng)過不同濃度的篩選培養(yǎng)基(含卡那霉素)的篩選和生根培養(yǎng)基中的誘導(dǎo)生根，開瓶鍛煉3～5天后移入花盆中，即獲得轉(zhuǎn)化植株。提取轉(zhuǎn)化植株的總DNA和RNA，PCR分析顯示在約520bp處出現(xiàn)一亮帶，說明SjFer已成功轉(zhuǎn)入油菜中；Northern-blot雜交呈陽性，說明SjFer已在轉(zhuǎn)錄水平上得到正確的表達(dá)。提取轉(zhuǎn)化植株的總蛋白，以日本血吸蟲鐵蛋白抗血清作為一抗，進(jìn)行western-blotting活性檢測(cè)，結(jié)果呈陽性，且定位于22kD處，與預(yù)期結(jié)果一致�？梢奡jFer已在油菜中正確表達(dá)，且具有良好的免疫原性。 本研究在構(gòu)建cDNA文庫的基礎(chǔ)上，成功地?cái)U(kuò)增了日本血吸蟲鐵蛋白基因，并在油菜中實(shí)現(xiàn)了表達(dá)，且具有免疫活性，該研究為進(jìn)一步研究日本血吸蟲基因工程疫苗奠定基礎(chǔ)。
[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.

【學(xué)位授予單位】：湖南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R346;S852.5;Q943.2

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 羅佳捷;彭瑛;羅銳;張彬;李麗立;吳力專;占今舜;邢月騰;;中國(guó)荷斯坦奶牛鐵蛋白基因3′端克隆及序列分析[J];草業(yè)學(xué)報(bào);2013年05期

本文編號(hào)：1463875