摘要
东毕吸虫病是由分体科东毕属的各种吸虫寄生于牛、羊等哺乳动物的门静脉和肠系膜静脉而引起的一种血吸虫病。该病在我国分布极其广泛，给畜牧业带来极大经济损失。同时东毕吸虫的尾蚴可使人患尾蚴性皮炎，也称稻田皮炎，严重影响人类的身体健康，是一种非常重要的人畜共患寄生虫病。
目前对东毕吸虫病的防制还存在诸多困难，首先，实践证明通过杀灭血吸虫的中间宿主——螺类来防治血吸虫感染的效果是不明显的；其次，通过吡喹酮治疗血吸虫病虽然效果很好，但也存在治疗费用高、不能防止重复感染以及耐药性的产生等缺点，现普遍认为有必要研究血吸虫的疫苗来预防此病。本研究克隆了土耳其斯坦东毕吸虫肌动蛋白基因(ACTIN)和磷酸丙糖异构酶基因(TPI)的全长序列，并将磷酸丙糖异构酶基因在毕赤酵母中高效表达。
首先，根据日本血吸虫和曼氏血吸虫肌动蛋白基因和磷酸丙糖异构酶基因核苷酸序列的保守区设计引物，利用RT-PCR从土耳其斯坦东毕吸虫成虫总RNA分别扩增ACTIN和TPI的中间大片段，再根据已知序列利用5RACE和3RACE技术分别扩增出ACTIN和TPI的5’和3’端，将三段序列拼接得到ACTIN和TPI的全长cDNA序列并登录GeneBank，登录号为：ACTIN为DQ017265，TPI为DQ092331。
其次，根据TPI全长cDNA序列设计两条加有EcoR I和Not I酶切位点的引物，利用RT-PCR扩增TPI全长序列并克隆到pMD18-T载体，EcoR I和Not I双酶切重组载体pMD18-T／TPI后将获得的全长TPI亚克隆到毕赤酵母表达载体pPIC9k构成重组表达载体pPIC9k／TPI，Sal I酶切重组表达载体pPIC9k／TPI后电击转化到酵母菌株GS115感受态细胞中，用组氨酸缺陷培养基和G418依次筛选多拷贝质粒整合的毕赤酵母菌株。重组酵母细胞在含甲醇的培养基中分泌表达目的蛋白，培养3d后收取上清，SDS-PAGE显示表达的蛋白分子量为43kDa，进行westernblot分析结果表明该蛋白被自然感染东毕吸虫的绵羊血清所识别。蛋白表达时相分析表明，甲醇诱导72h蛋白表达产量最高。表达产物通过葡聚糖凝胶G-75层析柱纯化，测定蛋白浓度为1200mg／L。
最后，用纯化的蛋白免疫新西兰大白兔，ELISA分析结果表明：空白组的抗体水平一直处于平稳的状态，而免疫组在免疫后抗体水平呈现不断上升的趋势，一次免疫组和加强免疫组的抗体水平均在第四周达到峰值，但加强免疫组较一次免疫组的免疫效果好。由此可见，TPI蛋白具有一定的免疫原性，可以作为东毕吸虫疫苗的候选基因。
Orientobilharziasis, a kind of schistosomiasis, is caused by the trematodes of genus Orientobilharzia parasitizing in portal veins and mesentery veins of mammals such as catties and sheep. It is widely distributed in China and brings animal husbandry a great economic loss. In addition, Orientobilharzia cercariae may make human suffer from cercarial dermatitis and seriously endanger people'health, so Orientobilharziasis is an important zoonoses parasitic disease.
At present, it is difficult in prevention and treatment of Orientobilharziasis. Firstly, the preventive effect is not remarkable by killing snails, the middle host of schistosomes. Secondly, although Praziquantel is very effective for cure of Orientobilharziasis, there are some weakness such as high cure cost, resistence and repeated infection. It is widely believed that development of preventive vaccine against schistosomiasis is very necessary.
In this research, the full-length cDNA of Actin and Triosephosphate Isomerase gene(TPI) of Orientobilharzia turkestanium were cloned, and the TPI was highly expressed in Picha Pastoris GS115 cells. At first, the partial coding region of Actin and TPI were cloned using RT-PCR. The 3'end fragment and 5'end fragment of Actin and TPI were obtained by 3RACE and 5RACE. The full-length cDNA sequence was gotten by splicing sequences of the three fragments. The cDNA sequences have been submitted to GeneBank and accession number of ACTIN is DQ017265 and accession number of TPI is DQ092331. Then, the full-length TPI was cloned by RT-PCR and it was cloned into pMD18-T vector, and the recombinant plasmid pMD18-T/TPI was digested with Not I and EcoK I and the digested full-length TPI was subcloned into espression vector pPIC9k.After electroporation into the GS115 cells, the recombinant plasmids lined by Sal I were screened out by His~- medium and G418. The protein was induced in the medium contains methanol. The supernatant was collected on the 3rd day, SDS-PAGE indicated that the target protein is around 43KD. Western-blot shows it can react with serum of goat naturally infected by orientobilharzia. The optimization experiments indicated that highest yield could be gotten by 0.5% methanol, and on the 72th hour. The TPI protein was purified by Superdex 75 column and protein concentration is around 1200mg/L.
At last, the rabbits were vaccined by the purified protein mixed with Montanide ISA 206 adjuvant(the ratio of the antigen to adjuvant is 1:1). ELISA result showed that the level of TPI antibody reached the highest titer on the 4th week, and the vaccined effect of enhanced vaccined group is better than one time vaccined group.
The study above facilitates that TPI is immunogenic and can become one of candidate genes for prevention of Orientobilharziasis.
引文
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