口蹄疫病毒空衣壳蛋白的表达加工及其免疫原性研究
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摘要
口蹄疫是一种严重危害畜牧业生产的烈性传染病,在世界上许多国家和地区都有该病的流行。疫苗接种是预防口蹄疫的有效手段。近年来,用口蹄疫空衣壳蛋白作为疫苗候选抗原成为研究的热点。本实验首先构建了C型口蹄疫病毒P1序列不同长度缺失的突变载体,初步研究了这些缺失突变对空衣壳形成的影响;然后构建了含A型口蹄疫病毒P1-2A和3C序列的逆转录病毒载体,利用逆转录病毒载体系统基因转移技术建立了稳定表达空衣壳蛋白的MDBK细胞系;最后用这些细胞的裂解物免疫豚鼠,免疫后豚鼠体内能产生抗口蹄疫病毒的特异性抗体,并能耐受病毒的攻击。这些结果为进一步研制口蹄疫亚单位疫苗奠定了基础。
1.构建了含有C型口蹄疫病毒P1序列不同长度缺失的载体。利用PCR方法分别将VP3-VP1连接处11个氨基酸、VP1 C端33个氨基酸、100个氨基酸、167个氨基酸缺失。在VP1 C端引入FLAG标签,以利于抗FLAG单抗的识别。使用T7 RNA聚合酶体外瞬时表达系统,在细胞中表达T7 RNA聚合酶以启动载体的体外转录和翻译。用脂质体介导的方法将P1序列载体单独转染或与3C序列载体共转染BHK-21细胞,转染后24h收获细胞,表达产物通过Western-blot和ELISA方法检测。结果显示用缺失突变的载体转染细胞后仅能检测到部分聚蛋白的表达而检测不到其裂解产物,提示这些位置氨基酸的缺失会严重影响空衣壳的组装。使用不同比例的P1序列载体和3C序列载体共转染细胞,结果显示当P1序列载体与3C序列载体质量比为40:1共转染细胞时,P1聚蛋白的裂解产物含量较高,提示空衣壳的形成效率也会较高。
2.采用PCR方法获得A型口蹄疫病毒P1-2A和3C序列,将两个扩增片段依次插入逆转录病毒载体pBABEpuro。重组载体pBABEpuro-P1-2A-3C和pVSV-G被膜载体共转染GP2-293细胞,包装形成有感染能力的假病毒粒子。假病毒感染MDBK细胞后使用嘌呤霉素(2.5μg/ml)筛选抗性细胞,大约10d后用克隆环套取单克隆细胞并扩大培养。用间接免疫荧光和ELISA方法检测MDBK细胞中目的蛋白的表达,并在电镜下观察空衣壳。结果显示MDBK细胞中有目的基因的整合;间接免疫荧光方法检测到目的蛋白的表达:ELISA方法检测目的蛋白表达最高OD值约为阳性对照最高OD值的1/30。将细胞连续传至35代,取不同代次的细胞进行检测,目的蛋白的表达量无显著差异,证实已建立了稳定表达空衣壳蛋白的MDBK细胞系。
3.将细胞裂解物浓缩后免疫豚鼠,第一次免疫后14d用间接ELISA方法未检测到FMDV特异性抗体,第二次免疫后14d,豚鼠血清抗体水平显著升高达到一个高峰,第三次免疫后血清抗体水平持续升高。第三次免疫后第14dpBABEpuro-P1-2A-3C病毒感染MDBK细胞裂解物免疫组有两只豚鼠中和抗体达到1:64。以200GID_(50)/0.2ml剂量攻毒,pBABEpuro-P1-2A-3C病毒感染MDBK细胞裂解物免疫组豚鼠全部保护,阴性对照组全部发病。
Foot-and-mouth disease(FMD) is a highly contagious viral disease of cloven-hoofed animals and severely threatens animal husbandry.FMD is endemic in large areas of Africa,Asia and South America and has shown an extraordinary ability to cross international boundaries and to cause epidemics in previously free areas. Vaccination is the major method to prevent FMD.However the conventional inactivated vaccines have many defects.Therefore,it is necessary to develop a safe and efficient FMD vaccine.These empty capsids are able to induce neutralizing antibodies of the same specificity as the whole virus and appear to be a good candidate for the development of a recombinant vaccine.
In this study,A series of deletion mutation vectors of P1 coding sequence were constructed and were tested whether they would effect empty capsid assembling.A retroviral vector containing the P1-2A and 3C coding sequences was constructed and MDBK cells line stable expressing empty capsid proteins was established. Subsequently,the lysate of MDBK cells infected by pBABEpuro-P1-2A-3C virus were used to immunize guinea pigs.All guinea pigs immunized with the lysate of MDBK cells infected by pBABE puro-P1-2A-3C virus developed specific anti-FMDV antibody and neutralizing antibody.These guinea pigs were completely protected from viral challenge.These results laid a foundation of development of FMD subunit vaccine.
(1) A series of deletion mutations were introduced into P1 coding sequence. These deletion sequences included sequence which 11 amino acids at VP3/VP1 junction were deleted and sequences which 33,100,167 amino acids at C-terminal of VP1 were deleted,respectively.The FLAG tag was introduced at C-terminal of VP1 by PCR in order to accurately test target proteins.A series of deleted sequences were obtained by PCR and were subcloned into expression vectors.Processing of the P1-polyprotein was verified by in vitro transcription/translation using T7 RNA polymerase transient expression system.The plasmids containing deleted P1 sequences and the plasmids containing 3C sequence were transferred into BHK-21 cells with liposome-mediated method.The transferred cells were harvested and lysated after 24h transfection.The products were analysed using Western-blot and ELISA.The result demonstrated deletion mutation of P1 sequence impaired its ability to act as substrates for 3C proteinase activity.The assembling efficiency was high when the ratio of plasmids was 40:1.
(2) P1-2A and 3C sequences were obtained by PCR.A recombinant retroviral vector pBABEpuro-P1-2A-3C was constructed by inserting P1-2A sequence and 3C sequence by turns.Both this vector and pVSV-G envelope vector were co-transfected into GP2-293 cells by liposome-mediated method.The recombinant pseudovirus were produced after 24h transfection.The pseudovirus infected the interesting MDBK cells. The infected MDBK cells were selected by puromycin(2.5μg/ml) for 10d and the monoclonal cells were selected using cloning rings.The expression of capsid proteins was detected by indirect immunofluorescence and sandwich-ELISA.The empty capsids of FMDV were observed under electron microscope.The results revealed the target gene sequences had been integrated into genome of MDBK cells and OD values of target proteins were lower than OD values of positive control.The expression levels in screened cells of various passages showed no significant difference.
(3) Subsequently,the lysate of MDBK cells infected by pBABEpuro-P1-2A-3C virus were used as antigen to immunize guinea pigs and evaluated for its ability to induce a humoral response of FMDV in guinea pigs.In addition,the ability to protect guinea pigs against homologous virus challenge was examined.Guinea pigs were given booster vaccination twice and guinea pigs were challenged 22 days after the third vaccination.Control groups included animals immunized with commercial vaccine,lysate of MDBK cells infected by pBABEpuro virus and lysate of uninfected MDBK cells.Anti-FMDV antibodys were elicited and reached a high level 14d after the second inoculation.Furthermore,guinea pigs developed a strong neutralizing antibody responses and antibodies increased further after the third inoculation.The neutralizing antibody titers of two guinea pigs immunized with the lysate of MDBK cells infected by pBABEpuro-P1-2A-3C virus reached 1:64.These guinea pigs were completely protected from viral challenge.
In conclusion,this study may provide the experimental bases of research for subunit vaccine.
1.构建了含有C型口蹄疫病毒P1序列不同长度缺失的载体。利用PCR方法分别将VP3-VP1连接处11个氨基酸、VP1 C端33个氨基酸、100个氨基酸、167个氨基酸缺失。在VP1 C端引入FLAG标签,以利于抗FLAG单抗的识别。使用T7 RNA聚合酶体外瞬时表达系统,在细胞中表达T7 RNA聚合酶以启动载体的体外转录和翻译。用脂质体介导的方法将P1序列载体单独转染或与3C序列载体共转染BHK-21细胞,转染后24h收获细胞,表达产物通过Western-blot和ELISA方法检测。结果显示用缺失突变的载体转染细胞后仅能检测到部分聚蛋白的表达而检测不到其裂解产物,提示这些位置氨基酸的缺失会严重影响空衣壳的组装。使用不同比例的P1序列载体和3C序列载体共转染细胞,结果显示当P1序列载体与3C序列载体质量比为40:1共转染细胞时,P1聚蛋白的裂解产物含量较高,提示空衣壳的形成效率也会较高。
2.采用PCR方法获得A型口蹄疫病毒P1-2A和3C序列,将两个扩增片段依次插入逆转录病毒载体pBABEpuro。重组载体pBABEpuro-P1-2A-3C和pVSV-G被膜载体共转染GP2-293细胞,包装形成有感染能力的假病毒粒子。假病毒感染MDBK细胞后使用嘌呤霉素(2.5μg/ml)筛选抗性细胞,大约10d后用克隆环套取单克隆细胞并扩大培养。用间接免疫荧光和ELISA方法检测MDBK细胞中目的蛋白的表达,并在电镜下观察空衣壳。结果显示MDBK细胞中有目的基因的整合;间接免疫荧光方法检测到目的蛋白的表达:ELISA方法检测目的蛋白表达最高OD值约为阳性对照最高OD值的1/30。将细胞连续传至35代,取不同代次的细胞进行检测,目的蛋白的表达量无显著差异,证实已建立了稳定表达空衣壳蛋白的MDBK细胞系。
3.将细胞裂解物浓缩后免疫豚鼠,第一次免疫后14d用间接ELISA方法未检测到FMDV特异性抗体,第二次免疫后14d,豚鼠血清抗体水平显著升高达到一个高峰,第三次免疫后血清抗体水平持续升高。第三次免疫后第14dpBABEpuro-P1-2A-3C病毒感染MDBK细胞裂解物免疫组有两只豚鼠中和抗体达到1:64。以200GID_(50)/0.2ml剂量攻毒,pBABEpuro-P1-2A-3C病毒感染MDBK细胞裂解物免疫组豚鼠全部保护,阴性对照组全部发病。
Foot-and-mouth disease(FMD) is a highly contagious viral disease of cloven-hoofed animals and severely threatens animal husbandry.FMD is endemic in large areas of Africa,Asia and South America and has shown an extraordinary ability to cross international boundaries and to cause epidemics in previously free areas. Vaccination is the major method to prevent FMD.However the conventional inactivated vaccines have many defects.Therefore,it is necessary to develop a safe and efficient FMD vaccine.These empty capsids are able to induce neutralizing antibodies of the same specificity as the whole virus and appear to be a good candidate for the development of a recombinant vaccine.
In this study,A series of deletion mutation vectors of P1 coding sequence were constructed and were tested whether they would effect empty capsid assembling.A retroviral vector containing the P1-2A and 3C coding sequences was constructed and MDBK cells line stable expressing empty capsid proteins was established. Subsequently,the lysate of MDBK cells infected by pBABEpuro-P1-2A-3C virus were used to immunize guinea pigs.All guinea pigs immunized with the lysate of MDBK cells infected by pBABE puro-P1-2A-3C virus developed specific anti-FMDV antibody and neutralizing antibody.These guinea pigs were completely protected from viral challenge.These results laid a foundation of development of FMD subunit vaccine.
(1) A series of deletion mutations were introduced into P1 coding sequence. These deletion sequences included sequence which 11 amino acids at VP3/VP1 junction were deleted and sequences which 33,100,167 amino acids at C-terminal of VP1 were deleted,respectively.The FLAG tag was introduced at C-terminal of VP1 by PCR in order to accurately test target proteins.A series of deleted sequences were obtained by PCR and were subcloned into expression vectors.Processing of the P1-polyprotein was verified by in vitro transcription/translation using T7 RNA polymerase transient expression system.The plasmids containing deleted P1 sequences and the plasmids containing 3C sequence were transferred into BHK-21 cells with liposome-mediated method.The transferred cells were harvested and lysated after 24h transfection.The products were analysed using Western-blot and ELISA.The result demonstrated deletion mutation of P1 sequence impaired its ability to act as substrates for 3C proteinase activity.The assembling efficiency was high when the ratio of plasmids was 40:1.
(2) P1-2A and 3C sequences were obtained by PCR.A recombinant retroviral vector pBABEpuro-P1-2A-3C was constructed by inserting P1-2A sequence and 3C sequence by turns.Both this vector and pVSV-G envelope vector were co-transfected into GP2-293 cells by liposome-mediated method.The recombinant pseudovirus were produced after 24h transfection.The pseudovirus infected the interesting MDBK cells. The infected MDBK cells were selected by puromycin(2.5μg/ml) for 10d and the monoclonal cells were selected using cloning rings.The expression of capsid proteins was detected by indirect immunofluorescence and sandwich-ELISA.The empty capsids of FMDV were observed under electron microscope.The results revealed the target gene sequences had been integrated into genome of MDBK cells and OD values of target proteins were lower than OD values of positive control.The expression levels in screened cells of various passages showed no significant difference.
(3) Subsequently,the lysate of MDBK cells infected by pBABEpuro-P1-2A-3C virus were used as antigen to immunize guinea pigs and evaluated for its ability to induce a humoral response of FMDV in guinea pigs.In addition,the ability to protect guinea pigs against homologous virus challenge was examined.Guinea pigs were given booster vaccination twice and guinea pigs were challenged 22 days after the third vaccination.Control groups included animals immunized with commercial vaccine,lysate of MDBK cells infected by pBABEpuro virus and lysate of uninfected MDBK cells.Anti-FMDV antibodys were elicited and reached a high level 14d after the second inoculation.Furthermore,guinea pigs developed a strong neutralizing antibody responses and antibodies increased further after the third inoculation.The neutralizing antibody titers of two guinea pigs immunized with the lysate of MDBK cells infected by pBABEpuro-P1-2A-3C virus reached 1:64.These guinea pigs were completely protected from viral challenge.
In conclusion,this study may provide the experimental bases of research for subunit vaccine.
引文
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