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標題: | 黃鰭鯛生長激素基因在昆蟲細胞之表現及其促進吳郭魚生長之研究 Epression of Acanthopagrus latus growth hormone cDNA in insect cells and its promoting the growth rate of ti1apia (Oreochrimos niloticus) |
作者: | Horng-Ming Chen 陳弘民 |
出版年 : | 1992 |
學位: | 碩士 |
摘要: | 黃鰭鯛(Acanthopagrus latus Houttuyn)(yp),為臺灣重要經濟海水養殖之魚種,其生長激素基因(ypGH cDNA)已從 cDNA 基因庫中篩選出。本論文以 ypGH cDNA 為模版(template),利用 polymerase chain reaction(PCR)合成四種不同組合之 ypGH cDNA :(l)為整段 cDNA,共 915 bp;(2)和(3)為含前導序列(leader sequence)cDNA 之 coding region,約 620 bp,其中(3)於 ATG 前多接上 AAT;(4)只有 mature cDNA,為 561bP;將其分別接於桿狀病毒表現載體 pAcT7 之核多角體核蛋白(polyhedrin;PH)的?動子後,然後與野生型病毒 (Autographa californica nuclear polyhedrosis virus;AcMNPV )共轉形(cotransfection)於昆蟲細胞株SF 21-AE (Spodoptera frugiperda cell line)。經電泳後銀染色,含有 leader sequence 之 ypGH cDNA 在 22 kda 處可見-清晰色帶,該色帶與 anti-chum salmon GH 抗體可產生免疫反應。recombinant GH(rGH)的產量約佔細胞所有蛋白質之 1 % 胞外約佔分泌性蛋白之 2-8 %。至於無前導序列之重組病毒的表現,則僅於細胞內可測得。以 Amicon 濃縮大量感染重組病毒之不含血清感染液,再冷凍乾燥,將此含 rGH 之粉末以生理食鹽水調配成每10μl含1、0.5、及0.1μg三種劑量,分別以腹腔注射法注入三組吳郭魚(Oreochrimos niloticus) 每-星期注射乙次,每次10μl,連續注射五星期。以注射生理食鹽水及全無處理的二組作為對照組。注射後之第五星期以注射 0.1μg 劑量的效果最好,體重增加率已達 120%,其次為注射 0.5μg劑量組,而以 1μg劑量最差,但其增加率達 92.2 %仍比對照組之 46%高。體長之增長,三組實驗組亦較二組對照組大,增長倍數為 1.8 ×(39.6 % / 22.25 %)。至於飼料利用效率,亦以注射 0.1μg組達 78%最高,因此顯然的利用昆蟲病毒表現載體系統所生產之 rGH 具有良好之生物活性。將表現之 rGH,由細胞懸浮液直接以 Amicon 濃縮後,當免疫蛋白注射於白色來亨雞(Gallus gallus,single comb white leghorn)其產生之抗體對於向昆蟲細胞及大腸桿菌所產生之 rGH 皆有良好抗原性反應,此法可提供了往後對於 rGH 之偵測有更好的專-性。本研究為首次以桿狀病毒表現系統表現魚類 GH cDNA 的報告,同時也提供了利用 rGH 應用於養殖之可行性。 The growth hormone cDNA of Acathopagrus latus Houttuyn (yp) has been isolated from cDNA library of pituitary glands. Using polymerase chain reaction (PCR) technique, four types of ypGH cDNA were constructed: (1) ypGH1, 915 bp of intact GH cDNA, (2) ypGH2, 620 bp of prehormone coding region,(3) ypGH3, adding AAT before ATG of prehormone coding region, and (4) ypGH4, 561 bp of mature cDNA. These cDNAs were inserted into baculoviral expression vector pAcT7 and cotransfected with wild type virus Autographa californica nuclear polyhedrosis virus ( AcMNPV) into insect cell, Spodoptera frugiperda cell line (SF21-AE), respectively. The recombinant virus was isolated by limiting dilution and screened by dot blotting. ypGH cDNA containing virus was further detected by PCR and Southern blot. From 12% of SDS polyacrylamide gel electrophoresis (PAGE), rGH corresponding region 22kD was presented in the cell lysate and supernatant, except rGH4, and had positive reaction with anti-chum salmon GH antibody. Using densitometer scanning, the production of rGH1 was 1% of total cellular protein and 2 - 8 % of secretory proteins. Supernatant of infected culture medium without serum was partial purified and concentrated by Amicon , then freeze dried. Three doses (0.1, 0.5 and 1 ug) of rGH1 were prepared in 10 ul of normal saline and injected into tilapia (Oreochrimos niloticus) from intraperitoneal injection once per week for five weeks. Injection of normal saline or without injection were used as control. The initial wet weight was about 8.38±0.4 (g) and body length was 7.52±0.38 (cm). After five weeks treatment, the wet weight of 0.1 ug group was increased 2.6 times than control. Gain (%) of 0.1 ug group was 120 %, 0.5 ug group was 112% and 1 ug group was 92.2%, while control group was only 46%. In body length and feed efficiency, the experimental groups were 1.8 and 2 times to two control groups, respectively. It is significant that rGH produced from insect baculovirus expression system has its biological activity. Finally, the rGH was also immunizied single comb white leghorn Gallus gallus. Either antibodies from chicken anti-yprGH or rabbit anti-chum salmon GH antibody can detect rGH which produced from E.coli and insect cells. This is the first report by using baculovirus expression system to produce fish GH cDNA. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75927 |
全文授權: | 未授權 |
顯示於系所單位: | 漁業科學研究所 |
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