猪带绦虫六钩蚴TSOL18重组基因工程疫苗的研制
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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(Cysticercus cellulosae)感染猪或人而引起的人畜共患寄生虫病。是联合国卫生组织列为需要根除的六大疾病之一。目前,研制有效的猪囊虫疫苗来控制和消除流行于世界各地的囊虫病是世界各国普遍关注的焦点。
本研究从孵化并激活的猪带绦虫六钩蚴中提取总RNA,利用RT-PCR技术在国内首次克隆了猪带绦虫六钩蚴疫苗侯选基因TSOL18基因。选用近年来发展最快和最具潜能的真核表达系统-毕赤酵母,构建了重组表达载体pPIC9K-TSOL18,电转化毕赤酵母菌种GS115,并用G418抗性梯度筛选法,获得多拷贝重组菌株。对重组菌株用甲醇进行诱导表达,SDS-PAGE和Western-blot分析结果表明,诱导表达的培养上清中表达出大小为12ku和16ku具有反应活性的重组蛋白。用糖苷内切酶H(Endo-H)对TSOL18蛋白分析后表明,目的蛋白进行了适度的糖基化修饰。通过对发酵条件如pH、温度和溶氧水平等的调整,重组毕赤酵母在5L发酵罐高细胞密度发酵中表达量达2.54g/L,用薄层扫描分析,目的蛋白约占培养上清蛋白总量的80%以上。
在猪带绦虫疫苗研究中,首次应用具有天然蛋白特性的真核表达产物进行动物保护试验。将TSOL18重组抗原与206佐剂结合,制成猪囊虫基因工程疫苗,对猪作了两批免疫试验。试验一中,重组抗原在用206作为佐剂的基础上,分别加上细胞因子(IL-4和IFN-γ)和CpG DNA作为免疫增强剂免疫仔猪,在免疫后15 d攻虫感染。在实验二中,加大了重组抗原TSOL18的免疫剂量,且选择在抗体水平较高时攻虫感染。结果显示,试验各组抗体水平在免疫后15d就呈强阳性,40d达到峰值,而且可保持抗体阳性达150d左右。从免疫保护效果来看,试验一中,TSOL18+206佐剂组减虫率仅为70%,远远低于粗制抗原组;而在试验二中,TSOL18+206佐剂组减虫率高达94%,高于粗制抗原组(89%)。这表明用毕赤酵母表达的猪带绦虫六钩蚴重组抗原疫苗具有广阔的推广应用前景,有望成为预防猪囊虫病的一种新型生物制剂。
用pcDNA3.1/IFN-γ重组质粒作为猪囊虫重组抗原免疫增强剂,抗体检测结果没有明显升高,但免疫猪后淋巴细胞增殖非常明显,并明显增强了疫苗的保护效果,该组免疫猪减虫率高达91%,与粗制抗原免疫组相同。而用pcDNA3.1/IL-4和pUC18/CpG作为免疫增强剂,虽然明显增强了疫苗的抗体水平,但免疫猪淋巴细胞增殖不太明显,且对免疫保护作用也没有明显的增强作用,两组减虫率分别为68.7%和67%,远远低于粗制抗原免疫组。说明IFN-γ对猪带绦虫六钩蚴TSOL18重组抗原具有疫苗增强效应,这为以后选择猪囊虫疫苗的免疫增强剂提供了理论依据。
Cysticercosis caused by Taenia solium larval Cysticercus cellulosae, which can infect human and pigs, is a parasitic zoonsis and this disease is one of six eradicated diseases issued by WHO. At present, it is extensively focused on development of vaccine against cysticercosis.Total RNA was extracted from Taenia solium oncospheres hatched and activated in vitro and vaccine candidate TSOL18 gene was amplified by RT-PCR. The positive recombinant plasmid, designed as pPIC9K-TSOL18, was constructed and transformed into GS115 by electroporation and selected using G418 with gradient concentration. The results of SDS-PAGE and Western blot of the positive GS115 induced with methanol indicated that there were 12ku and 16ku target proteins in supernatant and the latter was of glycosylation determined by Endo-H. Under adjustment of pH, temperature, oxygen content and so on, the amount of TSOL18 secreted by Pichia pastoris was up to 2.54 g/L, accounting for more than 80% in total supernatant proteins.In this studies, it was the first time that secreted TSOL18 antigen with native protein's traits had used for development of cysticercosis vaccines. There was high level of antibodies against recombinant TSOL18 in both 1 and 2 trails and the positive antibodies reached at peak 40d after immunization and retained for 150d. In trail 1, immune increasers cytokines (IL-4 and IFN-γ) and CpG DNA were added into TSOL18 vaccine emulsified with 206 adjuvant, respectively, and pigs were challenged 15d after immunization. Statistic data showed that the group of TSOL18 plus 206 was of 70% protection less than that of coarse antigens. In trail 2, the immunized dose of TSOL18 was increased and eggs challenge was performed during periods of high titer of antibodies and the TSOL18+206 group (94%) was more higher that coarse antigens group in protection (89%). These results suggest that TSOL18 vaccine has promising prospects and is a new bio-product for prevention of cysticercosis.The group, in which pcDNA3.1/IFN-γwas used as a immune increaser, had obvious lymphocyte proliferation and increase in protection (94%) as the same as the coarse antigens group but not high antibody level. The groups using pcDNA3.1/IL-4 and pUC18/CpG were just the way around, 68.7% and 67% protection, respectively. Together with above data, it is recommended that IFN-γis used for development ofTSOL18 vaccine against cysticercosis.
本研究从孵化并激活的猪带绦虫六钩蚴中提取总RNA,利用RT-PCR技术在国内首次克隆了猪带绦虫六钩蚴疫苗侯选基因TSOL18基因。选用近年来发展最快和最具潜能的真核表达系统-毕赤酵母,构建了重组表达载体pPIC9K-TSOL18,电转化毕赤酵母菌种GS115,并用G418抗性梯度筛选法,获得多拷贝重组菌株。对重组菌株用甲醇进行诱导表达,SDS-PAGE和Western-blot分析结果表明,诱导表达的培养上清中表达出大小为12ku和16ku具有反应活性的重组蛋白。用糖苷内切酶H(Endo-H)对TSOL18蛋白分析后表明,目的蛋白进行了适度的糖基化修饰。通过对发酵条件如pH、温度和溶氧水平等的调整,重组毕赤酵母在5L发酵罐高细胞密度发酵中表达量达2.54g/L,用薄层扫描分析,目的蛋白约占培养上清蛋白总量的80%以上。
在猪带绦虫疫苗研究中,首次应用具有天然蛋白特性的真核表达产物进行动物保护试验。将TSOL18重组抗原与206佐剂结合,制成猪囊虫基因工程疫苗,对猪作了两批免疫试验。试验一中,重组抗原在用206作为佐剂的基础上,分别加上细胞因子(IL-4和IFN-γ)和CpG DNA作为免疫增强剂免疫仔猪,在免疫后15 d攻虫感染。在实验二中,加大了重组抗原TSOL18的免疫剂量,且选择在抗体水平较高时攻虫感染。结果显示,试验各组抗体水平在免疫后15d就呈强阳性,40d达到峰值,而且可保持抗体阳性达150d左右。从免疫保护效果来看,试验一中,TSOL18+206佐剂组减虫率仅为70%,远远低于粗制抗原组;而在试验二中,TSOL18+206佐剂组减虫率高达94%,高于粗制抗原组(89%)。这表明用毕赤酵母表达的猪带绦虫六钩蚴重组抗原疫苗具有广阔的推广应用前景,有望成为预防猪囊虫病的一种新型生物制剂。
用pcDNA3.1/IFN-γ重组质粒作为猪囊虫重组抗原免疫增强剂,抗体检测结果没有明显升高,但免疫猪后淋巴细胞增殖非常明显,并明显增强了疫苗的保护效果,该组免疫猪减虫率高达91%,与粗制抗原免疫组相同。而用pcDNA3.1/IL-4和pUC18/CpG作为免疫增强剂,虽然明显增强了疫苗的抗体水平,但免疫猪淋巴细胞增殖不太明显,且对免疫保护作用也没有明显的增强作用,两组减虫率分别为68.7%和67%,远远低于粗制抗原免疫组。说明IFN-γ对猪带绦虫六钩蚴TSOL18重组抗原具有疫苗增强效应,这为以后选择猪囊虫疫苗的免疫增强剂提供了理论依据。
Cysticercosis caused by Taenia solium larval Cysticercus cellulosae, which can infect human and pigs, is a parasitic zoonsis and this disease is one of six eradicated diseases issued by WHO. At present, it is extensively focused on development of vaccine against cysticercosis.Total RNA was extracted from Taenia solium oncospheres hatched and activated in vitro and vaccine candidate TSOL18 gene was amplified by RT-PCR. The positive recombinant plasmid, designed as pPIC9K-TSOL18, was constructed and transformed into GS115 by electroporation and selected using G418 with gradient concentration. The results of SDS-PAGE and Western blot of the positive GS115 induced with methanol indicated that there were 12ku and 16ku target proteins in supernatant and the latter was of glycosylation determined by Endo-H. Under adjustment of pH, temperature, oxygen content and so on, the amount of TSOL18 secreted by Pichia pastoris was up to 2.54 g/L, accounting for more than 80% in total supernatant proteins.In this studies, it was the first time that secreted TSOL18 antigen with native protein's traits had used for development of cysticercosis vaccines. There was high level of antibodies against recombinant TSOL18 in both 1 and 2 trails and the positive antibodies reached at peak 40d after immunization and retained for 150d. In trail 1, immune increasers cytokines (IL-4 and IFN-γ) and CpG DNA were added into TSOL18 vaccine emulsified with 206 adjuvant, respectively, and pigs were challenged 15d after immunization. Statistic data showed that the group of TSOL18 plus 206 was of 70% protection less than that of coarse antigens. In trail 2, the immunized dose of TSOL18 was increased and eggs challenge was performed during periods of high titer of antibodies and the TSOL18+206 group (94%) was more higher that coarse antigens group in protection (89%). These results suggest that TSOL18 vaccine has promising prospects and is a new bio-product for prevention of cysticercosis.The group, in which pcDNA3.1/IFN-γwas used as a immune increaser, had obvious lymphocyte proliferation and increase in protection (94%) as the same as the coarse antigens group but not high antibody level. The groups using pcDNA3.1/IL-4 and pUC18/CpG were just the way around, 68.7% and 67% protection, respectively. Together with above data, it is recommended that IFN-γis used for development ofTSOL18 vaccine against cysticercosis.
引文
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