弓形虫—伪狂犬病毒重组二价基因工程疫苗研究
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摘要
弓形虫病(Toxoplasmosis)是由刚地弓形虫(Toxoplasma gondii)引起的、在世界范围内广泛流行的人兽共患寄生虫病。猫是弓形虫的终末宿主,会排出感染性的卵囊,卵囊被认为是弓形虫感染的主要来源之一。人经食物、饮水等途径摄入卵囊而感染。不过,也有越来越多的研究将目光关注于人通过食用含有T. gondii组织包囊的肉及肉制品而感染弓形虫的风险。免疫预防是控制弓形虫病的主要手段,但由于弓形虫复杂的生活史中有着多种多样的因素对弓形虫免疫产生影响,目前弓形虫的免疫预防研究进展不大。在弓形虫病诊断方面,尽管已有很多方法用于弓形虫的检测,但在临床实际应用中缺乏操作简便、快速且不需要昂贵仪器的病原学诊断方法。为此本研究对T. gondii LAMP病原学诊断方法及T. gondii-PRV二价基因工程疫苗进行了研究,主要研究结果如下：
1.弓形虫LAMP检测方法的建立
环介导等温扩增(loop-mediated isothermal amplification, LAMP)是由日本学者Notomi于2000年发明的一种新型体外恒温核酸扩增方法,主要是利用4种不同的特异性引物识别靶基因的6个特定区域,在等温条件下进行高效扩增反应。基因的扩增和产物的检测可一步完成。目前,该技术已被广泛应用于病毒、细菌、动物原虫等疾病的检测。
本研究中,我们针对弓形虫MIC3基因(GeneBank No. EU572718)设计了一组引物,包括有内引物对、外引物对以及促环引物对,通过一系列反应条件的筛选建立了弓形虫LAMP病原诊断方法。敏感性实验结果表明该方法能检测到的极限值为0.4个速殖子,特异性实验结果表明该方法具有特异性强的特点。利用该方法和B1-based Nested-PCR同时检测320份临床采集的猪的血液和组织样品,其阳性检出率分别为11.6%和9.69%,说明LAMP方法更为敏感。另外利用该方法检测猪肉样品中弓形虫的感染,在某屠宰场的45份样品中检测出9份阳性,而在5个市场收集的68份样品检出3份阳性,这些结果说明,猪肉中弓形虫感染的风险是切实存在的。上述结果表明基于弓形虫MIC3基因的LAMP诊断方法具有敏感性高、特异性强的特点,加上该方法具有操作省时、省力,不需要昂贵的仪器,因此具有很好的推广应用前景。
2.表达弓形虫SAG1、MIC3和GRA7基因的重组伪狂犬病病毒的构建
伪狂犬病病毒(pseudorabies virus, PRV)是一种能引起多种动物以发热、奇痒及脑脊髓炎为主要症状的病毒,由该病毒引起的猪伪狂犬病是目前危害全球养猪业的主要传染病之一。伪狂犬病病毒基因缺失标志疫苗是目前应用最为成功的动物标志疫苗之一,同时也是良好的基因工程重组疫苗载体,迄今为止,已有几十种外源基因在PRV中得到表达。
本研究中,分别将经PCR方法扩增的SAG1、MIC3和GRA7全基因插入到PRV gG-缺失通用转移载体pgG-Uni的多克隆位点中以构建转移质粒pgG-SAG1、pgG-MIC3和pgG-GRA7。然后再将上述质粒与PRV TK-/gG-/EGFP+基因组共转染IBRS-2细胞,病变后经空斑纯化得到重组病毒TK-/gG-/ SAG1(MIC3,GRA7)+。通过Western blotting检测重组病毒中外源基因的表达。结果表明重组病毒构建正确,且外源基因得到了正确表达。重组病毒的TCID50测定结果表明,外源基因的插入不影响重组病毒在IBRS-2细胞上的增殖。重组病毒传代30代后外源基因仍然存在,同时以不同剂量接种实验动物没有出现毒力增强现象,这些结果说明重组病毒具有很好的遗传稳定性和安全性。
3.重组病毒作为疫苗的免疫效果及保护力
利用构建的三株重组病毒单独或联合免疫小鼠,所有接种重组病毒的小鼠都产生对弓形虫裂解抗原(TLA)特异性的抗体,伴随着很强的淋巴细胞增殖反应,并且IFN-γ和IL-2的量明显上升。这些结果说明重组伪狂犬病毒能够诱导产生很强的体液免疫和Th1型的细胞免疫。免疫攻击实验结果表明重组伪狂犬病毒免疫后能使小鼠产生抵抗致死性RH株弓形虫攻击的保护力,明显延长攻虫小鼠的存活时间,并同时诱导产生较高水平PRV中和抗体和抵抗致死性PRV的攻击。而那些用两株和三株rPRV混合免疫的小鼠能够产生更高水平的T. gondii-specific IgG抗体、更强的淋巴细胞增殖反应,并且更有效的抵抗弓形虫的攻击。这些结果说明表达弓形虫保护性抗原的重组伪狂犬病毒具备开发成为一种新型疫苗的潜力,可用来同时预防动物伪狂犬病和弓形虫病。
Toxoplasmosis, caused by Toxoplasma gondii, is widely distributed in warm-blooded animals and has significant zoonotic importance. Cats are the definitive host of T. gondii, may produce infectious oocysts. Oocysts is considered the main way of T. gondii infection, people could be infected T. gondii by ingestion of environmentally resistant oocytes. However, there are more and more research focused on the risk of human Toxoplasma-infection by consumption of meat or meat products contains T. gondii tissue cysts. Immune prevention is the main route for contral of toxoplasmosis. As the life cycle of T. gondii have a wide variety of complex factors that affect on Toxoplasma-immunity, the immune prevention of T. gondii present little progress; and, although there are many methods for detection of Toxoplasma gondii, but lack of a simple and rapid pathogen-diagnostic method in clinical. In the present study, we explored a diagnostic method of LAMP for detecting T. gondii DNA and a bivalent genetic engineering vaccines against T.gondii and pseudorabies virus.
1. Development and application of LAMP assay for detecting T.gondii DNA
Loop-mediated isothermal amplification (LAMP) is invented by Notomi in 2000, as a new type method for nucleic acid amplification in vitro, mainly using 4 different primers identify the target genes of 6 a specific area, in isothermal conditions for efficient amplification. Gene amplification and product detection can be finished at one step, at present, the technology has been widely applied to viruses, bacteria, protozoa and other animal disease detection.
In the present study describes the development of loop-mediated isothermal amplification (LAMP) specific to the single-copy gene MIC3 as a diagnostic tool of toxoplasmosis. A set of primers, composed of outer primers, inner primers and loop primers was designed from a published sequence data (GeneBank No. EU572718). The results confirmed that this LAMP method has good sensitivity and specificity, which can detect the limit to 0.4 tachyzoites. This LAMP and B1-based Nested-PCR were used to detect the 320 clinical samples of pigs at the same time, the positive rates were 11.6% and 9.69% respectively. Then, the LAMP method has been used to detect Toxoplasma gondii infection in pork,9 samples(9/45) from a slaughterhouse were positive,3 samples(3/68) from five market were detected positive, these results showed that the risk of Toxoplasma infection in pork is the fact. All the results indicated that LAMP can be widely used not only for the diagnosis of toxoplasmosis in pig, but also in daily monitoring of the risk of meat infected with T. gondii.
2. Construction of recombinant pseudorabies viruses expressing SAG1, MIC3 and GRA7 gene of T.gondii
Pseudorabies virus(PRV) can cause a variety of animals with fever, itching, and encephalitis as the main symptoms. Pseudorabies caused by the pseudorabies virus is one of the major infectious diseases threatening the global pig industry. Currently, pseudorabies virus(virulence genes knock-out) vaccine is one of most successful vaccines against animal disesases and a good vector, dozens of foreign genes were expressed in PRV.
The DNA fragment containing the T.gondii SAG1(orMIC3, orGRA7) expression cassette [pCMV-SAG1(orMIC3,orGRA7)-BGH poly(A)] was released from pcDNA-SAG1(or pcDNA-MIC3, or pcDNA-GRA7) plasmid, and inserted into pgG-Uni, resulting in the recombinant transfer plasmids pgG-SAG1, pgG-MIC3 and pgG-GRA7. And then this plasmid was co-infected into IBRS-2 cells with genome of PRV TK-/gG-/EGFP+. When cytopathic effect was occurred in cells, recombinant virus TK-/gG-/SAG1(orMIC3, orGRA7)+was obtained from the cytopathic cells by plaque purification. The Three recombinant viruses expression of SAG1(orMIC3, orGRA7) protein was detected with Western blotting respectively. Results of TCID50 showed the insertion of ORF2 or ORF1-ORF2 gene had no influence on the propagation of recombinant viruses in IBRS-2 cells. And, the data also showed that the recombinant viruses are good genetic stability and safety.
3. Immune effect and protective efficacy of recombinant pseudorabies viruses
Three recombinant viruses were used to vaccinate mice with single or combined. All the mice inoculated with the recombinant virus produced specific antibodies to T. gondii lysis antigen (TLA), accompanied by a strong lymphocyte proliferation and the amounts of both IL-2 and IFN-y significantly increased. These results suggested that recombinant pseudorabies virus is capable of inducing strong humoral and Thl-type cellular immunity. Moreover, the mice immunization recombinant pseudorabies virus can produce protective immunity to resist lethal T. gondii RH strain attack, and can induce high levels of PRV neutralizing antibodies.Those with two or even three rPRV mixed immunized mice to produce higher levels of T. gondii-specific IgG antibody, lymphocyte proliferation stronger and more effective attacks against T. gondii. These results indicated that expression of protective antigen of T. gondii recombinant pseudorabies virus have developed the potential to become a new type of vaccine used to prevent and control both animals pseudorabies and toxoplasmosis.
1.弓形虫LAMP检测方法的建立
环介导等温扩增(loop-mediated isothermal amplification, LAMP)是由日本学者Notomi于2000年发明的一种新型体外恒温核酸扩增方法,主要是利用4种不同的特异性引物识别靶基因的6个特定区域,在等温条件下进行高效扩增反应。基因的扩增和产物的检测可一步完成。目前,该技术已被广泛应用于病毒、细菌、动物原虫等疾病的检测。
本研究中,我们针对弓形虫MIC3基因(GeneBank No. EU572718)设计了一组引物,包括有内引物对、外引物对以及促环引物对,通过一系列反应条件的筛选建立了弓形虫LAMP病原诊断方法。敏感性实验结果表明该方法能检测到的极限值为0.4个速殖子,特异性实验结果表明该方法具有特异性强的特点。利用该方法和B1-based Nested-PCR同时检测320份临床采集的猪的血液和组织样品,其阳性检出率分别为11.6%和9.69%,说明LAMP方法更为敏感。另外利用该方法检测猪肉样品中弓形虫的感染,在某屠宰场的45份样品中检测出9份阳性,而在5个市场收集的68份样品检出3份阳性,这些结果说明,猪肉中弓形虫感染的风险是切实存在的。上述结果表明基于弓形虫MIC3基因的LAMP诊断方法具有敏感性高、特异性强的特点,加上该方法具有操作省时、省力,不需要昂贵的仪器,因此具有很好的推广应用前景。
2.表达弓形虫SAG1、MIC3和GRA7基因的重组伪狂犬病病毒的构建
伪狂犬病病毒(pseudorabies virus, PRV)是一种能引起多种动物以发热、奇痒及脑脊髓炎为主要症状的病毒,由该病毒引起的猪伪狂犬病是目前危害全球养猪业的主要传染病之一。伪狂犬病病毒基因缺失标志疫苗是目前应用最为成功的动物标志疫苗之一,同时也是良好的基因工程重组疫苗载体,迄今为止,已有几十种外源基因在PRV中得到表达。
本研究中,分别将经PCR方法扩增的SAG1、MIC3和GRA7全基因插入到PRV gG-缺失通用转移载体pgG-Uni的多克隆位点中以构建转移质粒pgG-SAG1、pgG-MIC3和pgG-GRA7。然后再将上述质粒与PRV TK-/gG-/EGFP+基因组共转染IBRS-2细胞,病变后经空斑纯化得到重组病毒TK-/gG-/ SAG1(MIC3,GRA7)+。通过Western blotting检测重组病毒中外源基因的表达。结果表明重组病毒构建正确,且外源基因得到了正确表达。重组病毒的TCID50测定结果表明,外源基因的插入不影响重组病毒在IBRS-2细胞上的增殖。重组病毒传代30代后外源基因仍然存在,同时以不同剂量接种实验动物没有出现毒力增强现象,这些结果说明重组病毒具有很好的遗传稳定性和安全性。
3.重组病毒作为疫苗的免疫效果及保护力
利用构建的三株重组病毒单独或联合免疫小鼠,所有接种重组病毒的小鼠都产生对弓形虫裂解抗原(TLA)特异性的抗体,伴随着很强的淋巴细胞增殖反应,并且IFN-γ和IL-2的量明显上升。这些结果说明重组伪狂犬病毒能够诱导产生很强的体液免疫和Th1型的细胞免疫。免疫攻击实验结果表明重组伪狂犬病毒免疫后能使小鼠产生抵抗致死性RH株弓形虫攻击的保护力,明显延长攻虫小鼠的存活时间,并同时诱导产生较高水平PRV中和抗体和抵抗致死性PRV的攻击。而那些用两株和三株rPRV混合免疫的小鼠能够产生更高水平的T. gondii-specific IgG抗体、更强的淋巴细胞增殖反应,并且更有效的抵抗弓形虫的攻击。这些结果说明表达弓形虫保护性抗原的重组伪狂犬病毒具备开发成为一种新型疫苗的潜力,可用来同时预防动物伪狂犬病和弓形虫病。
Toxoplasmosis, caused by Toxoplasma gondii, is widely distributed in warm-blooded animals and has significant zoonotic importance. Cats are the definitive host of T. gondii, may produce infectious oocysts. Oocysts is considered the main way of T. gondii infection, people could be infected T. gondii by ingestion of environmentally resistant oocytes. However, there are more and more research focused on the risk of human Toxoplasma-infection by consumption of meat or meat products contains T. gondii tissue cysts. Immune prevention is the main route for contral of toxoplasmosis. As the life cycle of T. gondii have a wide variety of complex factors that affect on Toxoplasma-immunity, the immune prevention of T. gondii present little progress; and, although there are many methods for detection of Toxoplasma gondii, but lack of a simple and rapid pathogen-diagnostic method in clinical. In the present study, we explored a diagnostic method of LAMP for detecting T. gondii DNA and a bivalent genetic engineering vaccines against T.gondii and pseudorabies virus.
1. Development and application of LAMP assay for detecting T.gondii DNA
Loop-mediated isothermal amplification (LAMP) is invented by Notomi in 2000, as a new type method for nucleic acid amplification in vitro, mainly using 4 different primers identify the target genes of 6 a specific area, in isothermal conditions for efficient amplification. Gene amplification and product detection can be finished at one step, at present, the technology has been widely applied to viruses, bacteria, protozoa and other animal disease detection.
In the present study describes the development of loop-mediated isothermal amplification (LAMP) specific to the single-copy gene MIC3 as a diagnostic tool of toxoplasmosis. A set of primers, composed of outer primers, inner primers and loop primers was designed from a published sequence data (GeneBank No. EU572718). The results confirmed that this LAMP method has good sensitivity and specificity, which can detect the limit to 0.4 tachyzoites. This LAMP and B1-based Nested-PCR were used to detect the 320 clinical samples of pigs at the same time, the positive rates were 11.6% and 9.69% respectively. Then, the LAMP method has been used to detect Toxoplasma gondii infection in pork,9 samples(9/45) from a slaughterhouse were positive,3 samples(3/68) from five market were detected positive, these results showed that the risk of Toxoplasma infection in pork is the fact. All the results indicated that LAMP can be widely used not only for the diagnosis of toxoplasmosis in pig, but also in daily monitoring of the risk of meat infected with T. gondii.
2. Construction of recombinant pseudorabies viruses expressing SAG1, MIC3 and GRA7 gene of T.gondii
Pseudorabies virus(PRV) can cause a variety of animals with fever, itching, and encephalitis as the main symptoms. Pseudorabies caused by the pseudorabies virus is one of the major infectious diseases threatening the global pig industry. Currently, pseudorabies virus(virulence genes knock-out) vaccine is one of most successful vaccines against animal disesases and a good vector, dozens of foreign genes were expressed in PRV.
The DNA fragment containing the T.gondii SAG1(orMIC3, orGRA7) expression cassette [pCMV-SAG1(orMIC3,orGRA7)-BGH poly(A)] was released from pcDNA-SAG1(or pcDNA-MIC3, or pcDNA-GRA7) plasmid, and inserted into pgG-Uni, resulting in the recombinant transfer plasmids pgG-SAG1, pgG-MIC3 and pgG-GRA7. And then this plasmid was co-infected into IBRS-2 cells with genome of PRV TK-/gG-/EGFP+. When cytopathic effect was occurred in cells, recombinant virus TK-/gG-/SAG1(orMIC3, orGRA7)+was obtained from the cytopathic cells by plaque purification. The Three recombinant viruses expression of SAG1(orMIC3, orGRA7) protein was detected with Western blotting respectively. Results of TCID50 showed the insertion of ORF2 or ORF1-ORF2 gene had no influence on the propagation of recombinant viruses in IBRS-2 cells. And, the data also showed that the recombinant viruses are good genetic stability and safety.
3. Immune effect and protective efficacy of recombinant pseudorabies viruses
Three recombinant viruses were used to vaccinate mice with single or combined. All the mice inoculated with the recombinant virus produced specific antibodies to T. gondii lysis antigen (TLA), accompanied by a strong lymphocyte proliferation and the amounts of both IL-2 and IFN-y significantly increased. These results suggested that recombinant pseudorabies virus is capable of inducing strong humoral and Thl-type cellular immunity. Moreover, the mice immunization recombinant pseudorabies virus can produce protective immunity to resist lethal T. gondii RH strain attack, and can induce high levels of PRV neutralizing antibodies.Those with two or even three rPRV mixed immunized mice to produce higher levels of T. gondii-specific IgG antibody, lymphocyte proliferation stronger and more effective attacks against T. gondii. These results indicated that expression of protective antigen of T. gondii recombinant pseudorabies virus have developed the potential to become a new type of vaccine used to prevent and control both animals pseudorabies and toxoplasmosis.
引文
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