日本血吸蟲硫氧還蛋白谷胱甘肽還原酶（SjTGR）的克
發(fā)布時間：2019-03-01 21:21
【摘要】：血吸蟲病(schistosomiasis)是一種危害嚴重、分布廣泛的人畜共患病。半個多世紀以來,我國血吸蟲病防治取得了舉世矚目的成就,但由于重復感染嚴重,單靠治療藥物不能控制血吸蟲病的流行,加強血吸蟲病疫苗候選分子的篩選和疫苗的研制開發(fā)無疑是血吸蟲病防治的重要舉措。硫氧還蛋白谷胱甘肽還原酶是血吸蟲體內一種特殊的氧化還原酶類,對保護血吸蟲不受宿主免疫系統(tǒng)和自身新陳代謝而產生的游離氧的損害具有重要作用。本研究以SjTGR為研究對象,開展了該基因的克隆、表達及重組蛋白的免疫保護效果的研究,為深入探討SjTGR的生物學功能,評估其作為疫苗候選分子和藥靶的應用前景提供了基礎。 本實驗室前期應用biotin-avidin分析日本血吸蟲成蟲表面蛋白質組時發(fā)現(xiàn),SjTGR蛋白也出現(xiàn)在蟲體表面,推測SjTGR在血吸蟲中具有重要功能。本研究鑒于此發(fā)現(xiàn),并參考SmTGR基因的擴增方法,獲得SjTGR的全長cDNA序列。通過生物信息學分析,發(fā)現(xiàn)SjTGR基因的ORF為1791bp,編碼596個氨基酸,理論分子量為64940.25Da,理論等電點為6.38, SjTGR和SmTGR的氨基酸序列相似性為82%。本研究成功地構建了重組表達質粒SjTGR-pET-28a和SjTGR-pET-32a,兩種重組蛋白都在E.coli(DE3)中以可溶性蛋白形式表達,分子量分別69kD和72kD, Western blotting分析表明兩種重組蛋白具有較好的抗原性和免疫原性。用純化的SjTGR-pET-28a和SjTGR-pET-32a重組蛋白免疫BALB/c小鼠,結果與空白對照組相比,重組蛋白SjTGR-pET-28a在小鼠中分別誘導了91.24%的減蟲率和93.37%的肝臟減卵率,差異極顯著(p0.01)。重組蛋白SjTGR-pET-32a誘導了42.78%的減蟲率和41.29%的肝臟減卵率,差異顯著(p0.05)。用ELISA方法檢測小鼠血清中特異性IgG抗體水平變化,結果表明,重組蛋白可誘導小鼠體內抗重組抗原的特異性IgG抗體迅速產生,并且維持在一個較高的水平。表明該重組蛋白具有發(fā)展為抗血吸蟲病候選疫苗和新藥靶的潛力及深入研究的價值。 提取7d、14d、21d、28d、35d、42d和42d雌雄各個時期的蟲體蛋白,并用新鮮蟲體制作冰凍切片,以α-tublin為內參,通過Western blotting檢測各個時期蟲體中TGR蛋白的表達情況；經過免疫熒光檢測方法,對該蛋白的表達部位進行了分析。同時,以看家基因α-tublin為內參,應用熒光定量PCR技術分析TGR基因在日本血吸蟲各個時期蟲體內的轉錄情況。結果顯示,TGR蛋白在日本血吸蟲各個蟲體中均有表達,表達部位主要在體被表膜；該基因在日本血吸蟲感染宿主后7d、14d、21d、28d、35d、42d蟲體及42d雌蟲和雄蟲內均有轉錄,在35d-42d蟲體的表達量較高,雌蟲表達量高于雄蟲。在35d到42d這個階段,蟲體發(fā)育成熟并開始產卵,新陳代謝比較旺盛,蟲體產生的活性氧增多,TGR的高表達量可能與減少活性氧對蟲體的損害、與血吸蟲的生長發(fā)育和繁殖相關。本研究為深入研究SjTGR基因的生物學功能奠定了基礎,為篩選新的血吸蟲病疫苗候選分子和藥物靶標提供了新思路
[Abstract]:Schistosomiasis is a serious and widely distributed man-and-animal disease. Since more than half a century, the prevention and control of schistosomiasis in China has made great achievements, but because of the serious repetition of the infection, the treatment of the medicine alone cannot control the epidemic of the schistosomiasis, The research and development of the screening and vaccine for strengthening the candidate molecule of the schistosomiasis vaccine is undoubtedly an important measure of the prevention and control of the schistosomiasis. The thioredoxin glutathione reductase is a special redox enzyme in the body of the schistosome, and has an important role in protecting the schistosome from the damage of the free oxygen produced by the host immune system and the metabolism of the host. In order to study the biological function of SjTGR, this study provided a basis for exploring the biological function of SjTGR, and to evaluate its application prospect as a candidate molecule and target. It was found that the SjTGR protein also appeared on the surface of the worm body, and it was suggested that the SjTGR had important work in the schistosome. The full-length cDNA sequence of SjTGR was obtained in view of this finding and with reference to the amplification method of the SmTGR gene. The amino acid sequence similarity of SjTGR gene was found to be 1791 bp, encoding 596 amino acids, the theoretical molecular weight was 64940.25 Da, the theoretical isoelectric point was 6.38, and the similarity of the amino acid sequence of SjTGR and SmTGR was 82. %. The recombinant expression plasmid SjTGR-pET-28a and SjTGR-pET-32a were successfully constructed in this study. Both of the two recombinant proteins were expressed in the form of soluble protein in E. coli (DE3). The molecular weight was 69 kD and 72 kD, respectively. Western blotting analysis indicated that the two recombinant proteins had better antigenicity and immunogen. The recombinant protein SjTGR-pET-28a and SjTGR-pET-32a recombinant protein were used to immunize the BALB/ c mice. The recombinant protein SjTGR-pET-32a induced 42.78% of the reduction rate and 41.29% of the liver egg reduction rate, and the difference was significant (p0.05). The results showed that the recombinant protein could induce the rapid production of the specific IgG antibody against the recombinant antigen in the mouse, and it was maintained at a higher water level. It shows that the recombinant protein has the potential to develop the candidate vaccine for anti-schistosomiasis and the target of new drug, and the price of the in-depth study. The expression of TGR was detected by Western blotting, and the expression of TGR was detected by Western blotting. the measuring method is carried out on the expression part of the protein, The effects of TGR gene on the body of Schistosoma japonicum in various stages of Schistosoma japonicum were analyzed by using fluorescence quantitative PCR (PCR) as an internal reference at the same time. The results showed that the TGR protein was expressed in the various insect bodies of Schistosoma japonicum, and the expression site was mainly expressed in the surface of the body. The gene was transcribed in 7 days,14 days,21 days,28 days,35 days,42 days, and 42 days after the infection of the host of Schistosoma japonicum. the amount of the female worm is high, the expression amount of the female worm is high, in this stage of 35d to 42d, the insect body is mature and the oviposition is started, the metabolism is more vigorous, the active oxygen generated by the insect body is increased, the high expression quantity of the TGR may be related to the reduction of the damage of the active oxygen to the worm body, the development and the propagation of the schistosome, The study provides a basis for studying the biological function of the SjTGR gene and provides the candidate molecular and drug target for screening new schistosomiasis vaccine.
【學位授予單位】：南京農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392
本文編號：2432812
[Abstract]:Schistosomiasis is a serious and widely distributed man-and-animal disease. Since more than half a century, the prevention and control of schistosomiasis in China has made great achievements, but because of the serious repetition of the infection, the treatment of the medicine alone cannot control the epidemic of the schistosomiasis, The research and development of the screening and vaccine for strengthening the candidate molecule of the schistosomiasis vaccine is undoubtedly an important measure of the prevention and control of the schistosomiasis. The thioredoxin glutathione reductase is a special redox enzyme in the body of the schistosome, and has an important role in protecting the schistosome from the damage of the free oxygen produced by the host immune system and the metabolism of the host. In order to study the biological function of SjTGR, this study provided a basis for exploring the biological function of SjTGR, and to evaluate its application prospect as a candidate molecule and target. It was found that the SjTGR protein also appeared on the surface of the worm body, and it was suggested that the SjTGR had important work in the schistosome. The full-length cDNA sequence of SjTGR was obtained in view of this finding and with reference to the amplification method of the SmTGR gene. The amino acid sequence similarity of SjTGR gene was found to be 1791 bp, encoding 596 amino acids, the theoretical molecular weight was 64940.25 Da, the theoretical isoelectric point was 6.38, and the similarity of the amino acid sequence of SjTGR and SmTGR was 82. %. The recombinant expression plasmid SjTGR-pET-28a and SjTGR-pET-32a were successfully constructed in this study. Both of the two recombinant proteins were expressed in the form of soluble protein in E. coli (DE3). The molecular weight was 69 kD and 72 kD, respectively. Western blotting analysis indicated that the two recombinant proteins had better antigenicity and immunogen. The recombinant protein SjTGR-pET-28a and SjTGR-pET-32a recombinant protein were used to immunize the BALB/ c mice. The recombinant protein SjTGR-pET-32a induced 42.78% of the reduction rate and 41.29% of the liver egg reduction rate, and the difference was significant (p0.05). The results showed that the recombinant protein could induce the rapid production of the specific IgG antibody against the recombinant antigen in the mouse, and it was maintained at a higher water level. It shows that the recombinant protein has the potential to develop the candidate vaccine for anti-schistosomiasis and the target of new drug, and the price of the in-depth study. The expression of TGR was detected by Western blotting, and the expression of TGR was detected by Western blotting. the measuring method is carried out on the expression part of the protein, The effects of TGR gene on the body of Schistosoma japonicum in various stages of Schistosoma japonicum were analyzed by using fluorescence quantitative PCR (PCR) as an internal reference at the same time. The results showed that the TGR protein was expressed in the various insect bodies of Schistosoma japonicum, and the expression site was mainly expressed in the surface of the body. The gene was transcribed in 7 days,14 days,21 days,28 days,35 days,42 days, and 42 days after the infection of the host of Schistosoma japonicum. the amount of the female worm is high, the expression amount of the female worm is high, in this stage of 35d to 42d, the insect body is mature and the oviposition is started, the metabolism is more vigorous, the active oxygen generated by the insect body is increased, the high expression quantity of the TGR may be related to the reduction of the damage of the active oxygen to the worm body, the development and the propagation of the schistosome, The study provides a basis for studying the biological function of the SjTGR gene and provides the candidate molecular and drug target for screening new schistosomiasis vaccine.
【學位授予單位】：南京農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392
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