弓形虫CDPKs核酸疫苗及重组亚单位疫苗的免疫保护性研究
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摘要
弓形虫病是呈世界性分布的一种重要的人兽共患寄生虫病。该病由专性细胞内寄生的弓形虫所引起，可以导致婴儿脑炎、孕妇和家畜的流产以及免疫功能低下者的脑弓形虫病甚至死亡。由于药物治疗无法彻底治愈弓形虫病，因此免疫预防成为控制该病的首选。但目前为止，尚未有有效、安全的疫苗用来预防人类弓形虫病。
钙依赖蛋白激酶（Calcium-dependent protein kinases，CDPKs）是接收并传导胞外Ca2+信号的效应分子之一，广泛存在于各种植物、绿藻和顶复门原虫中。在弓形虫中，CDPKs参与调控虫体入侵和逸出细胞、蛋白分泌、运动和分化等多种生物学功能，是一类重要的信号通路调控分子。
为研究弓形虫CDPKs基因家族作为抗弓形虫疫苗的候选抗原的可行性和为下一步研制商用型抗弓形虫疫苗寻找思路，本研究首先通过分析TgCDPK2基因在不同宿主和地域来源的13株弓形虫虫株的遗传变异情况。发现TgCDPK2基因的基因组变异率为1.42%，cDNA序列变异率为0.66%，但基于此基因构建的系统发育树发现，该基因无法区分不同基因型的弓形虫虫株。同时对弓形虫CDPKs家族成员的的遗传变异情况研究发现，在弓形虫不同基因型虫株中，12个CDPKs家族成员基因均较保守，其变异率均很低，约在0-1.4%之间。因此，该研究结果提示我们弓形虫CDPKs基因家族成员较为保守，适合作为抗弓形虫疫苗的候选抗原分子，并可产生抵抗不同基因型弓形虫感染的免疫保护效果。
然后选取其中的TgCDPK3，TgCDPK5和TgCDPK6基因翻译成氨基酸序列利用生物信息学方法进行结构分析和抗原表位预测，结果表明在三个蛋白结构中含有数量不等的α-螺旋，β-折叠和无规则卷曲。同时通过综合分析三个蛋白的柔性区域和表面氨基酸序列等，预测三个蛋白中分别含有17、17和14个B淋巴细胞表位，提示其可能具有良好的免疫原性，能作为抗弓形虫疫苗的候选抗原。
分别将TgCDPK3，TgCDPK5和TgCDPK6三个基因连接真核表达载体pVAX I后制备核酸疫苗免疫小鼠，于三免后检测其诱导小鼠产生的免疫应答反应。结果表明，三种疫苗均可以引发免疫小鼠Th1型体液和细胞免疫应答反应。经弓形虫RH株致死性和PRU株慢性攻击感染后，TgCDPK3，TgCDPK5和TgCDPK6免疫小鼠，能显著延长小鼠的存活时间，分别为13.5d，9d和12.2d，脑包囊数量减少率分别为51.93%，46.81%和71.1%。说明三个抗原具有良好的免疫原性，能诱导小鼠产生较强的免疫应答以抵抗弓形虫的感染。
最后将弓形虫ROP18基因和CDPK6基因进行原核表达并纯化，混合后分别用206和聚D,L-乳酸-co-乙醇酸（PLG）为佐剂制备蛋白疫苗免疫小鼠。蛋白疫苗可以在二免后6周刺激机体产生较强的体液和细胞免疫应答反应和抗弓形虫感染效果，且以PLG为佐剂的疫苗效果优于用206为佐剂制备的疫苗。说明用PLG制备的微颗粒可以缓慢释放蛋白抗原，长时间刺激机体产生特异性免疫应答。本研究筛选的三个候选抗原和PLG缓释颗粒的应用，为下一步研制实用性抗弓形虫疫苗奠定基础。
Toxoplasmosis is an important zoonosis having a worldwide distribution. The disease is caused byToxoplasma gondii, an obligate intracellular parasite, which may lead to encephalitis in children,abortion and neonatal loss in pregnant women and in sheep and goats, and cerebral toxoplasmosis oreven death in immunodeficiency individuals. No available chemical treatments could completelyeliminate the parasite in vivo, thus T. gondii vaccine should be an optimal option for controlling thedisease. However, no available one could be used in humans until now.
Calcium-dependent protein kinases (CDPKs) are known as key effectors in regulating calciumrelated signaling pathways, which exist in plants, algae and apicomplexan protozoans. In T. gondii,CDPKs are recognized as significant signaling mediators, involved in control a diverse array offunctions including gliding motility, cell invasion, egress and some other developmental processes thatoccur at distinct stages in its complex life.
To evaluate whether T. gondii CDPKs could be used as vaccine candidates and provide the baselinedata for commercial vaccines against the parasite, we firstly examined the sequence variation inTgCDPK2genes among13T. gondii strains from different hosts and geographical locations in thepresent study. Results showed that the sequence variation of complete TgCDPK2DNA and cDNA were1.42%and0.66%, respectively, suggesting that the gene cannot be used as a proper marker for studyingthe genetic diversity among different T. gondii genotypes. Furthermore, the family of CDPKs was alsoquite conservative, and the low sequences variation ranging from0to1.40%.
We then translated TgCDPK3, TgCDPK5and TgCDPK6gene sequences into amino acidsequences and analyzed the structure and its potential epitopes using multiple bioinformatics approaches.The results showed that all the three proteins contained numbers of α-helixes, β-folds and random coils.A total of17,17and14potential epitopes were predicted in TgCDPK3, TgCDPK5and TgCDPK6proteins, suggesting that these three antigens are potential vaccine candidates.
T. gondii CDPK3, CDPK5and CDPK6genes were then amplified and linked into the eukaryoticexpression vector pVAX I to construct the DNA vaccines, respectively. After the third immunization, theprotective efficacy against T. gondii infection in Kunming mice was evaluated. The results showed thatall the three vaccines could develop Th1type response with a high level of specific antibodies andstrong lymphoproliferative responses. The mice immunized with TgCDPK3, TgCDPK5and TgCDPK6genes were prolonged the survival time to13.5d,9d and12.2d compared to the controls which diedwithin7.2days after lethal challenge with the tachyzoites of the virulent T. gondii RH strain. In chronicinfection, the numbers of brain cysts of the mice immunized with TgCDPK3, TgCDPK5and TgCDPK6were reduced significantly when compared with those in control groups (P <0.05), and the rate ofreduction could reach to51.93%,46.81%and71.1%, suggesting that TgCDPK3, TgCDPK5andTgCDPK6are promising vaccine candidates against T. gondii infection.
In the end, we amplified the TgROP18and TgCDPK6genes and ligated to pET-30a for prokaryotic expression. The purified ROP18protein and CDPK6protein were mixed and emulsifiedwith206and poly (lactide-co-glycolide)(PLG). The protein vaccines were used to immunize Kunmingmice. At6weeks after the last immunization, the vaccines could induce strong humoral and cellularimmune responses and generate protective immunity against acute and chronic T. gondii infection.Furthermore, protein plus PLG could elicit better protective efficacy compared to the protein plus the206adjuvant. The identification of the three new vaccine candidates and the evaluation of antigensencapsulated in PLG microparticles provide a valuable basis for developing new vaccines against T.gondii for future use in humans and animals.
钙依赖蛋白激酶（Calcium-dependent protein kinases，CDPKs）是接收并传导胞外Ca2+信号的效应分子之一，广泛存在于各种植物、绿藻和顶复门原虫中。在弓形虫中，CDPKs参与调控虫体入侵和逸出细胞、蛋白分泌、运动和分化等多种生物学功能，是一类重要的信号通路调控分子。
为研究弓形虫CDPKs基因家族作为抗弓形虫疫苗的候选抗原的可行性和为下一步研制商用型抗弓形虫疫苗寻找思路，本研究首先通过分析TgCDPK2基因在不同宿主和地域来源的13株弓形虫虫株的遗传变异情况。发现TgCDPK2基因的基因组变异率为1.42%，cDNA序列变异率为0.66%，但基于此基因构建的系统发育树发现，该基因无法区分不同基因型的弓形虫虫株。同时对弓形虫CDPKs家族成员的的遗传变异情况研究发现，在弓形虫不同基因型虫株中，12个CDPKs家族成员基因均较保守，其变异率均很低，约在0-1.4%之间。因此，该研究结果提示我们弓形虫CDPKs基因家族成员较为保守，适合作为抗弓形虫疫苗的候选抗原分子，并可产生抵抗不同基因型弓形虫感染的免疫保护效果。
然后选取其中的TgCDPK3，TgCDPK5和TgCDPK6基因翻译成氨基酸序列利用生物信息学方法进行结构分析和抗原表位预测，结果表明在三个蛋白结构中含有数量不等的α-螺旋，β-折叠和无规则卷曲。同时通过综合分析三个蛋白的柔性区域和表面氨基酸序列等，预测三个蛋白中分别含有17、17和14个B淋巴细胞表位，提示其可能具有良好的免疫原性，能作为抗弓形虫疫苗的候选抗原。
分别将TgCDPK3，TgCDPK5和TgCDPK6三个基因连接真核表达载体pVAX I后制备核酸疫苗免疫小鼠，于三免后检测其诱导小鼠产生的免疫应答反应。结果表明，三种疫苗均可以引发免疫小鼠Th1型体液和细胞免疫应答反应。经弓形虫RH株致死性和PRU株慢性攻击感染后，TgCDPK3，TgCDPK5和TgCDPK6免疫小鼠，能显著延长小鼠的存活时间，分别为13.5d，9d和12.2d，脑包囊数量减少率分别为51.93%，46.81%和71.1%。说明三个抗原具有良好的免疫原性，能诱导小鼠产生较强的免疫应答以抵抗弓形虫的感染。
最后将弓形虫ROP18基因和CDPK6基因进行原核表达并纯化，混合后分别用206和聚D,L-乳酸-co-乙醇酸（PLG）为佐剂制备蛋白疫苗免疫小鼠。蛋白疫苗可以在二免后6周刺激机体产生较强的体液和细胞免疫应答反应和抗弓形虫感染效果，且以PLG为佐剂的疫苗效果优于用206为佐剂制备的疫苗。说明用PLG制备的微颗粒可以缓慢释放蛋白抗原，长时间刺激机体产生特异性免疫应答。本研究筛选的三个候选抗原和PLG缓释颗粒的应用，为下一步研制实用性抗弓形虫疫苗奠定基础。
Toxoplasmosis is an important zoonosis having a worldwide distribution. The disease is caused byToxoplasma gondii, an obligate intracellular parasite, which may lead to encephalitis in children,abortion and neonatal loss in pregnant women and in sheep and goats, and cerebral toxoplasmosis oreven death in immunodeficiency individuals. No available chemical treatments could completelyeliminate the parasite in vivo, thus T. gondii vaccine should be an optimal option for controlling thedisease. However, no available one could be used in humans until now.
Calcium-dependent protein kinases (CDPKs) are known as key effectors in regulating calciumrelated signaling pathways, which exist in plants, algae and apicomplexan protozoans. In T. gondii,CDPKs are recognized as significant signaling mediators, involved in control a diverse array offunctions including gliding motility, cell invasion, egress and some other developmental processes thatoccur at distinct stages in its complex life.
To evaluate whether T. gondii CDPKs could be used as vaccine candidates and provide the baselinedata for commercial vaccines against the parasite, we firstly examined the sequence variation inTgCDPK2genes among13T. gondii strains from different hosts and geographical locations in thepresent study. Results showed that the sequence variation of complete TgCDPK2DNA and cDNA were1.42%and0.66%, respectively, suggesting that the gene cannot be used as a proper marker for studyingthe genetic diversity among different T. gondii genotypes. Furthermore, the family of CDPKs was alsoquite conservative, and the low sequences variation ranging from0to1.40%.
We then translated TgCDPK3, TgCDPK5and TgCDPK6gene sequences into amino acidsequences and analyzed the structure and its potential epitopes using multiple bioinformatics approaches.The results showed that all the three proteins contained numbers of α-helixes, β-folds and random coils.A total of17,17and14potential epitopes were predicted in TgCDPK3, TgCDPK5and TgCDPK6proteins, suggesting that these three antigens are potential vaccine candidates.
T. gondii CDPK3, CDPK5and CDPK6genes were then amplified and linked into the eukaryoticexpression vector pVAX I to construct the DNA vaccines, respectively. After the third immunization, theprotective efficacy against T. gondii infection in Kunming mice was evaluated. The results showed thatall the three vaccines could develop Th1type response with a high level of specific antibodies andstrong lymphoproliferative responses. The mice immunized with TgCDPK3, TgCDPK5and TgCDPK6genes were prolonged the survival time to13.5d,9d and12.2d compared to the controls which diedwithin7.2days after lethal challenge with the tachyzoites of the virulent T. gondii RH strain. In chronicinfection, the numbers of brain cysts of the mice immunized with TgCDPK3, TgCDPK5and TgCDPK6were reduced significantly when compared with those in control groups (P <0.05), and the rate ofreduction could reach to51.93%,46.81%and71.1%, suggesting that TgCDPK3, TgCDPK5andTgCDPK6are promising vaccine candidates against T. gondii infection.
In the end, we amplified the TgROP18and TgCDPK6genes and ligated to pET-30a for prokaryotic expression. The purified ROP18protein and CDPK6protein were mixed and emulsifiedwith206and poly (lactide-co-glycolide)(PLG). The protein vaccines were used to immunize Kunmingmice. At6weeks after the last immunization, the vaccines could induce strong humoral and cellularimmune responses and generate protective immunity against acute and chronic T. gondii infection.Furthermore, protein plus PLG could elicit better protective efficacy compared to the protein plus the206adjuvant. The identification of the three new vaccine candidates and the evaluation of antigensencapsulated in PLG microparticles provide a valuable basis for developing new vaccines against T.gondii for future use in humans and animals.
引文
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