猪瘟病毒石门株E2基因在永生化猪血管内皮细胞的表达
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摘要
猪瘟(Classical swine fever, CSF)是由猪瘟病毒(Classical swine fever virus, CSFV)引起的猪的急性高度接触性传染病,严重危害着世界养猪业的发展,被世界动物卫生组织(OIE)列为法定必须报告的传染病。CSFV为有囊膜的RNA病毒,病毒基因组仅有一个大的开放阅读框(ORF),编码E0(或Erns)、E1和E2 3种囊膜糖蛋白,其中E2基因是猪瘟病毒的主要保护性抗原,可以有效刺激机体产生中和抗体,抵抗CSFV强毒株的攻击。由于传统弱毒活疫苗接种后无法在血清学诊断上与自然感染强毒株相区别,弱毒活苗还能通过母体胎盘的感染造成死胎、弱仔、先天性感染仔猪,导致免疫耐受,而基因工程疫苗的出现为这一问题的解决提供了新的思路。因此,本试验选取E2基因在逆转录病毒的介导下转染永生化猪脐血管内皮细胞(swine umbilical vein endothelial cells, SUVECs),并表达在该细胞膜上,初步构建基因工程疫苗。且本试验所选用的永生化猪脐血管内皮细胞为一种生理状态下的细胞系,不具有致瘤性,不存在生物安全方面的问题,这在研究和开发猪瘟基因工程细胞苗方面具有相当大的潜力。本试验获得了以下结果:
1.重组逆转录病毒载体pBABE-puro-E2的构建。RT-PCR扩增猪瘟病毒石门株E2基因,将其连接到重组逆转录病毒载体pBABE-puro中,经PCR扩增和酶切鉴定,证明E2基因成功插入重组逆转录病毒载体pBABE-puro中,重组载体构建成功。聚乙二醇法提取和纯化质粒,以备包装假病毒用。
2.假病毒的包装。构建好的重组逆转录病毒载体pBABE-puro-E2与pVSV-G质粒经磷酸钙共转染法,转入293GP细胞中包装成逆转录病毒假病毒,36～48 h后收取上清,分装备用。
3.表达E2基因阳性细胞的筛选和初步免疫试验。包装好的假病毒转入永生化猪脐血管内皮细胞,嘌呤霉素筛选出阳性细胞,对筛选出的阳性细胞进行流式细胞分析和分选;经间接免疫荧光染色试验和RT-PCR鉴定,猪瘟病毒石门株E2基因在永生化猪脐血管内皮细胞膜上表达成功。将表达E2蛋白的永生化的猪脐血管内皮细胞作为基因工程细胞苗腹腔免疫4周龄的Balb/c小鼠,免疫3次后,用ELISA法检测免疫鼠的血清,小鼠产生了抗猪瘟病毒E2蛋白的抗体,效价达1∶3 500。
Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly contagious vital disease of pigs. It is harmful to the global pig farmings and is listeded among type-A diseases of Office International des Epizooties(OIE).
The genome of CSFV consists of a single-stranded positive RNA of about 12.3 kb, enco- ding three envelope glyprotein, i.e. E2, E0(Erns)and E1. E2 is the major protective antigen of the virion. It can stimulate organism to produce neutralizing antibody to resist CSFV velogenic strain aggression. Because it is impossible to discriminate between immunization with conventional vaccination and natural infection on serodiagnosis, and attenuated vaccines can through placenta to results in fetal death、weak piglet、congenitally infected pigs which can induce immunotolerance. But genetically engineering vaccine is a new method to solve these problems. So, the paper chose E2 as target gene to be expressed on immortality swine vein endothelial cells (SUVEC), to construct initial genetically engineering vaccine. And it has suitable developing potential on application, because the SUVEC is the physiological cell line without oncogenicity. The genetically engineering vaccines have no problem on biosafty. Main research contents include:
1. The construction of recombinant retroviral vector pBABE-puro-E2. By a series of molecular biological methods, E2 gene of CSFV Shimen strain was inserted into retroviral vector pBABE-puro named pBABE-puro-E2. And the result of enzyme digestion and PCR identification showed the construction was successful. So, the recombinant plasmid was extracted with alkaline lysis method and purified with PEG 8 000, for the next transfection to procure retrovirus pseudovirion.
2. Packing retrovirus pseudovirion. The recombinant plasmid pBABE-puro-E2 and the pVSV-G plasmid were transferred into 293GP cells by calcium phosphate cotransfection, and procured the packing retrovirus pseudovirion.
3. Selected the positive cells of expressing E2 protein and rudiment immunity test. Packing retrovirus pseudovirion infected the SUVEC and the positive cells were gotten by optimal concentration of puromycin, then the cells were analyzed by flow cytometry. Identified by indirect immunofluorescence test and RT-PCR, the CSFV shimen strain of E2 proteins were successfully expressed on SUVEC. The positive SUVEC as a gene engineering vaccine were inoculated into the Balb/c mice by intraperitoneal injection. After the third immunization, the mice were killed to collected sera and detected for anti-CSFV antibodies by ELISA. The result indicated that the sera of mice contained anti-CSFV antibodies and the valence of antibody is 1:3 500.
1.重组逆转录病毒载体pBABE-puro-E2的构建。RT-PCR扩增猪瘟病毒石门株E2基因,将其连接到重组逆转录病毒载体pBABE-puro中,经PCR扩增和酶切鉴定,证明E2基因成功插入重组逆转录病毒载体pBABE-puro中,重组载体构建成功。聚乙二醇法提取和纯化质粒,以备包装假病毒用。
2.假病毒的包装。构建好的重组逆转录病毒载体pBABE-puro-E2与pVSV-G质粒经磷酸钙共转染法,转入293GP细胞中包装成逆转录病毒假病毒,36～48 h后收取上清,分装备用。
3.表达E2基因阳性细胞的筛选和初步免疫试验。包装好的假病毒转入永生化猪脐血管内皮细胞,嘌呤霉素筛选出阳性细胞,对筛选出的阳性细胞进行流式细胞分析和分选;经间接免疫荧光染色试验和RT-PCR鉴定,猪瘟病毒石门株E2基因在永生化猪脐血管内皮细胞膜上表达成功。将表达E2蛋白的永生化的猪脐血管内皮细胞作为基因工程细胞苗腹腔免疫4周龄的Balb/c小鼠,免疫3次后,用ELISA法检测免疫鼠的血清,小鼠产生了抗猪瘟病毒E2蛋白的抗体,效价达1∶3 500。
Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly contagious vital disease of pigs. It is harmful to the global pig farmings and is listeded among type-A diseases of Office International des Epizooties(OIE).
The genome of CSFV consists of a single-stranded positive RNA of about 12.3 kb, enco- ding three envelope glyprotein, i.e. E2, E0(Erns)and E1. E2 is the major protective antigen of the virion. It can stimulate organism to produce neutralizing antibody to resist CSFV velogenic strain aggression. Because it is impossible to discriminate between immunization with conventional vaccination and natural infection on serodiagnosis, and attenuated vaccines can through placenta to results in fetal death、weak piglet、congenitally infected pigs which can induce immunotolerance. But genetically engineering vaccine is a new method to solve these problems. So, the paper chose E2 as target gene to be expressed on immortality swine vein endothelial cells (SUVEC), to construct initial genetically engineering vaccine. And it has suitable developing potential on application, because the SUVEC is the physiological cell line without oncogenicity. The genetically engineering vaccines have no problem on biosafty. Main research contents include:
1. The construction of recombinant retroviral vector pBABE-puro-E2. By a series of molecular biological methods, E2 gene of CSFV Shimen strain was inserted into retroviral vector pBABE-puro named pBABE-puro-E2. And the result of enzyme digestion and PCR identification showed the construction was successful. So, the recombinant plasmid was extracted with alkaline lysis method and purified with PEG 8 000, for the next transfection to procure retrovirus pseudovirion.
2. Packing retrovirus pseudovirion. The recombinant plasmid pBABE-puro-E2 and the pVSV-G plasmid were transferred into 293GP cells by calcium phosphate cotransfection, and procured the packing retrovirus pseudovirion.
3. Selected the positive cells of expressing E2 protein and rudiment immunity test. Packing retrovirus pseudovirion infected the SUVEC and the positive cells were gotten by optimal concentration of puromycin, then the cells were analyzed by flow cytometry. Identified by indirect immunofluorescence test and RT-PCR, the CSFV shimen strain of E2 proteins were successfully expressed on SUVEC. The positive SUVEC as a gene engineering vaccine were inoculated into the Balb/c mice by intraperitoneal injection. After the third immunization, the mice were killed to collected sera and detected for anti-CSFV antibodies by ELISA. The result indicated that the sera of mice contained anti-CSFV antibodies and the valence of antibody is 1:3 500.
引文
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