猪带绦虫六钩蚴45W-4B和TSO18重组基因工程疫苗的研究
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摘要
囊虫病(Cysticercosis)是由猪带绦虫(Taenia solium)的幼虫猪囊尾蚴寄生于人或猪等中间宿主丽引起的人畜共患寄生虫病,在中国大部分地区尤其在少数民族地区是一个重要的公共卫生问题,对人的健康危害很大。不仅如此,囊虫病的广泛存在还造成畜牧业的重大经济损失,极大影响了我国畜产品在国际市场上的竞争力,因而是公认的社会经济病之一。猪囊尾蚴病的免疫预防之所以一直困扰着兽医工作者,主要原因是抗原来源非常有限。羊绦虫45W重组基因工程疫苗的研制成功,为猪囊尾蚴重组基因工程疫苗的研制提供了典范。为了探索猪带绦虫45W-4B和TSO18重组抗原对猪囊尾蚴感染的免疫保护效果,获得具有良好免疫保护作用的重组抗原,用做猪囊虫病的免疫预防,我们进行了本项研究。
收集成熟的猪带绦虫虫卵,经孵化和激活后,提取六钩蚴总RNA,设计特异性引物RT-PCR扩增45W-4B和TSO18基因,测序证实扩增产物与国外克隆的猪带绦虫45W-4B和TSOL18的同源性分别为99.5%和99.6%,说明TSO18和45W-4B在不同虫株间相对保守。用蛋白质专家系统对TSO18和45W-4B进行结构预测,发现它们都有分泌信号序列、FnⅢ结构域以及糖基化、磷酸化位点。根据预测结果PCR扩增截去45W-4B基因的N端信号肽和C端疏水氨基酸序列,获得45W-4BX。将TSO18和45W-4BX经限制性内切酶处理后与相同处理的表达载体pGEX-4T-1连接,转化BL_(21)感受态细胞,酶切及PCR扩增鉴定获得两基因的重组表达菌株。经测序证明阅读框完全正确,用IPTG诱导表达,产物进行SDS—PAGE分析,发现在合适的诱导温度和IPTG浓度下,分别获得了高效表达且以可溶性形式存在的45W-4BX和以包涵体形式存在的TSO18重组蛋白。Westernblot分析表明无论是感染初期的猪囊尾蚴血清还是人囊虫阳性血清均可被两种重组蛋白所识别。
分别用纯化的GST、TSO18、45W-4BX重组蛋白和猪囊尾蚴粗提物为抗原,以206为佐剂制成疫苗免疫猪,用25000枚猪带绦虫虫卵攻击感染,ELISA测定不同时间各组的抗体水平,同时用MTT法分析免疫和感染后猪外周血淋巴细胞增殖情况。结果表明,免疫后15d抗体水平开始上升,在感染后30d左右达到峰值,且至少在免疫后90d内保持阳性水平。猪免疫后外周血淋巴细胞略有增殖,而在感染后10d迅速升高,21d时基本降至未免疫对照组的水平。感染后90d剖检猪,计数各组的囊尾蚴感染数,结果发现TS018和45W-4BX单独免疫组的减虫率分别达95%和94%,与猪囊尾蚴粗抗原免疫组(减虫率为92%)的保护效果相当。而TSO18+4BX+IL4免疫组的减虫率为99.7%,完全保护率达80%,保护效果明显优于粗抗原对照组。在补体参与下,TSO18和45W-4BX抗体对六钩蚴的杀伤作用大大增强,六钩蚴的存活率分别从90%和85%下降到55%和70%。TSO18在补体存在下杀伤六钩蚴的作用与未加补体对照间存在显著差异(P=0.024<0.05)。囊尾蚴抗体水平的测定表明,虫体负荷与抗体水平间不存在相关性。重组抗原的稳定性分析发现,TSO18保存3个月后免疫猪减虫率降低了51%,保存两个月后含量检测发现1/3的蛋白已经降解,表明TSO18
Cysticercosis is an important zoonosis caused by Taenia solium larval cysticercus cellulosae that can infect intermediate hosts such as pigs or human, which is a major public health problem in most areas of China, especially in minority regions, and a great potential threat to human security. This disease, whose prevention is always an obstacle for veterinarians mainly because of limitation of protective antigens, is known as an economic disease for it attributes not only to great economic loses but also to decrease of international competition on meat products. The success of commercialization of a recombinant 45W vaccine against Taenia ovis provides a bright way to development of such a vaccine against cysticercosis.In the studies, we attempt to develop an effective vaccine based on candidates 45W-4B and TSO18 through evaluation of protection of recombinant antigens in experimental animals.Taenia solium eggs were collected and total RNA was extracted from hatched and activated eggs in vitro. Genes 45W-4B and TSO18, which showed high homology with the published, were cloned and predicted with regard to their structure traits using web sources. The truncated 45W-4B with removal of a signal peptide at N terminal and 19 hydrophobic amino acids at C terminal, designed as 45W-4BX, was amplified by PCR according to predictive results. The interest fragments TSO18 and 45W-4BX were ligated with an expression vector pGEX-4T-l with the same cohesive ends, transformed into competent bacteria BL_21 and identified with enzyme restriction and PCR and sequencing. Soluble 45W-4BX and TSO18 in the form of inclusive bodies were detected on SDS-PAGE, induced under suitable conditions of temperature and concentration of IPTG. Western blot result indicated that they could be recognized by both sera from pig infected by early developed cysticercus cellulosae and cysticercosis patients.To evaluate the efficacy of protection through lymphocyte proliferation and specific antibody level at different infective periods using MTT method and ELISA, respectively, and cyst determined 90 days postinfection, purified antigens 45W-4BX, TSO18 and GST and cysticercus cellulosae coarse extract were immunized pigs, which were experimentally challenged with 25000 eggs 28 days postimmunization, and 206 was used as adjuvant. Data suggested that antibody level would start to raise and reach at peak 15 and 45 days postimmunization, respectively. MTT analysis indicated that a little proliferation of lymphocyte existed postimmunization and peaked at 10th days after challenge. Trials in vitro certified that complements seemed involved in killing effects of antigens 45W-4BX and TSO18 on oncospheres. Moreover, animal experiments showed the groups immunized with recombinant proteins alone were a 94% and 95% reduction in the number of cysticerci, respectively. Level
收集成熟的猪带绦虫虫卵,经孵化和激活后,提取六钩蚴总RNA,设计特异性引物RT-PCR扩增45W-4B和TSO18基因,测序证实扩增产物与国外克隆的猪带绦虫45W-4B和TSOL18的同源性分别为99.5%和99.6%,说明TSO18和45W-4B在不同虫株间相对保守。用蛋白质专家系统对TSO18和45W-4B进行结构预测,发现它们都有分泌信号序列、FnⅢ结构域以及糖基化、磷酸化位点。根据预测结果PCR扩增截去45W-4B基因的N端信号肽和C端疏水氨基酸序列,获得45W-4BX。将TSO18和45W-4BX经限制性内切酶处理后与相同处理的表达载体pGEX-4T-1连接,转化BL_(21)感受态细胞,酶切及PCR扩增鉴定获得两基因的重组表达菌株。经测序证明阅读框完全正确,用IPTG诱导表达,产物进行SDS—PAGE分析,发现在合适的诱导温度和IPTG浓度下,分别获得了高效表达且以可溶性形式存在的45W-4BX和以包涵体形式存在的TSO18重组蛋白。Westernblot分析表明无论是感染初期的猪囊尾蚴血清还是人囊虫阳性血清均可被两种重组蛋白所识别。
分别用纯化的GST、TSO18、45W-4BX重组蛋白和猪囊尾蚴粗提物为抗原,以206为佐剂制成疫苗免疫猪,用25000枚猪带绦虫虫卵攻击感染,ELISA测定不同时间各组的抗体水平,同时用MTT法分析免疫和感染后猪外周血淋巴细胞增殖情况。结果表明,免疫后15d抗体水平开始上升,在感染后30d左右达到峰值,且至少在免疫后90d内保持阳性水平。猪免疫后外周血淋巴细胞略有增殖,而在感染后10d迅速升高,21d时基本降至未免疫对照组的水平。感染后90d剖检猪,计数各组的囊尾蚴感染数,结果发现TS018和45W-4BX单独免疫组的减虫率分别达95%和94%,与猪囊尾蚴粗抗原免疫组(减虫率为92%)的保护效果相当。而TSO18+4BX+IL4免疫组的减虫率为99.7%,完全保护率达80%,保护效果明显优于粗抗原对照组。在补体参与下,TSO18和45W-4BX抗体对六钩蚴的杀伤作用大大增强,六钩蚴的存活率分别从90%和85%下降到55%和70%。TSO18在补体存在下杀伤六钩蚴的作用与未加补体对照间存在显著差异(P=0.024<0.05)。囊尾蚴抗体水平的测定表明,虫体负荷与抗体水平间不存在相关性。重组抗原的稳定性分析发现,TSO18保存3个月后免疫猪减虫率降低了51%,保存两个月后含量检测发现1/3的蛋白已经降解,表明TSO18
Cysticercosis is an important zoonosis caused by Taenia solium larval cysticercus cellulosae that can infect intermediate hosts such as pigs or human, which is a major public health problem in most areas of China, especially in minority regions, and a great potential threat to human security. This disease, whose prevention is always an obstacle for veterinarians mainly because of limitation of protective antigens, is known as an economic disease for it attributes not only to great economic loses but also to decrease of international competition on meat products. The success of commercialization of a recombinant 45W vaccine against Taenia ovis provides a bright way to development of such a vaccine against cysticercosis.In the studies, we attempt to develop an effective vaccine based on candidates 45W-4B and TSO18 through evaluation of protection of recombinant antigens in experimental animals.Taenia solium eggs were collected and total RNA was extracted from hatched and activated eggs in vitro. Genes 45W-4B and TSO18, which showed high homology with the published, were cloned and predicted with regard to their structure traits using web sources. The truncated 45W-4B with removal of a signal peptide at N terminal and 19 hydrophobic amino acids at C terminal, designed as 45W-4BX, was amplified by PCR according to predictive results. The interest fragments TSO18 and 45W-4BX were ligated with an expression vector pGEX-4T-l with the same cohesive ends, transformed into competent bacteria BL_21 and identified with enzyme restriction and PCR and sequencing. Soluble 45W-4BX and TSO18 in the form of inclusive bodies were detected on SDS-PAGE, induced under suitable conditions of temperature and concentration of IPTG. Western blot result indicated that they could be recognized by both sera from pig infected by early developed cysticercus cellulosae and cysticercosis patients.To evaluate the efficacy of protection through lymphocyte proliferation and specific antibody level at different infective periods using MTT method and ELISA, respectively, and cyst determined 90 days postinfection, purified antigens 45W-4BX, TSO18 and GST and cysticercus cellulosae coarse extract were immunized pigs, which were experimentally challenged with 25000 eggs 28 days postimmunization, and 206 was used as adjuvant. Data suggested that antibody level would start to raise and reach at peak 15 and 45 days postimmunization, respectively. MTT analysis indicated that a little proliferation of lymphocyte existed postimmunization and peaked at 10th days after challenge. Trials in vitro certified that complements seemed involved in killing effects of antigens 45W-4BX and TSO18 on oncospheres. Moreover, animal experiments showed the groups immunized with recombinant proteins alone were a 94% and 95% reduction in the number of cysticerci, respectively. Level
引文
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