土耳其斯坦东毕吸虫cDNA文库的构建及原肌球蛋白基因的克隆和表达
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摘要
东毕吸虫病(orientobilharziasis)是由东毕属吸虫寄生于牛、羊等多种动物的门静脉和肠系膜静脉内引起的一种血吸虫病，其尾蚴也可感染人类引起尾蚴性皮炎(cercarial dermatitis)，是一种重要的人兽共患寄生虫病。我国以土耳其斯坦东毕吸虫为主，该病在我国分布广泛，常呈地方和季节性流行，严重危害人们的健康并给畜牧业带来巨大经济损失。由于东毕吸虫感染人后特殊的发育过程，从而引起了人们广泛关注。目前东毕吸虫研究相对滞后于曼氏、日本血吸虫，因此深入研究东毕吸虫就显得尤为重要。
本文利用SMART cDNA文库构建试剂盒成功构建了土耳其斯坦东毕吸虫成虫cDNA文库。采用RT-PCR结合RACE和已构建的cDNA文库克隆了土耳其斯坦东毕吸虫原肌球蛋白(tropomyosin，TM)基因，提交GenBank，登录号为AY560898。TM部分编码序列亚克隆至原核表达载体pET28a(+)，重组质粒pET28-TM在大肠杆菌BL21中经IPTG诱导表达出可溶性融合蛋白，其分子量为37.5KDa左右，利用Ni-NTA亲和层析纯化目的蛋白。Western-blotting结果表明纯化蛋白可以识别自然感染东毕吸虫的山羊血清；将纯化蛋白与法国Montanide ISA 206佐剂等体积混合免疫家兔，并收集血清到免疫后第10周。Western-blotting结果表明纯化蛋白可以被特异性免疫后血清识别，表明纯化蛋白可以刺激实验兔产生抗TM抗体。ELISA结果表明抗体在第5周时达到峰值，从第7周到第10周抗体一直保持较高水平。原肌球蛋白较好的免疫原性表明其在疫苗和诊断试剂研制方面有重要的应用前景。上述结果为进一步研究东毕吸虫病的免疫提供重要的基础。
Orientobilharziasis, a kind of schistosomiasis, is caused by the trematodes of genus Orientobilharzia parasitizing in the portal veins and mesentery veins of many animals including cattle and sheep. In addition, Orientobilharzia cercariae may also make human suffer from cercarial dermatitis. Hence Orientobilharziasis is an important zoonose parasitic disease, Orientobilharzia turkestanicum is a main species in China. Orientobilharziasis is widely distributed in China and prevailed in some regions and seasons. Not only does it seriously endanger people's health, but brings animal husbandry a great economic loss. It is because of the special development process of Orientobilharzia after infecting human that people pay a widely attention to Orientobilharzia. At present, study on it falls behind other schistosome species. So it is very important for us to deeply research Orientobilharzia.
In this thesis, cDNA library for mature worm of Orientobilharzia turkestanicum was successfully constructed using the SMART cDNA library construction kit. Besides, the full-length cDNA of tropomyosin (TM) of Orientobilharzia turkestanicum was cloned using RT-PCR, RACE and cDNA library. The cDNA sequence has been submitted to GeneBank and its accession number is AY560898. In addition, The partial coding sequence of TM gene was subcloned into expression vector pET28a ( + ) , the recombinant plasmid pET28-TM was transformed into BL21 and expressed the dissoluble fusion proteins induced by IPTG. The molecular weight of the fusion protein is around 37.5KD. The fusion proteins were purified using Ni-NTA chromagraphy column. Western-blotting results showed that the purified proteins can react with serum of goat infected naturally by Orientobilharzia and stimulate the rabbit to produce the relevant antibody recognized specifically by the antiserum. The purified proteins were mixed with Montanide ISA 206 adjuvant (th
e ratio of the antigen to oil adjuvant =1:1) . The prepared mixture immunized the rabbit and the serum of the rabbit was collected from first week to 10th week. ELISA results show that the level of TM antibody reached the highest titer on the 5th week, and kept a higher liter from 7th week to 1 Oth week. Better immunogenicity of TM suggests that it has a good prospect in developing vaccine and diagnostic reagent. Our results lay a foundation to research the immunity of the Orientbilharziasis further.
本文利用SMART cDNA文库构建试剂盒成功构建了土耳其斯坦东毕吸虫成虫cDNA文库。采用RT-PCR结合RACE和已构建的cDNA文库克隆了土耳其斯坦东毕吸虫原肌球蛋白(tropomyosin，TM)基因，提交GenBank，登录号为AY560898。TM部分编码序列亚克隆至原核表达载体pET28a(+)，重组质粒pET28-TM在大肠杆菌BL21中经IPTG诱导表达出可溶性融合蛋白，其分子量为37.5KDa左右，利用Ni-NTA亲和层析纯化目的蛋白。Western-blotting结果表明纯化蛋白可以识别自然感染东毕吸虫的山羊血清；将纯化蛋白与法国Montanide ISA 206佐剂等体积混合免疫家兔，并收集血清到免疫后第10周。Western-blotting结果表明纯化蛋白可以被特异性免疫后血清识别，表明纯化蛋白可以刺激实验兔产生抗TM抗体。ELISA结果表明抗体在第5周时达到峰值，从第7周到第10周抗体一直保持较高水平。原肌球蛋白较好的免疫原性表明其在疫苗和诊断试剂研制方面有重要的应用前景。上述结果为进一步研究东毕吸虫病的免疫提供重要的基础。
Orientobilharziasis, a kind of schistosomiasis, is caused by the trematodes of genus Orientobilharzia parasitizing in the portal veins and mesentery veins of many animals including cattle and sheep. In addition, Orientobilharzia cercariae may also make human suffer from cercarial dermatitis. Hence Orientobilharziasis is an important zoonose parasitic disease, Orientobilharzia turkestanicum is a main species in China. Orientobilharziasis is widely distributed in China and prevailed in some regions and seasons. Not only does it seriously endanger people's health, but brings animal husbandry a great economic loss. It is because of the special development process of Orientobilharzia after infecting human that people pay a widely attention to Orientobilharzia. At present, study on it falls behind other schistosome species. So it is very important for us to deeply research Orientobilharzia.
In this thesis, cDNA library for mature worm of Orientobilharzia turkestanicum was successfully constructed using the SMART cDNA library construction kit. Besides, the full-length cDNA of tropomyosin (TM) of Orientobilharzia turkestanicum was cloned using RT-PCR, RACE and cDNA library. The cDNA sequence has been submitted to GeneBank and its accession number is AY560898. In addition, The partial coding sequence of TM gene was subcloned into expression vector pET28a ( + ) , the recombinant plasmid pET28-TM was transformed into BL21 and expressed the dissoluble fusion proteins induced by IPTG. The molecular weight of the fusion protein is around 37.5KD. The fusion proteins were purified using Ni-NTA chromagraphy column. Western-blotting results showed that the purified proteins can react with serum of goat infected naturally by Orientobilharzia and stimulate the rabbit to produce the relevant antibody recognized specifically by the antiserum. The purified proteins were mixed with Montanide ISA 206 adjuvant (th
e ratio of the antigen to oil adjuvant =1:1) . The prepared mixture immunized the rabbit and the serum of the rabbit was collected from first week to 10th week. ELISA results show that the level of TM antibody reached the highest titer on the 5th week, and kept a higher liter from 7th week to 1 Oth week. Better immunogenicity of TM suggests that it has a good prospect in developing vaccine and diagnostic reagent. Our results lay a foundation to research the immunity of the Orientbilharziasis further.
引文
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