弓形虫疫苗候选抗原IMP1生物学特性及其免疫原性研究
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摘要
弓形虫是一种专性细胞内寄生繁殖的机会性致病病原体,可以感染包括人在内的几乎所有的温血动物,严重威胁人类及动物健康。正常人感染弓形虫后,大多数不表现出明显的临床症状,呈隐性感染状态,但对婴儿或机体免疫力低下或免疫抑制时(AIDS、骨髓移植、器官移植等)可表现出严重的临床症状,甚至可以引起死亡。妊娠期母畜感染该病,常可引起流产,死产,木乃伊胎以及其它一些严重的临床症状,造成严重的经济损失。目前,对于弓形虫病的防治仍以预防为主,无特效药物。因此,研发切实有效的疫苗是防治的关键。IMP1蛋白是2011年刚发现的蛋白,被认为在弓形虫病疫苗开发方面具有重要潜力。本研究通过分子生物学、基因工程学、免疫生物学等方法对弓形虫IMP1的表达特性及免疫原性进行研究,探讨IMP1及其相关蛋白的功能,为弓形虫病防控提供新的思路与方法。
1.浙江省动物弓形虫病血清流行病学调查
为了解浙江省动物弓形虫病的流行现状,更好地为弓形虫病的防治提供科学的依据,本项目组成员采集了来自浙江省十一地市的813份猪血清、70份奶牛血清、151份山羊血清、213份犬血清及60份猫血清样品,进行了弓形虫病的血清流行病学调查。结果表明,猪的感染率最高,平均血清阳性率高达53.4%,其次为犬16.0%,猫13.3%及山羊2.7%。在对70份奶牛的血清进行检测时,未有阳性样品检出。对采样背景较为详细的猪、犬、猫样品的试验数据进行统计学分析,结果显示,该病的血清阳性率随动物的年龄增长而显著性升高。弓形虫血清阳性率与饲养环境、动物品种等相关。本试验首次对浙江省猪、奶牛、山羊、犬、猫弓形虫血清阳性率进行较为详细的调查,调查结果显示浙江省动物特别是猪的弓形虫感染较为严重,鉴于目前尚无有效的疫苗用于该病的治疗,因此制定合理的方案控制该病的传播在浙江省动物弓形虫的防控方面具有重要意义。
2.基于SAG1基因弓形虫荧光定量PCR检测方法的建立
荧光定量PCR因其高效敏感的特性在各种病原的检测中得到广泛应用,然而其检测效果与所选择的目的基因密切相关。本研究根据GenBank上公布的弓形虫RH株SAG1(又称P30)基因(X14080)保守区域序列设计特异性引物,采用SYBR(?) green I荧光染料法建立弓形虫的荧光定量PCR检测体系。与传统的PCR方法相比,该方法表现出更高的敏感性,最低可以检测到一个弓形虫速殖子。对人工感染小鼠的不同组织进行检测,1天后各样品即可被检出(血液25%、脾脏50%、肺脏50%)。本方法特异性好,与新孢子虫、大肠杆菌、牛巴贝斯虫、布氏锥虫、隐孢子虫、犬弓首蛔虫DNA均无交叉反应。用建立的方法对临床181份猪血进行检测,有11份样品检测结果为阳性,比普通PCR的9份表现出更高的敏感性。研究结果表明,基于SAG1基因建立了一种可靠的弓形虫SYBR(?) green I荧光定量PCR检测方法,该方法的建立可为弓形虫病理学、免疫学及治疗方面的研究提供重要的技术支持。
3.弓形虫IMP1的表达特性研究
弓形虫IMP1是一个新发现的蛋白,在鸡球虫、犬新孢子虫中都有高度保守的同源序列,本研究通过基因克隆技术用弓形虫RH株为模板成功获得了弓形虫IMP1序列,将所扩增基因与GenBank上公布的弓形虫ME49株(XM_002370108;2e-36)的序列进行相似性分析比较,分别在114(A-G)与342(T-C)处各有一个突变,序列相似性为99.8%,但对编码氨基酸无影响。对所获得的序列进行原核表达、纯化,制备了兔源多克隆抗体。通过间接免疫荧光技术进一步对速殖子不同时期该蛋白的表达特性进行研究。研究结果发现在游离、吸附于细胞表面、入侵中、入侵后及纳虫空泡中的速殖子IMP1均有表达,均被定位于虫体表面,且在有些虫体的顶端表达更为强烈。该研究为进一步阐明IMP1的功能奠定了基础。
4.弓形虫二价重组亚单位疫苗rSAG1-IMP1的免疫原性研究
利用分子生物学技术分别构建BL21-pET30a-IMP1及BL21-pET30a-SAG1-IMP1原核表达系统,IPTG诱导重组蛋白的表达。用纯化后的重组蛋白免疫ICR小鼠,探讨弓形虫重组蛋白γSAG1-IMP1的免疫效果。按照每只小鼠100μg融合蛋白的剂量进行首免,2周后按照每只小鼠50μg融合蛋白的剂量进行二免,二免后两周再加强免疫一次。分别于免疫前和首免后每2周小鼠眼眶采血分离血清进行抗体检测,持续至三免后4周；三免后2周取脾脏,进行淋巴细胞增殖试验和细胞因子检测；三免后两周用新鲜传代的弓形虫按500个速殖子／只的剂量攻毒,评价其免疫保护效果。结果表明各重组蛋白均能显著提高机体对融合蛋白的免疫应答水平和淋巴细胞的增殖水平(P<0.05)：蛋白免疫组首免后4周抗体水平开始显著上升,首免后6周即可达到较高水平。其中二价亚单位疫苗rSAG1-IMP1的抗体水平要显著高于单价亚单位疫苗rIMP1和SAG1(P<0.05)。免疫后机体IgG1、IgG2a、IL-4、INF-γ水平均有所上升,但IgG1、IL-4更为明显。攻虫试验表明各蛋白免疫组均能显著延长小鼠的存活时间,弓形虫重组二价亚单位疫苗rSAG1-IMP1较单价亚单位疫苗具有更好的免疫效果。
5.减毒沙门氏菌为载体传递弓形虫IMP1基因的免疫原性研究
研究利用分子生物学技术构建了携带有弓形虫IMP1基因的减毒沙门氏菌活载体核酸疫苗ZJ111/pcDNA3.1-IMP1、ZJ111/pcDNA3.1-SAG1-IMP1。将构建的活载体核酸疫苗与空载减毒沙门氏菌ZJ111/pcDNA3.1免疫ICR小鼠,分别通过抗体水平检测、抗体亚类检测、淋巴细胞增殖试验、细胞因子检测以及攻虫试验等,探讨减毒沙门氏菌活载体核酸疫苗ZJ111/pcDNA3.1-IMP1、 ZJ111/pcDNA3.1-SAG1-IMP1的免疫效果。结果表明：减毒沙门氏菌活载体核酸疫苗ZJ111/pcDNA3.1-IMP1、ZJ111/pcDNA3.1-SAG1-IMP1具有良好的免疫原性,可以诱导小鼠产生较强的体液及细胞免疫应答,显著提高机体INF-γ的分泌及IgG2a水平(P<0.05),对小鼠抵抗弓形虫感染具有一定的免疫保护效果,可显著延长小鼠的存活时间(P<0.05)。而且二价核酸疫苗ZJ111/pcDNA3.1-SAG1-IMP1的免疫保护效果要显著的好于单价核酸疫苗ZJ111/pcDNA3.1-IMP1。
综上,本研究基于SAG1基因建立了一种可靠的弓形虫SYBR(?)green I荧光定量PCR检测方法,首次对浙江省猪、奶牛、山羊、犬、猫弓形虫血清阳性率进行较为详细的调查,通过制备IMP1多克隆抗体,应用间接免疫荧光技术对弓形虫速殖子感染不同阶段的IMP1蛋白的表达特性进行了研究,构建了弓形虫二价重组亚单位疫苗rSAG1-IMP1及减毒沙门氏菌活载体核酸疫苗ZJ111/pcDNA3.1-IMP1、ZJ111/pcDNA3.1-SAG1-IMP1并通过体液免疫和细胞免疫水平及小鼠攻虫试验等分析弓形虫IMP1蛋白作为疫苗的可能性及其前景,研究结果为进一步开发弓形虫病疫苗奠定了良好的基础。
Toxoplasma gondii (T. gondii), an opportunistic intracellular protozoan parasite, can infect almost all worm blood animals including humans and exists as a serious threat to public health. In humans, whilst infections often remain asymptomatic in immunocompetent individuals they can lead to mild or severe sequelae in congenitally infected infants, and can cause life-threatening encephalitis in immunocompromised patients (AIDS, bone marrow and organ transplant patients, etc.). In animals, T. gondii infection is a prominent cause of spontaneous abortion, stillbirth, or severe disease and associated economic losses. At present, there is still no effective medicine for Toxoplasmosis and the only method is still prevention. Therefore, vaccine development plays an important role in the disease prevention. IMP1is a newly discovered protein in2011and considered with great importance in the toxoplasmosis vaccine development. In this study, we used the method of molecular biology, gene engineering, immunobiology to do the further research on the distribution and immunogenicity of IMP1, attempting to know better about the function of IMP1and related proteins, so as to provide a novel strategy for Toxoplasmosis prevention.
1. Seroprevalence of Toxoplasma gondii infection in animals in Zhejiang province
Inorder to assess the seroprevalence of T. gondii infection in animals in Zhejiang province, a survey was carried out via enzyme linked immunosorbent assay (ELISA) using serum (813of pigs,131of cattle,151of goats213of dogs and60of cats) collected from11regions of Zhejiang province. Anti-Toxoplasma antibodies were found in53.4%for pigs,16.0%for dogs,13.3%for cats and2.7%(4/151) for goats. No cattle were found seropositive for T. gondii infection. For pigs and cats, results were statistically analyzed by chi-square (χ2) test. We found that seroprevalence in pigs increased progressively with age. Differences were observed in feeding environment, animals'species and so on. This is the first detailed study on seroprevalence of T. gondii infection in pigs, cattle, goats, pigs and cats in Zhejiang province. The result revealed that there was a high seroprevalence of T. gondii infection in animals especially pigs in the province of Zhejiang. Since there in no effective vaccine against this parasite, prevention of zoonotic transmission must be considered in Zhejiang province.
2. Establish of a real-time PCR assay based on the single-copy SAG1gene for the detection of Toxoplasma gondii
Real-time PCR-based detection of Toxoplasma gondii is very sensitive and convenient for diagnosing toxoplasmosis. However, the performance of the PCR assays could be influenced by the target gene chosen. Here we evaluate a real-time PCR assay using double-stranded DNA dyes (SYBR(?) Green I assay) with a new set of primers targeting the SAG1gene (X14080) for the fast and specific detection of T. gondii. The assay showed higher sensitivity than conventional PCR protocols using T. gondii DNA as template. The detection limit of the developed real-time PCR assay was in the order of1tachyzoite. The assay was also assessed by experimentally infected mice and showed positive results for blood (25%), spleen (50%) and lung (50%) as early as1dpi. The specificity of the assay was confirmed by using DNA from Neospora caninum, Escherichia coli, Babesia bovis, Tiypanosoma brucei, Cryptosporidium parvum, and Toxocara canis. Assay applicability was successfully tested in blood samples collected from slaughtered pigs. These results indicate that, based on SYBR(?) green I, the quantitative SAG1assay may also be useful in the study of the pathogenicity, immunoprophylaxis, and treatment of T. gondii.
3. The subcellular localization of Toxoplasma IMP1
IMP1is a newly discovered protein and has highly conserved homologues in Eimeria spp. and Neospora caninum. We got the IMP1gene by PCR using the genomic DNA of RH strain. Sequence analysis showed that the gene we cloned was99.8%identical to the published data of ME49strain (XM_002370108;2c-36).There arc two mutations, A changed to G in114and T changed to C in342, but they has no effect on the protein expression. The IMP1was expressed in E.coli and anti-IMP1antibody was collected from immunized rabbit. The subcellular localization of IMP1was carried out by indirect immunofluorescence technique, and was anchored on the membrane of the parasite in all the stage (free, absorbing, invading, after invading or in the PV), though some showed strong fluorescence signal on apical. This study is the foundation for deep research of the function of IMP1.
4. The immunogenicity of recombinant sub-unit vaccine rSAG1-IMP1of T. gondii
To evaluate the immunomodulatory potentials of the recombinant sub-unit vaccine rSAG1-IMP1, the BL21-pET30a-IMP1and BL21-pET30a-SAG1-IMP1Prokaryotic expression system were constructed. ICR mice were immunized subcutaneously with PBS,100μg rSAG1,100μg rIMP1,100μg rSAG1-IMP1on day1and immunized subcutaneously with PBS,50μg rSAGl,50μg rIMP1,50μg rSAG1-IMP1on week2and week4after first immune. After immunization, the immune response was evaluated by lymphoproliferative assay, antibody level detection and cytokine measurements. The mice were challenged with500tachyzoites (RH strain) and the survival times of mice were recorded. The results showed that all the recombinant sub-unit vaccines could elicite a high level of specific immune response against T. gondii, and the multiple antigens recombinant sub-unit vaccine rSAGl-IMP1could elicite a higher immune response. Our data demonstrate that the recombinant sub-unit vaccine rSAG1-IMP1can elicit a strong humoral and cellular response, and can effectively help immunized mice against T. gondii infection.
5. The immunogenicity of T. gondii IMP1gene carried by attenuated Salmonella typhimurium
To evaluate the immunomodulatory potentials of IMP1gene carried by attenuated Salmonella typhimurium, the ZJ111/pcDNA3.1-IMP1and ZJ111/pcDNA3.1-SAG1-IMP1DNA vaccine were constructed and immunized to ICR mice. After immunization, we evaluated the immune response by antibody measurements, lymphoproliferative assay, cytokine test and challenge assay in mice. The results showed that DNA vaccine delivered in attenuated Salmonella typhimurium, ZJ111/pcDNA3.1-IMP1、 ZJ111/pcDNA3.1-SAGl-IMPl could elicit a strong humoral and cellular response as well as provide effective protection against T. gondii infection in immunized mice. The multiple antigens vaccine ZJ111/pcDNA3.1-SAG1-IMP1could elicite a higher immune response than ZJ111/pcDNA3.1-IMP1.
In summary, Based on the single-copy SAG1gene, a real-time PCR assay was established for the detection of Toxoplasma gondii, the seroprevalence of T. gondii infection in pigs, cattle, goats, pigs and cats in Zhejiang province was carried out via enzyme linked immunosorbent assay (ELISA) using serum collected from11regions of Zhejiang province, The subcellular localization of IMP1was carried out by indirect immunofluorescence technique, The recombinant sub-unit vaccine rSAG1-IMP1and DNA vaccine ZJ111/pcDNA3.1-IMP1、ZJ111/pcDNA3.1-SAGMMP1against T. gondii were constructed. The possibility and the prospect of IMP1as the potent vaccine was evaluated by humoral and cellular response as well as effective protection against T. gondii infection in immunized mice, hoping to provide a new way for prevent Toxoplasmosis.
1.浙江省动物弓形虫病血清流行病学调查
为了解浙江省动物弓形虫病的流行现状,更好地为弓形虫病的防治提供科学的依据,本项目组成员采集了来自浙江省十一地市的813份猪血清、70份奶牛血清、151份山羊血清、213份犬血清及60份猫血清样品,进行了弓形虫病的血清流行病学调查。结果表明,猪的感染率最高,平均血清阳性率高达53.4%,其次为犬16.0%,猫13.3%及山羊2.7%。在对70份奶牛的血清进行检测时,未有阳性样品检出。对采样背景较为详细的猪、犬、猫样品的试验数据进行统计学分析,结果显示,该病的血清阳性率随动物的年龄增长而显著性升高。弓形虫血清阳性率与饲养环境、动物品种等相关。本试验首次对浙江省猪、奶牛、山羊、犬、猫弓形虫血清阳性率进行较为详细的调查,调查结果显示浙江省动物特别是猪的弓形虫感染较为严重,鉴于目前尚无有效的疫苗用于该病的治疗,因此制定合理的方案控制该病的传播在浙江省动物弓形虫的防控方面具有重要意义。
2.基于SAG1基因弓形虫荧光定量PCR检测方法的建立
荧光定量PCR因其高效敏感的特性在各种病原的检测中得到广泛应用,然而其检测效果与所选择的目的基因密切相关。本研究根据GenBank上公布的弓形虫RH株SAG1(又称P30)基因(X14080)保守区域序列设计特异性引物,采用SYBR(?) green I荧光染料法建立弓形虫的荧光定量PCR检测体系。与传统的PCR方法相比,该方法表现出更高的敏感性,最低可以检测到一个弓形虫速殖子。对人工感染小鼠的不同组织进行检测,1天后各样品即可被检出(血液25%、脾脏50%、肺脏50%)。本方法特异性好,与新孢子虫、大肠杆菌、牛巴贝斯虫、布氏锥虫、隐孢子虫、犬弓首蛔虫DNA均无交叉反应。用建立的方法对临床181份猪血进行检测,有11份样品检测结果为阳性,比普通PCR的9份表现出更高的敏感性。研究结果表明,基于SAG1基因建立了一种可靠的弓形虫SYBR(?) green I荧光定量PCR检测方法,该方法的建立可为弓形虫病理学、免疫学及治疗方面的研究提供重要的技术支持。
3.弓形虫IMP1的表达特性研究
弓形虫IMP1是一个新发现的蛋白,在鸡球虫、犬新孢子虫中都有高度保守的同源序列,本研究通过基因克隆技术用弓形虫RH株为模板成功获得了弓形虫IMP1序列,将所扩增基因与GenBank上公布的弓形虫ME49株(XM_002370108;2e-36)的序列进行相似性分析比较,分别在114(A-G)与342(T-C)处各有一个突变,序列相似性为99.8%,但对编码氨基酸无影响。对所获得的序列进行原核表达、纯化,制备了兔源多克隆抗体。通过间接免疫荧光技术进一步对速殖子不同时期该蛋白的表达特性进行研究。研究结果发现在游离、吸附于细胞表面、入侵中、入侵后及纳虫空泡中的速殖子IMP1均有表达,均被定位于虫体表面,且在有些虫体的顶端表达更为强烈。该研究为进一步阐明IMP1的功能奠定了基础。
4.弓形虫二价重组亚单位疫苗rSAG1-IMP1的免疫原性研究
利用分子生物学技术分别构建BL21-pET30a-IMP1及BL21-pET30a-SAG1-IMP1原核表达系统,IPTG诱导重组蛋白的表达。用纯化后的重组蛋白免疫ICR小鼠,探讨弓形虫重组蛋白γSAG1-IMP1的免疫效果。按照每只小鼠100μg融合蛋白的剂量进行首免,2周后按照每只小鼠50μg融合蛋白的剂量进行二免,二免后两周再加强免疫一次。分别于免疫前和首免后每2周小鼠眼眶采血分离血清进行抗体检测,持续至三免后4周；三免后2周取脾脏,进行淋巴细胞增殖试验和细胞因子检测；三免后两周用新鲜传代的弓形虫按500个速殖子／只的剂量攻毒,评价其免疫保护效果。结果表明各重组蛋白均能显著提高机体对融合蛋白的免疫应答水平和淋巴细胞的增殖水平(P<0.05)：蛋白免疫组首免后4周抗体水平开始显著上升,首免后6周即可达到较高水平。其中二价亚单位疫苗rSAG1-IMP1的抗体水平要显著高于单价亚单位疫苗rIMP1和SAG1(P<0.05)。免疫后机体IgG1、IgG2a、IL-4、INF-γ水平均有所上升,但IgG1、IL-4更为明显。攻虫试验表明各蛋白免疫组均能显著延长小鼠的存活时间,弓形虫重组二价亚单位疫苗rSAG1-IMP1较单价亚单位疫苗具有更好的免疫效果。
5.减毒沙门氏菌为载体传递弓形虫IMP1基因的免疫原性研究
研究利用分子生物学技术构建了携带有弓形虫IMP1基因的减毒沙门氏菌活载体核酸疫苗ZJ111/pcDNA3.1-IMP1、ZJ111/pcDNA3.1-SAG1-IMP1。将构建的活载体核酸疫苗与空载减毒沙门氏菌ZJ111/pcDNA3.1免疫ICR小鼠,分别通过抗体水平检测、抗体亚类检测、淋巴细胞增殖试验、细胞因子检测以及攻虫试验等,探讨减毒沙门氏菌活载体核酸疫苗ZJ111/pcDNA3.1-IMP1、 ZJ111/pcDNA3.1-SAG1-IMP1的免疫效果。结果表明：减毒沙门氏菌活载体核酸疫苗ZJ111/pcDNA3.1-IMP1、ZJ111/pcDNA3.1-SAG1-IMP1具有良好的免疫原性,可以诱导小鼠产生较强的体液及细胞免疫应答,显著提高机体INF-γ的分泌及IgG2a水平(P<0.05),对小鼠抵抗弓形虫感染具有一定的免疫保护效果,可显著延长小鼠的存活时间(P<0.05)。而且二价核酸疫苗ZJ111/pcDNA3.1-SAG1-IMP1的免疫保护效果要显著的好于单价核酸疫苗ZJ111/pcDNA3.1-IMP1。
综上,本研究基于SAG1基因建立了一种可靠的弓形虫SYBR(?)green I荧光定量PCR检测方法,首次对浙江省猪、奶牛、山羊、犬、猫弓形虫血清阳性率进行较为详细的调查,通过制备IMP1多克隆抗体,应用间接免疫荧光技术对弓形虫速殖子感染不同阶段的IMP1蛋白的表达特性进行了研究,构建了弓形虫二价重组亚单位疫苗rSAG1-IMP1及减毒沙门氏菌活载体核酸疫苗ZJ111/pcDNA3.1-IMP1、ZJ111/pcDNA3.1-SAG1-IMP1并通过体液免疫和细胞免疫水平及小鼠攻虫试验等分析弓形虫IMP1蛋白作为疫苗的可能性及其前景,研究结果为进一步开发弓形虫病疫苗奠定了良好的基础。
Toxoplasma gondii (T. gondii), an opportunistic intracellular protozoan parasite, can infect almost all worm blood animals including humans and exists as a serious threat to public health. In humans, whilst infections often remain asymptomatic in immunocompetent individuals they can lead to mild or severe sequelae in congenitally infected infants, and can cause life-threatening encephalitis in immunocompromised patients (AIDS, bone marrow and organ transplant patients, etc.). In animals, T. gondii infection is a prominent cause of spontaneous abortion, stillbirth, or severe disease and associated economic losses. At present, there is still no effective medicine for Toxoplasmosis and the only method is still prevention. Therefore, vaccine development plays an important role in the disease prevention. IMP1is a newly discovered protein in2011and considered with great importance in the toxoplasmosis vaccine development. In this study, we used the method of molecular biology, gene engineering, immunobiology to do the further research on the distribution and immunogenicity of IMP1, attempting to know better about the function of IMP1and related proteins, so as to provide a novel strategy for Toxoplasmosis prevention.
1. Seroprevalence of Toxoplasma gondii infection in animals in Zhejiang province
Inorder to assess the seroprevalence of T. gondii infection in animals in Zhejiang province, a survey was carried out via enzyme linked immunosorbent assay (ELISA) using serum (813of pigs,131of cattle,151of goats213of dogs and60of cats) collected from11regions of Zhejiang province. Anti-Toxoplasma antibodies were found in53.4%for pigs,16.0%for dogs,13.3%for cats and2.7%(4/151) for goats. No cattle were found seropositive for T. gondii infection. For pigs and cats, results were statistically analyzed by chi-square (χ2) test. We found that seroprevalence in pigs increased progressively with age. Differences were observed in feeding environment, animals'species and so on. This is the first detailed study on seroprevalence of T. gondii infection in pigs, cattle, goats, pigs and cats in Zhejiang province. The result revealed that there was a high seroprevalence of T. gondii infection in animals especially pigs in the province of Zhejiang. Since there in no effective vaccine against this parasite, prevention of zoonotic transmission must be considered in Zhejiang province.
2. Establish of a real-time PCR assay based on the single-copy SAG1gene for the detection of Toxoplasma gondii
Real-time PCR-based detection of Toxoplasma gondii is very sensitive and convenient for diagnosing toxoplasmosis. However, the performance of the PCR assays could be influenced by the target gene chosen. Here we evaluate a real-time PCR assay using double-stranded DNA dyes (SYBR(?) Green I assay) with a new set of primers targeting the SAG1gene (X14080) for the fast and specific detection of T. gondii. The assay showed higher sensitivity than conventional PCR protocols using T. gondii DNA as template. The detection limit of the developed real-time PCR assay was in the order of1tachyzoite. The assay was also assessed by experimentally infected mice and showed positive results for blood (25%), spleen (50%) and lung (50%) as early as1dpi. The specificity of the assay was confirmed by using DNA from Neospora caninum, Escherichia coli, Babesia bovis, Tiypanosoma brucei, Cryptosporidium parvum, and Toxocara canis. Assay applicability was successfully tested in blood samples collected from slaughtered pigs. These results indicate that, based on SYBR(?) green I, the quantitative SAG1assay may also be useful in the study of the pathogenicity, immunoprophylaxis, and treatment of T. gondii.
3. The subcellular localization of Toxoplasma IMP1
IMP1is a newly discovered protein and has highly conserved homologues in Eimeria spp. and Neospora caninum. We got the IMP1gene by PCR using the genomic DNA of RH strain. Sequence analysis showed that the gene we cloned was99.8%identical to the published data of ME49strain (XM_002370108;2c-36).There arc two mutations, A changed to G in114and T changed to C in342, but they has no effect on the protein expression. The IMP1was expressed in E.coli and anti-IMP1antibody was collected from immunized rabbit. The subcellular localization of IMP1was carried out by indirect immunofluorescence technique, and was anchored on the membrane of the parasite in all the stage (free, absorbing, invading, after invading or in the PV), though some showed strong fluorescence signal on apical. This study is the foundation for deep research of the function of IMP1.
4. The immunogenicity of recombinant sub-unit vaccine rSAG1-IMP1of T. gondii
To evaluate the immunomodulatory potentials of the recombinant sub-unit vaccine rSAG1-IMP1, the BL21-pET30a-IMP1and BL21-pET30a-SAG1-IMP1Prokaryotic expression system were constructed. ICR mice were immunized subcutaneously with PBS,100μg rSAG1,100μg rIMP1,100μg rSAG1-IMP1on day1and immunized subcutaneously with PBS,50μg rSAGl,50μg rIMP1,50μg rSAG1-IMP1on week2and week4after first immune. After immunization, the immune response was evaluated by lymphoproliferative assay, antibody level detection and cytokine measurements. The mice were challenged with500tachyzoites (RH strain) and the survival times of mice were recorded. The results showed that all the recombinant sub-unit vaccines could elicite a high level of specific immune response against T. gondii, and the multiple antigens recombinant sub-unit vaccine rSAGl-IMP1could elicite a higher immune response. Our data demonstrate that the recombinant sub-unit vaccine rSAG1-IMP1can elicit a strong humoral and cellular response, and can effectively help immunized mice against T. gondii infection.
5. The immunogenicity of T. gondii IMP1gene carried by attenuated Salmonella typhimurium
To evaluate the immunomodulatory potentials of IMP1gene carried by attenuated Salmonella typhimurium, the ZJ111/pcDNA3.1-IMP1and ZJ111/pcDNA3.1-SAG1-IMP1DNA vaccine were constructed and immunized to ICR mice. After immunization, we evaluated the immune response by antibody measurements, lymphoproliferative assay, cytokine test and challenge assay in mice. The results showed that DNA vaccine delivered in attenuated Salmonella typhimurium, ZJ111/pcDNA3.1-IMP1、 ZJ111/pcDNA3.1-SAGl-IMPl could elicit a strong humoral and cellular response as well as provide effective protection against T. gondii infection in immunized mice. The multiple antigens vaccine ZJ111/pcDNA3.1-SAG1-IMP1could elicite a higher immune response than ZJ111/pcDNA3.1-IMP1.
In summary, Based on the single-copy SAG1gene, a real-time PCR assay was established for the detection of Toxoplasma gondii, the seroprevalence of T. gondii infection in pigs, cattle, goats, pigs and cats in Zhejiang province was carried out via enzyme linked immunosorbent assay (ELISA) using serum collected from11regions of Zhejiang province, The subcellular localization of IMP1was carried out by indirect immunofluorescence technique, The recombinant sub-unit vaccine rSAG1-IMP1and DNA vaccine ZJ111/pcDNA3.1-IMP1、ZJ111/pcDNA3.1-SAGMMP1against T. gondii were constructed. The possibility and the prospect of IMP1as the potent vaccine was evaluated by humoral and cellular response as well as effective protection against T. gondii infection in immunized mice, hoping to provide a new way for prevent Toxoplasmosis.
引文
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