核衣壳靶向灭活策略筛选抗猪2型圆环病毒基因的初步研究
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摘要
猪2型圆环病毒(porcine circovirus type 2, PCV2)是猪断奶后多系统衰竭综合征(postweaning multisystemic wasting syndrome, PMWS)的原发病原,并与猪皮炎肾病综合征(PNDS)、猪呼吸道疾病综合征(PRDC)、增生性坏死性肺炎(PNP)、新生仔猪先天性震颤(CT-AII)等的发生密切相关,这些疾病被统称为猪2型圆环病毒病。猪2型圆环病毒病已成为严重危害养猪生产的重要的免疫抑制性疫病,给养猪业造成了巨大的经济损失。
面对PCV2给养猪业造成的重大损失,目前市场上却没有相应的有效疫苗和药物。因而探索抗猪2型圆环病毒感染的新策略成为当今研究的热点。衣壳蛋白靶向灭活(capsid targeted viral inactivation ,CTVI)策略是近年来新兴的基于蛋白水平的抗病毒策略,与其他基于核酸水平的抗病毒策略(如RNAi,反义核酸,核酶等)相比具有稳定、特异、高效、可有效避免因突变引起的免疫逃逸等优点。CTVI策略的出现为抗猪2型圆环病毒感染提供了新的思路。
本研究主要探索了CTVI策略应用于抗猪2型圆环病毒感染的可行性,构建了衣壳蛋白靶向核酸酶融合基因结构,并对基因结构的抗病毒效果进行了检测。
从金黄色葡萄球菌中直接扩增获得了金黄色葡萄球菌核酸酶的编码序列,通过拼装获得了将该核酸酶置于PCV2核衣壳蛋白氨基端(JNPC)和羧基端(PNJC)的两种融合蛋白基因。以pET-20b(+)原核表达载体为骨架对融合基因进行原核表达,并对表达产物生物学活性进行了分析。结果表明构建的两个融合蛋白基因表达的蛋白分子量大小约47KD,与预期大小相一致。使用特异性抗体进行Western Blot分析显示蛋白产物为融合目的蛋白。表达产物具有降解核酸的核酸酶活性,能够对DNA进行切割。这些结果为进一步探讨猪2型圆环病毒衣壳蛋白靶向抗PCV2感染作用的研究奠定了基础。
以pIRESneo载体为骨架构建了JNPC、PNJC、Cap基因真核表达载体,通过细胞转染和G418筛选获得了稳定表达上述三个基因以及携带pIRESneo载体的细胞系。采用PCR,RT-PCR,Northern Blot、Western Blot、DNA消化实验对转基因细胞系和蛋白表达产物进行鉴定与检测。结果显示转基因细胞系中有外源基因整合,融合基因在mRNA水平表达水平较低,经验证主要由融合基因存在异常拼接所致,细胞中有融合蛋白表达,且融合蛋白产物具有良好的核酸酶活性。
上述四个转基因细胞系及未经转基因的细胞进行病毒传代实验,通过半定量PCR和荧光定量PCR检测各细胞系中PCV2基因组含量,分析抗病毒效果。结果表明,病毒传递10代后,两种检测方法中,JNPC转基因细胞系病毒滴度与对照组相比分别下降了约60.55%和53.46%,而其他组间病毒滴度非常接近。两种方法结果基本相符,说明了所得数据的准确性。这一结果证明了CTVI策略适用于PCV2的抗病毒基因的筛选,获得了具有一定抗病毒效果的基因结构JNPC。但与已有的关于CTVI策略在其他病毒中应用的研究结果相比还不够理想,抗病毒效率还比较低。
总之,本研究在细胞水平上证实了CTVI策略应用于抗猪2型圆环病毒感染的可行性。研究获得了抗PCV2感染的基因结构,为抗PCV2基因筛选及相关转基因猪新品种的培育奠定了基础。
Porcine circovirus type 2 (PCV2) is the primary pathogen of post-weaning pigs multisystemic wasting syndrome (PMWS), and is also intimately associated with Porcine dermatitis and nephropathy syndrome (PDNS), Porcine respiratory disease complex (PRDC), Proliferative and necrotizing Pneumonia (PNP), congenital tremor AII (CT-AII) and etc . All these diseases are called PCV2 associated disease. PCV2 associated disease, as a sort of severe immunosuppressive disease, has given rise enormous economic loss in pig industry.
In the face of the enormous loss in pig industry, there is no yet corresponding effective vaccine and druggery at the market. So it has become the present research hotspots to quest for new strategies in anti-PCV2 infection. Capsid targeted viral inactivation (CTVI) is a newly emerging protein-based antiviral strategy. Compared with other nucleic-acid-based antiviral strategies, CTVI is more steady, specific and can efficiently avoid the immune escape resulted by pathogen mutation. The emergence of CTVI provided a new train of thought in confronting PCV2 infection.
To explore the feasibility of CTVI in anti-PCV2, we constructed the capsid targeted nucleases fusion gene and examined its antiviral effect.
We directly amplified and obtained the coding sequence of staphylococcal nuclease from staphylococcus aureus; and got two kinds of capsid protein fusion gene via recombination, one of which has staphylococcal nuclease gene at amino terminal (JNPC), the other one at carboxyl terminal(PNJC). The fusion gene has been expressed in E. coli. using pET-20b(+) as expression vector, and the nuclease activity of the fusion protein has been examined. Results showed that the molecular masses of two constructed fusion proteins are both about 47KD, just as expected. Western Blot analysis with specific antibodies showed that the products are target fusion proteins. The products have the nuclease activity, capable of cutting and degrading nucleic acid. These results laid the foundation for further investigating the effect of CTVI strategy on anti-PCV2 infection.
The eukaryotic expression cassette for JNPC, PNJC and Cap gene respectively had been constructed using pIRESneo as the vector. After cell transfection and G418 screening, steadly expression PK15 cell lines for the three genes mentioned above had been obtained (pIRESneo transgenic cell line as negative control). Then the integration and expression status of foreign genes in these transgenic cell lines had been anlaysed through PCR, RT-PCR, Northern Blot and Western Blot which showed that the steadly expression cell line for the genes mentioned above had been obtained and the fousion protein genes being expressed at a relatively lower level for abnormal splicing of the fusion genes. The fusion proteins can be detected by Western Blot and they show good nuclease activity in DNA digestion test. All these indicate that these cell lines can be used in the next experiment to verify the feasibility of CTVI in anti-PCV2.
The four cell lines mentioned above and the untransgenic cells all experienced the virus passage experiment, then the PCV2 genome content had been measured for the anti-viral effect analysis, which showed that after passage of 10 generations, compared with the control group, the virus titer of JNPC transgenic cell line reduced about 60.55℅ and 53.46℅, respectively via semi-quantitative PCR and fluorescence quantitative PCR, with the virus titers between other control groups extremely approximate. The good accordance of the two quantitative PCR results has confirmed the accuracy of the data. This result preliminarily bore out the applicability of CTVI strategy in screening the PCV2 resistant genes. We obtained the capsid protein fusion gene JNPC with a degree of antiviral effect. But compared with other applications of CTVI in virology, this result is not desirable enough yet. The antiviral efficiency is relatively lower.
In conclusion, we have borne out the applicability of CTVI strategy in treatment of PCV2 infection and laid the foundation for screening PCV2 resistant gene and subsequentaly breeding anti-PCV2 transgenic pigs.
面对PCV2给养猪业造成的重大损失,目前市场上却没有相应的有效疫苗和药物。因而探索抗猪2型圆环病毒感染的新策略成为当今研究的热点。衣壳蛋白靶向灭活(capsid targeted viral inactivation ,CTVI)策略是近年来新兴的基于蛋白水平的抗病毒策略,与其他基于核酸水平的抗病毒策略(如RNAi,反义核酸,核酶等)相比具有稳定、特异、高效、可有效避免因突变引起的免疫逃逸等优点。CTVI策略的出现为抗猪2型圆环病毒感染提供了新的思路。
本研究主要探索了CTVI策略应用于抗猪2型圆环病毒感染的可行性,构建了衣壳蛋白靶向核酸酶融合基因结构,并对基因结构的抗病毒效果进行了检测。
从金黄色葡萄球菌中直接扩增获得了金黄色葡萄球菌核酸酶的编码序列,通过拼装获得了将该核酸酶置于PCV2核衣壳蛋白氨基端(JNPC)和羧基端(PNJC)的两种融合蛋白基因。以pET-20b(+)原核表达载体为骨架对融合基因进行原核表达,并对表达产物生物学活性进行了分析。结果表明构建的两个融合蛋白基因表达的蛋白分子量大小约47KD,与预期大小相一致。使用特异性抗体进行Western Blot分析显示蛋白产物为融合目的蛋白。表达产物具有降解核酸的核酸酶活性,能够对DNA进行切割。这些结果为进一步探讨猪2型圆环病毒衣壳蛋白靶向抗PCV2感染作用的研究奠定了基础。
以pIRESneo载体为骨架构建了JNPC、PNJC、Cap基因真核表达载体,通过细胞转染和G418筛选获得了稳定表达上述三个基因以及携带pIRESneo载体的细胞系。采用PCR,RT-PCR,Northern Blot、Western Blot、DNA消化实验对转基因细胞系和蛋白表达产物进行鉴定与检测。结果显示转基因细胞系中有外源基因整合,融合基因在mRNA水平表达水平较低,经验证主要由融合基因存在异常拼接所致,细胞中有融合蛋白表达,且融合蛋白产物具有良好的核酸酶活性。
上述四个转基因细胞系及未经转基因的细胞进行病毒传代实验,通过半定量PCR和荧光定量PCR检测各细胞系中PCV2基因组含量,分析抗病毒效果。结果表明,病毒传递10代后,两种检测方法中,JNPC转基因细胞系病毒滴度与对照组相比分别下降了约60.55%和53.46%,而其他组间病毒滴度非常接近。两种方法结果基本相符,说明了所得数据的准确性。这一结果证明了CTVI策略适用于PCV2的抗病毒基因的筛选,获得了具有一定抗病毒效果的基因结构JNPC。但与已有的关于CTVI策略在其他病毒中应用的研究结果相比还不够理想,抗病毒效率还比较低。
总之,本研究在细胞水平上证实了CTVI策略应用于抗猪2型圆环病毒感染的可行性。研究获得了抗PCV2感染的基因结构,为抗PCV2基因筛选及相关转基因猪新品种的培育奠定了基础。
Porcine circovirus type 2 (PCV2) is the primary pathogen of post-weaning pigs multisystemic wasting syndrome (PMWS), and is also intimately associated with Porcine dermatitis and nephropathy syndrome (PDNS), Porcine respiratory disease complex (PRDC), Proliferative and necrotizing Pneumonia (PNP), congenital tremor AII (CT-AII) and etc . All these diseases are called PCV2 associated disease. PCV2 associated disease, as a sort of severe immunosuppressive disease, has given rise enormous economic loss in pig industry.
In the face of the enormous loss in pig industry, there is no yet corresponding effective vaccine and druggery at the market. So it has become the present research hotspots to quest for new strategies in anti-PCV2 infection. Capsid targeted viral inactivation (CTVI) is a newly emerging protein-based antiviral strategy. Compared with other nucleic-acid-based antiviral strategies, CTVI is more steady, specific and can efficiently avoid the immune escape resulted by pathogen mutation. The emergence of CTVI provided a new train of thought in confronting PCV2 infection.
To explore the feasibility of CTVI in anti-PCV2, we constructed the capsid targeted nucleases fusion gene and examined its antiviral effect.
We directly amplified and obtained the coding sequence of staphylococcal nuclease from staphylococcus aureus; and got two kinds of capsid protein fusion gene via recombination, one of which has staphylococcal nuclease gene at amino terminal (JNPC), the other one at carboxyl terminal(PNJC). The fusion gene has been expressed in E. coli. using pET-20b(+) as expression vector, and the nuclease activity of the fusion protein has been examined. Results showed that the molecular masses of two constructed fusion proteins are both about 47KD, just as expected. Western Blot analysis with specific antibodies showed that the products are target fusion proteins. The products have the nuclease activity, capable of cutting and degrading nucleic acid. These results laid the foundation for further investigating the effect of CTVI strategy on anti-PCV2 infection.
The eukaryotic expression cassette for JNPC, PNJC and Cap gene respectively had been constructed using pIRESneo as the vector. After cell transfection and G418 screening, steadly expression PK15 cell lines for the three genes mentioned above had been obtained (pIRESneo transgenic cell line as negative control). Then the integration and expression status of foreign genes in these transgenic cell lines had been anlaysed through PCR, RT-PCR, Northern Blot and Western Blot which showed that the steadly expression cell line for the genes mentioned above had been obtained and the fousion protein genes being expressed at a relatively lower level for abnormal splicing of the fusion genes. The fusion proteins can be detected by Western Blot and they show good nuclease activity in DNA digestion test. All these indicate that these cell lines can be used in the next experiment to verify the feasibility of CTVI in anti-PCV2.
The four cell lines mentioned above and the untransgenic cells all experienced the virus passage experiment, then the PCV2 genome content had been measured for the anti-viral effect analysis, which showed that after passage of 10 generations, compared with the control group, the virus titer of JNPC transgenic cell line reduced about 60.55℅ and 53.46℅, respectively via semi-quantitative PCR and fluorescence quantitative PCR, with the virus titers between other control groups extremely approximate. The good accordance of the two quantitative PCR results has confirmed the accuracy of the data. This result preliminarily bore out the applicability of CTVI strategy in screening the PCV2 resistant genes. We obtained the capsid protein fusion gene JNPC with a degree of antiviral effect. But compared with other applications of CTVI in virology, this result is not desirable enough yet. The antiviral efficiency is relatively lower.
In conclusion, we have borne out the applicability of CTVI strategy in treatment of PCV2 infection and laid the foundation for screening PCV2 resistant gene and subsequentaly breeding anti-PCV2 transgenic pigs.
引文
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