猪圆环病毒2型Cap蛋白的家蚕表达及亚单位疫苗在小鼠和仔猪的免疫效力试验
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摘要
猪圆环病毒2型(Porcine circovirus type2, PCV2)是猪断奶后多系统衰竭综合征(Postweaning multisystemic wasting syndrome, PMWS)和其他猪圆环病毒相关疾病的原发性病原。免疫预防是控制该类疾病的重要手段之一。本研究成功构建可表达PCV2空衣壳粒子的家蚕表达体系,并利用表达产物制备出PCV2Cap蛋白灭活疫苗,利用小鼠和猪分析了疫苗的免疫原性。
采集临床疑似断奶仔猪多系统衰竭综合征发病仔猪的淋巴结等脏器,采用PCR技术检测PCV2,阳性样本用PK-15细胞进行病毒分离。经间接免疫荧光、免疫过氧化物酶单层细胞试验(IPMA)、免疫电镜观察对分离物进行鉴定。最终分离出5株PCV2,分别命名为PCV2-1、PCV2-2、PCV2-3、PCV2-4和PCV2-5。
首先将去核定位信号的Cap基因克隆到大肠杆菌表达载体,分离纯化目的蛋白,经质谱鉴定后用于抗体制备。抗体效价达到1：3200以上者,可用于真核表达Cap蛋白产物的检测。用PCR扩增PCV2Cap基因,或将经人工改造构建Cap突变体基因,克隆到家蚕杆状病毒转移载体,经同源重组获得重组杆状病毒。用重组杆状病毒感染昆虫细胞和家蚕,收集并纯化表达产物。鉴定结果表明,纯化蛋白主要为病毒空衣壳粒子。经检测,推算PCV2空衣壳在家蚕中的表达量可达1.2-1.6mg/蛹。电镜观察可见到完整的空衣壳粒子。胶体金标记免疫电镜观察可见到完整的胶体金粒子,金粒大小为10nm。
将重组病毒培养液按105pfu/头注射五日龄幼蚕,收集发病的幼虫血淋巴或蚕蛹,按1：10(g/v)比例加入PBS液,经破碎、过滤、离心、灭活等程序,制备猪圆环病毒2型cap蛋白家蚕抗原。用206佐剂,按照疫苗乳化程序,将家蚕抗原进行乳化,制备出猪圆环病毒2型cap蛋白亚单位疫苗。
用制备的猪圆环病毒2型Cap蛋白亚单位疫苗免疫小鼠,通过检测小鼠PCV2抗体和免疫小鼠攻毒后组织PCV2分离,评价疫苗的免疫原性。同时,设勃林格殷格翰动物保健有限公司生产的猪圆环病毒2型杆状病毒载体灭活疫苗对照、空白蚕蛹处理液疫苗(206佐剂)对照、非免疫小鼠对照。试验结果表明,201001批疫苗免疫小鼠均出现了血清学阳转(10/10),PCV2ELISA抗体滴度可达1：800-1：3200,与勃林格殷格翰动物保健有限公司生产的猪圆环病毒2型杆状病毒灭活疫苗对照组相当,而空白蚕蛹处理液疫苗组和非免疫组小鼠抗体均为阴性;3批疫苗免疫小鼠组织的PCV2病毒分离均为阴性(0/10),而非免疫对照组小鼠组织PCV2病毒分离均为阳性(10/10)。
用制备的猪圆环病毒2型Cap蛋白亚单位疫苗免疫14-21日龄的PCV2抗体阴性仔猪,通过检测免疫仔猪PCV2抗体和免疫仔猪攻毒后组织PCV2病毒分离,评价疫苗的免疫原性。试验结果表明,免疫仔猪均呈现抗体阳转(5/5),PCV2ELISA抗体滴度可达1：320-1：640,略低于勃林格殷格翰动物保健有限公司生产的猪圆环病毒2型杆状病毒灭活疫苗免疫组,而非免疫组仔猪抗体均为阴性；201001批疫苗仔猪组织PCV2病毒分离均为阴性(0/5),而非免疫对照组仔猪组织PCV2病毒分离均为阳性(5/5)。
上述试验结果表明,制备的猪圆环病毒2型cap蛋白亚单位疫苗能刺激小鼠和仔猪产生特异性抗体,且可以有效地抑制PCV2在小鼠和仔猪体内的复制,说明制备的家蚕表达的PCV2Cap蛋白具有良好的免疫原性,具有开发成猪圆环病毒2型疫苗的潜力。
Porcine circovirus type2(PCV2) is the primary pathogen of postweaning multisystemic wasting syndrome (PMWS) and other PCV2associated disease (PCVAD). Vaccination is one of major measures for preventing these diseases. In this study, silkworm system was constructed to express Cap protein of PCV2and the expressed proteins were used in order to prepare the inactived vaccine against PCV2. Meanwhile, the immunogenicity of the prepared vaccine was analyzed in pigs and mice.
Lungs, spleeds, livers and lymph nodes were collected from the pigs with clinical symptoms of PMWS and PCR was performed for PCV2detection in these tissues. PCV1-free PK-15cells were inoculated with the PCV2-positive samples to isolate PCV2. Five isolates of PCV2, which were designated as PCV2-1, PCV2-2, PCV2-3, PCV2-4, PCV2-5respectively, were obtained and confirmed by IFA, IPMA, and immunoelectron microscopy.
The cap gene without nucleus-located signs was cloned and expressed in E.coli fistly.Then the expressed protein was purified and used to prepare the antibody in rabbit.The antibodies with the titer more than1:3200were used to detection of expressed Cap protein in silkworm. The cap gene of PCV2was amplified by PCR and the cap gene mutant was reconstructed artificially. The mutant of cap gene was cloned into the baculovirus carrying vector of silkworm system. The constructured vectors were homologously recombinated with the parental viruses in order to acquire the recombinant baculovirus. Then the Cap protein was expressed in the silkworm which was infected by the recombinant baculovirus containing the cap gene of PCV2. The purified protein was confirmed to be a fusion protein with a titer of1:3200by ELISA. The expression were proved to be PCV2empty particles mainly. It was estimated that the expression concentration of the empty particle of PCV2in the skillworm could be1.2-1.6mg per silkworm. The complete empty particle with a diameter of10nm was also observed by electron microscopy and colliadal gold immuno electron microscopy.
Five-day-old baby silkworms or the lymph-blood were inoculated with105pfu recombination baculovirus. The infected silkworms expressing the Cap protein were collected and mixed with PBS by1:10(g/v), and then broken, and further subjected to filtration, centrifugation and inactivation. Inactivated vaccine was prepared by means of emulsification with206adjuvant.
Mice were vaccinated by the PCV2Cap protein sub-unit vaccine. Antibody in sera and PCV2antigen in tissues of the vaccinated mice were detected to evaluate the immunogenicity of the vaccine.Meanwhile, porcine circovirus type2baculovirus vector vaccine produced by Boehringer Ingelhein Vetmedica, Inc, the healthy silkworm and non-vaccinated mice were served as controls. The results showed that the inoculated mice could generate specific antibody against PCV2, with a positive rate of10/10and a ELISA titer ranging from1:800to1:3200, which was similar to those of baculovirus vector vaccine group. PCV2antigen in tissues of the vaccinated mice showed negative in all vaccinated groups, and10/10positve rate in control group.
14to21-day-old pigs without PCV2antibody were also vaccinated. Antibody in sera and PCV2antigen in tissues of the vaccinated pigs were detected. Porcine circovirus type2baculovirus vector vaccine produced by Boehringer Ingelhein Vetmedica,Inc and the healthy silkworm were served as controls.The results showed that the vaccinated pigs could induce specific antibody against PCV2. The positive rate of PCV2antibody was5/5in group administered with the vaccine of batch201001, with a lightly lower titer ranging from1:320to1:640, than group given with baculovirus vector vaccine. PCV2antigen detection in tissues of the immunized pigs showed0/5positive in group given with the vaccine of batch201001and5/5positive in control group.
As a whole, PCV2Cap protein sub-unit vaccine could induce specific antibodies against PCV2, which was able to inhibit the replication of PCV2in mice and pigs, indicating that the porcine circovirus type2empty capsid particle expressed by silkworm system presents better immunogenicity and potential as effective vaccine.
采集临床疑似断奶仔猪多系统衰竭综合征发病仔猪的淋巴结等脏器,采用PCR技术检测PCV2,阳性样本用PK-15细胞进行病毒分离。经间接免疫荧光、免疫过氧化物酶单层细胞试验(IPMA)、免疫电镜观察对分离物进行鉴定。最终分离出5株PCV2,分别命名为PCV2-1、PCV2-2、PCV2-3、PCV2-4和PCV2-5。
首先将去核定位信号的Cap基因克隆到大肠杆菌表达载体,分离纯化目的蛋白,经质谱鉴定后用于抗体制备。抗体效价达到1：3200以上者,可用于真核表达Cap蛋白产物的检测。用PCR扩增PCV2Cap基因,或将经人工改造构建Cap突变体基因,克隆到家蚕杆状病毒转移载体,经同源重组获得重组杆状病毒。用重组杆状病毒感染昆虫细胞和家蚕,收集并纯化表达产物。鉴定结果表明,纯化蛋白主要为病毒空衣壳粒子。经检测,推算PCV2空衣壳在家蚕中的表达量可达1.2-1.6mg/蛹。电镜观察可见到完整的空衣壳粒子。胶体金标记免疫电镜观察可见到完整的胶体金粒子,金粒大小为10nm。
将重组病毒培养液按105pfu/头注射五日龄幼蚕,收集发病的幼虫血淋巴或蚕蛹,按1：10(g/v)比例加入PBS液,经破碎、过滤、离心、灭活等程序,制备猪圆环病毒2型cap蛋白家蚕抗原。用206佐剂,按照疫苗乳化程序,将家蚕抗原进行乳化,制备出猪圆环病毒2型cap蛋白亚单位疫苗。
用制备的猪圆环病毒2型Cap蛋白亚单位疫苗免疫小鼠,通过检测小鼠PCV2抗体和免疫小鼠攻毒后组织PCV2分离,评价疫苗的免疫原性。同时,设勃林格殷格翰动物保健有限公司生产的猪圆环病毒2型杆状病毒载体灭活疫苗对照、空白蚕蛹处理液疫苗(206佐剂)对照、非免疫小鼠对照。试验结果表明,201001批疫苗免疫小鼠均出现了血清学阳转(10/10),PCV2ELISA抗体滴度可达1：800-1：3200,与勃林格殷格翰动物保健有限公司生产的猪圆环病毒2型杆状病毒灭活疫苗对照组相当,而空白蚕蛹处理液疫苗组和非免疫组小鼠抗体均为阴性;3批疫苗免疫小鼠组织的PCV2病毒分离均为阴性(0/10),而非免疫对照组小鼠组织PCV2病毒分离均为阳性(10/10)。
用制备的猪圆环病毒2型Cap蛋白亚单位疫苗免疫14-21日龄的PCV2抗体阴性仔猪,通过检测免疫仔猪PCV2抗体和免疫仔猪攻毒后组织PCV2病毒分离,评价疫苗的免疫原性。试验结果表明,免疫仔猪均呈现抗体阳转(5/5),PCV2ELISA抗体滴度可达1：320-1：640,略低于勃林格殷格翰动物保健有限公司生产的猪圆环病毒2型杆状病毒灭活疫苗免疫组,而非免疫组仔猪抗体均为阴性；201001批疫苗仔猪组织PCV2病毒分离均为阴性(0/5),而非免疫对照组仔猪组织PCV2病毒分离均为阳性(5/5)。
上述试验结果表明,制备的猪圆环病毒2型cap蛋白亚单位疫苗能刺激小鼠和仔猪产生特异性抗体,且可以有效地抑制PCV2在小鼠和仔猪体内的复制,说明制备的家蚕表达的PCV2Cap蛋白具有良好的免疫原性,具有开发成猪圆环病毒2型疫苗的潜力。
Porcine circovirus type2(PCV2) is the primary pathogen of postweaning multisystemic wasting syndrome (PMWS) and other PCV2associated disease (PCVAD). Vaccination is one of major measures for preventing these diseases. In this study, silkworm system was constructed to express Cap protein of PCV2and the expressed proteins were used in order to prepare the inactived vaccine against PCV2. Meanwhile, the immunogenicity of the prepared vaccine was analyzed in pigs and mice.
Lungs, spleeds, livers and lymph nodes were collected from the pigs with clinical symptoms of PMWS and PCR was performed for PCV2detection in these tissues. PCV1-free PK-15cells were inoculated with the PCV2-positive samples to isolate PCV2. Five isolates of PCV2, which were designated as PCV2-1, PCV2-2, PCV2-3, PCV2-4, PCV2-5respectively, were obtained and confirmed by IFA, IPMA, and immunoelectron microscopy.
The cap gene without nucleus-located signs was cloned and expressed in E.coli fistly.Then the expressed protein was purified and used to prepare the antibody in rabbit.The antibodies with the titer more than1:3200were used to detection of expressed Cap protein in silkworm. The cap gene of PCV2was amplified by PCR and the cap gene mutant was reconstructed artificially. The mutant of cap gene was cloned into the baculovirus carrying vector of silkworm system. The constructured vectors were homologously recombinated with the parental viruses in order to acquire the recombinant baculovirus. Then the Cap protein was expressed in the silkworm which was infected by the recombinant baculovirus containing the cap gene of PCV2. The purified protein was confirmed to be a fusion protein with a titer of1:3200by ELISA. The expression were proved to be PCV2empty particles mainly. It was estimated that the expression concentration of the empty particle of PCV2in the skillworm could be1.2-1.6mg per silkworm. The complete empty particle with a diameter of10nm was also observed by electron microscopy and colliadal gold immuno electron microscopy.
Five-day-old baby silkworms or the lymph-blood were inoculated with105pfu recombination baculovirus. The infected silkworms expressing the Cap protein were collected and mixed with PBS by1:10(g/v), and then broken, and further subjected to filtration, centrifugation and inactivation. Inactivated vaccine was prepared by means of emulsification with206adjuvant.
Mice were vaccinated by the PCV2Cap protein sub-unit vaccine. Antibody in sera and PCV2antigen in tissues of the vaccinated mice were detected to evaluate the immunogenicity of the vaccine.Meanwhile, porcine circovirus type2baculovirus vector vaccine produced by Boehringer Ingelhein Vetmedica, Inc, the healthy silkworm and non-vaccinated mice were served as controls. The results showed that the inoculated mice could generate specific antibody against PCV2, with a positive rate of10/10and a ELISA titer ranging from1:800to1:3200, which was similar to those of baculovirus vector vaccine group. PCV2antigen in tissues of the vaccinated mice showed negative in all vaccinated groups, and10/10positve rate in control group.
14to21-day-old pigs without PCV2antibody were also vaccinated. Antibody in sera and PCV2antigen in tissues of the vaccinated pigs were detected. Porcine circovirus type2baculovirus vector vaccine produced by Boehringer Ingelhein Vetmedica,Inc and the healthy silkworm were served as controls.The results showed that the vaccinated pigs could induce specific antibody against PCV2. The positive rate of PCV2antibody was5/5in group administered with the vaccine of batch201001, with a lightly lower titer ranging from1:320to1:640, than group given with baculovirus vector vaccine. PCV2antigen detection in tissues of the immunized pigs showed0/5positive in group given with the vaccine of batch201001and5/5positive in control group.
As a whole, PCV2Cap protein sub-unit vaccine could induce specific antibodies against PCV2, which was able to inhibit the replication of PCV2in mice and pigs, indicating that the porcine circovirus type2empty capsid particle expressed by silkworm system presents better immunogenicity and potential as effective vaccine.
引文
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