猪瘟病毒的shRNA干扰、强弱毒鉴别及基因工程疫苗研究
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摘要
猪瘟(Classical swine fever ,CSF)是严重危害养猪业的传染性疾病,病原为猪瘟病毒(Classical swine fever virus, CSFV)。CSFV为单股正链RNA病毒,研究发现其3′非翻译区(3′-non-translation region,3′-NTR)在病毒的复制中有重要作用,为了研究CSFV Shimen株3′-NTR的不同位点在病毒复制中的作用,筛选3个靶位点设计了短发夹RNA(shRNA),构建了针对shimen株CSFV 3′-NTR 3个靶位点的shRNA表达质粒;将重组质粒转染PK-15细胞,获得3株稳定靶向CSFV Shimen株3′-NTR 3个靶位点的干扰细胞株,接种CSFV后,检测干扰细胞株对病毒复制的影响。
目前CSF防控上采取的是以疫苗免疫接种为主的策略,弱毒疫苗的大范围使用,使病毒变异、毒力返强的风险增高,当前临床上“非典型”CSF的大量出现,被怀疑与弱毒疫苗的大量使用有关,新型CSF疫苗的研制是防控CSF的需要;在分析国内外有关猪瘟基因工程疫苗研究进展的基础上,构建了表达CSFV Shimen株E2基因的“自杀性”DNA疫苗和同时表达CSFV Shimen株E0和E2基因的重组鸡痘病毒,对构建的疫苗进行了初步评价。
CSF弱毒疫苗的大量使用,使在生产中难以有效地用血清学方法区分野毒感染猪和疫苗免疫猪,对生猪及其产品的贸易造成严重影响;针对这个问题,在借鉴已有研究的基础上,建立了区分CSFV强/弱毒株的RT-PCR方法,通过对临床病料的检测表明,该方法的特异性较强,可用于临床上对CSFV野毒感染和弱毒疫苗免疫猪的鉴别检测。
本研究取得以下结果:
1.通过对CSFV Shimen株3′-NTR核苷酸序列的分析,分别设计了靶向CSFV Shimen株3′-NTR 115～132位、137～154位和210～228位mRNA的短发夹RNA(shRNA)的单链核苷酸,经退火后形成双链shRNA,将其分别与siRNA表达质粒载体连接,构建了编码CSFV Shimen株3′-NTR 3个位点shRNA的表达质粒载体;转染细胞后筛选获得了稳定转录靶向CSFV Shimen株3′-NTR shRNA PK-15细胞株(P-1、P-2和P-3);接毒试验表明,细胞株P-1、P-2及P-3在转录水平和蛋白表达水平上均可干扰CSFV Shimen株在感染细胞中的增殖(P<0.05)。
2.建立了可鉴别CSFV强毒和弱毒的RT-PCR方法。在CSFV强毒中可扩增得到187 bp的片段,弱毒可获得492 bp的片段;在对PCV2、TGEV、PRRSV等猪源病毒的检测,均未有PCR产物出现,说明方法的特异性较高。对临床疑是CSF病料的检测证明,建立的方法可有效地区分CSFV野毒感染猪和弱毒疫苗免疫猪,本方法可用于临床上对CSFV强弱毒的鉴别检测。
3.构建了CSFV E2基因“自杀性”DNA疫苗表达质粒载体PSCA1-E2。在转染细胞中可检测到E2蛋白的表达;免疫小鼠的脾淋巴细胞增殖刺激指数(SI)在二免后10d与对照组差异显著(p<0.05),20d和30d差异极显著(p<0.01);试验猪二免后21d能检测到抗CSFV特异性血清抗体,说明构建的表达CSFV E2基因的“自杀性”DNA疫苗能诱导试验动物的免疫反应。
4.将CSFV E0和E2基因及报告基因LacZ插入到鸡痘病毒FV282的复制非必需区,经同源重组构建了一株含CSFV E0和E2基因的重组鸡痘病毒(FV282-E0-E2)。用重组鸡痘病毒免疫接种试验小鼠和试验猪,检测发现,免疫动物均能产生特异性抗体,说明E0和E2基因在机体细胞内得以有效表达,并能激发机体产生免疫反应;对免疫猪的攻毒试验表明,重组鸡痘病毒免疫猪能获得部分的免疫保护,抵抗CSFV强毒的攻击,免疫保护率为75%(6/8)。
Classical swine fever (CSF) is a serious contagious disease of pig caused by classical swine fever virus (CSFV). CSFV is a single-strain positive RNAvirus, the 3′-nontranslation region(3′-NTR)plays an important role in virus replication. Three sites in 3′-NTR of CSFV shimen strain were selected by sequence analysis to study the function of 3′-NTR on virus replication. Three recombinant plasmids expressing the shRNAs target to 3′-NTR of CSFV shimen strain were constructed and tranfected into PK-15 cells. Three recombinant PK-15 cell strains targeting the 3′-NTR of CSFV were obtained. The interferences of CSFV replication were researched in the recombinant PK-15 cells infected with CSFV.
Vaccination-based strategies are the main ways for prevention and control CSF at present. The riskes of virus variation and virulence increasing with the CSFV attenuated vaccine are widely used at present. Some people suspected largely to use attenuated vaccine are the one of reasons of more cases of atypical CSF are found in swine population. It needs to develop more new typic vaccines to control CSF. A suicide DNA vaccine of CSFV E2 gene and a recombinant fowlpox virus harboring E0 and E2 genes of CSFV were constructed (FPV-pSY-E0-E2) by analysis of the advances of CSFV enginerred vaccine researches. The preliminary immune effect of the constrcted vaccines were assessed in this study.
Attenuated vaccines of CSF are widely used in the swine population in some countries. It is difficult to effectively differentiate the pigs or their products infected with CSFV wild virus or vaccine virus just by determining the CSF antibody in swine sera. This would has an influence on the trade of pigs and their products. A novel RT-PCR was developed for detecting and differentiating the wild and vaccine types of CSFV. The results that the specificity of developed RT-PCR was high and could be used to differentiate the CSFV infected pigs and vaccinated pigs.
1. Three short hairpin RNAs (shRNAs) of single-stranded nucleotides target to the sequences at 115-132,137-154 and 210-228 of CSFV shimen strain 3′-NTR mRNA were designed according the sequences analysis respectively. The double-stranded shRNAs were formed by annealing and inserted into plasmid expressing vectors. Three recombinant plasmids expressing the shRNAs target to 3′-NTR of CSFV were obtained. There positive PK-15 cell lines target to 3′-NTR shRNA were developed by transfecting PK-15cells with recombinant plasmid respectively. The positive cells (P1, P2, P3) were infected with CSFV shimen virus respectively. The detection results showed that all of the three cell lines could interfere with proliferation of CSFV at transcriptional and protein level.
2. RT-PCRs were developed for detecting and differentiating the wild CSFV from vaccine virus. A 187 bp PCR fragment response to CSFV Shimen strain and a 492 bp fragment response to vaccine virus were obntained using this PCR. No PCR products could be amplified in PCV2, TGEV and PRRSV, showed the designed primers were speicific to CSFV. The RT-PCRs can be used to detect and differentiate the wild from vaccine CSFV in the field.
3. The suicidal vectored DNA vaccine (pSCA1-E2) was constructed by inserting E2 gene of CSFV into pSCA1 plasmid vector. E2 protein was determined in PK15 cells transfected with purified pSCA1-E2.The stimulation index (SI) of T lymphoid cell of mice were significant different between the experimental groups and the control group at10d (p<0.05), 20d and 30d (p<0.01) after the second immunization. The anti-CSFV serum antibodies could be screened in experimental pigs at 21d after the second immunization with pSCA1-E2.
4. The E0 and E2 genes of CSFV as well as a LacZ reporter gene were all inserted into a non essential gene of fowlpox virus (FV282) by homologous recombinant and the recombinant fowlpox virus FV282-E0-E2 was constructed. The experimental mice and pigs immunized with FV282-E0-E2 could induce a high anti-CSFV antibody level, also showed that the E0 and E2 genes were expressed in experimental animals. Partial protective immunity was observed in the experimental pigs immunized with FV282-E0-E2 when they challenged with the highly virulence CSFV, 75% (6/8) immunoprotection was observed.
目前CSF防控上采取的是以疫苗免疫接种为主的策略,弱毒疫苗的大范围使用,使病毒变异、毒力返强的风险增高,当前临床上“非典型”CSF的大量出现,被怀疑与弱毒疫苗的大量使用有关,新型CSF疫苗的研制是防控CSF的需要;在分析国内外有关猪瘟基因工程疫苗研究进展的基础上,构建了表达CSFV Shimen株E2基因的“自杀性”DNA疫苗和同时表达CSFV Shimen株E0和E2基因的重组鸡痘病毒,对构建的疫苗进行了初步评价。
CSF弱毒疫苗的大量使用,使在生产中难以有效地用血清学方法区分野毒感染猪和疫苗免疫猪,对生猪及其产品的贸易造成严重影响;针对这个问题,在借鉴已有研究的基础上,建立了区分CSFV强/弱毒株的RT-PCR方法,通过对临床病料的检测表明,该方法的特异性较强,可用于临床上对CSFV野毒感染和弱毒疫苗免疫猪的鉴别检测。
本研究取得以下结果:
1.通过对CSFV Shimen株3′-NTR核苷酸序列的分析,分别设计了靶向CSFV Shimen株3′-NTR 115～132位、137～154位和210～228位mRNA的短发夹RNA(shRNA)的单链核苷酸,经退火后形成双链shRNA,将其分别与siRNA表达质粒载体连接,构建了编码CSFV Shimen株3′-NTR 3个位点shRNA的表达质粒载体;转染细胞后筛选获得了稳定转录靶向CSFV Shimen株3′-NTR shRNA PK-15细胞株(P-1、P-2和P-3);接毒试验表明,细胞株P-1、P-2及P-3在转录水平和蛋白表达水平上均可干扰CSFV Shimen株在感染细胞中的增殖(P<0.05)。
2.建立了可鉴别CSFV强毒和弱毒的RT-PCR方法。在CSFV强毒中可扩增得到187 bp的片段,弱毒可获得492 bp的片段;在对PCV2、TGEV、PRRSV等猪源病毒的检测,均未有PCR产物出现,说明方法的特异性较高。对临床疑是CSF病料的检测证明,建立的方法可有效地区分CSFV野毒感染猪和弱毒疫苗免疫猪,本方法可用于临床上对CSFV强弱毒的鉴别检测。
3.构建了CSFV E2基因“自杀性”DNA疫苗表达质粒载体PSCA1-E2。在转染细胞中可检测到E2蛋白的表达;免疫小鼠的脾淋巴细胞增殖刺激指数(SI)在二免后10d与对照组差异显著(p<0.05),20d和30d差异极显著(p<0.01);试验猪二免后21d能检测到抗CSFV特异性血清抗体,说明构建的表达CSFV E2基因的“自杀性”DNA疫苗能诱导试验动物的免疫反应。
4.将CSFV E0和E2基因及报告基因LacZ插入到鸡痘病毒FV282的复制非必需区,经同源重组构建了一株含CSFV E0和E2基因的重组鸡痘病毒(FV282-E0-E2)。用重组鸡痘病毒免疫接种试验小鼠和试验猪,检测发现,免疫动物均能产生特异性抗体,说明E0和E2基因在机体细胞内得以有效表达,并能激发机体产生免疫反应;对免疫猪的攻毒试验表明,重组鸡痘病毒免疫猪能获得部分的免疫保护,抵抗CSFV强毒的攻击,免疫保护率为75%(6/8)。
Classical swine fever (CSF) is a serious contagious disease of pig caused by classical swine fever virus (CSFV). CSFV is a single-strain positive RNAvirus, the 3′-nontranslation region(3′-NTR)plays an important role in virus replication. Three sites in 3′-NTR of CSFV shimen strain were selected by sequence analysis to study the function of 3′-NTR on virus replication. Three recombinant plasmids expressing the shRNAs target to 3′-NTR of CSFV shimen strain were constructed and tranfected into PK-15 cells. Three recombinant PK-15 cell strains targeting the 3′-NTR of CSFV were obtained. The interferences of CSFV replication were researched in the recombinant PK-15 cells infected with CSFV.
Vaccination-based strategies are the main ways for prevention and control CSF at present. The riskes of virus variation and virulence increasing with the CSFV attenuated vaccine are widely used at present. Some people suspected largely to use attenuated vaccine are the one of reasons of more cases of atypical CSF are found in swine population. It needs to develop more new typic vaccines to control CSF. A suicide DNA vaccine of CSFV E2 gene and a recombinant fowlpox virus harboring E0 and E2 genes of CSFV were constructed (FPV-pSY-E0-E2) by analysis of the advances of CSFV enginerred vaccine researches. The preliminary immune effect of the constrcted vaccines were assessed in this study.
Attenuated vaccines of CSF are widely used in the swine population in some countries. It is difficult to effectively differentiate the pigs or their products infected with CSFV wild virus or vaccine virus just by determining the CSF antibody in swine sera. This would has an influence on the trade of pigs and their products. A novel RT-PCR was developed for detecting and differentiating the wild and vaccine types of CSFV. The results that the specificity of developed RT-PCR was high and could be used to differentiate the CSFV infected pigs and vaccinated pigs.
1. Three short hairpin RNAs (shRNAs) of single-stranded nucleotides target to the sequences at 115-132,137-154 and 210-228 of CSFV shimen strain 3′-NTR mRNA were designed according the sequences analysis respectively. The double-stranded shRNAs were formed by annealing and inserted into plasmid expressing vectors. Three recombinant plasmids expressing the shRNAs target to 3′-NTR of CSFV were obtained. There positive PK-15 cell lines target to 3′-NTR shRNA were developed by transfecting PK-15cells with recombinant plasmid respectively. The positive cells (P1, P2, P3) were infected with CSFV shimen virus respectively. The detection results showed that all of the three cell lines could interfere with proliferation of CSFV at transcriptional and protein level.
2. RT-PCRs were developed for detecting and differentiating the wild CSFV from vaccine virus. A 187 bp PCR fragment response to CSFV Shimen strain and a 492 bp fragment response to vaccine virus were obntained using this PCR. No PCR products could be amplified in PCV2, TGEV and PRRSV, showed the designed primers were speicific to CSFV. The RT-PCRs can be used to detect and differentiate the wild from vaccine CSFV in the field.
3. The suicidal vectored DNA vaccine (pSCA1-E2) was constructed by inserting E2 gene of CSFV into pSCA1 plasmid vector. E2 protein was determined in PK15 cells transfected with purified pSCA1-E2.The stimulation index (SI) of T lymphoid cell of mice were significant different between the experimental groups and the control group at10d (p<0.05), 20d and 30d (p<0.01) after the second immunization. The anti-CSFV serum antibodies could be screened in experimental pigs at 21d after the second immunization with pSCA1-E2.
4. The E0 and E2 genes of CSFV as well as a LacZ reporter gene were all inserted into a non essential gene of fowlpox virus (FV282) by homologous recombinant and the recombinant fowlpox virus FV282-E0-E2 was constructed. The experimental mice and pigs immunized with FV282-E0-E2 could induce a high anti-CSFV antibody level, also showed that the E0 and E2 genes were expressed in experimental animals. Partial protective immunity was observed in the experimental pigs immunized with FV282-E0-E2 when they challenged with the highly virulence CSFV, 75% (6/8) immunoprotection was observed.
引文
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