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华支睾吸虫病严重危害着人们的身体健康 ,研制快速诊断试剂盒对于华支睾吸虫病的防治显得非常重要。目前 ,市场上销售的快速诊断试剂盒 ,其诊断抗原主要来自华支睾吸虫成虫的粗抗原 ,抗原敏感性高 ,特异性却不高 ,所以寻找并生产基因工程重组抗原有着非常重要的意义。半胱氨酸蛋白酶是一类含有半胱氨酸残基的蛋白水解酶 ,多种寄生虫都能产生或分泌半胱氨酸蛋白酶 ,它对寄生虫的生存、发育以及在寄生虫与宿主的相互关系上均有着极其重要的作用。越来越多的科研工作者开始重视它的免疫诊断价值 ,本文研究的目的是为华支睾吸虫快速诊断试剂盒寻找基因工程重组抗原。我们根据已知华支睾吸虫半胱氨酸蛋白酶基因序列设计一对引物 ,用文库构建试剂盒提取总RNA ,并反转录成cDNA ,以cDNA为模板 ,通过PCR技术从cDNA中扩增出目的基因。PCR产物通过测序鉴定 ,用工具酶BamHI和XholI同时消化目的基因和pGEX - 4T - 1原核表达载体 ,消化后的产物用连接酶连接 ,构建成 pGEX - 4T - 1-CP重组体 ,并将其转入Jm10 9大肠杆菌中。用PCR初步筛选阳性克隆 ,共获得阳性克隆 8个 ,再用双酶切和测序进一步鉴定初步筛选的阳性克隆。挑取阳性克隆 ,37℃条件下用IPTG诱导重组体表达 ,表达产物通过SDS -PAGE电泳鉴定。通过上述实验 ,获
Clonorchis sinensis is seriously endangering people’s health. It is very important to develop a rapid diagnostic kit for the prevention and treatment of Clonorchiasis. At present, the rapid diagnostic kits marketed mainly contain the crude antigen of adult Clonorchis sinensis, which has high antigen sensitivity and low specificity. Therefore, it is very important to find and produce the recombinant antigen. Cysteine protease is a class of cysteine residues containing proteolytic enzymes, a variety of parasites can produce or secrete cysteine protease, its parasite survival and development, as well as in parasites and host Mutual relations have an extremely important role. More and more researchers begin to value its immunodiagnosis value. The purpose of this study is to search for the recombinant antigen for the rapid diagnosis of Clonorchis sinensis. We designed a pair of primers based on the known cysteine protease gene sequence of Clonorchis sinensis, extracted the total RNA using a library construction kit and reverse transcribed it into cDNA. The cDNA was used as a template and amplified from the cDNA by PCR target gene. The PCR products were identified by sequencing. Simultaneous digestion of the target gene and pGEX - 4T - 1 prokaryotic expression vector with the tool enzymes BamHI and XholI, the digested product was ligated with ligase to construct the recombinant pGEX - 4T - 1 - CP. Transferred to Jm10 9 Escherichia coli. Positive clones were screened by PCR and 8 positive clones were obtained. Positive clones were screened by double enzyme digestion and sequencing. The positive clones were picked and induced with IPTG at 37 ℃. The expressed products were identified by SDS-PAGE. Through the above experiment, obtained