猪繁殖与呼吸综合征病毒多基因重组真核表达质粒的构建及免疫学研究
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摘要
本研究试图应用分子生物学试验方法构建表达猪繁殖与呼吸综合征病毒ORF3、ORF5、ORF6基因的真核重组质粒,并进行小鼠的免疫试验,以探讨所构建核酸疫苗的免疫保护作用。试验由3部分构成:
1.采用RT-PCR方法克隆得到猪繁殖与呼吸综合征病毒ORF3、ORF5的基因序列,将ORF3和ORF5基因在克隆载体pMD18-T上串联后,将其定向插入真核表达质粒pcDNA4.0多克隆位点,琼脂糖电泳和测序表明成功构建包含猪繁殖与呼吸综合征病毒ORF3、ORF5基因的重组质粒pcDNA4.0-ORF3-ORF5。
2.将ORF6和ORF3-ORF5基因串联后,定向克隆到真核表达质粒pcDNA4.0上,构建了重组真核表达质粒pcDNA4.0-ORF6-ORF3-ORF5。序列测定证明,重组质粒pcDNA4.0-ORF6-ORF3-ORF5包含完整的ORF6、ORF3和ORF5基因,从而为其免疫试验的研究奠定了基础。
3.重组真核表达质粒pcDNA4.0-ORF3-ORF5、pcDNA4.0-ORF6-ORF3-ORF5经转化、提取和纯化后,小鼠后肢胫前肌注射100μg/只(50μg/腿),共2次,间隔2周。每隔7 d采血1次,检测ELISA抗体效价和血清中和抗体水平。并分不同时间段采集组织样品,通过PCR检测pcDNA4.0-ORF6-ORF3-ORF5质粒在小鼠体内的动态分布情况。结果显示,血清抗体水平首免后逐渐增高,2免后14～21 d逐渐达到高峰,且重组质粒组产生的抗体水平明显高于空载体对照组。重组质粒免疫组小鼠于2免后第7 d就能检测到中和抗体,随后快速上升,于第42 d达到最高。说明所构建的重组质粒能够诱导小鼠产生特异性免疫反应。重组真核表达质粒pcDNA4.0-ORF6-ORF3-ORF5肌注小鼠后7 d可在肌肉、心、血液、肝、脾、肺、肾及脑组织中检测到,在血液、心肌中可存留84 d,注射部位肌肉存留时间最长,98 d时仍可检测到。
In order to explore new method to control the porcine reproductive and respiratory syndrome virus(PRRSV), the recombinant plasmid was constructed containing the ORF3、ORF5 and ORF6 of the porcine reproductive and respiratory syndrome virus(PRRSV) envelope protein genes and test the immunoreaction on mice. This study consists of three parts:
1. The reverse transcription and polymerase chain reaction(RT-PCR) was used to amplify PRRSV ORF3、ORF5 genes. The RT-PCR products were cloned into pMD18-T in series, and were ligated into the eukaryotic expression vector pcDNA4.0. Then the recombinant plasmid pcDNA4.0-ORF3-ORF5 was obtained.
2. The ORF6 were connected in series with ORF3-ORF5, and were cloned into the eukaryotic expression vector pcDNA4.0 to construct recombinant plasmid pcDNA4.0-ORF6 -ORF3-ORF5. Then the recombinant plasmid pcDNA4.0-ORF6- ORF3-ORF5 was obtained.
3. Then, 10 kunming mice were inoculated with the purified pcDNA4.0-ORF3-ORF5 and another with pcDNA4.0-ORF6-ORF3-ORF5 into hind legs tibial muscle(50μg per leg) by intramuscular injection, and were boosted on week 2 post-inoculation. The levels of serum antibodies and neutralizing antibodies were detected in peripheral bloods of the inoculated mice or the boosted mice every 7 days post-inoculation or post-booster-inoculation by indirect ELISA. Then ORF6-ORF3-ORF5 gene was detected by PCR from heart, brain, blood, liver, lung, spleen, kidney and muscle of mice and piglets different time post immunization (PI). The results showed that the expressed envelope protein from the plasmid could induce to produce high levels of serum antibodies and neutralizing antibodies. Recombinant plasmid pcDNA4.0-ORF6-ORF3-ORF5 was detected in the brain 7d-42d PI, in the liver, lung, spleen, kidney from 7 d to 70 d PI, in the blood and heart 7d to 84d PI, and was detected from the muscle till 98d PI.
1.采用RT-PCR方法克隆得到猪繁殖与呼吸综合征病毒ORF3、ORF5的基因序列,将ORF3和ORF5基因在克隆载体pMD18-T上串联后,将其定向插入真核表达质粒pcDNA4.0多克隆位点,琼脂糖电泳和测序表明成功构建包含猪繁殖与呼吸综合征病毒ORF3、ORF5基因的重组质粒pcDNA4.0-ORF3-ORF5。
2.将ORF6和ORF3-ORF5基因串联后,定向克隆到真核表达质粒pcDNA4.0上,构建了重组真核表达质粒pcDNA4.0-ORF6-ORF3-ORF5。序列测定证明,重组质粒pcDNA4.0-ORF6-ORF3-ORF5包含完整的ORF6、ORF3和ORF5基因,从而为其免疫试验的研究奠定了基础。
3.重组真核表达质粒pcDNA4.0-ORF3-ORF5、pcDNA4.0-ORF6-ORF3-ORF5经转化、提取和纯化后,小鼠后肢胫前肌注射100μg/只(50μg/腿),共2次,间隔2周。每隔7 d采血1次,检测ELISA抗体效价和血清中和抗体水平。并分不同时间段采集组织样品,通过PCR检测pcDNA4.0-ORF6-ORF3-ORF5质粒在小鼠体内的动态分布情况。结果显示,血清抗体水平首免后逐渐增高,2免后14～21 d逐渐达到高峰,且重组质粒组产生的抗体水平明显高于空载体对照组。重组质粒免疫组小鼠于2免后第7 d就能检测到中和抗体,随后快速上升,于第42 d达到最高。说明所构建的重组质粒能够诱导小鼠产生特异性免疫反应。重组真核表达质粒pcDNA4.0-ORF6-ORF3-ORF5肌注小鼠后7 d可在肌肉、心、血液、肝、脾、肺、肾及脑组织中检测到,在血液、心肌中可存留84 d,注射部位肌肉存留时间最长,98 d时仍可检测到。
In order to explore new method to control the porcine reproductive and respiratory syndrome virus(PRRSV), the recombinant plasmid was constructed containing the ORF3、ORF5 and ORF6 of the porcine reproductive and respiratory syndrome virus(PRRSV) envelope protein genes and test the immunoreaction on mice. This study consists of three parts:
1. The reverse transcription and polymerase chain reaction(RT-PCR) was used to amplify PRRSV ORF3、ORF5 genes. The RT-PCR products were cloned into pMD18-T in series, and were ligated into the eukaryotic expression vector pcDNA4.0. Then the recombinant plasmid pcDNA4.0-ORF3-ORF5 was obtained.
2. The ORF6 were connected in series with ORF3-ORF5, and were cloned into the eukaryotic expression vector pcDNA4.0 to construct recombinant plasmid pcDNA4.0-ORF6 -ORF3-ORF5. Then the recombinant plasmid pcDNA4.0-ORF6- ORF3-ORF5 was obtained.
3. Then, 10 kunming mice were inoculated with the purified pcDNA4.0-ORF3-ORF5 and another with pcDNA4.0-ORF6-ORF3-ORF5 into hind legs tibial muscle(50μg per leg) by intramuscular injection, and were boosted on week 2 post-inoculation. The levels of serum antibodies and neutralizing antibodies were detected in peripheral bloods of the inoculated mice or the boosted mice every 7 days post-inoculation or post-booster-inoculation by indirect ELISA. Then ORF6-ORF3-ORF5 gene was detected by PCR from heart, brain, blood, liver, lung, spleen, kidney and muscle of mice and piglets different time post immunization (PI). The results showed that the expressed envelope protein from the plasmid could induce to produce high levels of serum antibodies and neutralizing antibodies. Recombinant plasmid pcDNA4.0-ORF6-ORF3-ORF5 was detected in the brain 7d-42d PI, in the liver, lung, spleen, kidney from 7 d to 70 d PI, in the blood and heart 7d to 84d PI, and was detected from the muscle till 98d PI.
引文
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