柔嫩艾美耳球虫YL株3-1E和TA4基因的原核共表达
摘要
艾美耳属(Eimeria)的寄生原虫常常会给家禽养殖业造成巨大的经济损失。柔嫩艾美耳球虫(E. tenella)是感染鸡的7种艾美耳球虫中毒力最强的一种。它寄生于盲肠内,可引起盲肠出血,严重的则因为失血和休克而造成贫血甚至死亡。它的寄生会影响鸡只的生长发育和饲料的利用率,造成养鸡生产中死亡率上升,给家禽养殖业造成巨大的经济损失。艾美耳球虫复杂的生命周期包括在环境中的体外生殖阶段,在此期间排出卵囊进行孢子化。通过摄入孢子化卵囊获得感染后,在鸡肠道内进行无性繁殖(裂殖生殖)和性分化(配子生殖),随后受精,卵囊脱落。目前主要以药物治疗为主,由于长年大量的使用抗球虫药物造成耐药性的出现,基因工程苗或核酸疫苗成为防治鸡球虫病的新的研究方向。本试验在3-1E和TA4两个单个基因重组蛋白能对鸡产生一定的免疫保护力的基础上,对鸡E. tenella杨凌(YL)株相关抗原基因3-1E和TA4进行了重组克隆共表达研究。本研究获得了以下结果:
(1)应用RT-PCR方法从E. tenella YL株总RNA中扩增出3-1E基因,克隆的E. tenella YL 3-1E基因ORF为513个碱基,共编码170个氨基酸,与其它虫株相关基因比较,核苷酸同源性为99.22%~99.81%,氨基酸同源性为98%~99.33%。研究表明,3-1E基因的7处易变位点导致的氨基酸改变基本都不在3-1E蛋白表面暴露区,也都不在可能的优势抗原表位区,由此可推测3-1E基因的蛋白抗原性比较保守,并且具有很好的交叉保护性。将3-1E基因定向克隆到pET-32a(+)表达载体中,经诱导表达,成功地表达出蛋白分子量为42.7 ku的融合蛋白。
(2)将3-1E基因和TA4基因融合基因插入到pET-32a(+)载体中,构建成了pET-32a-TA4-3-1E共表达载体,转化BL21宿主菌并诱导表达后,通过SDS-PAGE检测,表明成功地表达出融合蛋白,蛋白分子量为59.7 ku,与预测的蛋白大小相符。
Protozoan parasites of the genus Eimeria cause great economic losses in the poultry industry. Eimeria tenella, one of the most virulent of seven species of Eimeria that infect chickens, parasitizes in the caeca and provokes haemorrhage and, in severe cases, anaemia and death due to blood loss and shock. It impairs bird growth and feed utilization, causes high mortality, and leads to tremendous economic losses in the poultry industry. The complex life cycle of Eimeria comprises an exogenous phase in the environment during which excreted oocysts undergo sporulation. After infection via ingestion of sporulated oocysts, an endogenous phase in the chicken intestine consisting of asexual reproduction (schizogony) and sexual differentiation (gamogony) takes place, followed by fertilization and shedding of unsporulated oocysts. At present, it mainly depends on drug treatment. The extensive use of anticoccidial drugs over the years has led to the emergence of drug resistance, so that the recombinant vaccines and/or DNA vaccines is becoming the new way to control the avain coccidiosis. For the 3-1E or TA4 recombinant protein can cause a determinate immunoprotection, the reaserch constructed a recombinant vector to co-express 3-1E and TA4 genes of E. tenella YL strain. The results as follows:
(1) The 3-1E gene was amplified by RT-PCR. The ORF of this gene is consisted of 513 nucleotides encoding 170 amino acids. Compared with other strains, the homology of nucleotides acid sequence was 99.22%~99.81%, and amino acid encoded was 98%~99.33%. The research showed that the seven labilization amino acid sequences are neither in the exposed area of the 3-1E protein surface nor in the possible advantage epitope area. The antigenicity of 3-1E protein is conservative, and the protein has a good cross-protection. The amplicons of 3-1E gene were cloned into pET-32a(+) vector, then the expression was induced with IPTG . It showed that the fusion protein was about 42.7 ku.
(2) The fusion gene of 3-1E and TA4 was cloned into pET-32a(+) vector, the recombinant vector of pET-32a-TA4-3-1E was constructed correctly. Then it was transformed into E.coli BL21 (DE3) and induced with IPTG. The expressed protein was detected by SDS-PAGE, with the expected molecular sizes of 59.7 ku. The expressed protein was consistent with the prediction.
(1)应用RT-PCR方法从E. tenella YL株总RNA中扩增出3-1E基因,克隆的E. tenella YL 3-1E基因ORF为513个碱基,共编码170个氨基酸,与其它虫株相关基因比较,核苷酸同源性为99.22%~99.81%,氨基酸同源性为98%~99.33%。研究表明,3-1E基因的7处易变位点导致的氨基酸改变基本都不在3-1E蛋白表面暴露区,也都不在可能的优势抗原表位区,由此可推测3-1E基因的蛋白抗原性比较保守,并且具有很好的交叉保护性。将3-1E基因定向克隆到pET-32a(+)表达载体中,经诱导表达,成功地表达出蛋白分子量为42.7 ku的融合蛋白。
(2)将3-1E基因和TA4基因融合基因插入到pET-32a(+)载体中,构建成了pET-32a-TA4-3-1E共表达载体,转化BL21宿主菌并诱导表达后,通过SDS-PAGE检测,表明成功地表达出融合蛋白,蛋白分子量为59.7 ku,与预测的蛋白大小相符。
Protozoan parasites of the genus Eimeria cause great economic losses in the poultry industry. Eimeria tenella, one of the most virulent of seven species of Eimeria that infect chickens, parasitizes in the caeca and provokes haemorrhage and, in severe cases, anaemia and death due to blood loss and shock. It impairs bird growth and feed utilization, causes high mortality, and leads to tremendous economic losses in the poultry industry. The complex life cycle of Eimeria comprises an exogenous phase in the environment during which excreted oocysts undergo sporulation. After infection via ingestion of sporulated oocysts, an endogenous phase in the chicken intestine consisting of asexual reproduction (schizogony) and sexual differentiation (gamogony) takes place, followed by fertilization and shedding of unsporulated oocysts. At present, it mainly depends on drug treatment. The extensive use of anticoccidial drugs over the years has led to the emergence of drug resistance, so that the recombinant vaccines and/or DNA vaccines is becoming the new way to control the avain coccidiosis. For the 3-1E or TA4 recombinant protein can cause a determinate immunoprotection, the reaserch constructed a recombinant vector to co-express 3-1E and TA4 genes of E. tenella YL strain. The results as follows:
(1) The 3-1E gene was amplified by RT-PCR. The ORF of this gene is consisted of 513 nucleotides encoding 170 amino acids. Compared with other strains, the homology of nucleotides acid sequence was 99.22%~99.81%, and amino acid encoded was 98%~99.33%. The research showed that the seven labilization amino acid sequences are neither in the exposed area of the 3-1E protein surface nor in the possible advantage epitope area. The antigenicity of 3-1E protein is conservative, and the protein has a good cross-protection. The amplicons of 3-1E gene were cloned into pET-32a(+) vector, then the expression was induced with IPTG . It showed that the fusion protein was about 42.7 ku.
(2) The fusion gene of 3-1E and TA4 was cloned into pET-32a(+) vector, the recombinant vector of pET-32a-TA4-3-1E was constructed correctly. Then it was transformed into E.coli BL21 (DE3) and induced with IPTG. The expressed protein was detected by SDS-PAGE, with the expected molecular sizes of 59.7 ku. The expressed protein was consistent with the prediction.
引文
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