柔嫩艾美耳球虫cDNA文库的构建及其主要抗原基因的克隆与表达
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摘要
顶复门都是由原生动物组成的寄生虫,它包含了一些重要的兽医寄生虫和医学寄生虫(如艾美耳球虫、疟原虫、巴贝斯虫、弓形虫、隐孢子虫、住肉孢子虫)。在各种寄生虫感染中,寄生在细胞内的艾美耳属球虫是主要制约现代家禽生产的一种原虫病。艾美耳球虫病不仅威胁家禽的健康和生命安全,而且在全球范围内可导致严重的经济损失。在各种艾美耳球虫中,寄生在盲肠中的柔嫩艾美耳球虫致病性最强。尽管采用了一些免疫学、生物技术学和遗传学的方法,但目前控制球虫病仍然主要依靠抗球虫的化学药物来预防。然而,由于许多抗球虫药出现了严重的抗药性而使用受限。加之药物或抗生素在家禽产品中的残留问题越来越受到消费者的关注,迫使人们寻求防治的新途径。在众多控制球虫的方法中,免疫学防治前景诱人。本研究选取柔嫩艾美耳球虫(Eimeria tenella)子孢子构建其cDNA表达文库,从中筛选、克隆免疫原基因,并进行表达、功能分析和动物免疫保护试验,获得了以下结果。
1.用纯化的E. tenella杨凌株(YL)孢子化卵囊口服接种感染雏鸡,制备鸡抗E. tenella YL株阳性血清;用纯化的E. tenella YL株孢子化卵囊作为抗原,裂解后加佐剂乳化后免疫家兔,制备兔抗鸡E. tenella YL株阳性血清。结果表明,二者均可用于E. tenella cDNA表达文库的免疫学筛选。
2.以E. tenella YL株孢子化卵囊为材料,首次用λSCREEN载体成功构建了E. tenella子孢子cDNA表达文库。经测定,库容量约为4×106 pfu/mL。能用特异性引物扩增出E. tenella子孢子表面抗原3-1E基因,说明文库质量高、代表性强。
3.从cDNA表达文库中扩增得到鸡E. tenella YL 3-1E基因。利用生物信息学和分子生物学软件对3-1E基因编码的蛋白进行结构预测,表明该蛋白为一结构松散的球状蛋白。将3-1E基因亚克隆到表达载体pGEX-4T-1,构建pGEX-3-1E重组质粒并在大肠杆菌BL21中进行表达,表达产物经SDS-PAGE分析,表明成功的表达出了分子质量为44.7 ku的融合蛋白。该研究为球虫基因工程疫苗的研制奠定了基础,也表明了所构建的E. tenella YL子孢子cDNA表达文库质量较高。
4.利用E. tenella YL株阳性血清和兔抗鸡E. tenella YL株阳性血清对E. tenella子孢子cDNA表达文库进行免疫学筛选,共获得3个阳性克隆,经鉴定,其中一个阳性克隆基因的阅读框(ORF)长为1 029 bp,共编码342个氨基酸。登陆GenBank比较,和已发表的MIC-2基因有很高的同源性,与已报道的E. tenella豪顿株(HT)和E. tenella北京株(BJ)的MIC-2基因ORF序列同源性分别为99.7%和99.8%,与二者氨基酸的
The Phylum Apicomplexa is composed entirely of parasitic protists, including many pathogens of veterinary and medical importance (e.g., Eimeria, Plasmodium, Babesia, Toxoplasma, Cryptosporidium and Sarcocystis). Among various parasitic infections, coccidiosis caused by obligate intracellular protozoan parasite of the genus Eimeria is the major constraint for modern poultry production. Coccidiosis, caused by various Eimeria spp., not only threatens the health and welfare of poultries, but also causes significant economic losses in poultry industries around the world. Among various Eimeria spp., Eimeria tenella, which causes caecal coccidiosis, is highly pathogenic. In spite of advances in immunological, biotechnological and genetical methods, control of coccidiosis chiefly depends upon prophylactic chemotherapy with anticoccidial drugs. However, the emergence of drug resistance in coccidia is a great problem with most of the drugs, which, in due course, limits their use. Furthermore, drug- or antibiotic-residue in the poultry product is potentially harmful to consumers. These limitations have necessitated the search for alternative Eimeria control measures. Among the several alternative Eimeria control measures only host vaccination against Eimeria appears promising. In this research, our strategy was constructing the cDNA expression library of the sporozoites by Eimeria tenella sporulated oocysts, and screening, cloning the immunogen genes, as well as expressing the interesting genes, analysing the protective immunity. Results of studies displayed as following.
1. Chickens was orally infected with sporulated oocysts of E. tenella YL strain for preparations of anti-E. tenella YL strain positive serum. Rabbit was infected with sporulated oocysts of E. tenella YL strain for preparations of anti-E. tenella YL strain positive serum. The results showed that two kinds of positive sera could be use for immunoscreening from cDNA expression library.
2. We first constructed successfully cDNA expression library of the E. tenella sporozoites by sporulated oocysts of E. tenella YL strain. The results showed that the transformation efficacy of library was 4×106 pfu/mL. We cloned the surface antigen 3-1E
1.用纯化的E. tenella杨凌株(YL)孢子化卵囊口服接种感染雏鸡,制备鸡抗E. tenella YL株阳性血清;用纯化的E. tenella YL株孢子化卵囊作为抗原,裂解后加佐剂乳化后免疫家兔,制备兔抗鸡E. tenella YL株阳性血清。结果表明,二者均可用于E. tenella cDNA表达文库的免疫学筛选。
2.以E. tenella YL株孢子化卵囊为材料,首次用λSCREEN载体成功构建了E. tenella子孢子cDNA表达文库。经测定,库容量约为4×106 pfu/mL。能用特异性引物扩增出E. tenella子孢子表面抗原3-1E基因,说明文库质量高、代表性强。
3.从cDNA表达文库中扩增得到鸡E. tenella YL 3-1E基因。利用生物信息学和分子生物学软件对3-1E基因编码的蛋白进行结构预测,表明该蛋白为一结构松散的球状蛋白。将3-1E基因亚克隆到表达载体pGEX-4T-1,构建pGEX-3-1E重组质粒并在大肠杆菌BL21中进行表达,表达产物经SDS-PAGE分析,表明成功的表达出了分子质量为44.7 ku的融合蛋白。该研究为球虫基因工程疫苗的研制奠定了基础,也表明了所构建的E. tenella YL子孢子cDNA表达文库质量较高。
4.利用E. tenella YL株阳性血清和兔抗鸡E. tenella YL株阳性血清对E. tenella子孢子cDNA表达文库进行免疫学筛选,共获得3个阳性克隆,经鉴定,其中一个阳性克隆基因的阅读框(ORF)长为1 029 bp,共编码342个氨基酸。登陆GenBank比较,和已发表的MIC-2基因有很高的同源性,与已报道的E. tenella豪顿株(HT)和E. tenella北京株(BJ)的MIC-2基因ORF序列同源性分别为99.7%和99.8%,与二者氨基酸的
The Phylum Apicomplexa is composed entirely of parasitic protists, including many pathogens of veterinary and medical importance (e.g., Eimeria, Plasmodium, Babesia, Toxoplasma, Cryptosporidium and Sarcocystis). Among various parasitic infections, coccidiosis caused by obligate intracellular protozoan parasite of the genus Eimeria is the major constraint for modern poultry production. Coccidiosis, caused by various Eimeria spp., not only threatens the health and welfare of poultries, but also causes significant economic losses in poultry industries around the world. Among various Eimeria spp., Eimeria tenella, which causes caecal coccidiosis, is highly pathogenic. In spite of advances in immunological, biotechnological and genetical methods, control of coccidiosis chiefly depends upon prophylactic chemotherapy with anticoccidial drugs. However, the emergence of drug resistance in coccidia is a great problem with most of the drugs, which, in due course, limits their use. Furthermore, drug- or antibiotic-residue in the poultry product is potentially harmful to consumers. These limitations have necessitated the search for alternative Eimeria control measures. Among the several alternative Eimeria control measures only host vaccination against Eimeria appears promising. In this research, our strategy was constructing the cDNA expression library of the sporozoites by Eimeria tenella sporulated oocysts, and screening, cloning the immunogen genes, as well as expressing the interesting genes, analysing the protective immunity. Results of studies displayed as following.
1. Chickens was orally infected with sporulated oocysts of E. tenella YL strain for preparations of anti-E. tenella YL strain positive serum. Rabbit was infected with sporulated oocysts of E. tenella YL strain for preparations of anti-E. tenella YL strain positive serum. The results showed that two kinds of positive sera could be use for immunoscreening from cDNA expression library.
2. We first constructed successfully cDNA expression library of the E. tenella sporozoites by sporulated oocysts of E. tenella YL strain. The results showed that the transformation efficacy of library was 4×106 pfu/mL. We cloned the surface antigen 3-1E
引文
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