猪圆环病毒2型遗传演化及含CpG基序的核酸疫苗研制
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摘要
猪圆环病毒(Porcine circovirus,PCV)分为无致病性的PCV1和有致病性的PCV2两个基因型。PCV2能引起断奶仔猪多系统衰竭综合征（PMWS），猪皮炎与肾病综合征（PDNS）、猪呼吸道病复合征（PRDC）、猪的流产和死亡综合征（SAMS）、猪增生性和坏死性肺炎（PNP）和猪先天性脑震颤（CT）等，并能导致母猪繁殖障碍和引起免疫抑制，给养猪业造成严重的经济损失。该病已被世界各国的兽医与养猪业者公认为是继猪繁殖与呼吸综合征之后新发现的重要猪传染病。
近年来该病毒不断发生变异，新的毒株不断出现，给该病的防控带来了一定的压力。为了深入了解本病的流行情况及研制新型的基因工程疫苗用于该病的防治，本研究在流行病学调查的基础上，分离鉴定了3株PCV2，对17株病毒进行了全基因组克隆测序并进行了PCV2遗传演化分析；调查了PCV2同属的Torque teno virus (TTV)在山东省猪群中的流行情况；建立了荧光定量PCR检测方法和PCV2小鼠动物模型；研制了含CpG基序的PCV2核酸疫苗。
主要表现为：对山东省规模化猪场进行了猪圆环病毒感染率的流行病学调查，发现猪圆环病毒的感染率呈逐年上升趋势，2005-2011抗原阳性率由11.3%增加到40.38%，抗体阳性率由38.7%增加到72.99%。混合感染现象仍很严重，二重感染中PCV+CSFV所占比例最大，为33.81%；三重感染中PCV+PRRSV+CSFV所占比例最大，为4.32%。在此基础上完成3株PCV2的分离鉴定工作，并进行了系列生物学鉴定。其中SD株（JQ653449）传代至第20代病毒毒价为106.0TCID50/mL。目前该病毒传至75代，效价稳定可以作为灭活疫苗的候选毒株。完成17株猪圆环病毒的全基因克隆测序。通过与从已公布的国内外PCV2分离株的比较分析发现国内PCV2一直在不断的变异，PCV2b是目前国内流行株，出现PCV2a和PCV2b重组型病毒，其基因组特征为ORF1来自PCV2a，ORF2来自PCV2b。PCV2基因型与地域无明显相关性。国内流行的圆环病毒的遗传演化规律为PCV2a型到PCV2a和PCV2b重组型病毒再到PCV2b型，未发现PCV2c型。
对PCV2小鼠动物感染模型的构建进行了初步研究。根据攻毒组小鼠脾脏、淋巴结内持续的病毒复制、血清PCV2抗体生成、明显的病理损伤得出PCV2感染昆明小鼠的结论，本试验初步建立了PCV2感染昆明小鼠的动物感染模型，并发现证明了PCV2可在昆明小鼠中水平传播。这为PCV2的致病机理研究及疫苗免疫效果评价等奠定了理论基础。
建立了SYBR Green I荧光定量PCR检测PCV2的方法。建立的检测方法特异性强、重复性好；敏感性达10个拷贝；在应用于临床检测时，可将反应时间缩短为1.5小时。为PCV2的临床检测提供了新的方法，并制定了山东省地方标准：猪圆环病毒2型荧光PCR检测技术（DB37/T 1827-2011），在全省范围内推广应用。
克隆了PCV2核衣壳蛋白基因ORF2，并插入到真核表达载体pVAX1中，成功构建pVAX1-ORF2。在前期动物实验的基础上人工合成了2条对猪具有免疫增强作用的CpG基序并成功插入到pVAX1-ORF2真核表达载体，获得2种含CpG基序的CpG-pVAX1-PCV2 ORF2核酸疫苗。对2种核酸疫苗在猪体进行了动物实验，利用试剂盒检测了其抗体效价和IL-2产生水平，并用流式细胞术对其细胞免疫效果进行了测定，对其免疫原性进行了探讨。申报了猪圆环病毒核酸疫苗的转基因安全评价中试，并获批（农基安办字2010-T179）。
通过对2种含CpG基序的核酸疫苗猪体试验，确定了最佳的CpG-pVAX1-ORF2及免疫剂量，筛选出的最佳核酸疫苗能够增加猪的平均日增重，并能刺激机体产生较强的细胞免疫应答和体液免疫应答，可有效阻止病毒在体内的增殖。本研究的主要创新点：
1、病原流行病学调查结果表明，2005年至2011年，山东省PCV2的感染率呈逐年上升趋势。发现PCV2b是目前国内流行株，出现PCV2a和PCV2b重组型病毒。
2）建立了PCV2的荧光定量PCR诊断方法，并制定了山东省地方标准（DB37/T1827-2011），为诊断PCV2感染提供了技术手段。
3）建立了PCV2感染昆明小鼠的动物感染模型，发现并证明了PCV2可在昆明小鼠中水平传播。
4）在国内外率先研制含CpG基序的CpG-pVAX1-PCV2 ORF2（核酸疫苗），有效提高了核酸疫苗的免疫效果，取得农业部转基因产品中试批文（农基安办字2010-T179）。
Porcine circovirus (Porcine circovirus, PCV) is divided into two genotypes,non-pathogenic PCV1 and pathogenic PCV2. PCV2 can cause postweaning multisystemicwasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), porcinerespiratory disease complex (PRDC), sow abortion and mortality syndrome (SAMs), porcineproliferative and necrotizing pneumonia (PNP) and congenital tremors (CT). So PCV2 canlead to sow reproductive failure and cause immunosuppression, which results in seriouseconomic losses to the pig industry. The disease has been considered as the newly discoveredswine infectious disease after the porcine reproductive and respiratory syndrome byveterinarians and pig farmers of the world.
In recent years, PCV2 constantly mutates and the new strains continue to emerge, whichresults in a certain amount of pressure to the prevention and control of the disease. To betterunderstand the prevalence of the disease and the development of new genetically engineeredvaccine for the prevention and control of the disease, based on the epidemiologicalinvestigation, 3 strains of PCV2 were isolated and identified. The genome of 17 strains ofPCV2 was sequenced and the PCV2 genetic evolution was analyzed. The prevalence ofTorque teno virus (TTV) in the herd of Shandong Province was also surveyed in the sametime. The real-time PCR method and PCV2 mouse model were developed. The DNA vaccinecontaining the CpG motif was also developed.
The main work is as follows:
Based on the epidemiological survey of PCV2 in pig farms of Shandong Province, it wasfound that PCV2 infection rates showed an increasing trend, the antigen positive rate wasfrom 11.3% to 40.38% and the antibody positive rate was from 38.7% to 72.99% from 2005to 2011. The phenomenon of mixed infection was still very serious. The rate of superinfection of PCV2+CSFV was 33.81% and the rate of triple infection of PCV2+PRRSV+CSFV was4.32%, which were the largest proportion ones. 3 strains of PCV2 were isolated and identified.The biological identification was also conducted. Among the 3 strains, the SD strain of PCV2was passaged to 20 generations and the titer was stable with 106.0TCID50/mL. At present, thevirus was passaged to 75 generations, the titer was stable and the strain of PCV2 can be usedas inactivated vaccine candidate strains. The whole genome sequences of 17 strains of PCV2were completed. PCV2 in China has been in continuous variation, PCV2b is the epidemicstrain, the reorganization virus of PCV2a and PCV2b was also appeared with the geneticcharacteristics of ORF1 from PCV2a and ORF2 from PCV2b. The genotype of PCV2 had nosignificant correlation with the geographical area. The regular pattern of genetic evolution ofdomestic popular PCV2 was the type of PCV2a to recombinant virus of PCV2a and PCV2band then PCV2b type. The type of PCV2c was not found.
The PCV2 mice infection model was also conducted. Based on the continued virusreplication in spleens and lymph nodes, PCV2 antibody generated in the serum and thesignificant pathological damage, it was concluded that PCV2 could infect Kunming mice.This study established the animal infection model of PCV2 infection in Kunming mice anddemonstrated that PCV2 could be transmissed in Kunming mice. This laid the theoreticalfoundation of PCV2 pathogenesis studies and evaluation of immune effects of vaccines.
SYBR Green I real-time PCR detection of PCV2 was established. The establishedmethod was very specific and repeatable. The sensitivity was 10 copies. The reaction timecould be shorten to 1.5 hours in clinical testing. So this study provided a new method for theclinical detection of PCV2, and the local standards of Shandong Province: porcine circovirustype 2 fluorescent PCR technology (DB37 to/T 1827-2011) was developed, which would beused in Shandong Province.
The PCV2 nucleocapsid protein gene of ORF2 was cloned and inserted into theeukaryotic expression vector pVAX1, the pVAX1-ORF2 was successfully constructed. 2 CpGmotifs having the immune-enhancing effects on the basis of preliminary animal experimentswere synthesized and successfully inserted in pVAX1-ORF2 eukaryotic expression vector,two kinds of DNA vaccine CpG-pVAX1-PCV2 ORF2 containing CpG were constructed. The two kinds of DNA vaccine were tested in pigs, the antibody titer and IL-2 production levelwere detected using ELISA kits and the cellular immune effects were determined by flowcytometry. The safety assessment of genetically modified PCV2 DNA vaccine was declaredand approved.
The two kinds of DNA vaccine with CpG motifs were tested in pigs and the best ofCpG-pVAX1-ORF2 and immunization dose were screened. The screened DNA vaccine couldsignificantly increase the average daily gain of pigs and stimulate the pigs to produce goodcellular immune response and humoral immune responses and effectively prevent theproliferation of the virus in the body.
The main innovation of this study:
1) A pathogen epidemiological survey results show that the prevalence of porcinecircovirus in Shandong Province appeared an increasing trend in 2005-2011. The regularpattern of genetic evolution of domestic popular PCV2 was the type of PCV2a to recombinantvirus of PCV2a and PCV2b and then PCV2b type. The type of PCV2c was not found.
2) SYBR Green I real-time PCR detection of PCV2 was established, which provided anew method for the clinical detection of PCV2. Porcine circovirus type 2 fluorescent PCRtechnology (DB37 to/T 1827-2011) was developed.
3) The PCV2 mice infection model was established. It was found that PCV2 could infectKunming mice and be transmissed in Kunming mice.
4) Two kinds of DNA vaccine CpG-pVAX1-PCV2 ORF2 containing CpG wereconstructed, which were able to induce a good humoral and cellular immune responses. Thesafety assessment of genetically modified products in the Ministry of Agriculture (agriculturemethamphetamine Office word 2010-T179) were approved and obtained. The new DNAvaccine provided a material basis for prevention and control of PCV2.
近年来该病毒不断发生变异，新的毒株不断出现，给该病的防控带来了一定的压力。为了深入了解本病的流行情况及研制新型的基因工程疫苗用于该病的防治，本研究在流行病学调查的基础上，分离鉴定了3株PCV2，对17株病毒进行了全基因组克隆测序并进行了PCV2遗传演化分析；调查了PCV2同属的Torque teno virus (TTV)在山东省猪群中的流行情况；建立了荧光定量PCR检测方法和PCV2小鼠动物模型；研制了含CpG基序的PCV2核酸疫苗。
主要表现为：对山东省规模化猪场进行了猪圆环病毒感染率的流行病学调查，发现猪圆环病毒的感染率呈逐年上升趋势，2005-2011抗原阳性率由11.3%增加到40.38%，抗体阳性率由38.7%增加到72.99%。混合感染现象仍很严重，二重感染中PCV+CSFV所占比例最大，为33.81%；三重感染中PCV+PRRSV+CSFV所占比例最大，为4.32%。在此基础上完成3株PCV2的分离鉴定工作，并进行了系列生物学鉴定。其中SD株（JQ653449）传代至第20代病毒毒价为106.0TCID50/mL。目前该病毒传至75代，效价稳定可以作为灭活疫苗的候选毒株。完成17株猪圆环病毒的全基因克隆测序。通过与从已公布的国内外PCV2分离株的比较分析发现国内PCV2一直在不断的变异，PCV2b是目前国内流行株，出现PCV2a和PCV2b重组型病毒，其基因组特征为ORF1来自PCV2a，ORF2来自PCV2b。PCV2基因型与地域无明显相关性。国内流行的圆环病毒的遗传演化规律为PCV2a型到PCV2a和PCV2b重组型病毒再到PCV2b型，未发现PCV2c型。
对PCV2小鼠动物感染模型的构建进行了初步研究。根据攻毒组小鼠脾脏、淋巴结内持续的病毒复制、血清PCV2抗体生成、明显的病理损伤得出PCV2感染昆明小鼠的结论，本试验初步建立了PCV2感染昆明小鼠的动物感染模型，并发现证明了PCV2可在昆明小鼠中水平传播。这为PCV2的致病机理研究及疫苗免疫效果评价等奠定了理论基础。
建立了SYBR Green I荧光定量PCR检测PCV2的方法。建立的检测方法特异性强、重复性好；敏感性达10个拷贝；在应用于临床检测时，可将反应时间缩短为1.5小时。为PCV2的临床检测提供了新的方法，并制定了山东省地方标准：猪圆环病毒2型荧光PCR检测技术（DB37/T 1827-2011），在全省范围内推广应用。
克隆了PCV2核衣壳蛋白基因ORF2，并插入到真核表达载体pVAX1中，成功构建pVAX1-ORF2。在前期动物实验的基础上人工合成了2条对猪具有免疫增强作用的CpG基序并成功插入到pVAX1-ORF2真核表达载体，获得2种含CpG基序的CpG-pVAX1-PCV2 ORF2核酸疫苗。对2种核酸疫苗在猪体进行了动物实验，利用试剂盒检测了其抗体效价和IL-2产生水平，并用流式细胞术对其细胞免疫效果进行了测定，对其免疫原性进行了探讨。申报了猪圆环病毒核酸疫苗的转基因安全评价中试，并获批（农基安办字2010-T179）。
通过对2种含CpG基序的核酸疫苗猪体试验，确定了最佳的CpG-pVAX1-ORF2及免疫剂量，筛选出的最佳核酸疫苗能够增加猪的平均日增重，并能刺激机体产生较强的细胞免疫应答和体液免疫应答，可有效阻止病毒在体内的增殖。本研究的主要创新点：
1、病原流行病学调查结果表明，2005年至2011年，山东省PCV2的感染率呈逐年上升趋势。发现PCV2b是目前国内流行株，出现PCV2a和PCV2b重组型病毒。
2）建立了PCV2的荧光定量PCR诊断方法，并制定了山东省地方标准（DB37/T1827-2011），为诊断PCV2感染提供了技术手段。
3）建立了PCV2感染昆明小鼠的动物感染模型，发现并证明了PCV2可在昆明小鼠中水平传播。
4）在国内外率先研制含CpG基序的CpG-pVAX1-PCV2 ORF2（核酸疫苗），有效提高了核酸疫苗的免疫效果，取得农业部转基因产品中试批文（农基安办字2010-T179）。
Porcine circovirus (Porcine circovirus, PCV) is divided into two genotypes,non-pathogenic PCV1 and pathogenic PCV2. PCV2 can cause postweaning multisystemicwasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), porcinerespiratory disease complex (PRDC), sow abortion and mortality syndrome (SAMs), porcineproliferative and necrotizing pneumonia (PNP) and congenital tremors (CT). So PCV2 canlead to sow reproductive failure and cause immunosuppression, which results in seriouseconomic losses to the pig industry. The disease has been considered as the newly discoveredswine infectious disease after the porcine reproductive and respiratory syndrome byveterinarians and pig farmers of the world.
In recent years, PCV2 constantly mutates and the new strains continue to emerge, whichresults in a certain amount of pressure to the prevention and control of the disease. To betterunderstand the prevalence of the disease and the development of new genetically engineeredvaccine for the prevention and control of the disease, based on the epidemiologicalinvestigation, 3 strains of PCV2 were isolated and identified. The genome of 17 strains ofPCV2 was sequenced and the PCV2 genetic evolution was analyzed. The prevalence ofTorque teno virus (TTV) in the herd of Shandong Province was also surveyed in the sametime. The real-time PCR method and PCV2 mouse model were developed. The DNA vaccinecontaining the CpG motif was also developed.
The main work is as follows:
Based on the epidemiological survey of PCV2 in pig farms of Shandong Province, it wasfound that PCV2 infection rates showed an increasing trend, the antigen positive rate wasfrom 11.3% to 40.38% and the antibody positive rate was from 38.7% to 72.99% from 2005to 2011. The phenomenon of mixed infection was still very serious. The rate of superinfection of PCV2+CSFV was 33.81% and the rate of triple infection of PCV2+PRRSV+CSFV was4.32%, which were the largest proportion ones. 3 strains of PCV2 were isolated and identified.The biological identification was also conducted. Among the 3 strains, the SD strain of PCV2was passaged to 20 generations and the titer was stable with 106.0TCID50/mL. At present, thevirus was passaged to 75 generations, the titer was stable and the strain of PCV2 can be usedas inactivated vaccine candidate strains. The whole genome sequences of 17 strains of PCV2were completed. PCV2 in China has been in continuous variation, PCV2b is the epidemicstrain, the reorganization virus of PCV2a and PCV2b was also appeared with the geneticcharacteristics of ORF1 from PCV2a and ORF2 from PCV2b. The genotype of PCV2 had nosignificant correlation with the geographical area. The regular pattern of genetic evolution ofdomestic popular PCV2 was the type of PCV2a to recombinant virus of PCV2a and PCV2band then PCV2b type. The type of PCV2c was not found.
The PCV2 mice infection model was also conducted. Based on the continued virusreplication in spleens and lymph nodes, PCV2 antibody generated in the serum and thesignificant pathological damage, it was concluded that PCV2 could infect Kunming mice.This study established the animal infection model of PCV2 infection in Kunming mice anddemonstrated that PCV2 could be transmissed in Kunming mice. This laid the theoreticalfoundation of PCV2 pathogenesis studies and evaluation of immune effects of vaccines.
SYBR Green I real-time PCR detection of PCV2 was established. The establishedmethod was very specific and repeatable. The sensitivity was 10 copies. The reaction timecould be shorten to 1.5 hours in clinical testing. So this study provided a new method for theclinical detection of PCV2, and the local standards of Shandong Province: porcine circovirustype 2 fluorescent PCR technology (DB37 to/T 1827-2011) was developed, which would beused in Shandong Province.
The PCV2 nucleocapsid protein gene of ORF2 was cloned and inserted into theeukaryotic expression vector pVAX1, the pVAX1-ORF2 was successfully constructed. 2 CpGmotifs having the immune-enhancing effects on the basis of preliminary animal experimentswere synthesized and successfully inserted in pVAX1-ORF2 eukaryotic expression vector,two kinds of DNA vaccine CpG-pVAX1-PCV2 ORF2 containing CpG were constructed. The two kinds of DNA vaccine were tested in pigs, the antibody titer and IL-2 production levelwere detected using ELISA kits and the cellular immune effects were determined by flowcytometry. The safety assessment of genetically modified PCV2 DNA vaccine was declaredand approved.
The two kinds of DNA vaccine with CpG motifs were tested in pigs and the best ofCpG-pVAX1-ORF2 and immunization dose were screened. The screened DNA vaccine couldsignificantly increase the average daily gain of pigs and stimulate the pigs to produce goodcellular immune response and humoral immune responses and effectively prevent theproliferation of the virus in the body.
The main innovation of this study:
1) A pathogen epidemiological survey results show that the prevalence of porcinecircovirus in Shandong Province appeared an increasing trend in 2005-2011. The regularpattern of genetic evolution of domestic popular PCV2 was the type of PCV2a to recombinantvirus of PCV2a and PCV2b and then PCV2b type. The type of PCV2c was not found.
2) SYBR Green I real-time PCR detection of PCV2 was established, which provided anew method for the clinical detection of PCV2. Porcine circovirus type 2 fluorescent PCRtechnology (DB37 to/T 1827-2011) was developed.
3) The PCV2 mice infection model was established. It was found that PCV2 could infectKunming mice and be transmissed in Kunming mice.
4) Two kinds of DNA vaccine CpG-pVAX1-PCV2 ORF2 containing CpG wereconstructed, which were able to induce a good humoral and cellular immune responses. Thesafety assessment of genetically modified products in the Ministry of Agriculture (agriculturemethamphetamine Office word 2010-T179) were approved and obtained. The new DNAvaccine provided a material basis for prevention and control of PCV2.
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